人源化单链抗体噬菌体库的构建及抗TNF-α抗体的筛选

发布时间：2018-04-22 16:02

本文选题：噬菌体抗体库 + 单链抗体　；参考：《天津大学》2012年博士论文

【摘要】：抗体是体液免疫系统的重要组成部分，也在生命科学研究、疫病检测和医药领域发挥着重要作用。噬菌体展示技术是人源治疗性抗体的重要技术来源之一。TNF-α是一种前炎症因子，与风湿性关节炎等多种疾病的发生密切相关。本研究采用噬菌体抗体展示技术构建一个人源噬菌体抗体库，并从中筛选出具有TNF-α细胞毒活性抑制作用的人源单链抗体。依据文献报道的人抗体恒定区序列及人源抗体各胚系基因序列，本研究设计42条引物，以200份人外周血中分离的总RNA为模板，利用这些引物扩增出了抗体的重链（VH）和轻链（VL）可变区基因。抗体重链可变区的扩增和组装采用三轮OE-PCR反应完成：首先分别扩增VH的CDR1、CDR2、CDR3和FR3，然后采用OE-PCR拼接重链的CDR1和CDR2、CDR3和FR3的基因片段，再将两者拼接成完整的人抗体VH基因。VL基因的扩增采用PCR方法从总RNA中经多次反应获得，再将其与VH基因连接形成完整的scFv基因。噬菌体库的构建采用载体pCANTAB5E、E.coli TG1和辅助性噬菌体M13KO7。将scFv基因插入pCANTAB5E载体酶切位点Sfi I和Not I之间，并将新载体转化E.coli TG1。辅助噬菌体M13KO7侵染转化后的TG1，，构建scFv噬菌体库。梯度稀释法测得噬菌体库库容为2.5×10~7，噬菌体滴度为10~(12)pfu。基因测序显示E.coli TG1中转化DNA与预期相符，抗体重链各区域组合正确，VH和VL之间有柔性多肽编码基因相连。采用微孔板筛选法以人TNF-α、禽流感NS1蛋白、人血清白蛋白HSA、牛血清白蛋白OVA和PRRSV病毒GP5蛋白对文库进行淘选，结果显示五种蛋白在淘选过程中均能有效富集。为获得高亲和力的抗TNF-α抗体，依据phage ELISA实验结果从随机挑选的87株重组噬菌体中筛选出4株阳性克隆进行重组蛋白表达和抗TNF-α活性测试。重组蛋白的表达采用pET-28a载体和大肠杆菌BL21（DE3）菌株。IPTG诱导后SDS-PAGE电泳显示scFv重组蛋白在大肠杆菌中大量表达，重组蛋白的表达量占菌体量的40%左右，经镍柱纯化的蛋白纯度在90%以上。MTT法检测TNF-α杀伤L292细胞的活性，显示在放线菌素为10μg/mL时，TNF-α的ED50为0.01ng/mL。重组蛋白B11和A24对TNF-α的IC_(50)分别为70μg/mL和100μg/mL。本研究构建、鉴定了人源scFv噬菌体抗体库，并从中筛选了2株具有抗TNF-α活性的人源scFv，为抗TNF-α抗体药物的研发奠定了基础。
[Abstract]:Antibody is an important part of humoral immune system, and also plays an important role in life science research, disease detection and medicine. Phage display is one of the important technical sources of human therapeutic antibodies. TNF- 伪 is a proinflammatory factor, which is closely related to the occurrence of many diseases such as rheumatoid arthritis. In this study, a human phage antibody library was constructed by phage antibody display technique, from which human scFv with TNF- 伪 cytotoxic activity was screened. According to the reported sequence of human antibody constant region and human antibody genes, 42 primers were designed, using 200 samples of total RNA isolated from human peripheral blood as template. These primers were used to amplify the VH (heavy chain) and VLV (light chain) variable region genes of antibodies. Three rounds of OE-PCR reaction were used to amplify and assemble the variable region of heavy chain of antibody. First, CDR1, CDR2, CDR3 and FR3 of VH were amplified, and then the CDR1 and CDR2 CDR3 and FR3 fragments of heavy chain were spliced by OE-PCR. The VH gene. VL gene of human antibody was amplified from total RNA by PCR method. The VH gene was ligated with VH gene to form a complete scFv gene. The phage library was constructed by vector pCANTAB5 E.coli TG1 and auxiliary bacteriophage M13KO7. The scFv gene was inserted into the pCANTAB5E vector between Sfi I and Not I, and the new vector was transformed into E.coli TG1. The scFv phage library was constructed by assisting the phage M13KO7 to infect the transformed TG1. The phage library capacity was 2.5 脳 10 ~ (7) and the phage titer was 10 ~ (10) ~ (10) ~ (12) pfuu by gradient dilution method. Gene sequencing showed that the transformed DNA in E.coli TG1 was in accordance with expectations, and the correct combination of regions of antibody heavy chain showed that there was a flexible polypeptide coding gene link between VH and VL. Human TNF- 伪, avian influenza NS1 protein, human serum albumin (HSA), bovine serum albumin (BSA) OVA and PRRSV virus GP5 protein were screened by microplate screening method. The results showed that the five proteins were effectively enriched during panning. In order to obtain anti-TNF- 伪 antibody with high affinity, four positive clones were screened from 87 randomly selected recombinant phages according to the results of phage ELISA assay to detect the expression of recombinant protein and the activity of anti-TNF- 伪. The expression of recombinant protein was induced by pET-28a vector and E. coli BL21DE3 strain. SDS-PAGE electrophoresis showed that the recombinant scFv protein was expressed in a large amount in E. coli, and the expression of recombinant protein was about 40% of the total number of bacteria. The activity of TNF- 伪 in killing L292 cells was detected by MTT assay. The results showed that the ED50 of TNF- 伪 was 0.01 ng / mL when actinomycin was 10 渭 g/mL. The ICs of recombinant protein B11 and A24 for TNF- 伪 were 70 渭 g/mL and 100 渭 g / mL, respectively. In this study, the human scFv phage antibody library was constructed, and two human scFvs with anti-TNF- 伪 activity were screened, which laid a foundation for the development of anti-TNF- 伪 antibody drugs.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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