技术领域Technical field
本发明涉及生物技术领域，具体涉及Rab8蛋白的特异性抗体及其制备方法与应用。The present invention relates to the field of biotechnology, and specifically to specific antibodies for Rab8 protein and preparation methods and applications thereof.
背景技术Background technique
抗体是一种主要由浆细胞分泌，被免疫系统用来鉴别与中和外来物质如细菌、病毒等外来物的蛋白质，该外来物被称为抗原。抗体和抗原的结合完全依靠非共价键的相互作用，这种特异性的结合机制使得抗体可以捕获外来微生物以及受感染的细胞，进一步诱导其他免疫机制对其进行攻击，或直接中和其目标。抗体及抗体相关产品已经被广泛应用于生命科学与医学等研究领域，基于抗原-抗体特异性结合而衍生的诸多实验技术奠定了科学研究与临床治疗的重要基础，例如免疫诊断、免疫印迹、酶联免疫吸附、流式细胞分析等等。Antibodies are proteins mainly secreted by plasma cells and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. The foreign substances are called antigens. The binding of antibodies and antigens relies entirely on non-covalent interactions. This specific binding mechanism allows antibodies to capture foreign microorganisms and infected cells, further inducing other immune mechanisms to attack them, or directly neutralize their targets. . Antibodies and antibody-related products have been widely used in research fields such as life sciences and medicine. Many experimental technologies derived from the specific binding of antigens and antibodies have laid an important foundation for scientific research and clinical treatment, such as immunodiagnosis, immunoblotting, enzyme immunosorbent assay, flow cytometry, etc.
Rab8(Ras-related protein 8)是Ras超家族的成员，在蛋白质转运和膜重组中发挥作用。Rab家族蛋白质在非活性GDP结合形式和活性GTP结合形式之间循环，能够将不同组的下游效应子募集到膜上，直接负责囊泡的形成、运动、束缚和融合。Rab8的细胞生理功能包括参与极化囊泡运输和神经递质释放、与多种重要蛋白质共同促进转胞吞作用、调节高尔基体的紧凑形态、参与上皮细胞极化、参与膜运输到纤毛和纤毛发生、调节粘附连接组件等。Rab8 (Ras-related protein 8) is a member of the Ras superfamily and plays a role in protein transport and membrane reorganization. Rab family proteins cycle between the inactive GDP-bound form and the active GTP-bound form, can recruit different groups of downstream effectors to the membrane, and are directly responsible for the formation, movement, tethering and fusion of vesicles. The cellular physiological functions of Rab8 include participating in polarized vesicle transport and neurotransmitter release, cooperating with a variety of important proteins to promote transcytosis, regulating the compact morphology of the Golgi apparatus, participating in epithelial cell polarization, and participating in membrane transport to cilia and cilia. Occurrence, regulation of adherens junction components, etc.
在研究Rab8过程中，抗体是一个非常重要的研究工具，对于患者诊断、病毒分析与研究等都具有重大的价值和意义。然而，传统的鼠、兔等动物仅能识别抗原表面平展的多肽。因此，需要开发一种能够识别抗原表面复杂的空间结构，产生高度特异性、高亲和力的纳米抗体。In the process of studying Rab8, antibodies are a very important research tool and are of great value and significance for patient diagnosis, virus analysis and research, etc. However, traditional animals such as mice and rabbits can only recognize peptides with flat antigen surfaces. Therefore, there is a need to develop a nanobody that can recognize the complex spatial structure of the antigen surface and produce highly specific and high-affinity nanobodies.
发明内容Contents of the invention
为了解决现有技术中存在的上述技术问题之一，本发明提供了一种Rab8蛋白的特异性抗体及其制备方法与应用。In order to solve one of the above technical problems existing in the prior art, the present invention provides a specific antibody for Rab8 protein and its preparation method and application.
第一方面，本发明提供用Rab8蛋白免疫骆驼科动物所得的抗原结合蛋白、抗体或抗体活性片段。In a first aspect, the present invention provides antigen-binding proteins, antibodies or antibody active fragments obtained by immunizing camelids with Rab8 protein.
在一些实施方案中，所述骆驼科动物选自单峰驼、双峰驼、美洲驼、骆马、羊驼和小羊驼，优选为羊驼。In some embodiments, the camelid is selected from the group consisting of dromedaries, Bactrian camels, llamas, vicuñas, alpacas and vipacas, preferably alpacas.
在一些实施方案中，所述抗体为纳米抗体，所述抗体活性片段为纳米抗体活性片段。In some embodiments, the antibody is a Nanobody and the antibody active fragment is a Nanobody active fragment.
在一些实施方案中，所述抗体为单克隆抗体或者多克隆抗体。In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.
在一些实施方案中，所述抗原结合蛋白、抗体或抗体活性片段与所述Rab8蛋白结合的kd值在500nM以下，优选在200nM以下，更优选在100nM以下。In some embodiments, the kd value of the antigen-binding protein, antibody or antibody active fragment binding to the Rab8 protein is below 500 nM, preferably below 200 nM, more preferably below 100 nM.
在一些实施方案中，所述Rab8蛋白具有如SEQ ID NO:1所示的氨基酸序列。进一步优选地，所述Rab8蛋白由包括如下步骤的方法制备而成：将编码所述Rab8蛋白的核苷酸序列构建至载体质粒；将所述载体质粒转染至真核细胞系中表达，纯化。In some embodiments, the Rab8 protein has the amino acid sequence set forth in SEQ ID NO:1. Further preferably, the Rab8 protein is prepared by a method including the following steps: constructing the nucleotide sequence encoding the Rab8 protein into a vector plasmid; transfecting the vector plasmid into a eukaryotic cell line for expression, and purifying .
在一些实施方案中，所述Rab8蛋白可通过商业渠道获得。In some embodiments, the Rab8 protein is commercially available.
第二方面，本发明提供一种构建抗体文库的方法，所述方法包括如下步骤：In a second aspect, the present invention provides a method for constructing an antibody library, which method includes the following steps:
(1)将Rab8蛋白作为抗原免疫骆驼科动物，采集被免疫动物的静脉外周血，分离得到淋巴细胞；(1) Use Rab8 protein as an antigen to immunize camelids, collect the venous peripheral blood of the immunized animals, and isolate lymphocytes;
(2)提取所述淋巴细胞的总mRNA，反转录为cDNA并进行扩增；(2) Extract the total mRNA of the lymphocytes, reverse transcribe into cDNA and amplify it;
(3)将所述扩增得到的DNA插入病毒表达载体，转化入细菌，收集菌落，得抗体文库。(3) Insert the amplified DNA into a viral expression vector, transform it into bacteria, and collect bacterial colonies to obtain an antibody library.
在一些实施方案中，所述骆驼科动物选自单峰驼、双峰驼、美洲驼、骆马、羊驼和小羊驼，优选为羊驼。In some embodiments, the camelid is selected from the group consisting of dromedaries, Bactrian camels, llamas, vicuñas, alpacas and vipacas, preferably alpacas.
在一些实施方案中，步骤(1)所述免疫采用皮下注射方式。所述免疫的次数优选为3～5次。所述静脉外周血优选在最后一次免疫之前和之后分别采集。In some embodiments, the immunization in step (1) is performed by subcutaneous injection. The number of immunizations is preferably 3 to 5 times. The venous peripheral blood is preferably collected before and after the last immunization.
在一些实施方案中，步骤(3)所述病毒表达载体为噬菌体表达载体。In some embodiments, the viral expression vector in step (3) is a phage expression vector.
在一些实施方案中，步骤(3)所述细菌为TG1感受态细菌。In some embodiments, the bacteria in step (3) are TG1 competent bacteria.
第三方面，本发明提供上述构建抗体文库的方法获得的抗体文库，或由所述抗体文库表达产生的多克隆抗体。In a third aspect, the present invention provides an antibody library obtained by the above method of constructing an antibody library, or a polyclonal antibody produced by expression of the antibody library.
第四方面，本发明提供一种构建抗原特异性抗体文库的方法，所述方法包括如下步骤：对第三方面所述的抗体文库进行筛选，获得抗原特异性抗体文库。In a fourth aspect, the present invention provides a method for constructing an antigen-specific antibody library, which method includes the following steps: screening the antibody library described in the third aspect to obtain an antigen-specific antibody library.
在一些实施方案中，所述构建抗原特异性抗体文库的方法包括如下步骤：In some embodiments, the method of constructing an antigen-specific antibody library includes the following steps:
(i)对所述抗体文库进行培养，释放病毒；(i) culturing the antibody library to release viruses;
(ii)将所述病毒与抗原进行孵育，去掉与抗原非特异性结合的病毒，保留与抗原特异性结合的病毒；(ii) incubate the virus with the antigen, remove the virus that non-specifically binds to the antigen, and retain the virus that specifically binds to the antigen;
(iii)用所述与抗原特异性结合的病毒侵染细菌，收集菌落，获得抗原特异性抗体文库。(iii) Infect bacteria with the virus that specifically binds to the antigen, collect bacterial colonies, and obtain an antigen-specific antibody library.
在一些实施方案中，步骤(iii)所述细菌为大肠杆菌。In some embodiments, the bacterium of step (iii) is E. coli.
第五方面，本发明提供上述构建抗原特异性抗体文库的方法获得的抗原特异性抗体文库，或由所述抗原特异性抗体文库表达产生的与抗原特异性结合的多克隆抗体。In a fifth aspect, the present invention provides an antigen-specific antibody library obtained by the above method of constructing an antigen-specific antibody library, or a polyclonal antibody specifically binding to an antigen produced by expression of the antigen-specific antibody library.
第六方面，本发明提供一种制备抗原结合蛋白、抗体或抗体活性片段的方法，所述方法包括如下步骤：对第三方面所述的抗体文库进行筛选，获得与抗原特异性结合的抗原结合蛋白、抗体或抗体活性片段。In a sixth aspect, the present invention provides a method for preparing antigen-binding proteins, antibodies or antibody active fragments. The method includes the following steps: screening the antibody library described in the third aspect to obtain antigen-binding proteins that specifically bind to the antigen. Protein, antibody or antibody active fragment.
在一些实施方案中，所述制备抗原结合蛋白、抗体或抗体活性片段的方法包括如下步骤：In some embodiments, the method of preparing an antigen-binding protein, antibody or antibody active fragment includes the steps of:
(a)对所述抗体文库进行培养，释放病毒；(a) culturing the antibody library to release viruses;
(b)将所述病毒与抗原进行孵育，去掉与抗原非特异性结合的病毒，保留与抗原特异性结合的病毒；(b) incubate the virus with the antigen, remove the virus that non-specifically binds to the antigen, and retain the virus that specifically binds to the antigen;
(c)用所述与抗原特异性结合的病毒侵染细菌，将被侵染的细菌涂抹至平板培养基培养，挑选单一菌落。(c) Infect bacteria with the virus that specifically binds to the antigen, smear the infected bacteria onto a plate culture medium for culture, and select a single colony.
在一些实施方案中，步骤(c)所述细菌为大肠杆菌。In some embodiments, the bacterium of step (c) is E. coli.
在一些实施方案中，可以对所述单一菌落进行扩大培养，之后进行抗原特异性结合鉴定。In some embodiments, the single colony can be expanded and cultured, followed by identification of antigen-specific binding.
在一些实施方案中，可以对所述单一菌落进行扩大培养，然后进行步骤(d)：提取DNA，转化至宿主细胞并表达，以获得单克隆抗体。In some embodiments, the single colony can be expanded and cultured, and then step (d) is performed: extracting DNA, transforming into host cells, and expressing to obtain monoclonal antibodies.
第七方面，本发明提供采用上述制备抗原结合蛋白、抗体或抗体活性片段的方法获得的抗原结合蛋白、抗体或抗体活性片段。In a seventh aspect, the present invention provides an antigen-binding protein, antibody or antibody active fragment obtained by using the above method for preparing an antigen-binding protein, antibody or antibody active fragment.
第八方面，本发明提供特异性识别和/或结合Rab8蛋白的抗原结合蛋白、抗体或抗体活性片段；所述抗原结合蛋白、抗体或抗体活性片段包含至少一个重链可变区；所述重链可变区具有：In the eighth aspect, the present invention provides antigen-binding proteins, antibodies or antibody active fragments that specifically recognize and/or bind Rab8 protein; the antigen-binding proteins, antibodies or antibody active fragments comprise at least one heavy chain variable region; the heavy chain The chain variable region has:
如SEQ ID NO:2或SEQ ID NO:3所示的CDR1；CDR1 as shown in SEQ ID NO:2 or SEQ ID NO:3;
如SEQ ID NO:4或SEQ ID NO:5所示的CDR2；和CDR2 as set forth in SEQ ID NO:4 or SEQ ID NO:5; and
如SEQ ID NO:6或SEQ ID NO:7所示的CDR3。CDR3 as shown in SEQ ID NO:6 or SEQ ID NO:7.
在一些实施方案中，所述重链可变区具有：如SEQ ID NO:2所示的CDR1、如SEQ IDNO:4所示的CDR2和如SEQ ID NO:6所示的CDR3。In some embodiments, the heavy chain variable region has: CDR1 as set forth in SEQ ID NO:2, CDR2 as set forth in SEQ ID NO:4, and CDR3 as set forth in SEQ ID NO:6.
在一些实施方案中，所述重链可变区具有：如SEQ ID NO:3所示的CDR1、如SEQ IDNO:5所示的CDR2和如SEQ ID NO:7所示的CDR3。In some embodiments, the heavy chain variable region has: CDR1 as set forth in SEQ ID NO:3, CDR2 as set forth in SEQ ID NO:5, and CDR3 as set forth in SEQ ID NO:7.
在一些实施方案中，所述重链可变区具有：如SEQ ID NO:8所示的氨基酸序列，或SEQ ID NO:8所示的氨基酸序列经过一个或多个氨基酸添加、删除、替换或修饰获得的保守性变异体。In some embodiments, the heavy chain variable region has: the amino acid sequence set forth in SEQ ID NO:8, or the amino acid sequence set forth in SEQ ID NO:8 with one or more amino acid additions, deletions, substitutions, or Conservative variants obtained through modification.
在一些实施方案中，所述重链可变区具有：如SEQ ID NO:9所示的氨基酸序列，或SEQ ID NO:9所示的氨基酸序列经过一个或多个氨基酸添加、删除、替换或修饰获得的保守性变异体。In some embodiments, the heavy chain variable region has: the amino acid sequence set forth in SEQ ID NO:9, or the amino acid sequence set forth in SEQ ID NO:9 with one or more amino acid additions, deletions, substitutions, or Conservative variants obtained through modification.
在一些实施方案中，所述抗原结合蛋白、抗体或抗体活性片段包含一个所述重链可变区且缺失轻链。In some embodiments, the antigen binding protein, antibody or antibody active fragment comprises one of the heavy chain variable regions and is missing the light chain.
在一些实施方案中，所述抗体为纳米抗体，所述抗体活性片段为纳米抗体活性片段。In some embodiments, the antibody is a Nanobody and the antibody active fragment is a Nanobody active fragment.
第九方面，本发明提供编码SEQ ID NO:2～SEQ ID NO:9任意一条序列所示氨基酸序列或上文所述抗原结合蛋白、抗体或抗体活性片段的核苷酸序列。In the ninth aspect, the present invention provides a nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NO: 2 to SEQ ID NO: 9 or the above-mentioned antigen-binding protein, antibody or antibody active fragment.
在一些实施方案中，编码所述抗原结合蛋白、抗体或抗体活性片段的核苷酸序列如SEQ ID NO:10所示。In some embodiments, the nucleotide sequence encoding the antigen-binding protein, antibody, or antibody active fragment is set forth in SEQ ID NO: 10.
在一些实施方案中，编码所述抗原结合蛋白、抗体或抗体活性片段的核苷酸序列如SEQ ID NO:11所示。In some embodiments, the nucleotide sequence encoding the antigen-binding protein, antibody, or antibody active fragment is set forth in SEQ ID NO: 11.
第十方面，本发明提供含有上文所述核苷酸序列的表达载体。In a tenth aspect, the present invention provides expression vectors containing the nucleotide sequences described above.
在一些实施方案中，所述表达载体为噬菌体表达载体，优选为噬菌体表面展示筛选载体。In some embodiments, the expression vector is a phage expression vector, preferably a phage surface display screening vector.
在一些实施方案中，所述表达载体中还含有编码噬菌体包膜蛋白pIII的核苷酸序列。In some embodiments, the expression vector also contains a nucleotide sequence encoding the phage envelope protein pill.
第十一方面，本发明提供外源转入了上文所述表达载体的病毒。In an eleventh aspect, the present invention provides a virus into which the expression vector described above is exogenously transferred.
在一些实施方案中，所述病毒为噬菌体。In some embodiments, the virus is a bacteriophage.
第十二方面，本发明提供外源转入了上文所述表达载体的宿主细胞，或者被上文所述病毒侵染的宿主细胞。In a twelfth aspect, the present invention provides a host cell into which the expression vector described above has been exogenously transferred, or a host cell infected by the virus described above.
在一些实施方案中，所述宿主细胞为大肠杆菌。In some embodiments, the host cell is E. coli.
第十三方面，本发明提供利用上文所述宿主细胞表达抗原结合蛋白、抗体或抗体活性片段的方法。In a thirteenth aspect, the present invention provides methods for expressing antigen-binding proteins, antibodies or antibody active fragments using the host cells described above.
第十四方面，本发明提供利用上文所述宿主细胞表达获得的抗原结合蛋白、抗体或抗体活性片段。In a fourteenth aspect, the present invention provides antigen-binding proteins, antibodies or antibody active fragments obtained by expression in the host cells described above.
第十五方面，本发明提供所述上文所述抗原结合蛋白、抗体或抗体活性片段经人源化后获得的人源化抗原结合蛋白、抗体或抗体活性片段。In a fifteenth aspect, the present invention provides a humanized antigen-binding protein, antibody or antibody active fragment obtained after humanization of the above-mentioned antigen-binding protein, antibody or antibody active fragment.
第十六方面，本发明提供一种蛋白偶联物，其包含上文所述抗原结合蛋白、抗体或抗体活性片段或者上文所述人源化抗原结合蛋白、抗体或抗体活性片段以及配体。In a sixteenth aspect, the present invention provides a protein conjugate comprising the above-mentioned antigen-binding protein, antibody or antibody active fragment or the above-mentioned humanized antigen-binding protein, antibody or antibody active fragment and a ligand .
在一些实施方案中，所述配体选自放射性同位素、荧光基团和递送载体。In some embodiments, the ligand is selected from the group consisting of radioisotopes, fluorophores, and delivery vehicles.
第十七方面，本发明提供检测样品中Rab8蛋白含量的试剂盒，其包含上文所述抗原结合蛋白、抗体或抗体活性片段或者上文所述人源化抗原结合蛋白、抗体或抗体活性片段。In the seventeenth aspect, the present invention provides a kit for detecting Rab8 protein content in a sample, which includes the above-mentioned antigen-binding protein, antibody or antibody active fragment or the above-mentioned humanized antigen-binding protein, antibody or antibody active fragment. .
在一些实施方案中，所述抗原结合蛋白、抗体或抗体活性片段经标记物标记。优选地，所述标记物选自酶、化学发光基团和同位素基团。In some embodiments, the antigen binding protein, antibody or antibody active fragment is labeled with a label. Preferably, the label is selected from the group consisting of enzymes, chemiluminescent groups and isotopic groups.
在一些实施方案中，所述样品为动物血清，优选为人血清。In some embodiments, the sample is animal serum, preferably human serum.
第十八方面，本发明提供上文所述抗原结合蛋白、抗体或抗体活性片段、上文所述人源化抗原结合蛋白、抗体或抗体活性片段、上文所述蛋白偶联物或者上文所述试剂盒在检测样品中Rab8蛋白含量中的应用。In the eighteenth aspect, the present invention provides the above-described antigen-binding protein, antibody or antibody active fragment, the above-described humanized antigen-binding protein, antibody or antibody active fragment, the above-described protein conjugate or the above-described Application of the kit in detecting Rab8 protein content in samples.
在一些实施方案中，所述样品为动物血清，优选为人血清。In some embodiments, the sample is animal serum, preferably human serum.
第十九方面，本发明提供利用上文所述抗原结合蛋白、抗体或抗体活性片段、上文所述人源化抗原结合蛋白、抗体或抗体活性片段、上文所述蛋白偶联物或者上文所述试剂盒检测样品中Rab8蛋白含量的方法。In the nineteenth aspect, the present invention provides the use of the above-described antigen-binding proteins, antibodies or antibody active fragments, the above-described humanized antigen-binding proteins, antibodies or antibody active fragments, the above-described protein conjugates, or the above-described The kit described in this article is a method for detecting the Rab8 protein content in samples.
在一些实施方案中，所述样品为动物血清，优选为人血清。In some embodiments, the sample is animal serum, preferably human serum.
与现有技术相比，本发明提供的技术方案具有如下显著优势：本发明提供的抗体可用于检测血清中目标抗原的含量，进一步用于检测原癌基因，判断病变，有潜在临床诊断和治疗价值；本发明提供的抗体结构简单，容易进行基因工程改造，具有成熟的优化策略用于增强纳米抗体亲和力、延长体内半衰期以及与其它分子偶联用于药物开发，如连接放射性同位素，偶联传递药物、CART和荧光标记高分辨成像等；本发明提供的抗体序列与人IgG的VH区域序列同源性高，少数氨基酸突变即可以实现单域抗体的人源化；且本发明提供的抗体稳定性高，能避免常规抗体需要低温储存和运输的要求，有利于大规模普及应用，量产成本较低，易于大规模重组制备。本发明所设计的单克隆纳米抗体在成本低廉的大肠杆菌表达系统就可以很好的重组表达，量产成本低、产量可以高达数十毫克/升大肠杆菌。大肠杆菌重组表达系统技术成熟，质量控制简单，有利于降低生产成本、实现规模化生产。Compared with the existing technology, the technical solution provided by the present invention has the following significant advantages: the antibody provided by the present invention can be used to detect the content of the target antigen in serum, and further used to detect proto-oncogenes, determine lesions, and has potential clinical diagnosis and treatment Value; The antibody provided by the invention has a simple structure, is easy to carry out genetic engineering, and has mature optimization strategies for enhancing nanobody affinity, extending half-life in vivo, and coupling with other molecules for drug development, such as connecting radioactive isotopes and coupling delivery. Drugs, CART and fluorescent label high-resolution imaging, etc.; the antibody sequence provided by the invention has high homology with the VH region sequence of human IgG, and a few amino acid mutations can realize the humanization of single domain antibodies; and the antibody provided by the invention is stable It has high stability and can avoid the requirements of low-temperature storage and transportation of conventional antibodies, which is conducive to large-scale popularization and application. The cost of mass production is low and it is easy to prepare large-scale recombinant preparations. The monoclonal nanobody designed in the present invention can be well recombinantly expressed in the low-cost Escherichia coli expression system, the mass production cost is low, and the output can be as high as tens of mg/liter of Escherichia coli. The E. coli recombinant expression system has mature technology and simple quality control, which is conducive to reducing production costs and achieving large-scale production.
不同于传统技术依赖于鼠、兔、猴、羊等经典的模式动物，本发明的技术方案是依靠羊驼的免疫系统产生的抗体，被成为“纳米抗体”。纳米抗体是从骆驼、鲨鱼等动物体内免疫球蛋白分离出的微小抗体片段，它具有与完整抗体相同的抗原结合能力和结构稳定性，是现有可结合目标抗原的最小的单位，相对分子质量仅为15kD。相比于传统的鼠、兔等动物仅能识别抗原表面平展的多肽，羊驼等动物体内的免疫系统能够识别抗原表面复杂的空间结构，能够产生高度特异性、高亲和力的纳米抗体。Unlike traditional technologies that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of the present invention relies on antibodies produced by the immune system of alpacas, which are called "nanobodies." Nanobodies are tiny antibody fragments isolated from immunoglobulins in animals such as camels and sharks. They have the same antigen-binding ability and structural stability as intact antibodies. They are the smallest existing units that can bind target antigens and have a relative molecular mass of Only 15kD. Compared with traditional mice, rabbits and other animals that can only recognize flat peptides on the antigen surface, the immune system in animals such as alpacas can recognize the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanobodies.
附图说明Description of the drawings
图1为单抗Rab8_9F3与抗原亲和力检测结果示意图。Figure 1 is a schematic diagram of the detection results of monoclonal antibody Rab8_9F3 and antigen affinity.
图2为单抗Rab8_10B4与抗原亲和力检测结果示意图。Figure 2 is a schematic diagram of the detection results of monoclonal antibody Rab8_10B4 and antigen affinity.
具体实施方式Detailed ways
为使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步的详细说明。此处所描述的具体实施例仅用于解释本发明，并不用于构成对本发明的任何限制。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本公开的概念。这样的结构和技术在许多出版物中也进行了描述。In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the embodiments. The specific embodiments described here are only used to explain the present invention and are not intended to constitute any limitation on the present invention. Furthermore, in the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and techniques are also described in many publications.
定义definition
除非另有定义，否则本发明使用的所有技术术语和科技术语都具有如在本发明所属领域中通常使用的相同含义。出于解释本说明书的目的，将应用以下定义，并且在适当时，以单数形式使用的术语也将包括复数形式，反之亦然。Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly used in the art to which this invention belongs. For the purposes of interpreting this specification, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural and vice versa.
除非上下文另有明确说明，否则本文所用的表述“一种”和“一个”包括复数指代。例如，提及“一个细胞”包括多个这样的细胞及本领域技术人员可知晓的等同物等等。As used herein, the expressions "a" and "an" include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells and equivalents known to those skilled in the art, and the like.
本文所用的术语“约”表示其后的数值的±20％的范围。在一些实施方式中，术语“约”表示其后的数值的±10％的范围。在一些实施方式中，术语“约”表示其后的数值的±5％的范围。The term "about" as used herein means a range of ±20% of the numerical value that follows. In some embodiments, the term "about" means a range of ±10% of the numerical value that follows. In some embodiments, the term "about" means a range of ±5% of the numerical value that follows.
Rab8：Rab8(Ras-related protein 8)是Ras超家族的成员，在蛋白质转运和膜重组中发挥作用。Rab家族蛋白质在非活性GDP结合形式和活性GTP结合形式之间循环，能够将不同组的下游效应子募集到膜上，直接负责囊泡的形成、运动、束缚和融合。Rab8的细胞生理功能包括参与极化囊泡运输和神经递质释放、与多种重要蛋白质共同促进转胞吞作用、调节高尔基体的紧凑形态、参与上皮细胞极化、参与膜运输到纤毛和纤毛发生、调节粘附连接组件等。Rab8: Rab8 (Ras-related protein 8) is a member of the Ras superfamily and plays a role in protein transport and membrane reorganization. Rab family proteins cycle between the inactive GDP-bound form and the active GTP-bound form, can recruit different groups of downstream effectors to the membrane, and are directly responsible for the formation, movement, tethering and fusion of vesicles. The cellular physiological functions of Rab8 include participating in polarized vesicle transport and neurotransmitter release, cooperating with a variety of important proteins to promote transcytosis, regulating the compact morphology of the Golgi apparatus, participating in epithelial cell polarization, and participating in membrane transport to cilia and cilia. Occurrence, regulation of adherens junction components, etc.
kd值：解离常数(dissociation constant，kd)是一种特定类型的平衡常数，用于衡量一较大物体与另一较小组分分开(解离)的倾向，是缔合常数的倒数，单位为mol/L(M)或nmol/L(nM)。kd值越小说明两个物质的结合能力越强。kd value : The dissociation constant (kd) is a specific type of equilibrium constant, used to measure the tendency of a larger object to separate (dissociate) from another smaller component. It is the reciprocal of the association constant, unit It is mol/L(M) or nmol/L(nM). The smaller the kd value, the stronger the binding ability of the two substances.
纳米抗体：在骆驼科动物外周血液中存在的天然缺失轻链的抗体，该抗体只包含一个重链可变区(VHH)和两个常规的CH2与CH3区，但却不像人工改造的单链抗体片段那样容易相互沾粘，甚至聚集成块；单独克隆并表达出来的VHH结构具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，是已知的可结合目标抗原的最小单位；VHH晶体为2.5nm，长4nm，分子量只有15KDa，因此也被称作纳米抗体(Nanobody，Nb)。相比于传统的鼠、兔等动物仅能识别抗原表面平展的多肽，骆驼科动物体内的免疫系统能够识别抗原表面复杂的空间结构，能够产生高度特异性、高亲和力的纳米抗体。Nanobodies : Antibodies that naturally lack the light chain and exist in the peripheral blood of camelids. This antibody only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not like artificially modified monoclonal antibodies. Chain antibody fragments easily stick to each other and even aggregate into blocks; the VHH structure cloned and expressed separately has structural stability and antigen-binding activity comparable to that of the original heavy chain antibody, and is the smallest known structure that can bind the target antigen. Unit; VHH crystal is 2.5nm, 4nm long, and has a molecular weight of only 15KDa, so it is also called Nanobody (Nb). Compared with traditional mice, rabbits and other animals that can only recognize flat peptides on the antigen surface, the immune system of camelids can recognize the complex spatial structure of the antigen surface and produce highly specific and high-affinity nanobodies.
根据本发明的技术方案，在不改变蛋白的活性或功能的情况下，可以对氨基酸序列中的某些氨基酸进行保守取代，参见下表1：According to the technical solution of the present invention, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, see Table 1 below:
表1Table 1
此外，因为碱基的简并性，在不改变多核苷酸序列的活性或功能的情况下，可以对多核苷酸序列的碱基进行取代，参见下表2：In addition, because of the degeneracy of the bases, the bases of the polynucleotide sequence can be substituted without changing the activity or function of the polynucleotide sequence, see Table 2 below:
表2Table 2
下面提供实施例和附图以帮助理解本发明。但应理解，这些实施例和附图仅用于说明本发明，但不构成任何限制。本发明的实际保护范围在权利要求书中进行阐述。应理解，在不脱离本发明精神的情况下，可以进行任何修改和改变。Examples and figures are provided below to aid understanding of the present invention. However, it should be understood that these embodiments and drawings are only used to illustrate the present invention and do not constitute any limitation. The actual scope of the invention is set forth in the claims. It is understood that any modifications and changes may be made without departing from the spirit of the invention.
实施例1.制备抗原Example 1. Preparation of antigens
重组人Rab8蛋白购于PROSPEC(货号：PRO-1528)。Recombinant human Rab8 protein was purchased from PROSPEC (catalog number: PRO-1528).
所述重组人Rab8蛋白抗原具有如SEQ ID NO:1所示的氨基酸序列，具体为：The recombinant human Rab8 protein antigen has the amino acid sequence shown in SEQ ID NO: 1, specifically:
MGSSHHHHHHSSGLVPRGSHMGSMAKTYDYLFKLLLIGDSGVGKTCLLFRFSEDAFNTTFISTIGIDFKIRTIELDGKKIKLQIWDTAGQERFRTITTAYYRGAMGIMLVYDITNEKSFDNIKNWIRNIEEHASSDVERMILGNKCDMNDKRQVSKERGEKLAIDYGIKFLETSAKSSANVEEAFFTLARDIMTKLNRKMNDSNSAGAGGPVKITENRSKKTSFFRC(SEQ ID NO:1)。MGSSHHHHHHSSGLVPRGSHMGSMAKTYDYLFKLLLIGDSGVGKTCLLFRFSEDAFNTTFISTIGIDFKIRTIELDGKKIKLQIWDTAGQERFRTITTAYYRGAMGIMLVYDITNEKSFDNIKNWIRNIEEHASSDVERMILGNKCDMNDKRQVSKERGEKLAIDYGIKFLETSAKSSANVEEAFFTLARDIMTKLNRKMNDNSSAGAGG PVKITENRSKKTSFFRC (SEQ ID NO: 1).
实施例2.羊驼免疫注射Example 2. Immunization injection of alpaca
本实施例将实施例1的抗原免疫羊驼。具体步骤如下：In this example, alpacas were immunized with the antigen of Example 1. Specific steps are as follows:
(1)将实施例1中的抗原平均分装成4份，每份约0.25mg；累计对羊驼进行4次免疫，将抗原经皮下注射至动物体内，记第一次免疫为第一天，后续的免疫分别于第10天、第19天、第28天；(1) Divide the antigen in Example 1 into 4 equal portions, each portion is about 0.25 mg; immunize alpacas 4 times in total, subcutaneously inject the antigen into the animals, and record the first immunization as the first day , subsequent immunizations were on the 10th, 19th, and 28th days respectively;
(2)第28天，于第四次免疫注射前，采集约200mL羊驼静脉外周血液；(2) On day 28, before the fourth immune injection, collect approximately 200 mL of alpaca venous peripheral blood;
(3)第42天，即第四次免疫之后14天，采集约200mL羊驼静脉外周血液。(3) On day 42, that is, 14 days after the fourth immunization, approximately 200 mL of alpaca venous peripheral blood was collected.
相比于传统鼠、兔等动物抗体的免疫技术方案，本技术优势在于采集大量的羊驼静脉外周血液，有利于后续筛选得到高度多样性的纳米抗体。Compared with traditional immunization technology solutions for animal antibodies such as mice and rabbits, the advantage of this technology is to collect a large amount of alpaca venous peripheral blood, which is conducive to subsequent screening to obtain highly diverse nanobodies.
实施例3.构建羊驼的纳米抗体文库Example 3. Construction of alpaca Nanobody library
以实施例2中采集的两批次羊驼静脉外周血液为原料，构建高多样性的纳米抗体文库。两批次羊驼静脉外周血液的处理方法相同，具体步骤如下：Two batches of alpaca venous peripheral blood collected in Example 2 were used as raw materials to construct a high-diversity Nanobody library. The two batches of alpaca venous peripheral blood are processed in the same way. The specific steps are as follows:
(1)使用密度梯度离心等方法，从羊驼静脉外周血液中分离得到淋巴细胞；(1) Use density gradient centrifugation and other methods to isolate lymphocytes from alpaca venous peripheral blood;
(2)提取淋巴细胞的总mRNA，并反转录为cDNA；(2) Extract total mRNA from lymphocytes and reverse-transcribe into cDNA;
(3)使用适当的DNA引物，以上述cDNA为模板，经聚合酶链式反应(PCR)扩增得到羊驼免疫球蛋白IgG2和IgG3的VHH片段，即纳米抗体的DNA片段；(3) Use appropriate DNA primers and use the above cDNA as a template to amplify the VHH fragments of alpaca immunoglobulin IgG2 and IgG3 through polymerase chain reaction (PCR), that is, the DNA fragment of the nanobody;
(4)将VHH的DNA连接至噬菌体表面展示筛选载体，构成VHH-pIII融合蛋白表达载体质粒库；其中，pIII是存在于噬菌体表面鞭毛上的蛋白质；(4) Connect the DNA of VHH to the phage surface display screening vector to form a VHH-pIII fusion protein expression vector plasmid library; where pIII is a protein present on the flagellum on the surface of the phage;
(5)将DNA连接产物经电转化方法，转化至TG1感受态细菌，适当培养后收集全部菌落，即为羊驼的纳米抗体文库。(5) Transform the DNA ligation product into TG1 competent bacteria through electroconversion method. After appropriate cultivation, collect all colonies to form the alpaca Nanobody library.
相比于传统的从鼠、兔等动物血清或淋巴细胞中分离得到抗体的方法，本技术方案能够长期保存持有羊驼的全部纳米抗体片段(即文库)，能够持续地支撑后续不断地进行纳米抗体的筛选与开发。Compared with the traditional method of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, this technical solution can preserve all the nanobody fragments (i.e., libraries) of alpacas for a long time, and can continuously support subsequent progress. Screening and development of nanobodies.
实施例4.噬菌体表面展示筛选特异性纳米抗体Example 4. Phage surface display screening of specific Nanobodies
本实施例以实施例3得到的纳米抗体文库为来源，经噬菌体表面展示筛选得到抗原特异性的纳米抗体。具体步骤如下：This example uses the Nanobody library obtained in Example 3 as a source, and obtains antigen-specific Nanobodies through phage surface display screening. Specific steps are as follows:
(1)取适量冻存的纳米抗体文库，接种至细菌培养基，经适当培养后加入适量的辅助噬菌体，继续于适量条件下培养；(1) Take an appropriate amount of frozen Nanobody library, inoculate it into a bacterial culture medium, add an appropriate amount of helper phage after appropriate culture, and continue culturing under appropriate conditions;
(2)以PEG-NaC法提取细菌培养上清中扩增的噬菌体；(2) Extract the amplified phage from the bacterial culture supernatant using the PEG-NaC method;
(3)将噬菌体与抗原适当孵育，Rab8抗原预先固定于免疫试管(Maxisorp免疫试管，ThermoFisher Scientific)；(3) Incubate the phage and antigen appropriately, and the Rab8 antigen is pre-fixed in an immune test tube (Maxisorp immune test tube, ThermoFisher Scientific);
(4)淘洗：弃去噬菌体，再以适当的缓冲液(如PBS等)润洗抗原适当次数，淘洗、除去与抗原非特异性结合的噬菌体，保留与抗原特异性结合的噬菌体；(4) Elutriation: Discard the phage, and then rinse the antigen with an appropriate buffer (such as PBS, etc.) for an appropriate number of times. Eluate and remove the phages that non-specifically bind to the antigen, while retaining the phages that specifically bind to the antigen;
(5)洗脱：以合适的方法处理与抗原特异性结合的噬菌体(如酸性甘氨酸溶液等)，使噬菌体与抗原解离并保留。(5) Elution: Use appropriate methods to treat the phage that specifically binds to the antigen (such as acidic glycine solution, etc.) to dissociate the phage from the antigen and retain it.
至此，即得到了表达有特异性纳米抗体的噬菌体，这些噬菌体可进行下述操作：At this point, phages expressing specific Nanobodies have been obtained. These phages can perform the following operations:
(6)转变为特异性的纳米抗体文库：将噬菌体再次侵染培养至合适状态的大肠杆菌，但不再加入辅助噬菌体，待噬菌体侵染完全后，特异性的纳米抗体即以DNA质粒的形式存在于大肠杆菌中。收集这些全部的大肠杆菌，即成为抗原特异性的纳米抗体文库，可以此文库为原料，返回步骤(1)进行下一轮的噬菌体表面展示筛选；(6) Convert to a specific Nanobody library: Re-infect the phage into E. coli cultured to a suitable state, but no longer add helper phage. After the phage infection is complete, the specific Nanobody will be in the form of a DNA plasmid Found in E. coli. Collect all these E. coli to form an antigen-specific Nanobody library. This library can be used as raw material and return to step (1) for the next round of phage surface display screening;
(7)转变为单克隆纳米抗体菌落：取少量步骤(5)得到的噬菌体(如0.5％)，稀释后再次侵染培养至合适状态的大肠杆菌，但不再加入辅助噬菌体，待噬菌体侵染完全后，将这些大肠杆菌均匀涂抹于细菌培养皿，适当调节下培养即可得到含有纳米抗体DNA质粒的单克隆菌落。以这些单克隆菌落为原料，可进行阳性单克隆纳米抗体的鉴定。(7) Transform into a monoclonal nanobody colony: take a small amount of the phage obtained in step (5) (such as 0.5%), dilute it and infect the E. coli cultured to a suitable state again, but no longer add the helper phage until the phage is infected. After completion, spread these E. coli evenly on the bacterial culture dish, and culture under appropriate adjustments to obtain a single clone colony containing the Nanobody DNA plasmid. Using these monoclonal colonies as raw materials, positive monoclonal Nanobodies can be identified.
实施例5.鉴定阳性单克隆纳米抗体Example 5. Identification of positive monoclonal Nanobodies
本实施例利用实施例4步骤(7)得到长有单克隆菌落的细菌培养皿，进行阳性单克隆纳米抗体的鉴定。具体步骤如下：In this example, step (7) of Example 4 is used to obtain a bacterial culture dish with monoclonal colonies, and positive monoclonal Nanobodies are identified. Specific steps are as follows:
(1)挑取单克隆菌落于微孔板进行培养；(1) Pick single clone colonies and culture them in microplates;
(2)加入IPTG诱导VHH-pIII(即含有纳米抗体的融合蛋白质)表达；(2) Add IPTG to induce the expression of VHH-pIII (i.e., fusion protein containing Nanobodies);
(3)收集含有纳米抗体的细菌培养上清，与抗原Rab8孵育，抗原预先固定于96微孔板(Maxisorp透明微孔板，ThermoFisher Scientific)；(3) Collect the bacterial culture supernatant containing Nanobodies and incubate it with the antigen Rab8. The antigen is pre-fixed on a 96-well microplate (Maxisorp transparent microplate, ThermoFisher Scientific);
(4)利用酶联免疫吸附检测(ELISA)，检测单克隆纳米抗体是否与Rab8抗原结合；(4) Use enzyme-linked immunosorbent assay (ELISA) to detect whether the monoclonal nanobody binds to the Rab8 antigen;
(5)对于可以与抗原结合的单克隆纳米抗体微生物菌落，再次适当培养后，提取DNA质粒并进行DNA测序获得纳米抗体核酸序列，翻译后即可得到纳米抗体的完整氨基酸序列，如表3-4所示。(5) For monoclonal Nanobody microbial colonies that can bind to antigens, after proper cultivation again, extract the DNA plasmid and perform DNA sequencing to obtain the Nanobody nucleic acid sequence. After translation, the complete amino acid sequence of the Nanobody can be obtained, as shown in Table 3- 4 shown.
表3：单抗Rab8_9F3的氨基酸序列和核苷酸序列Table 3: Amino acid sequence and nucleotide sequence of monoclonal antibody Rab8_9F3
表4：单抗Rab8_10B4的氨基酸序列和核苷酸序列Table 4: Amino acid sequence and nucleotide sequence of monoclonal antibody Rab8_10B4
实施例6.小批量单克隆纳米抗体重组表达与纯化Example 6. Recombinant expression and purification of small batch monoclonal Nanobodies
(1)经实施例5得到了能够特异性识别并结合抗原的单克隆纳米抗体，将纳米抗体的DNA质粒转化至BL21(DE3)感受态细胞，借助大肠杆菌表达系统即可小批量表达、纯化单克隆纳米抗体，批产能约数毫克。(1) Monoclonal Nanobodies that can specifically recognize and bind to antigens were obtained in Example 5. The DNA plasmids of the Nanobodies were transformed into BL21 (DE3) competent cells, and small batch expression and purification could be achieved with the help of the E. coli expression system. Monoclonal Nanobodies can be produced in batches of several milligrams.
(2)利用ELISA方法，孵育不同浓度的纳米抗体，根据纳米抗体与Rab8的结合能力测量纳米抗体与抗原的亲和力大小。(2) Use the ELISA method to incubate different concentrations of Nanobodies, and measure the affinity between Nanobodies and antigens based on the binding ability of Nanobodies to Rab8.
得到两组单克隆菌落对应抗体，与Rab8蛋白抗原的亲和力大小的检测结果如图1～2所示，亲和力数值kd的结果如下表5所示。Two groups of antibodies corresponding to monoclonal colonies were obtained. The detection results of the affinity to Rab8 protein antigen are shown in Figures 1 to 2. The results of the affinity value kd are shown in Table 5 below.
表5：亲和力测试结果Table 5: Affinity test results
由以上结果可知，本实施例挑选得到单克隆菌落所得抗体与所述Rab8蛋白结合的kd值在500nM以下，优选在200nM以下，更优选在150nM以下。It can be seen from the above results that the kd value of the antibody obtained by selecting monoclonal colonies in this example and binding to the Rab8 protein is below 500 nM, preferably below 200 nM, and more preferably below 150 nM.
本发明的技术方案不限于上述具体实施例的限制，凡是根据本发明的技术方案做出的技术变形，均落入本发明的保护范围之内。The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical modifications made based on the technical solution of the present invention fall within the protection scope of the present invention.
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| CN202310360867.7ACN116813765B (en) | 2023-04-06 | 2023-04-06 | Specific antibody for Rab8 protein and preparation method and application thereof |
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| CN116640209A (en)* | 2023-04-06 | 2023-08-25 | 生物岛实验室 | Rab11 protein specific antibody and its preparation method and application |
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| CN116640209B (en)* | 2023-04-06 | 2025-04-15 | 生物岛实验室 | Specific antibody for Rab11 protein and preparation method and application thereof |
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