技术领域technical field
本发明属于生物技术领域，更具体地，本发明公开了一类双特异重组抗HBsAg抗体、其制备方法及其在预防、治疗乙型肝炎病毒感染中的应用。The invention belongs to the field of biotechnology, and more specifically, the invention discloses a bispecific recombinant anti-HBsAg antibody, its preparation method and its application in preventing and treating hepatitis B virus infection.
背景技术Background technique
乙型肝炎病毒（Hepatitis B virus，HBV）是双链DNA病毒，引起慢性乙型肝炎，而导致肝衰竭、肝硬化和肝癌的发生。全球大约有三亿五千万HBV感染者，而这也成为世界公共健康问题。最近几十年，乙肝疫苗的研制成功，给乙肝的预防带来了很大的希望。然而，确有少部分人接种乙肝疫苗却不能激发免疫系统产生抗乙肝抗体，这一部分人群一旦接触到乙肝的高危因素，只能注射人乙肝免疫球蛋白来预防。但人免疫球蛋白成分相对复杂，使用的危险因素相对不确定。因此迫切需要开发一种人来源的、危险因素相对可控的生物制品替代人乙肝免疫球蛋白。Hepatitis B virus (HBV) is a double-stranded DNA virus that causes chronic hepatitis B, leading to liver failure, cirrhosis and liver cancer. There are approximately 350 million HBV-infected people in the world, and this has become a world public health problem. In recent decades, the successful development of hepatitis B vaccine has brought great hope to the prevention of hepatitis B. However, there are indeed a small number of people who receive hepatitis B vaccine but cannot stimulate the immune system to produce anti-hepatitis B antibodies. Once this part of the population is exposed to high-risk factors for hepatitis B, they can only be prevented by injection of human hepatitis B immune globulin. However, the composition of human immunoglobulin is relatively complex, and the risk factors for its use are relatively uncertain. Therefore, there is an urgent need to develop a human-derived biological product with relatively controllable risk factors to replace human hepatitis B immunoglobulin.
随着生物技术的发展，基因工程抗体的发展日新月异。人源单克隆抗体产生包括EBV永生化B淋巴细胞，人B淋巴细胞杂交瘤技术，其它种属抗体的人源化，基于噬菌体库展示的全人抗体技术，基于人源化小鼠的全人抗体技术和Single cell RT-PCR单细胞抗体技术。由于EBV永生化B淋巴细胞转化率低，严重限制了这一技术的推广[Guan Y, Sajadi MM,Kamin-Lewis R, et al. Discordant memory B cell and circulating anti-Envantibody responses in HIV-1 infection. Proc Natl Acad Sci U S A 2009;106:3952-7]。人B淋巴细胞与骨髓瘤细胞融合效率极低，导致人B淋巴细胞杂交瘤技术很少被用于制备人源单克隆抗体。噬菌体展示库全人抗体技术是基于人免疫球蛋白轻链和重链基因的随机配对，未经历体细胞突变和免疫系统的双性选择，导致其筛选得到的抗体普遍亲和力低下（KD＞10-7），解离速率快（V＜60min），抗体Tm值低（＜65℃），热稳定性差，潜在的过敏和自身免疫风险[Mohapatra S, Juan HS. Designer monoclonal antibodies as drugs:the state of the art. Expert Rev Clin Immunol 2008;4:305-7]。由于最具潜质的人源抗体技术-人源化小鼠的专利仅掌握在少数国际制药巨头手中，且开发成本高昂，严重制约了这一技术的推广。With the development of biotechnology, the development of genetically engineered antibodies is changing rapidly. Human monoclonal antibody production includes EBV immortalized B lymphocytes, human B lymphocyte hybridoma technology, humanization of antibodies from other species, fully human antibody technology based on phage library display, fully human antibody technology based on humanized mice Antibody technology and Single cell RT-PCR single cell antibody technology. Due to the low transformation rate of EBV immortalized B lymphocytes, the promotion of this technology is severely limited [Guan Y, Sajadi MM, Kamin-Lewis R, et al. Discordant memory B cell and circulating anti-Envantibody responses in HIV-1 infection. Proc Natl Acad Sci USA 2009;106:3952-7]. The fusion efficiency of human B lymphocytes and myeloma cells is extremely low, so that human B lymphocyte hybridoma technology is rarely used to prepare human monoclonal antibodies. The phage display library fully human antibody technology is based on the random pairing of human immunoglobulin light chain and heavy chain genes, without somatic mutation and bisexual selection by the immune system, resulting in generally low affinity (KD>10- 7 ), fast dissociation rate (V<60min), low antibody Tm value (<65°C), poor thermal stability, potential allergy and autoimmune risks [Mohapatra S, Juan HS. Designer monoclonal antibodies as drugs: the state of the art. Expert Rev Clin Immunol 2008;4:305-7]. Because the most potential human antibody technology - the patent of humanized mice is only in the hands of a few international pharmaceutical giants, and the development cost is high, which seriously restricts the promotion of this technology.
Single cell RT-PCR单细胞抗体技术直接从人外周血中分离含目的抗体的B细胞，运用单细胞RT-PCR技术直接克隆抗体轻链和重链，经过体外重组来制备抗体。所制备的抗体来源天然，危险因素可控。由于单克隆抗体都是从人外周血的B淋巴细胞中克隆而来，经历了人体免疫系统的双性选择、长期的体液循环和体细胞突变，因此，显示出其极好的亲和力（KD＜10-9），解离速率慢（V＞6hours），热稳定性好，潜在的过敏和自身免疫风险低[Smith K, Garman L, Wrammert J, et al. Rapid generation of fully humanmonoclonal antibodies specific to a vaccinating antigen. Nat Protoc 2009;4:372-84]。Single cell RT-PCR单细胞抗体技术开发成本低廉，尤其针对外来抗原更能显示其优越性。Single cell RT-PCR single cell antibody technology directly isolates B cells containing the target antibody from human peripheral blood, uses single cell RT-PCR technology to directly clone the light chain and heavy chain of the antibody, and prepares the antibody through in vitro recombination. The prepared antibodies come from natural sources, and the risk factors are controllable. Since the monoclonal antibodies are all cloned from B lymphocytes in human peripheral blood, they have undergone bisexual selection by the human immune system, long-term humoral circulation and somatic mutation, so they show excellent affinity (KD< 10-9 ), slow dissociation rate (V>6hours), good thermal stability, low risk of potential allergy and autoimmunity [Smith K, Garman L, Wrammert J, et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 2009;4:372-84]. Single cell RT-PCR monoclonal antibody technology has low development cost, especially for foreign antigens, which can show its superiority.
由于疾病的多因素性，单一靶点治疗已经不能满足临床的需求。在临床试验中已有联合运用两种单克隆抗体临床前实验的报道，但这种联合应用单克隆抗体却有很多限制性[Kou G, Shi J, Chen L, et al. A bispecific antibody effectively inhibitstumor growth and metastasis by simultaneous blocking vascular endothelialgrowth factor A and osteopontin. Cancer Lett 2010;299:130-6]。例如，联合应用两种单克隆抗体的的安全性和效果需要分别去评价，由此产生的长周期，高昂的开发费用限制其研制进程，双特异性抗体恰好解决了这一瓶颈[Wu C, Ying H, Grinnell C, et al.Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat Biotechnol 2007;25:1290-7]。此外，双特异抗体有时还能获得其亲本单克隆抗体所不具备的一些新奇的特性：如往往具有比原亲本抗体更低的解离速度，更强的封闭作用等[Lu D, Kotanides H, Jimenez X, et al. Acquiredantagonistic activity of a bispecific diabody directed against two differentepitopes on vascular endothelial growth factor receptor 2. J Immunol Methods1999;230:159-71]。Due to the multifactorial nature of the disease, single-target therapy can no longer meet clinical needs. There have been reports of preclinical experiments on the combined use of two monoclonal antibodies in clinical trials, but this combined use of monoclonal antibodies has many limitations [Kou G, Shi J, Chen L, et al. A bispecific antibody effectively inhibits tumor growth and metastasis by simultaneous blocking vascular endothelial growth factor A and osteopontin. Cancer Lett 2010;299:130-6]. For example, the safety and efficacy of the combined application of two monoclonal antibodies need to be evaluated separately. The resulting long cycle and high development costs limit the development process. Bispecific antibodies just solve this bottleneck[Wu C, Ying H, Grinnell C, et al. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat Biotechnol 2007;25:1290-7]. In addition, bispecific antibodies can sometimes acquire some novel properties that their parental monoclonal antibodies do not have: for example, they often have lower dissociation speeds and stronger blocking effects than the original parental antibodies [Lu D, Kotanides H, Jimenez X, et al. Acquired antagonistic activity of a bispecific diabetes directed against two different epitopes on vascular endothelial growth factor receptor 2. J Immunol Methods 1999;230:159-71].
发明内容Contents of the invention
针对以上问题，本发明设计基于Single Cell RT-PCR单细胞抗体技术开发一株有极好中和HBV病毒的Anti-HBsAg双特异性抗体。本发明采用超高速流式分选系统从接种乙肝疫苗的健康志愿者外周血中分离HBsAg特异的记忆性B细胞(HBsAg+IgG+CD19+)，并制备成单细胞样本。采用Single Cell RT-PCR技术克隆每一个单细胞样本中的抗体相关的基因，并克隆入哺乳动物高效表达载体中，体外重组表达相应的抗体。In view of the above problems, the present invention designs and develops an Anti-HBsAg bispecific antibody with excellent neutralization of HBV virus based on Single Cell RT-PCR single cell antibody technology. The invention uses an ultra-high-speed flow sorting system to separate HBsAg-specific memory B cells (HBsAg+ IgG+ CD19+ ) from the peripheral blood of healthy volunteers vaccinated with hepatitis B vaccine, and prepare them into single-cell samples. Single Cell RT-PCR technology is used to clone the antibody-related genes in each single-cell sample, and clone them into mammalian high-efficiency expression vectors, and express the corresponding antibodies in vitro.
本发明公开了：The present invention discloses:
1.一类双特异性抗体，它含有两个不同的重链可变区、轻链可变区，可以分别结合HBsAg不同的功能表位。1. A type of bispecific antibody, which contains two different heavy chain variable regions and light chain variable regions, which can respectively bind to different functional epitopes of HBsAg.
2.所述的一类双特异性抗体，其特征是重链可变区具有SEQ ID NO：2所示的氨 基酸序列，轻链可变区具有SEQ ID NO：4所示的氨基酸序列。2. The above-mentioned class of bispecific antibodies is characterized in that the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 4.
3.一种分离的核苷酸分子，编码所述氨基酸序列，具有SEQ ID NO：1所示核苷 酸序列及SEQ ID NO：3所示核苷酸序列。3. An isolated nucleotide molecule encoding the amino acid sequence, having the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 3.
4.一种载体，含有所述的核苷酸分子，和所述核苷酸分子的序列操作性相连的表达调控序列，其中载体可以为pDR1，pcDNA3.1（+）， pcDNA3.1/ZEO（+），pDHFR之一。4. A vector containing the nucleotide molecule and an expression control sequence operably linked to the sequence of the nucleotide molecule, wherein the vector can be pDR1, pcDNA3.1 (+), pcDNA3.1/ZEO (+), one of pDHFR.
5.所述的载体为pcDNA3.1（+）。5. The vector is pcDNA3.1 (+).
6.一种宿主细胞，含有所述载体，为真核细胞。6. A host cell containing the vector, which is a eukaryotic cell.
7.所述的宿主细胞，为哺乳动物细胞。7. The host cell is a mammalian cell.
8.所述的宿主细胞，为Freestyle 293F细胞。8. The host cell is Freestyle 293F cell.
9.一种制备所述的双特异性抗体的方法，该方法包括：9. A method for preparing the bispecific antibody, the method comprising:
a)采用超高流速流式分选系统分离接种乙肝疫苗的志愿者外周血中HBsAg特异的记忆性B细胞，并制备成单细胞样本；a) Separating HBsAg-specific memory B cells in the peripheral blood of volunteers vaccinated with hepatitis B vaccine using an ultra-high flow rate flow sorting system, and preparing them into single-cell samples;
b)采用Single Cell RT-PCR技术克隆每个样本中抗体相关基因，体外重组表达相应抗体；b) Use Single Cell RT-PCR technology to clone antibody-related genes in each sample, and express corresponding antibodies in vitro;
c)经过功能表位测定、亲和力测定及中和HBV活性测定，筛选出亲和力强、活性高的结合不同功能表位的两个抗HBsAg的单克隆抗体C4G4和D2H2；c) After functional epitope determination, affinity determination and neutralization HBV activity determination, two anti-HBsAg monoclonal antibodies C4G4 and D2H2 with strong affinity and high activity binding different functional epitopes were screened out;
d)利用生物工程技术将C4G4、D2H2 这两个单克隆抗体的重链、轻链可变区分别经过重链linker、轻链linker连接，制备成具有双功能的抗体；d) Using bioengineering technology, the heavy chain and light chain variable regions of the two monoclonal antibodies C4G4 and D2H2 are connected through a heavy chain linker and a light chain linker, respectively, to prepare antibodies with dual functions;
e)在表达条件下，培养任一所述的宿主细胞，表达双特异性抗体C4D2-BsAb，分离纯化。e) under expression conditions, culture any one of the host cells described above, express the bispecific antibody C4D2-BsAb, separate and purify.
10.一种组合物，含有所述的双特异性抗体，和药学上可接受的载体。10. A composition comprising the bispecific antibody and a pharmaceutically acceptable carrier.
11.所述的双特异性抗体在制备抗HBV感染中的应用。11. The application of the bispecific antibody in preparation of anti-HBV infection.
12.所述的组合物在制备抗HBV感染中的应用。12. The application of the composition in the preparation of anti-HBV infection.
13.任一所述的用途，还包括和其他的抗HBV感染药物联合使用。13. Any of the uses described above also includes combined use with other anti-HBV infection drugs.
在本发明中，成功克隆了42株全新的Anti-HBsAg单克隆抗体，采用ELISA的方法鉴定了其功能表位，亲和力及中和HBV活性。经一系列检测实验，从其中选择了2株中和HBV活性最好的Anti-HBsAg单克隆抗体C4G4和D2H2，利用生物工程技术将2株抗体制备成双特异性抗体C4D2-BsAb。In the present invention, 42 strains of brand new Anti-HBsAg monoclonal antibodies were successfully cloned, and their functional epitopes, affinity and neutralizing HBV activity were identified by ELISA. After a series of detection experiments, two anti-HBsAg monoclonal antibodies C4G4 and D2H2 with the best neutralizing HBV activity were selected, and the two antibodies were prepared into bispecific antibody C4D2-BsAb by bioengineering technology.
在HepaRG细胞模型中，C4D2-BsAb显示出比C4G4和D2H2联用更强的中和HBV的活性。此外，研究显示FcRn介导C4D2-BsAb内吞到肝细胞内抑制HBsAg和病毒颗粒的释放。In the HepaRG cell model, C4D2-BsAb showed stronger HBV neutralizing activity than the combination of C4G4 and D2H2. In addition, studies have shown that FcRn-mediated endocytosis of C4D2-BsAb into hepatocytes inhibits the release of HBsAg and viral particles.
附图说明Description of drawings
图1、流式CD19+IgG+HBsAg+ B cell分析。Figure 1. Flow CD19+ IgG+ HBsAg+ B cell analysis.
图2、PCR扩增抗体可变区基因图。Fig. 2. The gene map of variable region of antibody amplified by PCR.
图3、还原状态下SDS-PAGE分析重组人单克隆抗体电泳图。Figure 3. SDS-PAGE analysis of recombinant human monoclonal antibody electrophoresis in reduced state.
图4、双特异性抗体结构示意图。Figure 4. Schematic diagram of the bispecific antibody structure.
图5、还原条件下，SDS-PAGE分析纯化的双特异性抗体电泳图。Figure 5. SDS-PAGE analysis of the electrophoresis of the purified bispecific antibody under reducing conditions.
图6、anti-HBsAg 单克隆抗体的特征，合成肽在HBsAg上的位置。Figure 6. Characteristics of anti-HBsAg monoclonal antibody, and the position of the synthetic peptide on HBsAg.
图7、HepaRG细胞评估单克隆抗体中和HBV的活性。Figure 7. HepaRG cells evaluate the neutralizing activity of monoclonal antibodies against HBV.
图8、单克隆抗体C4G4和D2H2协同抑制作用。Figure 8. Synergistic inhibitory effect of monoclonal antibody C4G4 and D2H2.
图9、双特异性抗体C4D2-BsAb和D2C4-BsAb结合合成多肽P2、P3活性分析。Figure 9. Activity analysis of bispecific antibodies C4D2-BsAb and D2C4-BsAb combined with synthetic polypeptides P2 and P3.
图10、双特异性抗体C4D2-BsAb和D2C4-BsAb结合HBsAg活性分析。Figure 10. Analysis of the binding activity of bispecific antibodies C4D2-BsAb and D2C4-BsAb to HBsAg.
图11、双特异性抗体C4D2-BsAb中和HBV活性分析。Figure 11. Analysis of neutralizing activity of bispecific antibody C4D2-BsAb against HBV.
具体实施方式detailed description
以下结合实施例、实验例进一步对本发明进行说明，这些实施例、实验例不应理解为对本发明的限制。The present invention will be further described below in conjunction with examples and experimental examples, which should not be construed as limiting the present invention.
实施例1：HBsAg特异性单个B细胞的制备Example 1: Preparation of HBsAg-specific single B cells
通过分离注射过乙肝疫苗志愿者的外周血的淋巴细胞，用CD19-PE/Cy5, IgG-PE，HBsAg-biotin，Streptavidin-FITC荧光抗体分离HBsAg+CD19+IgG+B细胞。如图1所示，可见一小群HBsAg阳性的细胞（约占整个B细胞群0.01-0.02%）。MoFlo XDP超速流式细胞分选系统分选的每个HBsAg+CD19+IgG+B细胞注入96-well Single cell PCR plates中，确保每个PCR孔中一个细胞。然后加入裂解液，液氮速冻后，存储于-80℃冰中备用。By separating lymphocytes from peripheral blood of volunteers who had been injected with hepatitis B vaccine, HBsAg+ CD19+ IgG+ B cells were separated with CD19-PE/Cy5, IgG-PE, HBsAg-biotin, Streptavidin-FITC fluorescent antibodies. As shown in Figure 1, a small population of HBsAg-positive cells (approximately 0.01-0.02% of the total B cell population) can be seen. Each HBsAg+ CD19+ IgG+ B cell sorted by the MoFlo XDP ultrafast flow cytometry system is injected into 96-well Single cell PCR plates to ensure that there is one cell in each PCR well. Then, the lysate was added, frozen in liquid nitrogen, and stored in ice at -80°C for later use.
实施例2：RT-PCR和巢氏PCR克隆抗体可变区基因及基因表达Embodiment 2: RT-PCR and nested PCR clone antibody variable region gene and gene expression
取冻于-80℃冰箱中96-well Single cell PCR plates，在冰上使慢慢融化，加入逆转录酶合成cDNA。由于单个细胞合成的cDNA特别稀少，必须采用巢氏PCR的方法扩增其含有的抗体基因。两轮PCR后可见明显的PCR条带扩增（大小约400bp）。轻重链成对的PCR条带回收后测序。如图2所示，分析其序列后特异性PCR扩增抗体可变区条带，并引入限制性酶切位点。扩增抗体可变区PCR产物与抗体恒定区连接后插入表达载体pcDNA3.1(+)。Take the 96-well Single cell PCR plates frozen in a -80°C refrigerator, thaw slowly on ice, and then add reverse transcriptase to synthesize cDNA. Since the cDNA synthesized by a single cell is extremely rare, the antibody gene contained in it must be amplified by nested PCR. After two rounds of PCR, obvious PCR band amplification (about 400bp in size) can be seen. The paired PCR bands of the light and heavy chains were recovered and sequenced. As shown in Figure 2, after analyzing its sequence, specific PCR amplifies the variable region band of the antibody, and introduces a restriction enzyme cutting site. The PCR product of the amplified variable region of the antibody was connected with the constant region of the antibody and then inserted into the expression vector pcDNA3.1(+).
实施例3：单克隆抗体基因表达Example 3: Monoclonal Antibody Gene Expression
成对的抗体轻链和重链质粒瞬时转染无血清培养基培养的Freestyle 293F细胞，转染7天后收集细胞上清，Protein A纯化细胞上清中IgG抗体。共获得抗HBsAg单克隆抗体42株。SDS-PAGE电泳鉴定如图3所示，纯化的IgG抗体在10% SDS-PAGE显示出二条带，其中一条为50KD左右（重链），另外一条为25KD左右（轻链）。Paired antibody light chain and heavy chain plasmids were transiently transfected into Freestyle 293F cells cultured in serum-free medium, and the cell supernatant was collected 7 days after transfection, and the IgG antibody in the cell supernatant was purified by Protein A. A total of 42 anti-HBsAg monoclonal antibody strains were obtained. SDS-PAGE electrophoresis identification is shown in Figure 3. The purified IgG antibody showed two bands on 10% SDS-PAGE, one of which was about 50KD (heavy chain) and the other was about 25KD (light chain).
实施例4：抗HBsAg单克隆抗体HBsAg亲和力测定Embodiment 4: Anti-HBsAg monoclonal antibody HBsAg affinity determination
取一定浓度的单克隆抗体与系列倍比稀释的抗原HBsAg混合，4℃过夜，从而保证反应达到平衡。抗原抗体复合物转入到包被HBsAg酶标板中，37℃孵育1小时。洗涤后加入HRP标记的羊抗人κ链抗体孵育1小时。洗涤后TMB显色450nm波长测定各孔的吸收率，计算抗体的亲和力，结果如表1所示。A certain concentration of monoclonal antibody was mixed with serially diluted antigen HBsAg, and left overnight at 4°C to ensure that the reaction reached equilibrium. The antigen-antibody complex was transferred to the coated HBsAg microtiter plate and incubated at 37°C for 1 hour. After washing, add HRP-labeled goat anti-human κ chain antibody and incubate for 1 hour. After washing, TMB was developed at a wavelength of 450 nm to measure the absorbance of each well, and the affinity of the antibody was calculated. The results are shown in Table 1.
实施例5：抗体表位鉴定Example 5: Antibody epitope identification
合成生物素标记的乙肝表面抗原短肽测定anti-HBsAg单克隆抗体在HBsAg上的结合区域。如图6所示，合成的四条生物素标记乙肝表面抗原短肽P1（aa:104-120），P2（aa:121-137），P3（aa:139-148），P4（aa:149-163），并在HBsAg模式图上标注抗原短肽P1，P2，P3，P4所对应的区域的结构；其中抗原短肽P1和P4为线性结构，P2和P3为环状结构。[Jin A,Ozawa T, Tajiri K, et al. A rapid and efficient single-cell manipulationmethod for screening antigen-specific antibody-secreting cells from humanperipheral blood. Nat Med 2009;15:1088-92]。Synthetic biotin-labeled HBsAg short peptide was used to determine the binding region of anti-HBsAg monoclonal antibody on HBsAg. As shown in Figure 6, four synthetic biotin-labeled HBsAg short peptides P1 (aa:104-120), P2 (aa:121-137), P3 (aa:139-148), P4 (aa:149- 163), and mark the structures of the regions corresponding to the antigenic short peptides P1, P2, P3, and P4 on the HBsAg model map; the antigenic short peptides P1 and P4 are linear structures, and P2 and P3 are circular structures. [Jin A, Ozawa T, Tajiri K, et al. A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood. Nat Med 2009;15:1088-92].
采用ELISA的方法分析了42株Anti-HBsAg单克隆抗体的结合的抗原表位。如表1所示，结果显示结合在P1的占7.1% (3/42), 结合在P2的占38.1% (16/42), 结合在P3的占31.0% (13/42)和结合在P4的占9.5% (4/42) ，但是有14.3% (6/42)的单克隆抗体不与四个合成肽结合。The combined epitopes of 42 Anti-HBsAg monoclonal antibodies were analyzed by ELISA. As shown in Table 1, the results showed that 7.1% (3/42) bound to P1, 38.1% (16/42) bound to P2, 31.0% (13/42) bound to P3 and 31.0% (13/42) bound to P4 accounted for 9.5% (4/42), but 14.3% (6/42) of the monoclonal antibodies did not bind to the four synthetic peptides.
实施例6：抗HBsAg单克隆抗体体外中和HBV活性的鉴定Example 6: Identification of anti-HBsAg monoclonal antibody neutralizing HBV activity in vitro
HepaRG细胞系是当前唯一被广泛认可的HBV体外感染模型。因此，我们采用HepaRG细胞测定42株Anti-HBsAg 单克隆抗体的中和HBV感染HepaRG细胞的能力。用Anti-HBsAg单克隆抗体（1μg）和HBV（1×106 copies）预孵育，再加到DMSO和氢化可的松诱导分化的HepaRG细胞，测定其中和HBV活性的能力。感染第7天我们测定了细胞培养上清中HBsAg的含量和细胞中HBV-DNA拷贝数。如图8A所示，感染第7天，大部分细胞细胞培养上清中HBsAg出现明显下降(*P＜0.05)。同样如图7所示，感染第7天，大部分细胞细胞中HBV-DNA 拷贝数出现明显下降(*P＜0.05)。如表1所示，综合多因素进行42株Anti-HBsAg 单克隆抗体的中和HBV感染HepaRG细胞能力分析，最终选择效果较好的两个单克隆C4G4和D2H2。The HepaRG cell line is currently the only widely recognized HBV infection model in vitro. Therefore, we used HepaRG cells to measure the ability of 42 strains of Anti-HBsAg monoclonal antibodies to neutralize HBV infection in HepaRG cells. Anti-HBsAg monoclonal antibody (1μg) and HBV (1×106 copies) were pre-incubated, and then DMSO and hydrocortisone were added to induce differentiation of HepaRG cells, and the ability to neutralize HBV activity was determined. On the seventh day of infection, we measured the content of HBsAg in the cell culture supernatant and the copy number of HBV-DNA in the cells. As shown in FIG. 8A , on day 7 of infection, HBsAg in cell culture supernatants of most cells decreased significantly (*P <0.05). Also as shown in Figure 7, on the 7th day of infection, the copy number of HBV-DNA in most cells decreased significantly (*P <0.05). As shown in Table 1, the ability of 42 Anti-HBsAg monoclonal antibodies to neutralize HBV-infected HepaRG cells was analyzed comprehensively, and two monoclonal C4G4 and D2H2 with better effects were finally selected.
实施例7：抗HBsAg单克隆抗体协同中和HBV活性测定Embodiment 7: Anti-HBsAg monoclonal antibody synergistically neutralizes HBV activity assay
选择二株中和HBV活性最好的单克隆抗体（C4G4和D2H2）检测其协同效应。Two monoclonal antibodies (C4G4 and D2H2) with the best neutralizing HBV activity were selected to detect their synergistic effect.
HBV病毒颗粒(1×106copies)分别与1μg单克隆抗体C4G4、D2H2和Control humanIgG，预孵育1小时，加入到诱导分化好的HepaRG细胞系中（培养基中含4%PEG8000）过夜培养，次日洗PBS三次，换新鲜的培养基。在第6天检测细胞中的HBV-DNA拷贝数和培养上清中HBsAg含量。HBV-DNA检测使用HBV-DNA荧光定量PCR试剂盒（上海科华生物）。HBsAg测定收集培养上清，用乙肝表面抗原定量试剂盒（电化学发光法）（北京科美生物）检测。如图8所示，C4G4和D2H2联用与C4G4和D2H2单用相比，能显著减少细胞上清中HBsAg的表达和细胞中HBV-DNA拷贝数的（*P＜0.05）。HBV virus particles (1×106 copies) were pre-incubated with 1 μg monoclonal antibodies C4G4, D2H2 and Control humanIgG for 1 hour, and then added to the differentiated HepaRG cell line (medium containing 4% PEG8000) for overnight culture. The next day, wash with PBS three times and replace with fresh medium. On the 6th day, the HBV-DNA copy number in the cells and the HBsAg content in the culture supernatant were detected. HBV-DNA detection using HBV-DNA fluorescent quantitative PCR kit (Shanghai Kehua Bio). Determination of HBsAg The culture supernatant was collected and detected with the Hepatitis B Surface Antigen Quantitative Kit (electrochemiluminescence method) (Beijing Kemei Biology). As shown in Figure 8, the combined use of C4G4 and D2H2 can significantly reduce the expression of HBsAg in the cell supernatant and the copy number of HBV-DNA in the cells compared with the single use of C4G4 and D2H2 (*P <0.05).
实施例8：双特异性抗体的构建和鉴定Example 8: Construction and identification of bispecific antibodies
选择中和HBV活性最好的两株抗体C4G4和D2H2，设计双特异抗体，抗体结构如图4。一个单克隆抗体的重链可变区和轻链可变区分别通过重链连接肽和轻链接肽融合到另一个单克隆抗体的重链可变区和轻链可变区的5’端。重链接肽氨基酸序列为ASTKGPSVFPLAP，轻链接肽氨基酸序列为TVAAPSVFIFPP。将正确的双特异抗体重链、轻链可变区基因分别克隆入IgG1-AbVec质粒和Igκ-AbVec质粒中。在Freestyle 293F细胞系中表达双特异抗体C4D2-BsAb和D2C4-BsAb。SDS-PAGE电泳鉴定双特异性抗体C4D2-BsAb和D2C4-BsAb，结果如图5。The two antibodies C4G4 and D2H2 with the best neutralizing HBV activity were selected to design bispecific antibodies. The antibody structure is shown in Figure 4. The heavy chain variable region and the light chain variable region of one monoclonal antibody are fused to the 5' ends of the heavy chain variable region and light chain variable region of another monoclonal antibody through the heavy chain connecting peptide and the light chain peptide, respectively. The amino acid sequence of the heavy link peptide is ASTKGPSVFPLAP, and the amino acid sequence of the light link peptide is TVAAPSVFIFPP. The correct heavy chain and light chain variable region genes of the bispecific antibody were cloned into IgG1-AbVec plasmid and Igκ-AbVec plasmid respectively. The bispecific antibodies C4D2-BsAb and D2C4-BsAb were expressed in Freestyle 293F cell line. The bispecific antibodies C4D2-BsAb and D2C4-BsAb were identified by SDS-PAGE electrophoresis, and the results are shown in Figure 5.
实验例1：双特异性抗体的亲和力测定Experimental Example 1: Affinity Determination of Bispecific Antibody
分别以合成肽P2和P3包被ELISA板，使用ELISA方法检测两种不同结构的双特异性抗体同亲本单克隆抗体（C4G4和D2H2）之间的亲和力区别。如图9显示：当C4G4可变区放在D2H2可变区的前面时，构成的C4D2-BsAb对合成抗原短肽P2和P3均保持了与亲本单克隆抗体（C4G4和D2H2）相似的亲和力；而当将D2H2可变区放于C4G4之前构成D2C4-BsAb时，D2C4-BsAb和合成抗原短肽P2的结合活性与C4G4相比下降明显；D2C4-BsAb和合成抗原短肽P3的结合活性与D2H2无明显差别。ELISA plates were coated with synthetic peptides P2 and P3, respectively, and the difference in affinity between the two bispecific antibodies with different structures and the parental monoclonal antibodies (C4G4 and D2H2) was detected by ELISA method. As shown in Figure 9: when the C4G4 variable region is placed in front of the D2H2 variable region, the constructed C4D2-BsAb maintains a similar affinity to the parent monoclonal antibody (C4G4 and D2H2) for the synthetic antigen short peptides P2 and P3; When the D2H2 variable region was placed before C4G4 to form D2C4-BsAb, the binding activity of D2C4-BsAb and synthetic antigen short peptide P2 decreased significantly compared with C4G4; the binding activity of D2C4-BsAb and synthetic antigen short peptide P3 was similar to that of D2H2 No significant difference.
同样测定两个双特异性抗体C4D2-BsAb和D2C4-BsAb与HBsAg的亲和力。如图10所示，C4D2-BsAb和D2C4-BsAb都能很好的与HBsAg结合。The affinities of two bispecific antibodies, C4D2-BsAb and D2C4-BsAb, to HBsAg were also determined. As shown in Figure 10, both C4D2-BsAb and D2C4-BsAb can bind well to HBsAg.
实验例2：双特异性抗体C4D2-BsAb中和HBV活性测定Experimental example 2: Determination of neutralizing HBV activity of bispecific antibody C4D2-BsAb
HBV病毒颗粒(1×106copies)分别与不同浓度的C4G4、D2H2、C4G4+D2H2 和C4D2-BsAb预孵育1小时，加入到诱导分化好的HepaRG细胞系中（培养基中含4%PEG8000）过夜培养，次日洗PBS三次，换新鲜的培养基。在第6天检测细胞中的HBV-DNA拷贝数和培养上清中HBsAg含量。HBV-DNA检测使用HBV-DNA荧光定量PCR试剂盒（上海科华生物）。HBsAg测定收集培养上清，用乙肝表面抗原定量试剂盒（电化学发光法）（北京科美生物）检测。如图11，结果显示双特异性抗体C4D2-BsAb表现出极好的中和HBV的能力，超过了C4G4和D2H2联用的效果。HBV virus particles (1×106 copies) were pre-incubated with different concentrations of C4G4, D2H2, C4G4+D2H2 and C4D2-BsAb for 1 hour, and added to the differentiated HepaRG cell line (medium containing 4% PEG8000) Culture overnight, wash with PBS three times the next day, and replace with fresh medium. On the 6th day, the HBV-DNA copy number in the cells and the HBsAg content in the culture supernatant were detected. HBV-DNA detection using HBV-DNA fluorescent quantitative PCR kit (Shanghai Kehua Bio). Determination of HBsAg The culture supernatant was collected and detected with the Hepatitis B Surface Antigen Quantitative Kit (electrochemiluminescence method) (Beijing Kemei Biology). As shown in Figure 11, the results show that the bispecific antibody C4D2-BsAb exhibits an excellent ability to neutralize HBV, exceeding the effect of the combination of C4G4 and D2H2.
Characterization of anti-HBsAg antibodies.
aAffinities were analyzed with ELISA.Affinities were analyzed with ELISA.
bEpitopes were analyzed by ELISA using synthesized peptides (P1,amino acids 104–120; P2, amino acids 121–137; P3, amino acids 139–148; P4,amino acids 149–163) covering extracellular domain of HBsAg.b Epitopes were analyzed by ELISA using synthesized peptides (P1,amino acids 104–120; P2, amino acids 121–137; P3, amino acids 139–148; P4,amino acids 149–163) covering extracellular domain of HBsAg.
cHBV-neutralization activity : ++, more than 60% inhibition; +, 40%–60% inhibition; +/–, 20-40% inhibition at day 7 post-infection from that withcontrol IgG.c HBV-neutralization activity : ++, more than 60% inhibition; +, 40%–60% inhibition; +/–, 20-40% inhibition at day 7 post-infection from that with control IgG.
SEQUENCE LISTING SEQUENCE LISTING
<110> 上海张江生物技术有限公司 上海海思太科药业有限公司<110> Shanghai Zhangjiang Biotechnology Co., Ltd. Shanghai Haisi Taike Pharmaceutical Co., Ltd.
<120> 一类双特异重组抗HBsAg抗体、其制备方法及用途<120> A kind of bispecific recombinant anti-HBsAg antibody, its preparation method and use
<130> 2013<130> 2013
<160> 4<160> 4
<170> PatentIn version 3.5<170> PatentIn version 3.5
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<211> 1842<211> 1842
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atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt gcacagccag 60atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt gcacagccag 60
gtgcagctgg tggaaagcgg cggaggagtg gtgagacccg gaagaagcct gagactgagc 120gtgcagctgg tggaaagcgg cggaggagtg gtgagacccg gaagaagcct gagactgagc 120
tgcgccgcta gcggctttgc cttcagcgac tacagcatca actgggtgag gcaagctccc 180tgcgccgcta gcggctttgc cttcagcgac tacagcatca actgggtgag gcaagctccc 180
ggcaagggac tggaatgggt ggccatcatc agctacgacg gcaggatcac ctactacagg 240ggcaagggac tggaatgggt ggccatcatc agctacgacg gcaggatcac ctactacagg 240
gacagcgtga agggcaggtt caccatctcc agggacgaca gcaagaacac cctgtacctg 300gacagcgtga agggcaggtt caccatctcc agggacgaca gcaagaacac cctgtacctg 300
cagatgaaca gcctgaggac cgaggacacc gccgtgtatt actgcgccag gcagtactac 360cagatgaaca gcctgaggac cgaggacacc gccgtgtatt actgcgccag gcagtactac 360
gacttctgga gcggctcctc cgtgggcagg aactacgacg gaatggacgt gtggggcctg 420gacttctgga gcggctcctc cgtgggcagg aactacgacg gaatggacgt gtggggcctg 420
ggcaccacag tcaccgtgag ctcagctagc accaagggac cttctgtgtt ccctctggcc 480ggcaccacag tcaccgtgag ctcagctagc accaagggac cttctgtgtt ccctctggcc 480
cctcaggtgc agctggtgga aagcggagga ggcgtggtgc aacccggagg aagcctgagg 540cctcaggtgc agctggtgga aagcggagga ggcgtggtgc aacccggagg aagcctgagg 540
ctgagctgcg ctcctagcgg cttcgtgttc aggagctacg gcatgcactg ggtgaggcag 600ctgagctgcg ctcctagcgg cttcgtgttc aggagctacg gcatgcactg ggtgaggcag 600
acacccggca aaggcctgga gtgggtgagc ctgatctggc acgacggcag caacaggttc 660acacccggca aaggcctgga gtgggtgagc ctgatctggc acgacggcag caacaggttc 660
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tacctgcaga tgaacagcct gagggctgag gacaccgcca tgtacttctg cgccagggag 780tacctgcaga tgaacagcct gagggctgag gacaccgcca tgtacttctg cgccaggggag 780
aggctgattg ccgctcctgc cgctttcgac ctgtggggac agggcaccct ggtgaccgtc 840aggctgattg ccgctcctgc cgctttcgac ctgtggggac agggcaccct ggtgaccgtc 840
agctcggcgt cgaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 900agctcggcgt cgaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 900
tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 960tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 960
gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 1020gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 1020
tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 1080tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 1080
cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 1140cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 1140
gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 1200gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 1200
gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 1260gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 1260
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 1320acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 1320
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1380aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1380
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1440tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1440
ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1500ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1500
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1560atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1560
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1620gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1620
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1680gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1680
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1740cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1740
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1800aggtggcagc agggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1800
tacacgcaga agagcctctc cctgtctccg ggtaaatgat ga 1842tacacgcaga agagcctctc cctgtctccg ggtaaatgat ga 1842
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Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu AlaThr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
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Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro GlyPro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly
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Gly Ser Leu Arg Leu Ser Cys Ala Pro Ser Gly Phe Val Phe Arg SerGly Ser Leu Arg Leu Ser Cys Ala Pro Ser Gly Phe Val Phe Arg Ser
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Tyr Gly Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu TrpTyr Gly Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp
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Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrSer Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
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Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val ThrAla Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
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Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe ProVal Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
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Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val ThrAla Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
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Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnVal Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
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His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys SerHis Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
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Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu LeuCys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
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Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuGly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
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Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val SerMet Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
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His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val GluHis Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
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Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser ThrVal His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
450 455 460 450 455 460
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnTyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
465 470 475 480465 470 475 480
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala ProGly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
485 490 495 485 490 495
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro GlnIle Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
500 505 510 500 505 510
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln ValVal Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
515 520 525 515 520 525
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
530 535 540 530 535 540
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProGlu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
545 550 555 560545 550 555 560
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu ThrPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
565 570 575 565 570 575
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser ValVal Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
580 585 590 580 585 590
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser LeuMet His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
595 600 605 595 600 605
Ser Pro Gly LysSer Pro Gly Lys
610 610
<210> 3<210> 3
<211> 1080<211> 1080
<212> DNA<212>DNA
<213> 人工序列<213> Artificial sequence
<400> 3<400> 3
atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt gcacggcgac 60atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt gcacggcgac 60
atcgtcatga cccagagccc tctcagcctg tccgtgaccc ctggagaacc cgccagcatc 120atcgtcatga cccagagccc tctcagcctg tccgtgaccc ctggagaacc cgccagcatc 120
tcctgcagga gcagccagag cctgctgcac aggagcggca acaactacct ggactggtac 180tcctgcagga gcagccagag cctgctgcac aggagcggca acaactacct ggactggtac 180
ctgcagaagc ccggccatag cccccagctg ctgatctacg tgggcagcaa cagagcttcc 240ctgcagaagc ccggccatag cccccagctg ctgatctacg tgggcagcaa cagagcttcc 240
ggcgtgcccg acagattcag cggaagcgga tccggcaccg agtacaccct gaagatcagc 300ggcgtgcccg acagattcag cggaagcgga tccggcaccg agtacaccct gaagatcagc 300
agagtggagg ccgaggacgt gggcgtgtac tactgcatgc aggccctgca gacccccagg 360agagtggagg ccgaggacgt gggcgtgtac tactgcatgc aggccctgca gacccccagg 360
acattcggcc agggcaccaa gctggagatc aagcgtaccg tggctgcccc tagcgtgttc 420acattcggcc agggcaccaa gctggagatc aagcgtaccg tggctgcccc tagcgtgttc 420
atcttccctc ctgagctggt gatgacccag agcccttcca gcctgagcgc tagcgtggga 480atcttccctc ctgagctggt gatgacccag agcccttcca gcctgagcgc tagcgtggga 480
gacagggtga ccatcacctg cagggccagc cagggcatct acaacagcat cgcctggtac 540gacagggtga ccatcacctg cagggccagc cagggcatct acaacagcat cgcctggtac 540
cagcagaagc ccggcaaggc tcccaagctg ctgctgtaca gcaccagcac actgctgagc 600cagcagaagc ccggcaaggc tcccaagctg ctgctgtaca gcaccagcac actgctgagc 600
ggcgtgccca gcagattcag cggaagcggc agcggcaccg attacaccct gaccatcacc 660ggcgtgccca gcagattcag cggaagcggc agcggcaccg attacaccct gaccatcacc 660
aacctgcagc ctgaggactt cgccacctac tactgccagc agtacttcgt gacccccgag 720aacctgcagc ctgaggactt cgccacctac tactgccagc agtacttcgt gacccccgag 720
accttcggac agggcaccaa ggtggagatc aagcgtacgg tggctgcacc atctgtcttc 780accttcggac agggcaccaa ggtggagatc aagcgtacgg tggctgcacc atctgtcttc 780
atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 840atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 840
aataacttct accccagaga agccaaagtg cagtggaagg tggacaacgc cctgcagagc 900aataacttct accccagaga agccaaagtg cagtggaagg tggacaacgc cctgcagagc 900
ggaaacagcc aggaaagcgt gacagagcag gattccaagg attccacata cagcctgagc 960ggaaacagcc aggaaagcgt gacagagcag gattccaagg attccacata cagcctgagc 960
agcacactga cactgtccaa ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 1020agcacactga cactgtccaa ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 1020
acacaccagg gactgtcctc ccctgtgaca aagagcttca acagaggaga atgctgatga 1080acacaccagg gactgtcctc ccctgtgaca aagagcttca acagaggaga atgctgatga 1080
<210> 4<210> 4
<211> 358<211> 358
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<400> 4<400> 4
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr GlyMet Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 151 5 10 15
Val His Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Ser ValVal His Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val
20 25 30 20 25 30
Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser LeuThr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
35 40 45 35 40 45
Leu His Arg Ser Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys ProLeu His Arg Ser Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro
50 55 60 50 55 60
Gly His Ser Pro Gln Leu Leu Ile Tyr Val Gly Ser Asn Arg Ala SerGly His Ser Pro Gln Leu Leu Ile Tyr Val Gly Ser Asn Arg Ala Ser
65 70 75 8065 70 75 80
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Tyr ThrGly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Tyr Thr
85 90 95 85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr CysLeu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
100 105 110 100 105 110
Met Gln Ala Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys LeuMet Gln Ala Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu
115 120 125 115 120 125
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro ProGlu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
130 135 140 130 135 140
Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyGlu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
145 150 155 160145 150 155 160
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Asn SerAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Asn Ser
165 170 175 165 170 175
Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu LeuIle Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu
180 185 190 180 185 190
Tyr Ser Thr Ser Thr Leu Leu Ser Gly Val Pro Ser Arg Phe Ser GlyTyr Ser Thr Ser Thr Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly
195 200 205 195 200 205
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Thr Asn Leu Gln ProSer Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Thr Asn Leu Gln Pro
210 215 220 210 215 220
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Val Thr Pro GluGlu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Val Thr Pro Glu
225 230 235 240225 230 235 240
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala AlaThr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
245 250 255 245 250 255
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser GlyPro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
260 265 270 260 265 270
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu AlaThr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
275 280 285 275 280 285
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser GlnLys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
290 295 300 290 295 300
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu SerGlu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
305 310 315 320305 310 315 320
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val TyrSer Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
325 330 335 325 330 335
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys SerAla Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
340 345 350 340 345 350
Phe Asn Arg Gly Glu CysPhe Asn Arg Gly Glu Cys
355 355
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310521961.2ACN104592390B (en) | 2013-10-30 | 2013-10-30 | A kind of double special restructuring AntiHBsAg antibodies, preparation method and the usage |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310521961.2ACN104592390B (en) | 2013-10-30 | 2013-10-30 | A kind of double special restructuring AntiHBsAg antibodies, preparation method and the usage |
| Publication Number | Publication Date |
|---|---|
| CN104592390A CN104592390A (en) | 2015-05-06 |
| CN104592390Btrue CN104592390B (en) | 2017-11-10 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310521961.2AActiveCN104592390B (en) | 2013-10-30 | 2013-10-30 | A kind of double special restructuring AntiHBsAg antibodies, preparation method and the usage |
| Country | Link |
|---|---|
| CN (1) | CN104592390B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105001325A (en)* | 2015-07-31 | 2015-10-28 | 北京泰诺迪生物科技有限公司 | Total-humanized anti-hepatitis B virus neutralizing antibody and preparing method and application thereof |
| CN105061590B (en)* | 2015-08-03 | 2019-06-04 | 中国人民解放军第二军医大学 | Bispecific antibodies against hepatitis B surface protein and uses thereof |
| CN105481981B (en)* | 2016-01-27 | 2019-03-19 | 中国人民解放军第二军医大学 | Target VEGF bispecific antibody and application thereof |
| CN109021098B (en)* | 2018-08-06 | 2019-05-17 | 南京鼓楼医院 | Full Humanized monoclonal antibodies and its preparation method and application |
| CN119143869A (en)* | 2023-06-16 | 2024-12-17 | 复旦大学 | Hepatitis B virus surface antibody and application thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101370525A (en)* | 2005-08-19 | 2009-02-18 | 艾博特公司 | Dual variable domain immunoglobin and uses thereof |
| CN102757492A (en)* | 2011-04-26 | 2012-10-31 | 中国人民解放军第二军医大学 | Holistic hepatitis B surface protein monoclonal antibodies and application thereof to preparation of medicines for preventing HBV (hepatitis B virus) infection |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101370525A (en)* | 2005-08-19 | 2009-02-18 | 艾博特公司 | Dual variable domain immunoglobin and uses thereof |
| CN102757492A (en)* | 2011-04-26 | 2012-10-31 | 中国人民解放军第二军医大学 | Holistic hepatitis B surface protein monoclonal antibodies and application thereof to preparation of medicines for preventing HBV (hepatitis B virus) infection |
| Publication number | Publication date |
|---|---|
| CN104592390A (en) | 2015-05-06 |
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