抗堆型艾美耳球虫子孢子单链抗体的构建及表达

Construction and Expression of ScFv against Eimeria acervulina Sporozoite

应用RT-PCR技术,从稳定分泌抗堆型艾美耳球虫子孢子抗原的单克隆抗体(McAb)的杂交瘤细胞Easp-3G3中扩增出抗体VH和VL基因,通过在VH和VL可变区基因之间引入连接肽(Gly4Ser)3,体外构建抗堆型艾美耳球虫的单链抗体基因,并将其克隆至表达载体pET28a(+)载体中进行表达,用Ni-NTA的金属亲和层析法进行纯化,通过SDS-PAGE和ELISA测定其纯度和生物学活性.电泳结果分析表明重组蛋白的相对分子量为32kDa,纯度为94%;ELISA反应结果证明该单链抗体保持了单克隆抗体的生物学特性,并能与堆型艾美耳球虫子孢子抗原发生特异性反应.

VH and VL, genes were amplified from hybridoma cell Easp-3G3,secreting monoclonal antibody Easp-3G3, by RT-PER, and connected by linker （Gly4Ser）3 to form scFv gene,which was cloned into expression vector pET28a（ ＋ ） and finally expressed in E. coli. The expressed product Easp-3G3-scFv was purified by metal affinity chromatography using Ni-NTA, its purity and biological activity were determined using SDS-PAGE and ELISA. SDS-PAGE analysis showed that the relative molecular weight of recombinant protein was 32 kDa with the purity of 94%. ELISA assay revealed that Easp-3G3-scFv retained the immunoactivity of parent mAb, being capable of binding specitically to Eimeria acervulina sporozoite antigen.