人源抗狂犬病毒单链抗体基因原核表达质粒的构建及高效表达

The construction of prokaryotic expression plasmid and high-level expression of the human single chain Fv antibody fragment gene to rabies virus

在原核系统中高效表达抗狂犬病毒单链抗体scFv41,以便进一步研究其生物学功能,预测临床应用前景。以重组质粒pCANTABscFv41为模板,PCR扩增带NcoI和NotI位点的scFv41基因,克隆入原核表达载体pET-22b穴+雪,酶切鉴定重组表达质粒,转化大肠杆菌BL21穴DE3雪,IPTG诱导表达。竞争ELISA检测表达蛋白的特异结合活性。酶切鉴定证实scFv41基因已插入原核表达载体pET-22b穴+雪,重组表达质粒pET-scFv41在大肠杆菌BL21穴DE3雪中获得了高效表达,表达量约占菌体蛋白总量的30%。竞争ELISA检测结果表明scFv41表达蛋白可特异抑制抗狂犬病毒IGY与狂犬病毒的特异性结合。该实验为进一步研究scFv41的生物学特性和免疫保护作用,及基因工程抗体的制备奠定了基础。

To construct the prokaryotic expression plasmid of human scFv41 gene to rabies virus and prepare the scFv41 protein for identifying biological function. Using recombinant plasmid pCANTAB-scFv41 as template, the scFv41 gene with NcoI and NotI sites was amplified by PCR and cloned into the prokaryotic expression vector pET-22b(+). E.coli BL21(DE3) was transformed with the recombinant expression plasmid pET-scFv41 and protein expression was induced in Luria-Bertani medium by addition of IPTG. The recombinant expression plasmid pET-scFv41 successfully expressed scFv41 protein. The yield of the scFv41 fragment accounted for about 30% of the total bacterial protein and the protein showed specific immunological reaction with rabies virus in competition ELISA. The construction of the prokaryotic expression plasmid of scFv41 gene and preparation of scFv41 fragment established a solid basis for further studying the biological function 0f the human scFv41 to rabies virus.