PCV2Cap蛋白噬菌体单链抗体库的构建及淘选

摘要

Balb/c mice were immunized with recombinant PCV2 Cap protein,and the total RNA was isolated from spleen cells.The variable region of immunoglobulins heavy chain(VH) and light chain (VL) genes were amplified by RT-PCR.The ScFv sequence was constructed by fusing VH gene with VL gene via overlap-PCR,and then the sequence was cloned into the phagemid vector pCANTAB-5E.APCV2 Cap protein ScFv library,which consists of approximately 3.58× 106 individual colonies,was constructed by transforming the recombinant plasmid into the E.coli TG1 cells.Furthermore,the PCV2 Cap protein phage display library was constructed by infecting the transformed cells with M13K07 helper phage.After four rounds of panning,three Cap protein-specific phage clones were isolated.%用重组的PCV2 Cap蛋白免疫Balb/c小鼠,从免疫小鼠脾细胞中提取总RNA,经反转录后,扩增抗体重链可变区(VH)和轻链可变区(VL)基因,利用Overlap PCR方法将VH基因和VL基因组装成单链抗体(ScFv)基因,将ScFv基因克隆到噬菌粒载体pCANTAB-5E中,并将重组载体转化至感受态大肠杆菌TG1中,获得PCV2 Cap蛋白的单链抗体文库,经测定抗体库库容为3.58×106.通过辅助噬菌体M13K07拯救,构建鼠源PCV2 Cap蛋白的噬菌体单链抗体库.4轮生物淘选后,经ELISA方法检测,最终获得3株能与Cap蛋白特异性结合的噬菌体克隆.