猪传染性胃肠炎病毒猪源单链抗体的筛选与鉴定

Screening and identification of porcine-derived single-chain antibodies against transmissible gastroenteritis virus of swine

为了获得猪传染性胃肠炎病毒(TGEV)的单链抗体,首先利用噬菌体展示技术,构建猪源噬菌体单链抗体库,然后,通过微孔板筛选法对抗体库进行3轮富集淘筛,并用ELISA检测单链抗体与TGEV的亲和力,对亲和性较高的单链抗体进行基因测序与分析。结果表明,成功构建了猪源噬菌体单链抗体库,其库容为1.89×107 CFU/mL,通过Phage ELISA和Fab ELISA筛选得到4株与TGEV具有高亲和力的单链抗体,并通过桑格测序获得了与TGEV亲和性最佳的2株抗体的基因序列。利用在线生物信息学工具对所获得的抗体基因序列进行分析,结果表明,这2株单链抗体基因序列分别由猪源IgG的重链可变区基因(V_(H))和κ型轻链可变区基因(V_(L))构成,V_(H)和V_(L)均由3个CDR区和4个FR区构成,说明上述基因序列为完整的猪源TGEV抗体可变区。本研究的结果将为TGEV抗体药物的开发提供必要材料,为TGEV感染仔猪的临床治疗提供新的思路。

To obtain the single-chain fragment variable(ScFv)against transmissible gastroenteritis virus(TGEV),a porcine-origin phage single-chain antibody library was constructed by phage display technology.After three rounds of enrichment and panning on antibody library by microplate screening,the affinity between ScFv and TGEV was tested by ELISA.The high affinity ScFv was selected for further sequencing and analysis.We successfully constructed the porcine-derived phage single-chain antibody library with a volume of 1.89×107 CFU/mL.Through the screening from Phage ELISA and Fab ELISA,four ScFvs with high TGEV affinities were obtained.The gene sequences of the two antibodies with the highest affinity with TGEV were acquired by Sanger sequencing.The antibody sequences were analyzed by online bioinformatic tools,which showed the two ScFv gene sequences were composed of porcine-derived IgG heavy chain variable region gene(V_(H))andκ-type light chain variable region gene(V_(L)),both V_(H) and V_(L) consist of 3 CDR regions and 4 FR regions.These results indicate we obtained the complete variable region sequences for porcine-derived TGEV antibody.The research output of this study will lay the solid material basis for the development of TGEV antibody drugs and provide insight into the clinical treatment of TGEV-infected piglets.