堆型艾美耳球虫单链抗体-PE40重组免疫毒素的构建

Construction of ScFv-PE40 recombinant immunotoxin against Eimeria acervulina

用HindⅢ对已构建的质粒pT-PE40和原核表达载体pET22-ScFv进行单酶切,回收纯化1 101 bp的PE40基因片段并将其亚克隆至pET22-ScFv载体中,构建重组免疫毒素表达质粒pET22-ScFv-PE40。将该质粒转化入感受态大肠杆菌J M109中增殖,提取质粒后用SalⅠ和NotⅠ进行双酶切鉴定,得到了1 114 bp和6 181 bp目的基因片段。测序鉴定PE40的插入方向,选取以PE40基因5′端与ScFv基因3′端连接的重组质粒,构建了抗堆型艾美耳球虫重组免疫毒素质粒pET22-ScFv-PE40。抗堆型艾美耳球虫重组质粒pET22-ScFv-PE40经1 mmol/LIPTG诱导,在大肠杆菌中表达约68 000的目的蛋白。使用抗PE多克隆抗体对目的蛋白进行蛋白印迹分析,结果表达产物与抗PE多克隆抗体发生抗原抗体反应,证明融合基因得到表达。

In order to construct a prokaryotic expression vector containing the ScFv gene against Eimeria acervulina and the gene PE40,the simple digestion(HindⅢ) was performed to deal with pT-PE40 and pET22-ScFv respectively,and then PE40 gene was subcloned into pET22-ScFv.The recombinant plasmid was identified by double digestion(Sal Ⅰ and Not Ⅰ)and sequenced.Two objective gene fragments with the length of 1 114 bp and 6 181 bp were obtained.The sequence analysis has proved the correctness of the recombinant immunot...