核糖体展示口蹄疫单链抗体库的构建

Construction of Ribosome Display Single Chain Antibody Library of Foot and Mouth Disease

目的:构建库容量大、多样性好的核糖体展示口蹄疫单链抗体(scFv)库。方法:分离口蹄疫病毒免疫的兔脾细胞,提取总RNA,用RT-PCR扩增兔抗体的重链可变区(VH)基因和轻链可变区(VL)基因,同时扩增作为间隔区的兔抗体Ck基因;采用重叠延伸PCR(简称SOE-PCR)技术连接VH-VL基因,同时引入T7启动子和核糖体结合位点序列,体外构建核糖体展示scFv库模板,连接pMD18-T载体转化E.coli DH5α大肠杆菌,挑取阳性克隆测序以鉴定scFv组装。结果:成功构建了库容量达8.21×1013的兔源口蹄疫核糖体展示scFv库。结论:构建的大容量兔源性口蹄疫核糖体展示抗体库可以成为进一步筛选特异性口蹄疫单链抗体的实验平台,为开发诊断性口蹄疫单链抗体奠定了很好的实验基础。

Objective： To construct a rabbit source high-capacity and good-diversity ribosome display single chain antibody （scFv） library of FMDV. Methods ： The isolated spleen cells from rabbit immuned by the FMDV Virus,and extracted RNA for amplifying rabbit VH gene, VL gene and Ck gene as the spacer by RT-PCR, and then connect VH-VL gene by overlapping extension PCR （referred to as SOE-PCR）. After that, the elements for ribosome display such as T7 promoter and ribosome binding site were introduced after SOE products were amplified. Finally, the ribosome display scFv library template was constructed in vitro. Moreover, the ribosome display templates were connected to pMD18-T vector into E. coli DH5α, and the positive clones were picked sequencing to identify the ScFv assembly. Results： We successfully constructed a rabbit source ribosome display scFv library whose capacity is 8.21 × 10^13 of FMDV. Conclusion： The rabbit-derived high-capacity ribosome display antibody library can be a good platform for screening specific foot and mouth disease, and lay a good experimental basis for the measuring test of FMDV.