双峰驼天然单域抗体库的构建及重链抗体特异性多抗的制备

发布时间：2018-05-28 15:42

本文选题：双峰驼 + 单域抗体　；参考：《内蒙古农业大学》2016年硕士论文

【摘要】：骆驼血清中除了常规抗体IgG1,还存在两种天然缺失轻链的重链抗体IgG2和IgG3,其具有分子量小、稳定性强等优点,但市场上仍缺乏商品化的骆驼重链抗体特异性的抗体,使其研究和应用受到了限制,因此制备针对骆驼重链抗体的多抗将具有一定的应用前景。重链抗体的可变区VHH是目前发现的、具有完整功能的最小抗体分子片段,该抗体在基础研究、药物开发等领域应用前景极为广阔。将VHH展示在噬菌体表面,构建噬菌体抗体文库,再通过体外筛选技术将所需性质的多肽从含有大量变异体的集落中提取出来,为特异性抗体的制备提供了极大的便利。本研究的第一部分即结合骆驼重链抗体的优势与噬菌体展示技术的优点,从五峰未经免疫的健康双峰驼外周血淋巴细胞中提取总RNA,反转录为cDNA,利用PCR技术扩增编码抗体可变区的基因,通过与噬菌体载体连接,电转化E.coli TGI感受态细胞,构建了双峰驼天然单域抗体文库。经鉴定文库库容为4.4×107,转化阳性率为75%,多样性良好。本研究第二部分,我们利用分离淋巴细胞后的双峰驼血浆,经过Protein G/A Resin柱子对双峰驼三种亚型抗体进行了分离和纯化,以纯化的IgG2对家兔进行三次免疫制备了针对双峰驼重链抗体的多克隆抗体,并对其活性进行了鉴定。间接ELISA法测定多抗效价为1：4096；间接ELISA法和Western-blot方法检测多抗的特异性,结果显示制备的多抗与牛IgG以及双峰驼IgG1无交叉反应,而与IgG3抗体具有交叉反应性。本试验研究成功制备了双峰驼重链抗体特异性多克隆抗体。
[Abstract]:In addition to the routine antibody IgG1, there are two naturally absent heavy chain antibodies, IgG2 and IgG3, which have the advantages of low molecular weight and strong stability, but there is still a lack of commercial heavy chain antibodies in the market. Therefore, the preparation of multiple antibodies against camel heavy chain antibodies will have a certain application prospect. The variable region of heavy chain antibody (VHH) is the smallest fragment of antibody with complete function, which is widely used in basic research, drug development and other fields. VHH was displayed on the phage surface to construct phage antibody library, and then the polypeptide was extracted from the colony containing a large number of variants by in vitro screening technology, which provided a great convenience for the preparation of specific antibody. The first part of this study combines the advantages of camel heavy chain antibody and phage display technology. Total RNAs were extracted from unimmunized lymphocytes of healthy bactrian camels, and then reversely transcribed into cDNAs. The genes encoding variable regions of antibodies were amplified by PCR technique, and then electrotransformed into E.coli TGI receptive cells by ligation with phage vectors. The natural single domain antibody library of Bactrian camel was constructed. The library capacity was 4.4 脳 107 and the positive rate of transformation was 750.The diversity was good. In the second part of this study, we isolated and purified three subtypes of bactrian camel by Protein G / A Resin column. The polyclonal antibody against the heavy chain antibody of Bactrian camel was prepared by three times immunization with purified IgG2 and its activity was identified. The polyclonal antibody titer of indirect ELISA assay was 1: 4096, and the specificity of indirect ELISA and Western-blot methods was determined. The results showed that the prepared polyantibodies did not cross react with bovine IgG and IgG1, but had cross reactivity with IgG3 antibody. In this study, the specific polyclonal antibody against heavy chain antibody of Bactrian camel was successfully prepared.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S824

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