CN111363038A - anti-VEGF single domain antibody, humanization thereof, fusion protein constructed by single domain antibody and IgG1-Fc and application - Google Patents

Abstract

The invention discloses an anti-VEGF single domain antibody, a humanization method thereof, a fusion protein constructed by the single domain antibody and IgG1-Fc, and application thereof. The invention screens and obtains a group of VEGF single-domain antibodies, which have high activity and stronger neutralizing or binding capacity. The group of single-domain antibodies can specifically bind to human VEGF165 and VEGF121 antigens, bind to tumor cell strains expressing VEGF on the cell surface, and effectively block the combination of the VEGF antigens and VEGFR and generate corresponding signal cascade effects. The invention further carries out humanized modification on the single domain antibody to obtain the humanized antibody with improved affinity. The invention also discloses a fusion protein obtained by constructing the single-domain antibody or the humanized single-domain antibody and IgG-Fc. The humanized single domain antibody and/or the fusion protein can be used for detecting or diagnosing VEGF, blocking the interaction between VEGF and VEGFR and treating diseases related to abnormal expression of VEGF.

Description

anti-VEGF single domain antibody, humanization thereof, fusion protein constructed by single domain antibody and IgG1-Fc and application

Technical Field

The invention relates to a single domain antibody, in particular to a single domain antibody for resisting VEGF and a fusion protein constructed by fusing the single domain antibody or a humanized single domain antibody with IgG1-Fc, and further relates to applications of the single domain antibody or the humanized single domain antibody in detecting VEGF and treating diseases related to VEGF expression abnormality, belonging to the fields of the single domain antibody for resisting VEGF, the humanized single domain antibody and the application thereof.

Background

The Vascular Endothelial Growth Factor (VEGF) family is a multifunctional cytokine, has direct and indirect regulation and control effects in angiogenesis and lymphangiogenesis, and can promote Endothelial cell proliferation, angiogenesis and increase Vascular permeability. The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, etc., wherein the VEGF-A coding region, after transcription, splicing, etc., forms 5 monomers, which are named VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206, respectively, and contain 121, 145, 165, 189 and 206 amino acids, respectively, because of the number of amino acids contained in the monomers. Of the 8 exons, 1-5 are considered to be of great significance, because they can encode domains recognized by VEGF receptors, three monomers of VEGF121, VEGF145 and VEGF165 are secretory proteins and can be secreted from cytoplasm to outside cells. VEGF145 and VEGF165 can bind to KDR/flk-1 receptors of all endothelial cells. VEGF-B encodes VEGF-B mRNA that is spliced in a different manner, thus producing 2 different transcripts, which are designated VEGF-B167 and VEGF-B186, respectively, based on the number of amino acid residues they contain. The related receptor of VEGF-B is VEGFR-1/FLT-1, which can play a promoting role in the process of angiogenesis. VEGF-C, also known as related protein (VRP), is expressed in mRNA in human embryo and mature tissue, and most of adult VEGF-C is expressed in placenta, ovary, heart and gland except for small amount of VEGF-C expressed in brain, liver, thymus and peripheral blood leukocyte. VEGF-C is closely related to lymphatic vessels, including induction of lymphatic hyperplasia and effective regulation of lymphocyte endothelium, and tumors are usually first metastasized through lymphatic vessels, so VEGF-C is closely related to tumor metastasis. VEGF-C promotes differentiation and growth of blood vessels in developing embryos and thus acts earlier than other factors that promote angiogenesis. VEGF-C acts on VEGFR-2 and VEGFR-3 and induces endothelial cell proliferation, particularly microvascular cell proliferation. VEGF-D, the mRNA of which is expressed in humans at sites similar to VEGF-C, is expressed abundantly especially in skeletal muscle and colon, but rarely in placenta. VEGF-D has the function of promoting endothelial cell migration, can be used as ligands of VEGF receptors (VGEFRs), VEGFR-2(Flk-1) and VEGFR-3(Flt-4), and can play a role in promoting endothelial migration after being combined with VEGF-D, thereby promoting angiogenesis.

VEGF enhances microvascular permeability, which can lead to leakage of blood components, including fibrinogen and other coagulation proteins. When fibrin is accumulated outside blood vessels, the absorption and removal speed of intercellular edema liquid can be obviously slowed down, the environment around normal tissues is changed, the anti-angiogenesis function of the tissues is reduced, and the function of promoting angiogenesis is achieved. VEGF also increases vascular bed permeability of skin, pleura, peritoneum, mesentery, etc., and is a major cause of malignant pleural effusion and ascites. VEGF can cause cytoskeletal changes, thereby altering cell morphology. The release of nitric oxide and prostaglandins is an important way for VEGF to activate endothelial cells. Endothelial cells are activated and grow and migrate. VEGF, also an epithelial cytokinin, promotes mitosis in cells, and its principle may involve the protein kinase c pathway and the nitric oxide regulatory pathway. In the early stages of angiogenesis, the most critical changes are degradation of the basement membrane, enzymes and proteins required for the degradation process, most of which can be induced by VEGF. The matrix degradation metalloproteinases, the metalloproteinases interstitial collagenase and the serine proteinase are included, the substances are activated, the local microenvironment can be changed, and the migration condition suitable for endothelial cells is created. This increased receptor expression, which potentiates proteolytic action and tissue remodeling, also laterally demonstrates the angiogenic effect of VEGF. In addition, uPA itself can also induce increased VEGF expression, which forms an expression loop. While tumor growth requires a continuous blood supply, VEGF is essential in tumor growth because it promotes angiogenesis. VEGF, which is also an important factor mediating angiogenesis, strongly promotes the mitosis of vascular endothelial cells to form new blood vessels, has been considered as the most potent cytokine for promoting tumor angiogenesis and is essential for the neovascularization. VEGF-C induces almost the same number of vessels and lymphatic vessels, and endothelial penetration occurs only in capillaries, indicating that VEGF-C plays a role not only in promoting angiogenesis, but also in promoting lymphatic vessels. VEGF-C plays a certain role between tumor cells and lymphatic epithelial cells, so that the two cells are mutually regulated to promote the generation of new lymphatic vessels, the increase of the lymphatic vessels is that tumors are easier to metastasize in a plurality of solid tumors, and the malignancy degree of the tumors is positively correlated with the vascularization degree in tumor bodies. Overexpression of VEGF has been associated with a variety of neoplastic conditions and prognoses, including colon, breast, prostate, lung, and melanoma, among others. By blocking the VEGF signaling pathway, tumor growth and metastasis can be inhibited from both vascular and lymphatic vessels. VEGF has become an important therapeutic target for anti-angiogenesis.

The patent application WO9410202A discloses the Roche for the first time to screen the murine A4.6.1 antibody for VEGF, but the murine mAb has strong immunogenicity to human, may induce human anti-mouse antibody (HAMA) response, and is often rapidly cleared from the circulatory system of human. Humanized VEGF antibodies were constructed in WO9845331A and WO9845332A patent applications by combining 6 murine CDRs generated by A4.6.1 with human VLK1Cl light chain constant region and VH III-CH 1 heavy chain constant region. The obtained humanized anti-VEGF monoclonal antibody has similar affinity with VEGF to the original antibody. Bevacizumab of Rogowski is a recombinant humanized monoclonal antibody, has a molecular weight of about 149kD, and contains a framework region of a humanized antibody and a complementarity determining region of a murine antibody capable of binding VEGF. It can bind to VEGF and prevent it from binding to receptors on the surface of endothelial cells, thus blocking VEGF-induced endothelial cell expansion and neovascularization, reducing microangiogenesis and inhibiting the progression of metastatic lesions, and was approved by the U.S. Food and Drug Administration (FDA) in 2004 to become the first approved anti-tumor angiogenesis antibody drug to be marketed in the United states. FDA is currently approved for 6 indications, including metastatic colorectal cancer, non-small cell lung cancer, glioblastoma, metastatic renal cell carcinoma, cervical cancer, recurrent or metastatic cancer, recurrent epithelial ovarian cancer, fallopian tube cancer or primary peritoneal cancer. The product is approved by the national food and drug administration (CFDA) to enter China in 2010 and is named as avastin. In WO9845331A and WO9845332A patent applications, ranibizumab was prepared by fragmenting an anti-VEGF antibody and increasing its affinity by mutation for direct delivery of the drug into the eye. The ranibizumab is an antibody fragment of recombinant VEGF humanized monoclonal antibody IgG1, has no antibody Fc region and a molecular weight of about 48kD, treats wet age-related macular caused by excessive vascular proliferation, and is an antibody medicament for intraocular therapy. FDA approved for the treatment of age related macular in 2006, and FDA approved for the treatment of diabetic macular edema in 2012.

However, the monoclonal antibody has a complex structure, a long production period, high cost and high price. In 1993, Hamers-Casterman et al found that camel antibodies have a naturally deleted light chain, contain only a heavy chain, and the heavy chain lacks the CH1 region of the constant region, so that the variable region is directly linked to the hinge region, and are also called heavy chain antibodies. Cloning of the variable region of a heavy chain antibody yields a single domain antibody, called VHH antibody, consisting of only one heavy chain variable region (VHH). VHH crystals are 2.5nm in diameter and 4nm long, and therefore, also known as nanobodies (nanobodies), are the smallest naturally occurring fragment that can bind to an antigen. The nano antibody has an oval structure, is small in size, has molecular mass of 1/10 (about 15 kD) of the monoclonal antibody, has good stability and high affinity compared with a common antibody, overcomes the defects of a small-molecular functional antibody, has the advantages of small molecular mass, low immunogenicity, high tissue penetrating power, capability of being expressed in a large number of microorganisms such as saccharomycetes and escherichia coli, capability of large-scale production, easiness in popularization and application, low relative price and the like, and is not possessed by the monoclonal antibody and the polyclonal antibody, and is suitable for detection, disease treatment and the like. Therefore, the VEGF antibody developed by using the single domain antibody technology has wide application prospect.

Disclosure of Invention

One of the purposes of the invention is to provide a group of single domain antibodies of VEGF and coding genes thereof;

the other purpose of the invention is to carry out humanized transformation on the single domain antibody of anti-VEGF to obtain a humanized single domain antibody;

the third purpose of the invention is to fuse the single-domain antibody or humanized single-domain antibody with human IgG1-Fc to obtain a fusion protein;

the fourth purpose of the invention is to couple the single-domain antibody or the humanized single-domain antibody with one or more of enzyme phase, radioactive isotope, fluorescent compound or chemiluminescent compound to obtain a conjugate;

the fourth purpose of the invention is to apply the anti-VEGF single-domain antibody, the anti-VEGF humanized single-domain antibody, the fusion protein and the conjugate to the preparation of a reagent for detecting VEGF or the treatment of diseases related to abnormal expression of VEGF;

the above object of the present invention is achieved by the following technical solutions:

the invention firstly provides a single-domain antibody for resisting VEGF, wherein the single-domain antibody consists of a framework region and 3 complementarity determining regions; the single domain antibody is selected from any one of NBV1, NBV2, NBV3, NBV4, NBV5, NBV6, NBV7, NBV8, NBV9, NBV10, NBV11, NBV12, NBV13, NBV14, NBV15, NBV16, NB;

wherein, the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV1 are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV2 are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV3 are shown as SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV4 are shown as SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV5 are shown as SEQ ID No.13, SEQ ID No.14 and SEQ ID No.15 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV6 are shown as SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV7 are shown as SEQ ID No.19, SEQ ID No.20 and SEQ ID No.21 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV8 are shown as SEQ ID No.22, SEQ ID No.23 and SEQ ID No.24 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV9 are shown as SEQ ID No.25, SEQ ID No.26 and SEQ ID No.27 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV10 are shown as SEQ ID No.28, SEQ ID No.29 and SEQ ID No.30 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV11 are shown as SEQ ID No.31, SEQ ID No.32 and SEQ ID No.33 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV12 are shown as SEQ ID No.34, SEQ ID No.35 and SEQ ID No.36 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV13 are shown as SEQ ID No.37, SEQ ID No.38 and SEQ ID No.39 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV14 are shown as SEQ ID No.40, SEQ ID No.41 and SEQ ID No.42 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV15 are shown as SEQ ID No.43, SEQ ID No.44 and SEQ ID No.45 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV16 are shown as SEQ ID No.46, SEQ ID No.47 and SEQ ID No.48 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV17 are shown as SEQ ID No.49, SEQ ID No.50 and SEQ ID No.51 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV18 are shown as SEQ ID No.52, SEQ ID No.53 and SEQ ID No.54 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV19 are shown as SEQ ID No.55, SEQ ID No.56 and SEQ ID No.57 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV20 are shown as SEQ ID No.58, SEQ ID No.59 and SEQ ID No.60 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV21 are shown as SEQ ID No.61, SEQ ID No.62 and SEQ ID No.63 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV22 are shown as SEQ ID No.64, SEQ ID No.65 and SEQ ID No.66 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV23 are shown as SEQ ID No.67, SEQ ID No.68 and SEQ ID No.69 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV24 are shown as SEQ ID No.70, SEQ ID No.71 and SEQ ID No.72 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV25 are shown as SEQ ID No.73, SEQ ID No.74 and SEQ ID No.75 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV26 are shown as SEQ ID No.76, SEQ ID No.77 and SEQ ID No.78 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV27 are shown as SEQ ID No.79, SEQ ID No.80 and SEQ ID No.81 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV28 are shown as SEQ ID No.82, SEQ ID No.83 and SEQ ID No.84 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV29 are shown as SEQ ID No.85, SEQ ID No.86 and SEQ ID No.87 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV30 are respectively shown as SEQ ID No.88, SEQ ID No.89 and SEQ ID No. 90; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV31 are shown as SEQ ID No.91, SEQ ID No.92 and SEQ ID No.93 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV32 are shown as SEQ ID No.94, SEQ ID No.95 and SEQ ID No.96 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV33 are shown as SEQ ID No.97, SEQ ID No.98 and SEQ ID No.99 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV34 are shown as SEQ ID No.100, SEQ ID No.101 and SEQ ID No.102 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV35 are shown as SEQ ID No.103, SEQ ID No.104 and SEQ ID No.105 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV36 are shown as SEQ ID No.106, SEQ ID No.107 and SEQ ID No.108 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV37 are shown as SEQ ID No.109, SEQ ID No.110 and SEQ ID No.111 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV38 are shown as SEQ ID No.112, SEQ ID No.113 and SEQ ID No.114 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV39 are shown as SEQ ID No.115, SEQ ID No.116 and SEQ ID No.117 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV40 are shown as SEQ ID No.118, SEQ ID No.119 and SEQ ID No.120 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV41 are shown as SEQ ID No.121, SEQ ID No.122 and SEQ ID No.123 respectively; the amino acid sequences of the 3 complementarity determining regions of single domain antibody NBV42 are shown as SEQ ID No.124, SEQ ID No.125, and SEQ ID No.126, respectively.

Protein mutants obtained by deleting, substituting, inserting and/or adding one or more amino acids in any one of the amino acid sequences shown above, wherein the protein mutants have the same functions as the protein before mutation, and the protein mutants belong to the protection scope of the invention; in addition, amino acid sequences having at least 90% identity to any of the amino acid sequences shown above are also within the scope of the present invention.

The invention further provides an amino acid sequence of the single domain antibody, wherein the amino acid sequence of the single domain NBV1 is shown as SEQ ID No.127, the amino acid sequence of the single domain NBV2 is shown as SEQ ID No.128, the amino acid sequence of the single domain NBV3 is shown as SEQ ID No.129, the amino acid sequence of the single domain NBV4 is shown as SEQ ID No.130, the amino acid sequence of the single domain NBV5 is shown as SEQ ID No.131, the amino acid sequence of the single domain NBV6 is shown as SEQ ID No.132, the amino acid sequence of the single domain NBV7 is shown as SEQ ID No.133, the amino acid sequence of the single domain NBV8 is shown as SEQ ID No.134, the amino acid sequence of the single domain NBV9 is shown as SEQ ID No.135, the amino acid sequence of the single domain NBV10 is shown as SEQ ID No.136, the amino acid sequence of the single domain NBV11 is shown as SEQ ID No.137, the amino acid sequence of the single domain NBV12 is shown as SEQ ID No.139, the amino acid sequence of the single domain NBV13, the amino acid sequence of the single domain NBV14 is shown as SEQ ID No.140, the amino acid sequence of the single domain NBV15 is shown as SEQ ID No.141, the amino acid sequence of the single domain antibody NBV16 is shown as SEQ ID No.142, the amino acid sequence of the single domain antibody NBV17 is shown as SEQ ID No.143, the amino acid sequence of the single domain antibody NBV18 is shown as SEQ ID No.144, the amino acid sequence of the single domain antibody NBV19 is shown as SEQ ID No.145, the amino acid sequence of the single domain antibody NBV20 is shown as SEQ ID No.146, the amino acid sequence of the single domain antibody NBV21 is shown as SEQ ID No.147, the amino acid sequence of the single domain antibody NBV22 is shown as SEQ ID No.148, the amino acid sequence of the single domain antibody NBV23 is shown as SEQ ID No.149, the amino acid sequence of the single domain antibody NBV24 is shown as SEQ ID No.150, the amino acid sequence of the single domain antibody NBV25 is shown as SEQ ID No.151, the amino acid sequence of the single domain antibody NBV 152 is shown as SEQ ID No.27, the amino acid sequence NBV27 is shown as SEQ ID No.27, the amino acid sequence of the single domain antibody NBV28 is shown as SEQ ID No.154, the amino acid sequence of the single domain antibody NBV29 is shown as SEQ ID No.155, the amino acid sequence of the single domain antibody NBV30 is shown as SEQ ID No.156, the amino acid sequence of the single domain antibody NBV31 is shown as SEQ ID No.157, the amino acid sequence of the single domain antibody NBV32 is shown as SEQ ID No.158, the amino acid sequence of the single domain antibody NBV33 is shown as SEQ ID No.159, the amino acid sequence of the single domain antibody NBV34 is shown as SEQ ID No.160, the amino acid sequence of the single domain antibody NBV35 is shown as SEQ ID No.161, the amino acid sequence of the single domain antibody NBV36 is shown as SEQ ID No.162, the amino acid sequence of the single domain antibody NBV37 is shown as SEQ ID No.163, the amino acid sequence of the single domain antibody NBV38 is shown as SEQ ID No.166, the amino acid sequence of the single domain antibody NBV39 is shown as SEQ ID No.154, the amino acid sequence NBV40 is shown as SEQ ID No.166, the amino acid sequence of the single-domain antibody NBV41 is shown as SEQ ID No.167, and the amino acid sequence of the single-domain antibody NBV42 is shown as SEQ ID No. 168.

Protein mutants obtained by deleting, substituting, inserting and/or adding one or more amino acids in any one of the amino acid sequences shown above have the same functions as the protein before mutation, and the protein mutants belong to the protection scope of the invention; in addition, amino acid sequences having at least 90% identity to any of the amino acid sequences shown above are also within the scope of the present invention.

According to the experimental results of single-domain antibody affinity determination, the affinity of all VEGF single-domain antibodies of the invention is 50nM, 20nM, 10nM, 1nM, 0.1nM and 0.01nM, and the affinity of part of VEGF single-domain antibodies is in the range of 0.001-37.5 nM. According to the binding test with VEGF-165 antigen and other related antigens, the VEGF single-domain antibody disclosed by the invention is specifically bound with the human VEGF-165 antigen and basically not bound with other related proteins, and the VEGF single-domain antibody disclosed by the invention is good in specificity.

According to the experimental results of the binding competition inhibition effect of the VEGF single-domain antibody on VEGF and VEGFR, different VEGF single-domain antibodies can compete to inhibit the binding of VEGF protein and VEGFR protein, wherein the inhibition efficiency of the competition inhibition of 4 strains of single-domain antibodies (NBV5, NBV6, NBV14 and NBV16) and positive control bevacizumab on the binding of VEGF protein and VEGFR protein is the same.

The invention further carries out humanized transformation on single domain antibodies NBV11, NBV20, NBV32 and NBV35 respectively to obtain humanized antibodies; the affinity of all humanized VEGF single-domain antibodies is not obviously changed; wherein, the single domain antibody NBV11 is humanized and transformed to obtain a 4-humanized antibody, and the amino acid sequences of the antibody are respectively shown as SEQ ID No.169, SEQ ID No.170, SEQ ID No.171 and SEQ ID No. 172; the single domain antibody NBV20 is humanized and transformed to obtain a 4-humanized antibody, and the amino acid sequences of the antibody are respectively shown as SEQ ID No.173, SEQ ID No.174, SEQ ID No.175 and SEQ ID No. 176; the single domain antibody NBV32 is humanized and transformed to obtain a 4-humanized antibody, and the amino acid sequences of the antibody are respectively shown as SEQ ID No.177, SEQ ID No.178, SEQ ID No.179 and SEQ ID No. 180; the single domain antibody NBV35 is humanized and transformed to obtain a 3-humanized antibody, and the amino acid sequences of the 3-humanized antibody are respectively shown as SEQ ID No.181, SEQ ID No.182 and SEQ ID No. 183.

The invention further provides a coding gene sequence of the single domain antibody, wherein the nucleotide sequence of the coding gene of the single domain antibody NBV1 is shown as SEQ ID No.184, the nucleotide sequence of the coding gene of the single domain antibody NBV2 is shown as SEQ ID No.185, the nucleotide sequence of the coding gene of the single domain antibody NBV3 is shown as SEQ ID No.186, the nucleotide sequence of the coding gene of the single domain antibody NBV4 is shown as SEQ ID No.187, the nucleotide sequence of the coding gene of the single domain antibody NBV5 is shown as SEQ ID No.188, the nucleotide sequence of the coding gene of the single domain antibody NBV6 is shown as SEQ ID No.189, the nucleotide sequence of the coding gene of the single domain antibody NBV7 is shown as SEQ ID No.190, the nucleotide sequence of the coding gene of the single domain antibody NBV8 is shown as SEQ ID No.191, the nucleotide sequence of the coding gene of the single domain antibody NBV9 is shown as SEQ ID No.192, and the nucleotide sequence of the coding gene NBV10 is shown as SEQ ID No.193, the nucleotide sequence of the coding gene of the single domain antibody NBV11 is shown as SEQ ID No.194, the nucleotide sequence of the coding gene of the single domain antibody NBV12 is shown as SEQ ID No.195, the nucleotide sequence of the coding gene of the single domain antibody NBV13 is shown as SEQ ID No.196, the nucleotide sequence of the coding gene of the single domain antibody NBV14 is shown as SEQ ID No.197, the nucleotide sequence of the coding gene of the single domain antibody NBV15 is shown as SEQ ID No.198, the nucleotide sequence of the coding gene of the single domain antibody NBV16 is shown as SEQ ID No.199, the nucleotide sequence of the coding gene of the single domain antibody NBV17 is shown as SEQ ID No.200, the nucleotide sequence of the coding gene of the single domain antibody NBV18 is shown as SEQ ID No.201, the nucleotide sequence of the coding gene of the single domain antibody NBV19 is shown as SEQ ID No.202, the nucleotide sequence of the coding gene of the single domain antibody NBV20 is shown as SEQ ID No.203, the nucleotide sequence of the coding gene NBV21 is shown as SEQ ID No.204, the nucleotide sequence of the coding gene of the single domain antibody NBV22 is shown as SEQ ID No.205, the nucleotide sequence of the coding gene of the single domain antibody NBV23 is shown as SEQ ID No.206, the nucleotide sequence of the coding gene of the single domain antibody NBV24 is shown as SEQ ID No.207, the nucleotide sequence of the coding gene of the single domain antibody NBV25 is shown as SEQ ID No.208, the nucleotide sequence of the coding gene of the single domain antibody NBV26 is shown as SEQ ID No.209, the nucleotide sequence of the coding gene of the single domain antibody NBV27 is shown as SEQ ID No.210, the nucleotide sequence of the coding gene of the single domain antibody NBV28 is shown as SEQ ID No.211, the nucleotide sequence of the coding gene of the single domain antibody NBV29 is shown as SEQ ID No.212, the nucleotide sequence of the coding gene of the single domain antibody NBV30 is shown as SEQ ID No.213, the nucleotide sequence of the coding gene of the single domain antibody NBV31 is shown as SEQ ID No.214, and the nucleotide sequence of the coding gene NBV32 is shown as SEQ ID No.215, the nucleotide sequence of the coding gene of the single domain NBV33 is shown as SEQ ID No.216, the nucleotide sequence of the coding gene of the single domain antibody NBV34 is shown as SEQ ID No.217, the nucleotide sequence of the coding gene of the single domain antibody NBV35 is shown as SEQ ID No.218, the nucleotide sequence of the coding gene of the single domain antibody NBV36 is shown as SEQ ID No.219, the nucleotide sequence of the coding gene of the single domain antibody NBV37 is shown as SEQ ID No.220, the nucleotide sequence of the coding gene of the single domain antibody NBV38 is shown as SEQ ID No.221, the nucleotide sequence of the coding gene of the single domain antibody NBV39 is shown as SEQ ID No.222, the nucleotide sequence of the coding gene of the single domain antibody NBV40 is shown as SEQ ID No.223, the nucleotide sequence of the coding gene of the single domain antibody NBV41 is shown as SEQ ID No.224, and the nucleotide sequence of the coding gene of the single domain antibody NBV42 is shown as SEQ ID No. 225.

Wherein, the polynucleotide sequence capable of hybridizing with the complementary sequence of the polynucleotide sequence under the strict hybridization condition also belongs to the protection scope of the invention; also, polynucleotide sequences having at least 90% identity to any of the polynucleotide sequences shown above are within the scope of the present invention.

The present invention further provides a recombinant expression vector comprising one or more of the genes encoding the single domain antibody; preferably, the recombinant expression vector can be a recombinant prokaryotic cell expression vector, a recombinant yeast expression vector, a recombinant eukaryotic cell expression vector or other recombinant cell expression vectors.

The present invention also provides a recombinant host cell comprising the recombinant expression vector described above.

Preferably, the recombinant host cell is a recombinant prokaryotic expression cell, a recombinant eukaryotic expression cell, a recombinant fungal cell or a recombinant yeast cell, and the recombinant prokaryotic expression cell is preferably escherichia coli.

The invention further constructs a fusion protein of the anti-VEGF single-domain antibody or the humanized single-domain antibody and IgG-Fc; wherein, the Fc gene sequence can be derived from IgG, IgA and IgM or derived from IgG1, IgG2, IgG3 orIgG 4. The IgG is preferably human IgG and subclasses of IgG1, 2, 3 and 4, and may also be Fc fragment gene and amino acid sequence of human IgM, human IgA or other animal (such as mouse, rabbit, monkey) immunoglobulin.

The invention further couples the single-domain antibody or humanized single-domain antibody with one or more of enzyme phase (such as horseradish peroxidase, alkaline phosphatase, and the like), radioactive isotope, fluorescent compound or chemiluminescent compound to obtain conjugates, and the conjugates can be used for detecting VEGF or treating various diseases related to VEGF expression abnormality.

The conjugate obtained by coupling the anti-VEGF single-domain antibody, the humanized anti-VEGF single-domain antibody, the VEGF single-domain antibody or the humanized single-domain antibody with an IgG-Fc constructed fusion protein, the single-domain antibody or the humanized single-domain antibody with an enzyme phase, a radioisotope, a fluorescent compound or a chemiluminescent compound mainly has the following uses:

(1) preparing a drug or reagent related to the detection of VEGF;

(2) preparing a drug or reagent for blocking the interaction between VEGF and VEGFR.

(3) The application of preparing the medicine for treating the diseases related to the abnormal expression of VEGF. Preferably, the VEGF expression abnormality is related to different diseases, such as lung cancer, colorectal cancer, breast cancer, head and neck cancer and other tumor diseases, senile fundus macular degeneration and the like, and the detection and treatment applications of the diseases include but are not limited to the detection and treatment of the diseases.

The invention obtains a group of VEGF single-domain antibodies and humanized single-domain antibodies and fusion proteins thereof by screening. Compared with the existing antibody, the single-domain antibody for resisting VEGF obtained by screening has high activity and stronger neutralizing or binding capacity. The group of single-domain antibodies can specifically bind to human VEGF165 and VEGF121 antigens, bind to tumor cell strains expressing VEGF on the cell surface, effectively block the combination of the VEGF antigens and VEGFR and generate corresponding signal cascade effect, and can be used for detecting and/or treating various diseases related to VEGF expression abnormality. The VEGF humanized single-domain antibody and/or the fusion protein can be used for treating senile fundus wet macular degeneration diseases and can also be used for treating various related tumor diseases.

Definitions of terms to which the invention relates

The term "VEGF" as used herein is a family of Vascular Endothelial Growth Factors (VEGF), a multifunctional cytokine with direct and indirect regulation in angiogenesis and lymphangiogenesis, which promotes Endothelial cell proliferation, angiogenesis and increases Vascular permeability.

The term "single domain antibody (sdAb)" as used herein refers to a fragment comprising a single variable domain of an antibody, also known as a Nanobody. Like an intact antibody, it binds selectively to a particular antigen. The single domain antibody appears much smaller, approximately only 12-15 kDa, compared to the 150-160 kDa mass of the intact antibody. The first single domain antibody was artificially engineered from a camelid heavy chain antibody, referred to as a "VHH segment".

The term "identity" of sequences as used herein is used interchangeably with "identity" and refers to the degree of similarity between sequences as determined by sequence alignment software, such as BLAST. Methods and software for sequence alignment are well known to those skilled in the art. The modified nucleotide sequence may be obtained by substitution, deletion and/or addition of one or several amino acids or bases to a known sequence. For example, by conventional means (e.g., conservative substitutions, etc.), the sequences of SEQ ID NOs: 1-198, and can have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% sequence identity thereto, and substantially the same properties, all within the scope of the present invention. Preferably, the present invention obtains sequence identity by conservative substitutions, but is not limited to conservative substitutions.

The term "complementary" as used herein refers to two nucleotide sequences comprising antiparallel nucleotide sequences capable of pairing with each other upon hydrogen bonding between complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary strands are reverse complementary to each other when the sequences are viewed in both 5 'to 3' directions. It is also known in the art that two sequences that hybridize to each other under a given set of conditions do not necessarily have to be 100% perfectly complementary.

The term "amino acid sequence" refers to the sequence of amino acids linked together to form a peptide chain (or polypeptide), and the amino acid sequence can only be read in one orientation. There are more than 100 different types of amino acids, 20 of which are commonly used, and the present invention does not exclude other substances such as saccharides, lipids, etc. from the amino acid chain, nor is the present invention limited to the amino acids commonly used in 20.

The term "nucleotide sequence" refers to the order of bases in DNA or RNA, i.e., A, T, G, C in DNA or A, U, G, C in mRNA, and also includes the order of bases in rRNA, tRNA and mRNA. It is understood that the antibody genes claimed in the present invention also encompass RNA (rRNA, tRNA, mRNA) and their complementary sequences in addition to DNA sequences.

The substitutions described in the present invention may be conservative substitutions, i.e. the substitution of a specific amino acid residue for a residue having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include substitutions between amino acid residues containing aliphatic groups (e.g., substitutions between Ile, Val, Leu, or Ala), substitutions between polar residues (e.g., substitutions between Lys and Arg, Glu and Asp, Gln and Asn), and the like. Mutants resulting from deletion, substitution, insertion and/or addition of amino acids can be made by, for example, site-directed mutagenesis (see, for example, Nucleic acid research, Vol.10, No.20, p.6487-6500, 1982, which is incorporated herein by reference in its entirety) as a well-known technique on DNA encoding a wild-type protein.

In the present specification, "one or more amino acids" refers to amino acids that can be deleted, substituted, inserted, and/or added by a site-directed mutagenesis method, and is not limited, but is preferably 20 or less, 15 or less, 10 or less, or 7 or less, and more preferably 5 or less. In the case of site-directed mutagenesis, for example, in addition to the desired variation, i.e., a specific mismatch, synthetic oligonucleotide primers complementary to the single-stranded phage DNA to be mutated can be used in the following manner.

The term "Expression vectors" refers to vectors in which Expression elements (e.g., promoter, RBS, terminator, etc.) are added to the basic backbone of a cloning vector to enable the Expression of a desired gene. The expression vector comprises four parts: target gene, promoter, terminator and marker gene. The present invention includes, but is not limited to, prokaryotic, eukaryotic, or other cellular expression vectors.

The term "Framework region", i.e., a Framework region, has a large variation of about 110 amino acid sequences near the N-terminus of H and L chains of an immunoglobulin, and the amino acid sequences of the other portions are relatively constant, whereby the light chain and the heavy chain can be distinguished into a variable region (V) and a constant region (C). The variable region includes the hypervariable region HVR (hypervariable region) or Complementarity determining region CDR (complementary-determining region) and FR framework regions.

The term "humanized" antibody refers to the Fr region portion of the variable region (VH or VHH), the constant region portion (i.e., the CH and CL regions) or all of the antibody being encoded by human antibody genes. Humanized antibodies can greatly reduce the immune side effects of heterologous antibodies on the human body. Humanized antibodies include chimeric antibodies, modified antibodies, fully humanized antibodies, and the like. It will be appreciated that those skilled in the art will be able to prepare suitable humanized forms of the single domain antibodies of the invention as required and within the scope of the invention.

The term "stringent Hybridization conditions" means conditions of low ionic strength and high temperature known in the art. typically, under stringent conditions, a probe hybridizes to its target sequence to a greater extent than to other sequences (e.g., at least 2-fold over background). stringent Hybridization conditions are sequence dependent and will differ under different environmental conditions, longer sequences hybridize specifically at higher temperatures. by controlling the stringency or wash conditions of Hybridization, a target sequence that is 100% complementary to a probe can be identified. for exhaustive guidance of Nucleic acid Hybridization, reference may be made to the relevant literature (Tijssen, Techniques in biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "overture of Hybridization and the Hybridization of Nucleic acid assays.1993), more particularly, which are typically selected to be less than the thermal melting point (SDS) at a defined ionic strength (pH) and the target Nucleic acid Hybridization concentration of about 0.5-10% for a target Nucleic acid Hybridization concentration of at least 10.5 ℃ or less than about 10% under conditions including, where the probe can hybridize to a target Nucleic acid at a target Nucleic acid Hybridization concentration of less than 0.5% under a normal pH 5, 10.5-10% Hybridization condition (M.60) and a target Nucleic acid Hybridization condition (M.5-10.5) including, 10.5 to 10% Hybridization at a target Nucleic acid Hybridization temperature, which may be equal to 0.5.5.5.5% under normal conditions or less than a target Nucleic acid Hybridization condition (M) as a target Nucleic acid Hybridization condition, including but not less than a target Nucleic acid Hybridization condition (M) when added to a target Nucleic acid Hybridization condition of a target Nucleic acid Hybridization condition, where a target Nucleic acid Hybridization condition of a target Nucleic acid Hybridization may be added to a target Nucleic acid Hybridization at a target Nucleic acid Hybridization condition of a target Nucleic acid Hybridization at a target Nucleic acid Hybridization temperature, including but not less than a target Nucleic acid Hybridization at a target Nucleic acid Hybridization temperature, a target Nucleic acid Hybridization condition of a target Nucleic acid Hybridization at a target Nucleic acid Hybridization temperature, such as a target Nucleic acid Hybridization condition of a target Nucleic acid Hybridization at a target Nucleic acid Hybridization temperature, a target Nucleic acid Hybridization temperature.

The terms "mutation" and "mutant" have their usual meanings herein, and refer to a genetic, naturally occurring or introduced change in a nucleic acid or polypeptide sequence, which has the same meaning as is commonly known to those of skill in the art.

The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome.

Drawings

FIG. 1 shows SDS-PAGE electrophoresis results of expression test of anti-VEGF single domain antibody in E.coli, and the results show that 6 clones out of 8 clones can be efficiently expressed, and the other 2 clones need to be expressed after the expression conditions are optimized.

FIG. 2 shows that the purity of the purified VEGF single domain antibody reaches about 90% as a result of SDS-PAGE electrophoresis after nickel column purification after the VEGF single domain antibody is expressed in Escherichia coli.

FIG. 3 binding assay (ELISA) of purified VEGF single domain antibodies to human VEGF-165 antigen and other related antigens; the results show that the group of anti-VEGF single-domain antibodies have high specificity, only bind to the human VEGF165 protein and do not bind to other related factor proteins.

The results in fig. 4 show that VEGF single domain antibodies are able to compete for inhibition of binding of VEGF proteins to VEGFR proteins.

FIG. 5 expression purification of SDS-PAGE for VEGF humanized single domain antibody-HIgGfc fusion protein.

FIG. 6 shows the fluorescence detection results of eye tissuefrozen sections 12 hours after eye drop of fluorescein marker plus adjuvant polypeptide X eye drops of NBV11Hm4 protein.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Example 1 construction of Single Domain antibody libraries specific for anti-VEGF antigens

1) VEGF antigen immune alpaca

According to the conventional immunization method, the mature healthy alpaca is selected from a purchased VEGF antigen (Human VEGF Protein, Human, Recombinant, Beijing Yiqian Shenzhou, product number HPLC-11066-HNAH), the antigen is injected into the back of the neck at multiple points under the skin, the antigen and the equal volume of Freund's adjuvant are added, the immunization is divided into 4 to 8 times, and the absorption condition of a package block at an injection part is tracked and observed to confirm the immunization is correct. The immunization interval is 7-15 days, after 4 th immunization, blood is collected, the antigen immunity valence is measured, when the valence reaches more than 1-5 ten thousand times (ELISA method), about 100ml of whole blood is collected, lymphocytes are separated, and the whole blood is preserved at-80 ℃ for later use.

2) Separation of alpaca peripheral blood lymphocytes and extraction of RNA

Alpaca peripheral Blood leukocytes were isolated and treated with QIAGEN Kit (QIAamp RNA Blood Mini Kit (50), cat No. 52304) according to the instructions, briefly described below: adding 5-10 ml of erythrocyte lysate into 1.5ml of whole blood, uniformly mixing, placing in ice bath for 30 minutes, centrifuging for 10 minutes at 2000rpm after erythrocyte lysis, removing supernatant, adding 1-2 ml of erythrocyte lysate, uniformly mixing, placing in ice bath for 10 minutes to crack residual erythrocyte, centrifuging for 10 minutes at 2000rpm, removing supernatant, adding 0.3ml of lysate, uniformly mixing leukocytes, and storing at-80 ℃ for later use.

RNA was purified and the concentration of the obtained RNA was measured using QIAGEN Kit (QIAamp RNA Blood Mini Kit (50), cat # 52304) according to the instructions.

3) Heavy chain antibody variable region-VHH

First strand cDNA Synthesis: the procedure was followed using a cDNA synthesis kit (MiniBESTAgarose Gel DNA extraction kit Ver.4.0, TAKARA). With this template, two sets of primers were used to perform PCR amplification of heavy chain antibody VHH gene fragments, respectively. By adopting a nested PCR method, a common heavy chain gene fragment with the length of more than 800bp is obtained in the first PCR amplification, a heavy chain antibody gene fragment with the length of 800-500 bp is obtained in the deletion of a light chain, the heavy chain antibody gene fragment with the deletion of the light chain is recovered by cutting gel, and the gene fragment is used as a template and is subjected to PCR amplification by using a VHH specific primer to obtain a VHH target gene (500 bp).

The primers used were:

first round of PCRfd 5' primer YF: CGC CAT CAA GGT ACC AGT TGA

First round PCR Bd 3' primer YBN: CAG CCG GCC ATG GCC SMK GTR CAG CTG GTG GAKTCT GGG GGA G

Second round PCR primers:

YV-BACK：CAT GTG CATGGCCTA GAC TCG CGG CCCAGC CGG CCA TGG CC；YV-FOR：CAT GTG TAG ATT CCT GGC CGG CCT GGC CTG AGG AGA CGG TGA CCT GG

4) ligation of VHH fragments with phage display vectors and electrotransformation of TG1 competent

SfI VHH fragment and pHEN6 vector plasmid were digested singly, then the VHH fragment and pHEN6 vector (Concrath, KEMOther. anti-microbial Agents Chemothers (anti-microbial Chemothery) 2001,45 (10)2807-12, Chinese patent ZL20111028003.1) were ligated by ligase, electrotransformed into TG1 competent cells, plated and the antibody insertion rate was verified by colony PCR. Detecting the cloning efficiency of the recombinant gene: coating the electro-conversion bacterial liquid on an LB/Amp plate, culturing at 32 ℃ overnight, and verifying the connection efficiency of the antibody by a colony PCR method the next day, wherein the connection efficiency of a phage antibody library is more than 90%. The electrotransformation bacteria solution is spread on LB/Amp plate, cultured overnight at 32 ℃, washed with 2YT medium, added with 15% glycerol and stored at-80 ℃. Phage library approximately 0.5-1.0x10⁸(ii) a Randomly selecting 30-50 clones, and performing gene sequencing, wherein the repetition rate of three CDR sequences in the sequence of the VHH is less than 10%.

5) Preparation of VHH phage antibody libraries

The antibody library was rescued by adding the helper phage M13K07 (Invitrogen): phage antibody libraries were prepared according to conventional methods and stored at-80 ℃ until use.

Example 2 obtaining of Single Domain anti-VEGF antibodies

1. Screening for VEGF-specific Single Domain antibodies

The first round of VEGF protein concentration 150. mu.g/ml, 150. mu.l/well, 1 microwell, 4 ℃ overnight incubation.

The second round of incubation with VEGF protein concentration of 10-100. mu.g/ml, 150. mu.l/well, 5 microwells at 4 ℃ overnight.

The third round of incubation with VEGF protein concentration 10-50. mu.g/ml, 150. mu.l/well, 5 microwells at 4 ℃ overnight.

And (3) sealing: 1% CPBS, 300. mu.l/well, 37 ℃ and 2 hours incubation.

TABLE 1 screening results for VEGF-specific Single Domain antibodies

Number of screens	Adding the total amount of phage antibody library	Eluent + Tris-HCl	Number of single colony	Elution titer
					First wheel	5.6×1011	300μl+200μl	10	50/μl
Second wheel	5.25×1011	150 μ l/well +350 μ l	About 2500 a	2.5×104/μl
					Third wheel	5.32×1011	150 μ l/well +350 μ l	About 3100	3.1×104/μl

2. Selection of positive clones by phage ELISA

Individual colonies were randomly picked from agar plates fromround 3 screening for growing colonies, inoculated in 96-well plates containing Amp 2YT broth, and superinfected with helper phage to induce the expression of phage antibodies. The expression supernatant is harvested, ELISA assay is carried out by taking VEGF as an antigen, VEGF positive holes are selected, and DNA sequencing is carried out to identify the gene sequence of the anti-VEGF single-domain antibody clone. Obtaining a series of single domain antibody gene sequences including the gene sequences shown in the sequence table, and further expressing and screening the single domain antibody with specificity and high activity.

Example 3 construction of a plasmid expressing a specific VEGF Single Domain antibody

The specific VEGF single domain antibody gene obtained in example 3 was amplified by PCR to obtain PCR products with restriction enzymes BbsI and BamHI sites, and the PCR products and the vector (pSJF2 vector) (kim is. Biosic biochem.2002,66(5):1148-51, Chinese patent ZL 201110280031) were treated with restriction enzymes BbsI and BamHI, respectively, by T-T₄The plasmid sdAb-pSJF2 which can be efficiently expressed in Escherichia coli is obtained by ligase ligation and recombination, and the gene sequence is determined to determine the correctness of the sequence.

1) Obtaining the PCR amplification condition of the VHH target gene, and the composition of a 50-microliter PCR system:

5' primer-GAA GAAGAA GAC AA CAG GCC SVK GTG MAG CTG GWG GAK TCT

3' primer-gaagatctccggatccTGAGGAGACGGTGACCTGGGT

2) Carrying out enzyme digestion on a target gene and a vector, connecting the target gene and the vector, transforming TG1, identifying clone containing a target fragment by PCR, and carrying out gene sequencing to obtain a VEGF single-domain antibody expression plasmid with a correct gene sequence.

Example 4 expression and purification of anti-VEGF Single Domain antibodies

The strain containing plasmid VEGFsdAb-pSJF2 described in example 3 was inoculated onto LB plates containing ampicillin overnight at 37 ℃. Individual colonies were selected and inoculated into 15ml of LB medium containing ampicillin and shake-cultured overnight at 37 ℃. And (3) transferring 10ml of overnight culture into 1L of 2YT culture solution containing ampicillin, carrying out shake culture at 37 ℃, carrying out 240 r/min, adding 0.5-1.0 mMIPTG when OD value reaches 0.4-0.6, and continuing to culture overnight. And (4) centrifuging and collecting bacteria. Adding lysozyme to crack the bacteria, centrifuging, and collecting the soluble single-domain antibody protein in the supernatant. Obtaining the protein with the purity of more than 90 percent by Ni + ion affinity chromatography. FIG. 1 shows SDS-PAGE of the expressed anti-VEGF single domain antibody protein, and FIG. 2 shows SDS-PAGE of the expressed VEGF-sdAbs after nickel column purification.

Experimental example 1 affinity assay experiment for anti-VEGF Single Domain antibody

1) Sample preparation

Bio-VEGF was diluted to 10. mu.g/ml with 1 × dynamic buffer (1 × PBS, 0.05% Tween 20, 0.1% BSA, pH 7.2);

single-domain antibody is diluted with 1 × kinetic buffer solution to 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM in turn;

2) sample testing

The antigen to be detected is loaded through an SA sensor, the antigen is diluted by 5 dilutions, and the affinity of all VEGF single-domain antibodies is 50nm, 20nm, 10nm, 1nm, 0.1nm and 0.01 nm. The partial VEGF single domain antibody affinities are shown in Table 2, and range from 0.001-37.5 nM.

TABLE 2 partial VEGF single domain antibody affinity assay results

EXAMPLE 2 binding Experiment (ELISA) of purified VEGF Single Domain antibodies to VEGF-165 antigen and other related antigens

1 course of the experiment

Respectively coating Human VEGF-B Protein (Fc), Human VEGF-C Protein (His), Human VEGF-D Protein (His), Human PIGF Protein (Fc), Mouse VEGF164Protein, Mouse PIGFprotein, Rat VEGF164Protein and control VEGF165 (His) with the concentration of 2ug/ml, 100 ul/hole and 34 micropores, and incubating at 4 ℃ for overnight; add 1% CPBS blocking, 300 ul/well. Incubating for 2h at 37 ℃; diluting the different encoded VEGF single domain antibodies to a final concentration of 1ug/ml, 100 ul/well; diluting Anti-Myc tag antibody (HRP) (1:5000), 100 ul/well, incubating at 37 ℃ for 1 h; adding TMB color development solution, 100 ul/hole, and reacting for 10min in a dark place; add 50 ul/well 2M H₂SO₄Terminating the reaction; OD was measured at a wavelength of 450 nm.

2 results of the experiment

The experimental results are shown in FIG. 3, and it can be seen from the experimental results that the purified VEGF single domain antibody specifically binds to human VEGF-165 antigen and does not substantially bind to other related proteins.

Experimental example 3 Effect of anti-VEGF Single Domain antibody on competitive inhibition of binding between VEGF and VEGFR

Since VEGF is able to bind VEGFR, resulting in signaling and a subsequent series of biological functions and cell proliferation of vascular endothelial cells, a functional VEGF single domain antibody should be able to compete for inhibition of VEGF binding to VEGFR. VEGFR protein was coated at 1. mu.g/ml, 100. mu.l/well on a removable microplate and incubated overnight at 4 ℃. Blocking with 2% BSA was added at 300. mu.l/well and incubated at 37 ℃ for 2 hours. VEGF single domain antibody was diluted to a final concentration of 10. mu.g/ml. Mu.l of VEGF (10. mu.g/ml) single domain antibody was added, 2. mu.l of VEGF (5. mu.g/ml) protein was added to each well, and mixed well. Mouse anti-myc-IgG-HRP (1:5000) was diluted at 100. mu.l/well and incubated at 37 ℃ for 1 hour. Adding TMB color development solution, 100 mu l/hole, and reacting for 10 minutes in a dark place. Add 50. mu.l/well 2M H₂SO₄The reaction was terminated. OD was measured at a wavelength of 450 nm.

The results in fig. 4 show that VEGF single domain antibodies are able to compete for inhibition of binding of VEGF proteins to VEGFR proteins. Different VEGF single-domain antibodies can compete to inhibit the combination of VEGF protein and VEGFR protein, 4 strains of single-domain antibodies (NBV5, NBV6, NBV14 and NBV16) and positive control bevacizumab inhibit the combination of VEGF protein and VEGFR protein with the same inhibition efficiency, and 3 strains of single-domain antibodies have only weak competition inhibition effect.

EXPERIMENTAL EXAMPLE 4 binding Experiment (ELISA) of purified partial VEGF single domain antibody to human VEGF-165 antigen and human VEGF-121 antigen

The experimental procedure was the same as in example 2, and the experimental results are shown in Table 3. The experimental results show that the detected part of the VEGF single-domain antibody has basically the same binding capacity with the human VEGF-165 and human VEGF-121 antigens, and the part of the tested VEGF single-domain antibody is laterally proved to be bound with the antigen surface shared by the human VEGF-165 and human VEGF-121 antigens.

TABLE 3 Experimental results for the specific binding of anti-VEGF single domain antibodies to human VEGF-165 and human VEGF-121 antigens

Experimental example 5 humanization of anti-VEGF Single Domain antibodies

The humanization method is completed by adopting a protein surface amino acid humanization (Resurfacing) method and a VHH humanization general antigen binding complementary region grafting method (CDR grafting to a random frame).

The humanization procedure was as follows: anti-VEGF single domain antibodies NBV11, 20, 32 and 35 were modeled homologously with themodeling software Modeller 9. The anti-VEGF single domain antibodies NBV11, 20, 32 and 35 were humanized with reference to the amino acid sequence of the well soluble human antibody DP-47 and the homologous sequence NBBcII10 antibody, and the engineered sequences are shown in Table 4.

TABLE 4 humanized engineering of anti-VEGFsingle domain antibodies 11, 20, 32 and 35

X*: indicating that the amino acid may continue to humanize the change.

The above-mentioned VEGF single domain antibodies were humanized and tested for their binding properties to antigen, and the results are shown in Table 5.

TABLE 5 test results of the binding characteristics of VEGF single domain antibody to antigen after humanized modification

From the results in table 5, it can be seen that all of the humanized VEGF single domain antibodies have substantially no significant change in affinity, and the framework region humanization rate meets the design requirements.

Experimental example 6 construction of fusion protein by linking humanized VEGF Single Domain antibody to human IgG1-Fc

1. Constructing a step (1), and completely synthesizing NBV11Hm1 (or NBV11Hm4) + human IgG1-Fc (NBV 11Hmx-HIgG1Fc for short) genes; (2) NBV20Hm1 (or NBV20Hm3) and human IgG1-Fc (NBV20 Hmx + HIgGfc for short) gene total synthesis; (3) NBV32Hm1 (or NBV32Hm2) + human IgG1-Fc (NBV 32Hmx + HIgGfc for short) gene total synthesis; (4) NBV35Hm1 (or NBV35Hm2) + human IgG1-Fc (abbreviated as NBV35Hmx + HIgGfc) gene was completely synthesized and XhoI-EcoRI was added to double-cleave the gene, NBV11Hmx + HIgGfc, (2) NBV20Hmx + HIgGfc, (3) NBV32Hmx + HIgGfc, (4) NBV35Hmx + HIgGfc gene was ligated to p327.7 expression vector (patent publication No. CN 104195173A), and the corresponding cleavage site and stop codon were added to double-cleave with XbaI-SalI, and the other identical (1) (2) (3) (4) gene was ligated to the vector already containing the cleaved (1) NBV11Hmx + HIgGfc, (2) NBV20Hmx + HIgGfc, (3) NBV32Hmx + HIgGfc, (4) NBV35Hmx + HIgGfc, (2) NBV35 HgI + HIgGfc, (2) gene was ligated to the final NBV11 HgGfc + HIgGfc + NBGmV 2 (NBGx + HIgGfc).

Expression and purification of VEGF humanized single domain antibody Fc fusion protein

The expression vectors constructed above were transfected into CHO/K1 cells, MSX was used to screen stable protein high expression cell lines, 3 stable expression cell lines were screened altogether, and stable expression cell lines were used to perform protein expression by culturing in 500ml shake flasks.

Protein purification: the cell expression supernatant was purified by affinity chromatography using protein A strain, and the purified protein was replaced with a citric acid (0.05% Tween80, pH6.2) buffer. The purified protein expressed from the VEGF humanized single domain antibody Fc fusion protein is shown in FIG. 5 (electrophoresis of SDS-PAGE reducing gels and non-reducing gels).

From the results of fig. 5, it can be seen that the identification result of the VEGF humanized single domain antibody fusion protein after expression and purification is nearly consistent with the theoretical calculation value, and the protein band of the non-reducing gel after expression and purification is about 76KD compared with the molecular weight mark.

Experimental example 7 experiment of mixing VEGF single domain antibody or VEGF humanized single domain antibody with pharmaceutical adjuvant polypeptide for ophthalmic eye drops

The preparation process comprises the following steps: the fluorescein marker of the NBV11Hm 4protein expressed and purified by escherichia coli and the auxiliary material polypeptide X (see Chinese patent application publication No. CN107638405A) are prepared according to different proportions of 1:1/1:2/1:5/1:10/1:20 and the like.

The eyedrops prepared instep 1 are dripped into burned mouse eyes after Balb/c mouse eyeground is burned by laser, and are dripped into the burned mouse eyes for 1 time after 4 hours, eyeball frozen sections are taken after 12 hours, the fluorescence intensity of VEGF single domain antibody containing marked fluorescence and auxiliary material polypeptide X of different mouse eye tissues is tested by a fluorescence intensity tester, and the test is simultaneously provided with VEGF single domain antibody singly marked with fluorescence and a non-drug control group.

The results of the experiment are shown in FIG. 6. According to the fluorescence intensity detection results of the fluorescence marker of the NBV11Hm 4protein and the auxiliary material polypeptide X in the eye tissue section of the experimental animal group, the fluorescence intensity of the cornea and the retina of the eye is 5 times higher and 10-30 times higher than that of the fluorescence marker group of the NBV11Hm 4protein and that of the non-drug control group, and the fluorescence intensity is significantly statistically different.

Experimental example 8 experiment of VEGF humanized single domain antibody Fc fusion protein inhibiting tumor cell growth in mouse

1. Establishing an animal test model: tying the cultured human bodyIntestinal cancer cells (LS174T) are blown into single cell suspension, centrifuged at 800 rpm for 5 minutes, supernatant is discarded, cells are resuspended by PBS, and the cell concentration is adjusted to 2 x10 ^7 cells/mL for standby. Balb/c-nu nude mice neck and back skin are inoculated, a 1mL syringe is used for extracting cell suspension, and 100 mu L/mouse of cells are injected after blood-free is pumped back at the expected growth part of the tumor. After inoculation is completed, when the tumor volume is 100-³When 40% of the animals in the range were reached, mice with tumor volumes in this range were randomly grouped into 10 groups of 3 groups using RAND function of excel software, labeled as Z1(NBV20Hm1 group), Z2 (bevacizumab-positive control group), Z3(PBS control group), respectively. The day of grouping wasday 0 and dosing was started by intraperitoneal injection (I.P.) 2 times per week for 6 consecutive times, and the mice were sacrificed 3 days after the last dose and tumor tissue was weighed. The dosing regimen is shown in table 6.

TABLE 6 test grouping and administration mode

Group of	Number of animals	Injection sample	Route of administration	Dosage form	Frequency/period
						Z1
	10	NBV20Hm1	I.P.	5mg/Kg	2/week × 3weeks
						Z2
	10	Bevacizumab injection	I.P.	5mg/Kg	2/week × 3weeks
						Z3
	10	PBS	I.P.	---------	2/week × 3 weeks

2. Experimental animal observations and statistics of experimental results

Animal body weight, tumor size (longest and shortest path of tumor mass) were measured 2 times per week after tumor formation, tumors were dissected after animal sacrifice, tumor weights were weighed and photographed (1) tumor volume calculation by measuring longest and shortest paths (a) and (b) of tumors after tumor formation, 2 times per week by formula V1/2 × a × b²Tumor volumes were calculated. And (5) drawing a tumor growth curve by taking time as an abscissa and tumor volume as an ordinate.

(2) Relative tumor proliferation rate T/C (%): the antitumor effect of the drug was evaluated by calculating T/C (%), T/C (%) > 40% being ineffective, T/C (%) < 40%, and p < 0.05 being effective by statistical treatment. The T/C (%) was calculated as follows: T/C (%) ═ T_RTV/C_RTV×100％(T_RTVTreatment group RTV; c_RTVNegative control group RTV), relative tumor volume RTV ═ V_T/V₀，V₀Tumor volume, V, measured for cage administration (D0)_TThe resulting tumor volume was measured for each time. (3) Tumor weight inhibition ratio: mice were sacrificed after the end of dosing, tumor weights were weighed, and tumor weight inhibition calculatedThe tumor weight inhibition rate is (1-T/C) × 100% (T is the average tumor weight of a treatment group, C is the average tumor weight of a negative control group) (4) statistical analysis, the statistical analysis of tumor volume and tumor weight by using a TTEST function of excel software shows that p is less than 0.05 and is not more than 0.05, and the results of the table 9 show that the VEGF humanized single domain antibody Fc fusion protein-NBV 20Hm1 test animal group and the negative control animal group have significant difference in the tumor growth inhibition rate of mice and have no significant difference compared with the test results of the positive control group of monoclonal antibody injection of bevacizumab.

TABLE 7 results of antitumor Effect on Balb/c-nu nude mice transplanted with LS174T cells (statistical results of mean tumor volume)

)

Remarking: 1. when compared with Z3(PBS control group), a represents p < 0.05, and b represents p ≧ 0.05; c represents p < 0.05 and d represents p ≧ 0.05 when compared with Z2 (positive control); and (6) days: the number of days of administration; T/C (%): relative tumor proliferation rate.

SEQUENCE LISTING

<110> Beijing Newcastle Biotechnology Ltd

<120> anti-VEGF single domain antibody, humanization thereof, fusion protein constructed by single domain antibody and IgG1-Fc, and application

<130>BJ-3038-191217A

<160>225

<170>PatentIn version 3.5

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<400>7

Thr Gly Ser His Tyr Asp Leu Ala

1 5

<210>8

<211>16

<212>PRT

<213>Artifical sequence

<400>8

Val Ala Glu Phe Thr Trp Arg Ser Gly Pro Thr Ser Tyr Ala Glu Ser

1 5 10 15

<210>9

<211>15

<212>PRT

<213>Artifical sequence

<400>9

Ala Ser Arg Tyr Phe Tyr Thr Tyr Gly Asp Pro Leu Lys Tyr Asp

1 5 10 15

<210>10

<211>6

<212>PRT

<213>Artifical sequence

<400>10

Ser Ile Asn His Met Ala

1 5

<210>11

<211>14

<212>PRT

<213>Artifical sequence

<400>11

Ala Arg Ala Phe Ser Ser Gly Ser Thr Thr Tyr Ala Asp Ser

1 5 10

<210>12

<211>15

<212>PRT

<213>Artifical sequence

<400>12

Asn Gly Asp Gly Phe Leu Leu Tyr Asp Asp Thr Tyr Tyr Ser Asn

1 5 10 15

<210>13

<211>6

<212>PRT

<213>Artifical sequence

<400>13

Ser Val Phe Asp Met Ala

1 5

<210>14

<211>14

<212>PRT

<213>Artifical sequence

<400>14

Ala Glu Thr Thr Thr Ala Gly Ile Asn Thr Tyr Ala Asp Ser

1 5 10

<210>15

<211>9

<212>PRT

<213>Artifical sequence

<400>15

Asn Ala Lys Asn Gly Trp Arg Thr Leu

1 5

<210>16

<211>6

<212>PRT

<213>Artifical sequence

<400>16

Arg Asp Met Ser Phe Gly

1 5

<210>17

<211>14

<212>PRT

<213>Artifical sequence

<400>17

Ala Tyr Ile Ser Ser Ser Gly Tyr Thr Asn Tyr Val Asp Ala

1 5 10

<210>18

<211>4

<212>PRT

<213>Artifical sequence

<400>18

Asn Thr Leu Val

1

<210>19

<211>6

<212>PRT

<213>Artifical sequence

<400>19

Ser Val Phe Asp Met Ala

1 5

<210>20

<211>14

<212>PRT

<213>Artifical sequence

<400>20

Ala Arg Ile Thr Phe Asp Gly Ile Pro His Tyr Ala Asp Ser

1 5 10

<210>21

<211>9

<212>PRT

<213>Artifical sequence

<400>21

Asn Ala Lys Asn Gly Trp Arg Thr Leu

1 5

<210>22

<211>7

<212>PRT

<213>Artifical sequence

<400>22

Phe Ser Lys Tyr Ala Met Gly

1 5

<210>23

<211>15

<212>PRT

<213>Artifical sequence

<400>23

Gly Asn Ile Tyr Trp Ser Asp Gly Ser Thr His Tyr Gln Asp Ser

1 5 10 15

<210>24

<211>18

<212>PRT

<213>Artifical sequence

<400>24

Ala Ser Arg Gly Ser Asn Asn Gly Gly Ser Tyr Tyr Ser Glu Thr Gly

1 5 10 15

Tyr Asp

<210>25

<211>8

<212>PRT

<213>Artifical sequence

<400>25

Thr Ile Gly Ser Tyr Asp Met Gly

1 5

<210>26

<211>14

<212>PRT

<213>Artifical sequence

<400>26

Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser

1 5 10

<210>27

<211>14

<212>PRT

<213>Artifical sequence

<400>27

Ala Ala Arg Arg Trp His Arg Ser Ala Pro His Asp Tyr Glu

1 5 10

<210>28

<211>6

<212>PRT

<213>Artifical sequence

<400>28

Ala Arg Tyr Ser Met Gly

1 5

<210>29

<211>15

<212>PRT

<213>Artifical sequence

<400>29

Ala Gly Ile Ser Arg Ser Ser Gly Thr Ile Ile Tyr Gly Gly Ser

1 5 10 15

<210>30

<211>16

<212>PRT

<213>Artifical sequence

<400>30

Ala Ala Arg Glu Ser Leu Leu Ala Val Thr Thr Thr Arg Asp Tyr Pro

1 5 10 15

<210>31

<211>7

<212>PRT

<213>Artifical sequence

<400>31

Ile Pro Tyr Val Pro Asp Met

1 5

<210>32

<211>14

<212>PRT

<213>Artifical sequence

<400>32

Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser

1 5 10

<210>33

<211>13

<212>PRT

<213>Artifical sequence

<400>33

Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu

1 5 10

<210>34

<211>6

<212>PRT

<213>Artifical sequence

<400>34

Ser Asp Tyr Ala Met Ala

1 5

<210>35

<211>14

<212>PRT

<213>Artifical sequence

<400>35

Ala Gly Ile Ser Trp Thr Gly Gly Arg Tyr Tyr Ala Glu Ser

1 5 10

<210>36

<211>18

<212>PRT

<213>Artifical sequence

<400>36

Ala Thr Pro Asn Gln Ala Gly Leu Val Leu Leu Asp Asp Ala Glu Gly

1 5 10 15

Tyr Ala

<210>37

<211>7

<212>PRT

<213>Artifical sequence

<400>37

Val Ser Thr Arg Asn Met Gly

1 5

<210>38

<211>15

<212>PRT

<213>Artifical sequence

<400>38

Ala Arg Ile Gly Ser Asp Gly Ser Thr Tyr Asn Val Asp Ser Val

1 5 10 15

<210>39

<211>10

<212>PRT

<213>Artifical sequence

<400>39

Asn Thr Phe Pro Val Thr Lys Phe Tyr Asp

1 5 10

<210>40

<211>8

<212>PRT

<213>Artifical sequence

<400>40

Arg Phe Ser Ala Tyr Asp Met Gly

1 5

<210>41

<211>15

<212>PRT

<213>Artifical sequence

<400>41

Ala Thr Ile Asn Trp Ser Ala Leu Ser Arg Tyr Tyr Ala Asp Ser

1 5 10 15

<210>42

<211>18

<212>PRT

<213>Artifical sequence

<400>42

Ala Gly Gly Arg Ile Ser Ala Ala Leu Arg Val Pro Asp Arg Asp Ala

1 5 10 15

Tyr Ser

<210>43

<211>7

<212>PRT

<213>Artifical sequence

<400>43

Val Arg Asp Asp Ala Met Gly

1 5

<210>44

<211>16

<212>PRT

<213>Artifical sequence

<400>44

Ala Thr Ile Ser Trp Asn Gly Gly Ser Thr Tyr Tyr Ala Ala Ser Ala

1 5 10 15

<210>45

<211>18

<212>PRT

<213>Artifical sequence

<400>45

Ala Val Ala Ser Arg Tyr Arg Gln Ile Val Leu Asn Thr Glu Glu Lys

1 5 10 15

Tyr Asp

<210>46

<211>7

<212>PRT

<213>Artifical sequence

<400>46

Ser Phe Ser Val Pro His Met

1 5

<210>47

<211>15

<212>PRT

<213>Artifical sequence

<400>47

Ala Thr Ile Ser Arg Gly Gly Val Ser Thr Tyr Ala Ala Asp Ser

1 5 10 15

<210>48

<211>14

<212>PRT

<213>Artifical sequence

<400>48

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu

1 5 10

<210>49

<211>6

<212>PRT

<213>Artifical sequence

<400>49

Ser His Arg Thr Phe Ala

1 5

<210>50

<211>14

<212>PRT

<213>Artifical sequence

<400>50

Ala Thr Ile Gly Ser Tyr Gly Ser Thr Tyr Tyr Asp Glu Ser

1 5 10

<210>51

<211>4

<212>PRT

<213>Artifical sequence

<400>51

His Thr Gln Val

1

<210>52

<211>7

<212>PRT

<213>Artifical sequence

<400>52

Ser Thr Ser Tyr Val Tyr Asp

1 5

<210>53

<211>11

<212>PRT

<213>Artifical sequence

<400>53

Ala Ile Thr Asp Gly Gly Ser Thr Asp Asn Tyr

1 5 10

<210>54

<211>16

<212>PRT

<213>Artifical sequence

<400>54

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Asp

1 5 10 15

<210>55

<211>7

<212>PRT

<213>Artifical sequence

<400>55

Ser Thr Ser Tyr Val Tyr Asp

1 5

<210>56

<211>11

<212>PRT

<213>Artifical sequence

<400>56

Ala Ile Thr Asp Gly Gly Ser Thr Asp Asn Tyr

1 5 10

<210>57

<211>17

<212>PRT

<213>Artifical sequence

<400>57

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr

1 5 10 15

Asp

<210>58

<211>7

<212>PRT

<213>Artifical sequence

<400>58

Ile Pro Tyr Val Pro Asp Met

1 5

<210>59

<211>14

<212>PRT

<213>Artifical sequence

<400>59

Ala Thr Ile Thr Arg Gly Gly Val Thr Met Tyr Ala Asp Ser

1 5 10

<210>60

<211>14

<212>PRT

<213>Artifical sequence

<400>60

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu

1 5 10

<210>61

<211>7

<212>PRT

<213>Artifical sequence

<400>61

Ser Thr Ser Tyr Val Tyr Asp

1 5

<210>62

<211>12

<212>PRT

<213>Artifical sequence

<400>62

Ala Ala Ile Ser Arg Gly Gly Val Thr Asp Asn Tyr

1 5 10

<210>63

<211>17

<212>PRT

<213>Artifical sequence

<400>63

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr

1 5 10 15

Asp

<210>64

<211>7

<212>PRT

<213>Artifical sequence

<400>64

Ile Ser Tyr Val Pro Asp Met

1 5

<210>65

<211>14

<212>PRT

<213>Artifical sequence

<400>65

Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser

1 5 10

<210>66

<211>14

<212>PRT

<213>Artifical sequence

<400>66

Asn Ala Asp Val Trp Ser Ser Val Leu Phe Lys Leu Val Glu

1 5 10

<210>67

<211>7

<212>PRT

<213>Artifical sequence

<400>67

Ser Phe Ser Tyr Ile Tyr Asp

1 5

<210>68

<211>12

<212>PRT

<213>Artifical sequence

<400>68

Ala Ala Ile Ser Arg Gly Ser Ser Thr Asp Asn Tyr

1 5 10

<210>69

<211>17

<212>PRT

<213>Artifical sequence

<400>69

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr

1 5 10 15

Asp

<210>70

<211>7

<212>PRT

<213>Artifical sequence

<400>70

Ser Phe Ser Tyr Ile Pro Asp

1 5

<210>71

<211>12

<212>PRT

<213>Artifical sequence

<400>71

Ala Ala Ile Ser Arg Gly Ser Ser Thr Asp Asn Tyr

1 5 10

<210>72

<211>15

<212>PRT

<213>Artifical sequence

<400>72

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu

1 5 10 15

<210>73

<211>8

<212>PRT

<213>Artifical sequence

<400>73

Arg Phe Ser Ala Tyr Asp Met Gly

1 5

<210>74

<211>15

<212>PRT

<213>Artifical sequence

<400>74

Ala Thr Ile Asn Trp Ser Thr Leu Ser Arg Tyr Tyr Ala Asp Ser

1 5 10 15

<210>75

<211>18

<212>PRT

<213>Artifical sequence

<400>75

Ala Gly Gly Arg Ile Ser Ser Glu Leu Arg Val Thr Ala Arg Asp Ala

1 5 10 15

Tyr Thr

<210>76

<211>8

<212>PRT

<213>Artifical sequence

<400>76

Ile Ser Tyr Val Pro Asp Met Gly

1 5

<210>77

<211>12

<212>PRT

<213>Artifical sequence

<400>77

Ala Ala Ile Ser Arg Gly Gly Ser Thr Asp Asn Tyr

1 5 10

<210>78

<211>19

<212>PRT

<213>Artifical sequence

<400>78

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser

1 5 10 15

Ala Tyr Asp

<210>79

<211>8

<212>PRT

<213>Artifical sequence

<400>79

Ile Ser Tyr Val Pro Asp Met Gly

1 5

<210>80

<211>12

<212>PRT

<213>Artifical sequence

<400>80

Ala Thr Ile Ser Arg Gly Gly Val Thr Asp Asn Tyr

1 5 10

<210>81

<211>18

<212>PRT

<213>Artifical sequence

<400>81

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser Ala

1 5 10 15

Tyr Asp

<210>82

<211>8

<212>PRT

<213>Artifical sequence

<400>82

Thr Ser Tyr Val Pro Asp Met Gly

1 5

<210>83

<211>12

<212>PRT

<213>Artifical sequence

<400>83

Ala Thr Ile Ser Arg Gly Gly Val Thr Asp Tyr Tyr

1 5 10

<210>84

<211>18

<212>PRT

<213>Artifical sequence

<400>84

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser Ala

1 5 10 15

Tyr Asp

<210>85

<211>6

<212>PRT

<213>Artifical sequence

<400>85

Ser Ser Tyr His Met Asp

1 5

<210>86

<211>15

<212>PRT

<213>Artifical sequence

<400>86

Ala Ala Ile Ser Trp Thr Gly His Ser Thr Tyr Tyr Ala Asp Ser

1 5 10 15

<210>87

<211>14

<212>PRT

<213>Artifical sequence

<400>87

Ala Ala Thr Arg Arg Ala Thr Met Ile Ala Val Pro Ser Asp

1 5 10

<210>88

<211>7

<212>PRT

<213>Artifical sequence

<400>88

Phe Ile Ser Tyr Val Pro Asp

1 5

<210>89

<211>13

<212>PRT

<213>Artifical sequence

<400>89

Ala Ala Thr Ile Ser Arg Gly Gly Val Thr Asp Tyr Tyr

1 5 10

<210>90

<211>16

<212>PRT

<213>Artifical sequence

<400>90

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser Ala

1 5 10 15

<210>91

<211>7

<212>PRT

<213>Artifical sequence

<400>91

Phe Ile Ser Tyr Val Pro Asp

1 5

<210>92

<211>12

<212>PRT

<213>Artifical sequence

<400>92

Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Asp Tyr

1 5 10

<210>93

<211>17

<212>PRT

<213>Artifical sequence

<400>93

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser

1 5 10 15

Ala

<210>94

<211>7

<212>PRT

<213>Artifical sequence

<400>94

Tyr Thr Ser Tyr Leu Pro Asp

1 5

<210>95

<211>12

<212>PRT

<213>Artifical sequence

<400>95

Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr

1 5 10

<210>96

<211>18

<212>PRT

<213>Artifical sequence

<400>96

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro

1 5 10 15

Ser Asp

<210>97

<211>8

<212>PRT

<213>Artifical sequence

<400>97

Thr Phe Gly Ser Tyr Asp Met Ala

1 5

<210>98

<211>15

<212>PRT

<213>Artifical sequence

<400>98

Ala Ala Ile Asn Trp Tyr Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser

1 5 1015

<210>99

<211>22

<212>PRT

<213>Artifical sequence

<400>99

Ala Ala Glu Lys Gly Phe Ala Ser Leu Arg Leu Trp Ala Leu Ser His

1 5 10 15

Lys Pro His Glu Ser Arg

20

<210>100

<211>7

<212>PRT

<213>Artifical sequence

<400>100

Tyr Ile Pro Tyr Leu Pro Asp

1 5

<210>101

<211>11

<212>PRT

<213>Artifical sequence

<400>101

Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr

1 5 10

<210>102

<211>17

<212>PRT

<213>Artifical sequence

<400>102

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro Ser

1 5 1015

Asp

<210>103

<211>8

<212>PRT

<213>Artifical sequence

<400>103

Pro Ser Ser Tyr Tyr Gly Val Ser

1 5

<210>104

<211>15

<212>PRT

<213>Artifical sequence

<400>104

Ala Gly Ile Ser Arg Ser Gly Gly Thr Asp Asn Tyr Ala Asn Phe

1 5 10 15

<210>105

<211>16

<212>PRT

<213>Artifical sequence

<400>105

Ala Ala Ala Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Ala Tyr Asp

1 5 10 15

<210>106

<211>7

<212>PRT

<213>Artifical sequence

<400>106

Tyr Ile Pro Tyr Leu Pro Asp

1 5

<210>107

<211>11

<212>PRT

<213>Artifical sequence

<400>107

Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr

1 5 10

<210>108

<211>18

<212>PRT

<213>Artifical sequence

<400>108

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Glu Tyr

1 5 10 15

Ser Asp

<210>109

<211>7

<212>PRT

<213>Artifical sequence

<400>109

Tyr Ser Thr Tyr Leu Pro Asp

1 5

<210>110

<211>12

<212>PRT

<213>Artifical sequence

<400>110

Ala Ala Ile Thr Arg Gly Gly His Ser Thr Tyr Tyr

1 5 10

<210>111

<211>18

<212>PRT

<213>Artifical sequence

<400>111

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Glu Tyr

1 5 10 15

Ser Asp

<210>112

<211>7

<212>PRT

<213>Artifical sequence

<400>112

Val Ser Ser Tyr Asp Met Gly

1 5

<210>113

<211>14

<212>PRT

<213>Artifical sequence

<400>113

Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser

1 5 10

<210>114

<211>14

<212>PRT

<213>Artifical sequence

<400>114

Ala Ala Arg Arg Tyr Tyr Arg Ser Gly Ala Gln Asp Tyr Glu

1 5 10

<210>115

<211>7

<212>PRT

<213>Artifical sequence

<400>115

Ser Ser Thr Tyr Leu Pro Asp

1 5

<210>116

<211>11

<212>PRT

<213>Artifical sequence

<400>116

Ala Ile Thr Arg Gly Gly His Ser Thr Tyr Tyr

1 5 10

<210>117

<211>17

<212>PRT

<213>Artifical sequence

<400>117

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Ser

1 5 10 15

Asp

<210>118

<211>7

<212>PRT

<213>Artifical sequence

<400>118

Ile Gly Ser Tyr Asp Met Gly

1 5

<210>119

<211>14

<212>PRT

<213>Artifical sequence

<400>119

Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser

15 10

<210>120

<211>14

<212>PRT

<213>Artifical sequence

<400>120

Ala Ala Arg Arg Trp Tyr Arg Ser Ala Pro Gln Asp Tyr Glu

1 5 10

<210>121

<211>7

<212>PRT

<213>Artifical sequence

<400>121

Ser Ser Thr Tyr Leu Pro Asp

1 5

<210>122

<211>11

<212>PRT

<213>Artifical sequence

<400>122

Ala Ile Thr Arg Gly Gly Val Ser Thr Tyr Tyr

1 5 10

<210>123

<211>18

<212>PRT

<213>Artifical sequence

<400>123

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Pro

1 5 10 15

Ser Asp

<210>124

<211>7

<212>PRT

<213>Artifical sequence

<400>124

Ile Gly Ser Tyr Asp Met Gly

1 5

<210>125

<211>14

<212>PRT

<213>Artifical sequence

<400>125

Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser

1 5 10

<210>126

<211>14

<212>PRT

<213>Artifical sequence

<400>126

Ala Ala Arg Arg Tyr Tyr Arg Ser Ala Pro Gln Asp Tyr Asp

1 5 10

<210>127

<211>124

<212>PRT

<213>Vicugna pacos

<400>127

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Pro Ser Phe Ser Thr Tyr

20 25 30

Ala Val Gly Trp Phe Arg Gln Val Pro Arg Arg Glu Arg Ala Phe Val

35 40 45

Ala Gly Ile Asn Trp Ser Gly Glu Glu Thr Thr Tyr His Asn Ser Val

50 55 60

Asn Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asn Arg Arg Asn Tyr Ser Ser Thr Tyr Lys Gly Asn Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>128

<211>119

<212>PRT

<213>Vicugna pacos

<400>128

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

3540 45

Ala Ala Ile Arg Arg Asp Ala Val Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Met Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Met Gly Asp Asp Tyr Val Asp Glu Tyr Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210>129

<211>123

<212>PRT

<213>Vicugna pacos

<400>129

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Val Ala Ser Arg Val Thr Gly Ser His Tyr

20 25 30

Asp Leu Ala Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Glu Val Val

35 40 45

Ala Glu Phe Thr Trp Arg Ser Gly Pro Thr Ser Tyr Ala Glu Ser Val

5055 60

Lys Gly Arg Phe Ala Val Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Asn Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Arg Tyr Phe Tyr Thr Tyr Gly Asp Pro Leu Lys Tyr Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>130

<211>122

<212>PRT

<213>Vicugna pacos

<400>130

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn

20 25 30

His Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Arg Ala Phe Ser Ser Gly Ser Thr Thr Tyr Ala Asp Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gly Asp Gly Phe Leu Leu Tyr Asp Asp Thr Tyr Tyr Ser Asn Ala Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>131

<211>115

<212>PRT

<213>Vicugna pacos

<400>131

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Ala Phe Ser Val Phe

20 25 30

Asp Met Ala Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Glu Thr Thr Thr Ala Gly Ile Asn Thr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Ser Cys Asn

85 90 95

Ala Lys Asn Gly Trp Arg Thr Leu Trp Gly Gln Gly Thr Gln Val Thr

100 105 110

Val Ser Ser

115

<210>132

<211>111

<212>PRT

<213>Vicugna pacos

<400>132

Gly Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Val Ser Gly Ser Thr Phe Arg Asp Met

20 25 30

Ser Phe Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Arg Leu

35 40 45

Ala Tyr Ile Ser Ser Ser Gly Tyr Thr Asn Tyr Val Asp Ala Val Lys

50 55 60

Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Asn

85 90 95

Thr Leu Val Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

100 105 110

<210>133

<211>115

<212>PRT

<213>Vicugna pacos

<400>133

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Ala Phe Ser Val Phe

20 25 30

Asp Met Ala Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu Val

35 40 45

Ala Arg Ile Thr Phe Asp Gly Ile Pro His Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Ser Cys Asn

85 90 95

Ala Lys Asn Gly Trp Arg Thr Leu Trp Gly Gln Gly Thr Gln Val Thr

100 105 110

Val Ser Ser

115

<210>134

<211>126

<212>PRT

<213>Vicugna pacos

<400>134

Gln Val Gln Leu Val Asp Ser Gly Gly Gly Tyr Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Ser Phe Ser Lys Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val

35 40 45

Gly Asn Ile Tyr Trp Ser Asp Gly Ser Thr His Tyr Gln Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Leu Ser Lys Asp Tyr Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Glu Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Arg Gly Ser Asn Asn Gly Gly Ser Tyr Tyr Ser Glu Thr Gly

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>135

<211>121

<212>PRT

<213>Vicugna pacos

<400>135

Gln Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Ile Gly Ser Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser Val Glu

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Lys Asp Lys Thr Thr Met Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Arg Arg Trp His Arg Ser Ala Pro His Asp Tyr Glu Phe Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>136

<211>124

<212>PRT

<213>Vicugna pacos

<400>136

Gln Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Leu Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Phe Ala Arg Tyr

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ser Ser Gly Thr Ile Ile Tyr Gly Gly Ser Val

50 55 60

Lys Gly Arg Phe Ile Ile Phe Arg Asp Asn Val Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Gly Leu Lys Pro Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ala Arg Glu Ser Leu Leu Ala Val Thr Thr Thr Arg Asp Tyr Pro

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>137

<211>121

<212>PRT

<213>Vicugna pacos

<400>137

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>138

<211>125

<212>PRT

<213>Vicugna pacos

<400>138

Gln Val Gln Leu Val Asp Ser Gly Gly Gly Phe Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Trp Thr Gly Gly Arg Tyr Tyr Ala Glu Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Phe Leu

65 70 75 80

Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Gly Ile Tyr His Cys Ala

85 90 95

Thr Pro Asn Gln Ala Gly Leu Val Leu Leu Asp Asp Ala Glu Gly Tyr

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>139

<211>117

<212>PRT

<213>Vicugna pacos

<400>139

Gln Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Val Ser Thr Arg

20 25 30

Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Val Leu Val

35 40 45

Ala Arg Ile Gly Ser Asp Gly Ser Thr Tyr Asn Val Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ala Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Trp Cys Asn

85 90 95

Thr Phe Pro Val Thr Lys Phe Tyr Asp Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210>140

<211>126

<212>PRT

<213>Vicugna pacos

<400>140

Glu Val Lys Leu Val Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Arg Phe Ser Ala Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Asn Trp Ser Ala Leu Ser Arg Tyr Tyr Ala Asp Ser Val

5055 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser

65 70 75 80

Leu His Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gly Arg Ile Ser Ala Ala Leu Arg Val Pro Asp Arg Asp Ala

100 105 110

Tyr Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>141

<211>126

<212>PRT

<213>Vicugna pacos

<400>141

Gln Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Val Arg Asp Asp

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Asn Gly Gly Ser Thr Tyr Tyr Ala Ala Ser Ala

50 55 60

Lys Gly Arg Phe Ile Val Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Val Ala Ser Arg Tyr Arg Gln Ile Val Leu Asn Thr Glu Glu Lys

100 105 110

Tyr Asp Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>142

<211>122

<212>PRT

<213>Vicugna pacos

<400>142

Gln Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Ser Phe Ser Val Pro

20 25 30

His Met Asp Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Arg Gly Gly Val Ser Thr Tyr Ala Ala Asp Ser Val

50 55 60

Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>143

<211>111

<212>PRT

<213>Vicugna pacos

<400>143

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Thr Thr Phe Ser His Arg

20 25 30

Thr Phe Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Thr Ile Gly Ser Tyr Gly Ser Thr Tyr Tyr Asp Glu Ser Val Lys

50 55 60

Asp Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Gln Asp Thr Ala Val Tyr Tyr Cys His

8590 95

Thr Gln Val Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

100 105 110

<210>144

<211>124

<212>PRT

<213>Vicugna pacos

<400>144

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Tyr Val Tyr

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ala Ile Thr Asp Gly Gly Ser Thr Asp Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>145

<211>125

<212>PRT

<213>Vicugna pacos

<400>145

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Tyr Val Tyr

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ala Ile Thr Asp Gly Gly Ser Thr Asp Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>146

<211>121

<212>PRT

<213>Vicugna pacos

<400>146

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Val Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>147

<211>125

<212>PRT

<213>Vicugnapacos

<400>147

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Tyr Val Tyr

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ala Ile Ser Arg Gly Gly Val Thr Asp Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>148

<211>121

<212>PRT

<213>Vicugna pacos

<400>148

Glu Val Gln Leu Glu Asp SerGly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Tyr Val Pro

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Leu Phe Lys Leu Val Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>149

<211>125

<212>PRT

<213>Vicugna pacos

<400>149

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Phe Ser Tyr Ile Tyr

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ala Ile Ser Arg Gly Ser Ser Thr Asp Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>150

<211>123

<212>PRT

<213>Vicugna pacos

<400>150

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Phe Ser Tyr Ile Pro

2025 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ala Ile Ser Arg Gly Ser Ser Thr Asp Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>151

<211>126

<212>PRT

<213>Vicugna pacos

<400>151

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Arg Phe Ser Ala Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Asn Trp Ser Thr Leu Ser Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu His Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gly Arg Ile Ser Ser Glu Leu Arg Val Thr Ala Arg Asp Ala

100 105 110

Tyr Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>152

<211>127

<212>PRT

<213>Vicugna pacos

<400>152

Gln Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Tyr Val Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ala Ile Ser Arg Gly Gly Ser Thr Asp Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser

100 105 110

Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>153

<211>126

<212>PRT

<213>Vicugna pacos

<400>153

Gln Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Tyr Val Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Thr Ile Ser Arg Gly Gly Val Thr Asp Asn Tyr Ala Asp Ser Val

50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser Ala

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>154

<211>126

<212>PRT

<213>Vicugna pacos

<400>154

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Tyr Val Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Thr Ile Ser Arg Gly Gly Val Thr Asp Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser Ala

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>155

<211>122

<212>PRT

<213>Vicugna pacos

<400>155

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Ser Phe Ser Ser Tyr

20 25 30

His Met Asp Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Trp Thr Gly His Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Thr Arg Arg Ala Thr Met Ile Ala Val Pro Ser Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>156

<211>125

<212>PRT

<213>Vicugna pacos

<400>156

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Ser Tyr Val Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ala Thr Ile Ser Arg Gly Gly Val Thr Asp Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 9095

Cys Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>157

<211>125

<212>PRT

<213>Vicugna pacos

<400>157

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Ser Tyr Val Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>158

<211>126

<212>PRT

<213>Vicugna pacos

<400>158

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Tyr Leu Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro

100 105 110

Ser Asp TyrTrp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>159

<211>128

<212>PRT

<213>Vicugna pacos

<400>159

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Gly Ser Tyr

20 25 30

Asp Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Asn Trp Tyr Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ala Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Ser Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Glu Lys Gly Phe Ala Ser Leu Arg Leu Trp Ala Leu Ser His

100 105 110

Lys Pro His Glu Ser Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120125

<210>160

<211>124

<212>PRT

<213>Vicugna pacos

<400>160

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ile Pro Tyr Leu Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro Ser Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>161

<211>124

<212>PRT

<213>Vicugna pacos

<400>161

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Val Phe Ser Gly Arg Pro Ser Ser Tyr Tyr

20 25 30

Gly Val Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ser Gly Gly Thr Asp Asn Tyr Ala Asn Phe Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Lys Asp Asn Gly Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Ala Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>162

<211>125

<212>PRT

<213>Vicugna pacos

<400>162

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ile Pro Tyr Leu Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Glu Tyr Ser

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>163

<211>126

<212>PRT

<213>Vicugna pacos

<400>163

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Thr Tyr Leu Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ala Ile Thr Arg Gly Gly His Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Glu Tyr

100 105 110

Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>164

<211>121

<212>PRT

<213>Vicugna pacos

<400>164

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg LeuSer Cys Ala Ala Ser Gly Arg Thr Val Ser Ser Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Asp Arg Asp Lys Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Arg Arg Tyr Tyr Arg Ser Gly Ala Gln Asp Tyr Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>165

<211>124

<212>PRT

<213>Vicugna pacos

<400>165

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ser Thr Tyr Leu Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ile Thr Arg Gly Gly His Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Ser Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>166

<211>121

<212>PRT

<213>Vicugna pacos

<400>166

Asp Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Gly Thr Ile Gly Ser Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 4045

Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser Val Glu

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Ser Thr Met Tyr Leu

65 70 75 80

Gln Met His Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Arg Arg Trp Tyr Arg Ser Ala Pro Gln Asp Tyr Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>167

<211>125

<212>PRT

<213>Vicugna pacos

<400>167

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ser Thr Tyr Leu Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Ile Thr Arg Gly Gly Val Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Pro Ser

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>168

<211>121

<212>PRT

<213>Vicugna pacos

<400>168

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Ile Gly Ser Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser Val Glu

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Ser Thr Met Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Ala

85 90 95

Ala Arg Arg Tyr Tyr Arg Ser Ala Pro Gln Asp Tyr Asp Phe Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>169

<211>121

<212>PRT

<213>artifical sequence

<400>169

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

GlnMet Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>170

<211>121

<212>PRT

<213>artifical sequence

<400>170

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>171

<211>121

<212>PRT

<213>artifical sequence

<400>171

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly

100105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>172

<211>121

<212>PRT

<213>artifical sequence

<400>172

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>173

<211>121

<212>PRT

<213>artifical sequence

<400>173

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Val Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>174

<211>121

<212>PRT

<213>artifical sequence

<400>174

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Val Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>175

<211>121

<212>PRT

<213>artifical sequence

<400>175

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Val Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>176

<211>120

<212>PRT

<213>artifical sequence

<400>176

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 510 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gly Leu Gln Val Ala

35 40 45

Thr Ile Thr Arg Gly Gly Val Thr Met Tyr Ala Asp Ser Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln

65 70 75 80

Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala

85 90 95

Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>177

<211>126

<212>PRT

<213>artifical sequence

<400>177

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Tyr Leu Pro

20 25 30

Asp Met Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro

100 105 110

Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>178

<211>126

<212>PRT

<213>artifical sequence

<400>178

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Tyr Leu Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro

100 105 110

Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>179

<211>126

<212>PRT

<213>artifical sequence

<400>179

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Tyr Leu Pro

20 25 30

Asp Met Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val

35 40 45

Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro

100 105 110

Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>180

<211>126

<212>PRT

<213>artifical sequence

<400>180

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Tyr Leu Pro

20 25 30

Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val

35 40 45

Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro

100 105 110

Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>181

<211>124

<212>PRT

<213>artifical sequence

<400>181

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Val Phe Ser Gly Arg Pro Ser Ser Tyr Tyr

20 25 30

Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ser Gly Gly Thr Asp Asn Tyr Ala Asn Phe Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Ala Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>182

<211>124

<212>PRT

<213>artifical sequence

<400>182

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Val Phe Ser Gly Arg Pro Ser Ser Tyr Tyr

20 25 30

Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Ser Arg Ser Gly Gly Thr Asp Asn Tyr Ala Asn Phe Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr

65 7075 80

Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Ala Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>183

<211>124

<212>PRT

<213>artifical sequence

<400>183

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Val Phe Ser Gly Arg Pro Ser Ser Tyr Tyr

20 25 30

Gly Val Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val

35 40 45

Ala Gly Ile Ser Arg Ser Gly Gly Thr Asp Asn Tyr Ala Asn Phe Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Ala Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>184

<211>372

<212>DNA

<213>Vicugna pacos

<400>184

caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggacc atccttcagt acctatgccg tgggctggtt ccgccaggtt 120

ccccggagag agcgtgcgtt tgttgcaggt attaattgga gtggcgaaga aacaacatat 180

cataactccg tgaacggccg attcaccatc tccagagaca acgccaagaa tacggtgtat 240

ctacaaatga acagcctgca acctgaggac acggccgttt attactgtgc agccaaccgt 300

cggaattact ctagcaccta taaagggaac tatgactact ggggccaggg gacccaggtc 360

accgtctcct ca 372

<210>185

<211>357

<212>DNA

<213>Vicugna pacos

<400>185

caggtacagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcag cctctggacg caccttcagt agctatgcca tgggctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tgtagcagct attaggcggg atgctgttag cacatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagga tatggtgtat 240

ctgcaaatga acaacctgaa acctgaggac acggccgttt attactgtgc gatgggggat 300

gactatgtag atgagtatga ctactggggc cagggaaccc aggtcaccgt ctcctca 357

<210>186

<211>369

<212>DNA

<213>Vicugna pacos

<400>186

caggtgaagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

gcctgtgtag cctccagagt taccggtagt cactatgacc tggcctggtt ccgccagggt 120

ccagggaagg agcgtgaagt tgtagcagag tttacctggc gtagtggtcc cacatcgtat 180

gcagagtccg tgaagggccg attcgccgtc tccaaagata acgccaagaa cacactgtat 240

ctgcaaatgg acaacctgag acctgaggac acggccgttt attactgtgc atcccgatac 300

ttttacacct atggcgaccc cctgaagtat gactattggg gccagggaac ccaggtcacc 360

gtctcctca 369

<210>187

<211>366

<212>DNA

<213>Vicugna pacos

<400>187

caggtaaagc tggaggagtc tgggggaggc ttggtgcgcc ctggggggtc tctgagactc 60

tcctgtgcag cctccggaag catcttcagt atcaatcaca tggcctggta ccgccaggct 120

ccagggaagg agcgcgagtt cgtcgcacgt gcttttagta gtggttcgac aacctatgca 180

gactccgtga ggggccgatt caccatctcc agagacgacg ccaagaacac ggtgtatcta 240

caaatgaaca gtctgaaacc tgaggacacg gccgtctatt actgtaatgg agatgggttt 300

ctattatacg acgatactta ctactccaac gcctggggcc aggggaccca ggtcaccgtc 360

tcctca 366

<210>188

<211>345

<212>DNA

<213>Vicugna pacos

<400>188

caggtaaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaat tgccttcagt gtctttgaca tggcctggta ccgccaggct 120

ccagggaacc agcgcgagtt ggtcgcagag actactactg ctggtattaa cacatatgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240

cagatgaaca gcctgagacc cgaggacacg gccgtctata gctgtaatgc gaaaaatggg 300

tggcggaccc tctggggcca ggggacccag gtcaccgtct cctca 345

<210>189

<211>333

<212>DNA

<213>Vicugna pacos

<400>189

ggtgtgcagc tggtggagtc tgggggaggc ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgtag tttctggaag caccttcagg gacatgtcct tcggctggta ccgccaggct 120

ccagggaagc agcgcgagcg gctcgcatac atttcgagca gtggctacac aaactatgta 180

gatgctgtga aggaccgatt caccatctcc agagacaacg ccaagaacac ggtgtacctc 240

caaatgaaca gcctgaaacc tgaggacacg gccatctatt actgcaatac ccttgtgtac 300

tggggccagg ggacccaggt caccgtctcc tca 333

<210>190

<211>345

<212>DNA

<213>Vicugna pacos

<400>190

caggtaaagc tggaagagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaat tgccttcagt gtctttgaca tggcctggta ccgccaggct 120

ccagggaacc agcgcgagtt ggtcgcacgc atcacttttg atggtatccc gcactacgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240

cagatgaaca gcctgagacc cgaggacacg gccgtctata gctgtaatgc gaaaaatggg 300

tggcggaccc tctggggcca ggggacccag gtcaccgtct cctca 345

<210>191

<211>378

<212>DNA

<213>Vicugna pacos

<400>191

caggtacagc tggtggattc tgggggaggc tatgtgcagc cgggggggtc tctgagactc 60

tcctgtgcag cctctggagg cagcttcagt aaatatgcca tgggatggtt ccgccaggct 120

ccagggaagg agcgtcagtt tgtaggaaat atttactgga gtgatggtag cacacactat 180

caagactccg tgaagggccg attcaccctc tccaaagact acgccaagaa cacggtgtat 240

ctagaaatga acaacctgaa acctgaggac acggccgttt attactgcgc gtctcggggc 300

tccaataatg gtggttctta ctacagcgag actgggtatg attactgggg ccaggggact 360

caggtcaccg tctcctca 378

<210>192

<211>363

<212>DNA

<213>Vicugna pacos

<400>192

caggtgcagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcgg cctctggagg caccatcggt agctatgaca tgggctggtt ccgccaagct 120

ccagggaagg agcgcgagtt tgtagcagcc attacctgga tttctaacac aaactatgca 180

gactccgtgg agggccgatt caccatctcc agagacaaag acaagacgac gatgtatctg 240

caaatgaata gcctgaaacc tgaggacacg gccgtttatt actgtgcagc acgccgttgg 300

catcgctccg ccccacacga ctatgagttt tggggccagg ggacccaggt caccgtctcc 360

tca 363

<210>193

<211>372

<212>DNA

<213>Vicugna pacos

<400>193

caggtacagc tggtggattc tgggggagga ttggtgctgg ctgggggctc tctgagactc 60

tcctgtgcag cctctggaga caccttcgct aggtattcta tgggctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tgttgcaggt attagcaggt cgagcggtac cataatttat 180

ggaggctccg tgaagggccg attcatcatc ttcagagaca atgtcaagaa cacggtgtat 240

ctacaaatga acggtttgaa acctgacgac acggccgttt atttctgtgc agcccgagag 300

agcttactgg cagtgactac cacacgggat tatccgtact ggggccaggg gacccaggtc 360

accgtctcct ca 372

<210>194

<211>363

<212>DNA

<213>Vicugna pacos

<400>194

caggtgaagc tggaggagtc tgggggaggg ttggtgcagg ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag cataccatat gtccctgaca tgcactggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcaacac aatgtatgct 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatcta 240

caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300

tcgagtgttg cattgaagct tgtggaatac tggggccagg ggacccaggt caccgtctcc 360

tca 363

<210>195

<211>375

<212>DNA

<213>Vicugna pacos

<400>195

caggtacagc tggtggattc tgggggagga tttgtgcagg ctgggggctc tctgagactc 60

tcctgtgcag cctctggacg caccttcagt gactatgcca tggcctggtt ccgccaggct 120

ccagggaagg agcgtgaatt tgtagcaggt attagctgga ctggtggcag atactatgct 180

gagtccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggcgtttctg 240

caaatgaaca cgctgaaacc tgaggacacg ggcatttatc actgtgcaac tcccaaccaa 300

gccgggcttg tgctgttaga tgacgccgaa gggtacgcct actggggcca ggggacccag 360

gtcaccgtct cctca 375

<210>196

<211>351

<212>DNA

<213>Vicugna pacos

<400>196

caggtacagc tggtggattc tgggggaggg ttggtgcagg ctggggggtc tctgagactc 60

tcctgcgcag tctctggatt caccgtcagc acccggaaca tgggctggta ccgccaggct 120

ccagggaagc agcgtgtttt ggtcgcacgt attggtagtg atggtagtac ctacaatgta 180

gactccgtga agggccgatt caccatctcc agagacaact ctaagaacgc ggtgtatctg 240

caaatgaaca gcctgaagcc tgaggacacg gccgtgtact ggtgtaacac ctttcccgtc 300

acaaagttct atgactactg gggccagggg acccaggtca ccgtctcctc a 351

<210>197

<211>378

<212>DNA

<213>Vicugna pacos

<400>197

gaggtgaagc tggtggattc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcag cctctggact ccgcttcagt gcctatgaca tgggctggtt ccgtcaggct 120

ccagggaagg agcgtgagtt tgtagcaact attaactgga gtgctcttag ccgatactat 180

gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatct 240

ctgcacatga acagcctgaa atctgaggac acggccgttt attactgtgc aggagggaga 300

attagtgcgg cactacgtgt cccggaccgg gatgcgtata gctactgggg ccagggtacc 360

caggtcaccg tctcctca 378

<210>198

<211>378

<212>DNA

<213>Vicugna pacos

<400>198

caggtacagc tggtggattc tgggggaggt ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgtcg cttctggacg caccgtcagg gacgatgcca tgggctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tgtagcaact attagctgga atggtggtag tacatactat 180

gccgcctccg caaaggggcg tttcatagtc tctagagaca atgccaagaa caccatgtat 240

ctgcaaatga acagcctgaa acctgaggac acggccgtgt actactgtgc agtagcgtct 300

cgttatcggc agatagtact taataccgag gagaagtatg acgcctgggg ccaggggacc 360

caggtcaccg tctcctca 378

<210>199

<211>426

<212>DNA

<213>Vicugna pacos

<400>199

caggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc cctgagactc 60

tcctgtacag cctctggaag ctctttcagt gtccctcaca tggactggtt ccgccaggct 120

ccaggccagg agcgtgagtt tgtagcaact attagccgtg gaggtgtcag tacttacgca 180

gcagactccg tgaagggccg aagcaccatc tccagagaca acgccaaaaa cacgatgtac 240

cttcaaatga acaacctgaa acctgaggac acggccgttt attactgtaa tgcagacgtt 300

tggtcgagtg tttcattgaa attggtggag tactggggcc aggggaccca ggtcaccgtc 360

tcctcaggat ccgaacaaaa actgatcagc gaagaagatc tgaaccatca ccatcaccat 420

tagtga 426

<210>200

<211>333

<212>DNA

<213>Vicugna pacos

<400>200

caggtgaagc tggaggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60

tcctgtgcag cctctacaac caccttcagt caccgtactt ttgcctggta ccgccaggct 120

ccagggaagc agcgcgagtt ggtcgcaact attggaagtt acggcagtac gtactatgac 180

gagtccgtga aggaccgatt caccatcgcc agagacaacg ccaagaacac ggtgtatctg 240

caaatgaaca gcctgaaacc tcaggacacg gccgtctatt actgtcatac ccaggtgtac 300

tggggccagg ggacccaggt caccgtctcc tca 333

<210>201

<211>372

<212>DNA

<213>Vicugna pacos

<400>201

gaggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag cacttcctat gtctatgaca tgcactggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcagca attactgatg gaggctccac ggataactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtaa tgcagacgtt 300

tggtcgagtg tttcattgaa attggtggag tacgactact ggggccaggg gacccaggtc 360

accgtctcct ca 372

<210>202

<211>375

<212>DNA

<213>Vicugna pacos

<400>202

gaggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag cacttcctat gtctatgaca tgcactggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcagca attactgatg gaggctccac ggataactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300

gtttggtcga gtgtttcatt gaaattggtg gagtacgact actggggcca ggggacccag 360

gtcaccgtct cctca 375

<210>203

<211>363

<212>DNA

<213>Vicugna pacos

<400>203

caggtgaagc tggaggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag catcccctat gtccctgaca tgcactggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcgtcac gatgtatgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240

caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300

tcgagtgttt cattgaaatt ggtggagtac tggggccagg ggacccaggt caccgtctcc 360

tca 363

<210>204

<211>375

<212>DNA

<213>Vicugna pacos

<400>204

gaggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag cacttcctat gtctatgaca tgcactggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcagca atttctcgtg gaggcgtcac ggataactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300

gtttggtcga gtgtttcatt gaaattggtg gagtacgact actggggcca ggggacccag 360

gtcaccgtct cctca 375

<210>205

<211>363

<212>DNA

<213>Vicugna pacos

<400>205

gaggtgcagc tggaggattc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag catctcatat gtccctgaca tgcactggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcaacac aatgtatgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240

caaatgacctccctgaaacc tgaggacacg gccgtgtact actgtaatgc agacgtttgg 300

tcgagtgttt tattcaaact tgtggagtac tggggtcagg ggacccaggt caccgtctcc 360

tca 363

<210>206

<211>375

<212>DNA

<213>Vicugna pacos

<400>206

gaggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag cttctcctat atctatgaca tgcactggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcagca atttctcgtg gaagctccac ggataactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300

gtttggtcga gtgtttcatt gaaattggtg gagtacgact actggggcca ggggacccag 360

gtcaccgtct cctca 375

<210>207

<211>369

<212>DNA

<213>Vicugna pacos

<400>207

gaggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag cttctcctat atccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcagca atttctcgtg gaagctccac ggataactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300

gtttggtcga gtgtttcatt gaaattggtg gagtactggg gccaggggac ccaggtcacc 360

gtctcctca 369

<210>208

<211>378

<212>DNA

<213>Vicugna pacos

<400>208

caggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcag cctctggact ccgcttcagt gcctatgaca tgggctggtt ccgtcaggct 120

ccagggaagg agcgtgagtt tgtagcaact attaactgga gtactcttag ccgatattat 180

gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcacatga acagcctgaa acctgaggac acggccgttt attactgtgc aggagggaga 300

attagttcgg aactacgtgt cacggcccgg gatgcgtata cctactgggg ccagggtacc 360

caggtcaccg tctcctca 378

<210>209

<211>381

<212>DNA

<213>Vicugna pacos

<400>209

caggtgcagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag catctcctat gtccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcagca atttctcgtg gaggctccac ggataactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300

gtttggtcga gtgtttcatt gaaattggtg gagtccgcct atgattactg gggccagggg 360

acccaggtca ccgtctcctc a 381

<210>210

<211>378

<212>DNA

<213>Vicugna pacos

<400>210

caggtgcagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag catctcctat gtccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcaact atttctcgtg gaggcgtcac ggataactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtaa tgcagacgtt 300

tggtcgagtg tttcattgaa attggtggag tccgcctatg attactgggg ccaggggacc 360

caggtcaccg tctcctca 378

<210>211

<211>378

<212>DNA

<213>Vicugna pacos

<400>211

caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag cacctcctat gtccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcaact atttctcgtg gaggcgtcac ggattattat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtaa tgcagacgtt 300

tggtcgagtg tttcattgaa attggtggag tccgcctatg attactgggg ccaggggacc 360

caggtcaccg tctcctca 378

<210>212

<211>366

<212>DNA

<213>Vicugna pacos

<400>212

caggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc cctgagactc 60

tcctgtacag cctctggaag ctctttcagt agttatcaca tggactggtt ccgccaggct 120

ccaggccagg agcgtgagtt tgtagcagct attagctgga ctggtcatag tacttactat 180

gcagactccg tgaagggccg aagcaccatc tccagagaca acgccaaaaa cacgatgtac 240

cttcaaatga acaacctgaa acctgaggac acggccgttt attactgtgc agcgaccagg 300

cgcgcgacta tgatcgccgt accgtctgat tactggggcc aggggaccca ggtcaccgtc 360

tcctca 366

<210>213

<211>375

<212>DNA

<213>Vicugna pacos

<400>213

caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt catctcctat gtccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcagca actatttctc gtggaggcgt cacggattac 180

tatgcagact ccgtgaaggg ccgattcacc atctccagag acaacgccaa gaacacggta 240

tatctgcaaa tgacctccct gaaacctgag gacacggccg tctactactg taatgcagac 300

gtttggtcga gtgtttcatt gaaattggtg gagtccgcct attggggcca ggggacccag 360

gtcaccgtct cctca 375

<210>214

<211>375

<212>DNA

<213>Vicugna pacos

<400>214

caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt catctcctat gtccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcagca actattactc gtggaggcgt cacggattac 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300

gtttggtcga gtgtttcatt gaaattggtg gagtccgcct attggggcca ggggacccag 360

gtcaccgtct cctca 375

<210>215

<211>378

<212>DNA

<213>Vicugna pacos

<400>215

caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggata cacctcctat ctccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcagca actattactc gtggaggcgt cacggaatac 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300

gtttggtcga gtgtttcatt gaaattggcc gtgccgtccg actattgggg ccaggggacc 360

caggtcaccg tctcctca 378

<210>216

<211>383

<212>DNA

<213>Vicugna pacos

<400>216

caggtgaagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcag cctctggacg caccttcggt agctatgata tggcctggtt ccgccaggtt 120

ccagggaagg aacgagagtt tgtagcggct attaactggt acggtggcta cacatactac 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cgcggtgtat 240

ctacaaatga acaacctgaa accgtcggac acggccgttt attactgtgc agcggagaaa 300

ggcttcgcat cgctcaggct gtgggcatta tctcacaaac cccatgagtc aaggggccag 360

gggacccagg tcaccgtctc ctc 383

<210>217

<211>372

<212>DNA

<213>Vicugna pacos

<400>217

caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggata catcccctat ctccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcgtcac ggaatacgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240

caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300

tcgagtgttt cattgaaatt ggccgtgccg tccgactatt ggggccaggg gacccaggtc 360

accgtctcct ca 372

<210>218

<211>372

<212>DNA

<213>Vicugna pacos

<400>218

caggtgcagc tggtggagtc tgggggagga ttggtgcagc ctgggggctc tctgaccctc 60

tcctgtgtat tctctggccg tcccagtagc tactatggcg tgagctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tgtagcagga atttcgagga gtggtggtac tgacaactat 180

gcaaatttcg tgaagggccg attcaccgtc tccaaagaca acggcaagaa cacggtgtat 240

ctccaaatga acaatctgaa acctgaggac acggccgttt attactgtgc agccgctaca 300

aatgtctatg cctccgcgac gttgtccgcc tatgattatt ggggccaggg gacccaggtc 360

accgtctcct ca 372

<210>219

<211>375

<212>DNA

<213>Vicugna pacos

<400>219

caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggata catcccctat ctccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcgtcac ggaatacgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240

caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300

tcgagtgttt cattgaaatt ggccgtggag tactccgact attggggcca ggggacccag 360

gtcaccgtct cctca 375

<210>220

<211>378

<212>DNA

<213>Vicugna pacos

<400>220

caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggata ctccacctat ctccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcagct attactcgtg gaggccactc cacgtactac 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240

ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtaa tgcagacgtt 300

tggtcgagtg tttcattgaa attggccgtg gagtactccg actattgggg ccaggggacc 360

caggtcaccg tctcctca 378

<210>221

<211>363

<212>DNA

<213>Vicugna pacos

<400>221

caggtgaagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcgg cctcgggacg caccgtcagt agctatgaca tgggctggtt ccgccaatct 120

ccagggaagg agcgcgagtt tgtagcagcc attacctgga tttcaaacac aaactatgca 180

gactccgtga agggccgatt caccatcgac agagacaaag ccaagaacac ggtgtatctg 240

caaatgaaca gcctgaaacc tgaggacacg gccgtttatt actgtgcagc acgccgttat 300

tatcgctctg gcgcacaaga ctatgagtac tggggccagg ggacccaggt caccgtctcc 360

tca 363

<210>222

<211>372

<212>DNA

<213>Vicugna pacos

<400>222

caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatc atccacctat ctccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgctatt actcgtggag gccactccac gtactacgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240

caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300

tcgagtgttt cattgaaatt ggtggagtac tccgactatt ggggccaggg gacccaggtc 360

accgtctcct ca 372

<210>223

<211>363

<212>DNA

<213>Vicugna pacos

<400>223

gatgtgaagc tggaggagtc tgggggagga ttggtgcagg ctggggactc tctgagactc 60

tcctgtgtgg cctctggagg caccatcggt agctatgaca tgggctggtt ccgccaagct 120

ccagggaagg agcgcgagtt tgtagcagcc attacctgga tttctaacac aaactatgca 180

gactccgtgg agggccgatt caccatctcc agagacaaag ccaagagcac gatgtatctg 240

caaatgcata gcctgaaacc tgaggacacg gccgtttatt actgtgcagc acgccgttgg 300

tatcgctccg ccccacaaga ctatgagtac tggggccagg ggacccaggt caccgtctcc 360

tca 363

<210>224

<211>375

<212>DNA

<213>Vicugna pacos

<400>224

caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatc atccacctat ctccctgaca tgggctggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgctatt actcgtggag gcgtgtccac gtactacgca 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240

caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300

tcgagtgttt cattgaaatt ggtggagtac ccatccgact attggggcca ggggacccag 360

gtcaccgtct cctca 375

<210>225

<211>363

<212>DNA

<213>Vicugna pacos

<400>225

caggtgaagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcgg cctctggagg caccatcggt agctatgaca tgggctggtt ccgccaagct 120

ccagggaagg agcgcgagtt tgtagcagcc attacctgga tttctaacac aaactatgca 180

gactccgtgg agggccgatt caccatctcc agagacaaag ccaagagcac gatgtatctg 240

caaatgaata gcctgaaacc tgaggacacg gccgtttatg tctgtgcagc acgccgttat 300

tatcgctccg ccccacaaga ctatgacttt tggggccagg ggacccaggt caccgtctcc 360

tca 363

Claims (10)

1. A set of anti-VEGF single domain antibodies, each of which consists of a framework region and 3 complementarity determining regions, wherein said single domain antibodies are selected from NBV1, NBV2, NBV3, NBV4, NBV5, NBV6, NBV7, NBV8, NBV9, NBV10, NBV11, NBV12, NBV13, NBV14, NBV15, NBV16, nb;

wherein, the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV1 are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV2 are respectively shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV3 are shown as SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV4 are shown as SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV5 are shown as SEQ ID No.13, SEQ ID No.14 and SEQ ID No.15 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV6 are shown as SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV7 are shown as SEQ ID No.19, SEQ ID No.20 and SEQ ID No.21 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV8 are shown as SEQ ID No.22, SEQ ID No.23 and SEQ ID No.24 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV9 are shown as SEQ ID No.25, SEQ ID No.26 and SEQ ID No.27, respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV10 are shown as SEQ ID No.28, SEQ ID No.29 and SEQ ID No.30 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV11 are shown as SEQ ID No.31, SEQ ID No.32 and SEQ ID No.33 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV12 are shown as SEQ ID No.34, SEQ ID No.35 and SEQ ID No.36 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV13 are shown as SEQ ID No.37, SEQ ID No.38 and SEQ ID No.39 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV14 are respectively shown as SEQ ID No.40, SEQ ID No.41 and SEQ ID No. 42; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV15 are shown as SEQ ID No.43, SEQ ID No.44 and SEQ ID No.45 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV16 are shown as SEQ ID No.46, SEQ ID No.47 and SEQ ID No.48 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV17 are shown as SEQ ID No.49, SEQ ID No.50 and SEQ ID No.51 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV18 are shown as SEQ ID No.52, SEQ ID No.53 and SEQ ID No.54 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV19 are shown as SEQ ID No.55, SEQ ID No.56 and SEQ ID No.57 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV20 are shown as SEQ ID No.58, SEQ ID No.59 and SEQ ID No.60 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV21 are shown as SEQ ID No.61, SEQ ID No.62 and SEQ ID No.63 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV22 are shown as SEQ ID No.64, SEQ ID No.65 and SEQ ID No.66 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV23 are shown as SEQ ID No.67, SEQ ID No.68 and SEQ ID No.69 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV24 are shown as SEQ ID No.70, SEQ ID No.71 and SEQ ID No.72 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV25 are shown as SEQ ID No.73, SEQ ID No.74 and SEQ ID No.75 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV26 are shown as SEQ ID No.76, SEQ ID No.77 and SEQ ID No.78 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV27 are shown as SEQ ID No.79, SEQ ID No.80 and SEQ ID No.81 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV28 are shown as SEQ ID No.82, SEQ ID No.83 and SEQ ID No.84 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV29 are shown as SEQ ID No.85, SEQ ID No.86 and SEQ ID No.87 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV30 are shown as SEQ ID No.88, SEQ ID No.89 and SEQ ID No.90 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV31 are shown as SEQ ID No.91, SEQ ID No.92 and SEQ ID No.93 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV32 are shown as SEQ ID No.94, SEQ ID No.95 and SEQ ID No.96 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV33 are shown as SEQ ID No.97, SEQ ID No.98 and SEQ ID No.99 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV34 are shown as SEQ ID No.100, SEQ ID No.101 and SEQ ID No.102 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV35 are shown as SEQ ID No.103, SEQ ID No.104 and SEQ ID No.105 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV36 are shown as SEQ ID No.106, SEQ ID No.107 and SEQ ID No.108, respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV37 are shown as SEQ ID No.109, SEQ ID No.110 and SEQ ID No.111 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV38 are shown as SEQ ID No.112, SEQ ID No.113 and SEQ ID No.114 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV39 are shown as SEQ ID No.115, SEQ ID No.116 and SEQ ID No.117 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV40 are shown as SEQ ID No.118, SEQ ID No.119 and SEQ ID No.120 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV41 are shown as SEQ ID No.121, SEQ ID No.122 and SEQ ID No.123 respectively; the amino acid sequences of 3 complementarity determining regions of the single domain antibody NBV42 are shown as SEQ ID No.124, SEQ ID No.125 and SEQ ID No.126 respectively;

or a protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in any one of the amino acid sequences shown above, wherein the protein mutant has the same function as the protein before mutation; or an amino acid sequence which has at least 90% identity to any of the amino acid sequences shown above.

2. The single domain antibody according to claim 1, characterized in that the amino acid sequence of the single domain NBV1 is represented by SEQ ID No.127, the amino acid sequence of the single domain NBV2 is represented by SEQ ID No.128, the amino acid sequence of the single domain NBV3 is represented by SEQ ID No.129, the amino acid sequence of the single domain NBV4 is represented by SEQ ID No.130, the amino acid sequence of the single domain NBV5 is represented by SEQ ID No.131, the amino acid sequence of the single domain NBV6 is represented by SEQ ID No.132, the amino acid sequence of the single domain NBV7 is represented by SEQ ID No.133, the amino acid sequence of the single domain NBV8 is represented by SEQ ID No.134, the amino acid sequence of the single domain NBV9 is represented by SEQ ID No.135, the amino acid sequence of the single domain NBV10 is represented by SEQ ID No.136, the amino acid sequence of the single domain NBV11 is represented by SEQ ID No.137, the amino acid sequence of the single domain NBV 539V 539138, the amino acid sequence of the single domain NBV14 is represented by SEQ ID No.139, the amino acid sequence of the single domain NBV15 is shown as SEQ ID No.141, the amino acid sequence of the single domain antibody NBV16 is shown as SEQ ID No.142, the amino acid sequence of the single domain antibody NBV17 is shown as SEQ ID No.143, the amino acid sequence of the single domain antibody NBV18 is shown as SEQ ID No.144, the amino acid sequence of the single domain antibody NBV19 is shown as SEQ ID No.145, the amino acid sequence of the single domain antibody NBV20 is shown as SEQ ID No.146, the amino acid sequence of the single domain antibody NBV21 is shown as SEQ ID No.147, the amino acid sequence of the single domain antibody NBV22 is shown as SEQ ID No.148, the amino acid sequence of the single domain antibody NBV23 is shown as SEQ ID No.149, the amino acid sequence of the single domain antibody NBV24 is shown as SEQ ID No.150, the amino acid sequence of the single domain antibody NBV25 is shown as SEQ ID No.151, the amino acid sequence of the single domain antibody NBV26 is shown as SEQ ID No.152, the amino acid sequence of the single domain antibody NBV27 is shown as SEQ ID No.28, the amino acid sequence NBV28 is shown as SEQ ID No. 685, the amino acid sequence of the single domain antibody NBV29 is shown as SEQ ID No.155, the amino acid sequence of the single domain antibody NBV30 is shown as SEQ ID No.156, the amino acid sequence of the single domain antibody NBV31 is shown as SEQ ID No.157, the amino acid sequence of the single domain antibody NBV32 is shown as SEQ ID No.158, the amino acid sequence of the single domain antibody NBV33 is shown as SEQ ID No.159, the amino acid sequence of the single domain antibody NBV34 is shown as SEQ ID No.160, the amino acid sequence of the single domain antibody NBV35 is shown as SEQ ID No.161, the amino acid sequence of the single domain antibody NBV36 is shown as SEQ ID No.162, the amino acid sequence of the single domain antibody NBV37 is shown as SEQ ID No.163, the amino acid sequence of the single domain antibody NBV38 is shown as SEQ ID No.164, the amino acid sequence of the single domain antibody NBV39 is shown as SEQ ID No.165, the amino acid sequence of the single domain antibody NBV40 is shown as SEQ ID No.166, the amino acid sequence of the single domain antibody NBV41 is shown as SEQ ID No.167, the amino acid sequence of the single-domain antibody NBV42 is shown as SEQ ID No. 168;

or a protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in any one of the amino acid sequences shown above, wherein the protein mutant has the same function as the protein before mutation; or an amino acid sequence which has at least 90% identity to any of the amino acid sequences shown above.

3. The humanized single-domain antibody for resisting VEGF is characterized in that (1) the single-domain antibody NBV11 is humanized and transformed to obtain a 4-humanized antibody, and the amino acid sequences of the 4-humanized antibody are respectively shown as SEQ ID No.169, SEQ ID No.170, SEQ ID No.171 and SEQ ID No. 172; or (2) the single domain antibody NBV20 is humanized and transformed to obtain 4 humanized antibodies, and the amino acid sequences of the antibodies are respectively shown as SEQ ID No.173, SEQ ID No.174, SEQ ID No.175 and SEQ ID No. 176; or (3) the single domain antibody NBV32 is humanized and transformed to obtain 4 humanized antibodies, and the amino acid sequences of the antibodies are respectively shown as SEQ ID No.177, SEQ ID No.178, SEQ ID No.179 and SEQ ID No. 180; or (4) the single domain antibody NBV35 is humanized and transformed to obtain a 3-humanized antibody, and the amino acid sequences of the 3-humanized antibody are respectively shown as SEQ ID No.181, SEQ ID No.182 and SEQ ID No. 183.

4. A gene encoding the single domain antibody of any one of claims 1 or 2; preferably, the nucleotide sequence of the coding gene of the single domain antibody NBV1 is shown as SEQ ID No.184, the nucleotide sequence of the coding gene of the single domain antibody NBV2 is shown as SEQ ID No.185, the nucleotide sequence of the coding gene of the single domain antibody NBV3 is shown as SEQ ID No.186, the nucleotide sequence of the coding gene of the single domain antibody NBV4 is shown as SEQ ID No.187, the nucleotide sequence of the coding gene of the single domain antibody NBV5 is shown as SEQ ID No.188, the nucleotide sequence of the coding gene of the single domain antibody NBV6 is shown as SEQ ID No.189, the nucleotide sequence of the coding gene of the single domain antibody NBV7 is shown as SEQ ID No.190, the nucleotide sequence of the coding gene of the single domain antibody NBV8 is shown as SEQ ID No.191, the nucleotide sequence of the coding gene of the single domain antibody NBV9 is shown as SEQ ID No.192, the nucleotide sequence of the coding gene NBV10 is shown as SEQ ID No.193, and the nucleotide sequence of the coding gene NBV11 is shown as SEQ ID No.194, the nucleotide sequence of the coding gene of the single domain antibody NBV12 is shown as SEQ ID No.195, the nucleotide sequence of the coding gene of the single domain antibody NBV13 is shown as SEQ ID No.196, the nucleotide sequence of the coding gene of the single domain antibody NBV14 is shown as SEQ ID No.197, the nucleotide sequence of the coding gene of the single domain antibody NBV15 is shown as SEQ ID No.198, the nucleotide sequence of the coding gene of the single domain antibody NBV16 is shown as SEQ ID No.199, the nucleotide sequence of the coding gene of the single domain antibody NBV17 is shown as SEQ ID No.200, the nucleotide sequence of the coding gene of the single domain antibody NBV18 is shown as SEQ ID No.201, the nucleotide sequence of the coding gene of the single domain antibody NBV19 is shown as SEQ ID No.202, the nucleotide sequence of the coding gene of the single domain antibody NBV20 is shown as SEQ ID No.203, the nucleotide sequence of the coding gene of the single domain antibody NBV21 is shown as SEQ ID No.204, the nucleotide sequence of the coding gene NBV22 is shown as SEQ ID No.205, the nucleotide sequence of the coding gene of the single domain antibody NBV23 is shown as SEQ ID No.206, the nucleotide sequence of the coding gene of the single domain antibody NBV24 is shown as SEQ ID No.207, the nucleotide sequence of the coding gene of the single domain antibody NBV25 is shown as SEQ ID No.208, the nucleotide sequence of the coding gene of the single domain antibody NBV26 is shown as SEQ ID No.209, the nucleotide sequence of the coding gene of the single domain antibody NBV27 is shown as SEQ ID No.210, the nucleotide sequence of the coding gene of the single domain antibody NBV28 is shown as SEQ ID No.211, the nucleotide sequence of the coding gene of the single domain antibody NBV29 is shown as SEQ ID No.212, the nucleotide sequence of the coding gene of the single domain antibody NBV30 is shown as SEQ ID No.213, the nucleotide sequence of the coding gene of the single domain antibody NBV31 is shown as SEQ ID No.214, the nucleotide sequence of the coding gene NBV32 is shown as SEQ ID No.215, and the nucleotide sequence of the coding gene NBV33 is shown as SEQ ID No.216, the nucleotide sequence of the coding gene of the single domain antibody NBV34 is shown as SEQ ID No.217, the nucleotide sequence of the coding gene of the single domain antibody NBV35 is shown as SEQ ID No.218, the nucleotide sequence of the coding gene of the single domain antibody NBV36 is shown as SEQ ID No.219, the nucleotide sequence of the coding gene of the single domain antibody NBV37 is shown as SEQ ID No.220, the nucleotide sequence of the coding gene of the single domain antibody NBV38 is shown as SEQ ID No.221, the nucleotide sequence of the coding gene of the single domain antibody NBV39 is shown as SEQ ID No.222, the nucleotide sequence of the coding gene of the single domain antibody NBV40 is shown as SEQ ID No.223, the nucleotide sequence of the coding gene of the single domain antibody NBV41 is shown as SEQ ID No.224, and the nucleotide sequence of the coding gene of the single domain antibody NBV42 is shown as SEQ ID No. 225;

or a polynucleotide sequence capable of hybridising to the complement of any one of the polynucleotide sequences presented above under stringent hybridisation conditions; or a polynucleotide sequence having at least 90% identity to any of the polynucleotide sequences shown above.

5. A recombinant expression vector comprising one or more of the coding genes of claim 4 or 5.

6. A fusion protein constructed by combining the single domain antibody of any one of claims 1 to 3 with IgG-Fc; preferably, the Fc gene sequence is an Fc gene sequence derived from IgG, IgA, IgM or from IgG1, IgG2, IgG3 or IgG 4; the IgG is preferably human IgG and IgG1, subclasses 2, 3, 4, human IgM, human IgA or Fc fragment of other animal immunoglobulin.

7. A conjugate obtained by coupling a single domain antibody according to any one of claims 1 to 3 to one or more of an enzyme phase, a radioisotope, a fluorescent compound or a chemiluminescent compound.

8. Use of the single domain antibody of any one of claims 1 to 3, the encoding gene of claim 4, the fusion protein of claim 6 and the conjugate of claim 7 for the preparation of a medicament or reagent for the detection or diagnosis of VEGF-related diseases.

9. Use of the single domain antibody of any one of claims 1-3, the encoding gene of claim 4, the fusion protein of claim 6, and the conjugate of claim 7 for the manufacture of a medicament or agent for blocking the interaction between VEGF and VEGFR.

10. Use of the single domain antibody of any one of claims 1-3, the encoding gene of claim 4, the fusion protein of claim 6, and the conjugate of claim 7 for the manufacture of a medicament for the treatment of a disease associated with abnormal VEGF expression; preferably, the diseases related to abnormal expression of VEGF comprise various tumor diseases or senile fundus macular degeneration.

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