易错PCR提高人源抗狂犬病毒单链抗体的亲和力  

Affinity maturation of a single-chain antibody against rabies virus

作　　者：孙丽娜[1] 李川[1] 刘洋[1] 李德新[1] 梁米芳[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《中华实验和临床病毒学杂志》2016年第3期328-332,共5页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的 通过易错PCR技术提高1株人源抗狂犬病毒单链抗体RV08的亲和力。方法采用Agilent公司GeneMorphIIRandom MutagenesisKit对RV08轻、重链可变区进行易错PCR扩增，随机引入突变，通过单链抗体（Single—chainantibodyfragmentScFv）噬菌体呈现技术构建随机突变抗体文库，经狂犬病毒颗粒aG株固相富集筛选，通过ELISA和IFA鉴定阳性抗体克隆并进行序列测定。利用IgG表达载体VH／VK双质粒系统瞬时转染293T细胞实现IgG抗体的分泌型表达，通过非竞争ELISA测定突变抗体亲和力。结果RV08随机突变抗体库的库容为6×10^6efu／ml，目的基因插人率为100％。重链序列突变频率约为0．95％，轻链序列突变频率约为1．2％，最后获得5株高亲和力的抗体突变株，其中3株抗体HIA6、HL2和HL37亲和力分别为原始克隆RV08的1．79倍、1．47倍和1．23倍。结论易错PCR技术与噬菌体抗体库技术相结合使得抗体亲和力获得明显提高，为抗体的体外亲和力成熟提供了参考方法。Objective To obtain a human single-chain antibody against rabies virus （RV） with high affinity by error-prone PCR technology. Methods A combinatorial scFv antibody mutant library was constructed by error-prone PCR amplifying VH and VL of scFv RV08. After package by hyperphage, the scFv phage mutant library was panned by ELISA and IFA with purified RV AG. The selected mutant antibodies were converted to full human IgG antibodies with the VH and VK Express cassettes. The affinity of mutant antibodies was measured by non-competitive enzyme immunoassay. Results The mutant antibody library with capacity of 6 × 10^6 cfu/ml contained target gene insertion rate of 100%. Heavy chain and light chain were sequenced with mutation frequency of about 0.95% and 1.2% respectively. Finally 5 mutant antibodies with high affinity were obtained, of which HL46, HL2 and HL37 had higher affinity with 1.79 times, 1.47 times and 1.23 times of RV08 respectively. Conclusions In this study, error-prone PCR technology and phage display technology combination enables to obtain significantly improved affinity of antibody.

关 键 词：狂犬病毒 单链抗体 亲和力 

分 类 号：R440[医药卫生—诊断学]