论文部分内容阅读
目的:从大容量噬菌体抗体库中筛选人源性抗呼吸道合胞病毒F蛋白的单链抗体。方法:以RSV F蛋白为靶抗原,通过“吸附-洗涤-洗脱-扩增”过程从天然人源性噬菌体抗体库中筛选特异性抗F蛋白单链抗体。5轮筛选后,单克隆经ELISA检测,阳性克隆进行核酸序列分析,并将阳性克隆噬菌体感染E.coliHB2151,经IPTG诱导,制备抗RSV F蛋白的可溶性单链抗体,并进行Western blot及Dot blot分析。结果:经过筛选,获得了18株能与F蛋白特异性结合的阳性克隆,取OD值最高的克隆E4经测序并检索Kabat数据库分析,显示其基因与人免疫球蛋白可变区基因具有高度同源性,Western blot及Dot blot分析表明为单链抗体。结论:利用天然人源性噬菌体抗体库技术制备出高特异性的人源性抗RSV F蛋白单链抗体。
OBJECTIVE: To screen single chain antibodies against human respiratory syncytial virus F protein from large-capacity phage antibody library. Methods: The specific anti-F protein single chain antibody was screened from the natural human phage antibody library by the “adsorption-wash-elution-amplification” process using RSV F protein as target antigen. After 5 rounds of screening, the monoclonal antibodies were detected by ELISA, and the positive clones were analyzed by nucleic acid sequence. Positive clones were infected with E.coli HB2151 and induced by IPTG to prepare soluble single chain antibodies against RSV F protein. Western blot and Dot blot analysis. RESULTS: After screening, 18 positive clones that could bind to F protein were obtained. Clone E4 with the highest OD value was sequenced and analyzed by Kabat database. The result showed that the gene was highly homologous with human immunoglobulin variable region gene Source, Western blot and Dot blot analysis showed single chain antibody. Conclusion: The high-specificity human anti-RSV F protein single-chain antibody was prepared by using natural human phage antibody library technology.