二硫键稳定的抗HIV-1 gp41单链抗体的构建及原核表达

目的构建二硫键稳定的抗HIV-1 gp41单链抗体(dsFv)基因原核表达载体,并进行表达和鉴定。方法采用PCR定点突变的方法,构建含二硫键稳定的抗HIV-1 gp41单链抗体突变基因质粒pUC57-d41,BamHⅠ和HindⅢ双酶切后,定向插入pET-28a(+),转化大肠杆菌BL21(DE3),IPTG诱导表达,用SDS-PAGE、Western blot鉴定表达产物。对目的蛋白进行纯化和复性,并进行抗原结合活性及相对稳定性检测。结果重组载体pET-d41经酶切鉴定,证实构建正确。表达产物相对分子质量约为28000,与理论预期值完全相符。目的蛋白最高表达量可占菌体总蛋白的45.48%。经Ni-NTA亲和层析法纯化并复性后,蛋白纯度达95%以上,抗HIV-1 gp41 dsFv具有抗原结合活性,稳定性优于scFv。结论已成功构建了二硫键稳定的抗HIV-1 gp41单链抗体(dsFv)基因原核表达载体,并获得表达,为进一步研究其生物学功能奠定了基础。

Construction of Porkaryotic Expression Vector for Disulfide-stabilized HIV-1 gp41 Single Chain Antibody

Objective To construct a prokaryotic expression vector for disulfide-stablilized HIV-1 gp41 single chain antibody (dsFv) and identify the expressed product.Methods Amplify disulfide-stabilized HIV-1 gp41 single chain antibody gene by PCR-based point mutagenesis strategy and clone into plasmid pUC57.Digest the constructed recombinant plasmid pUC57-d41 with BamH Ⅰ and Hind Ⅲ and insert the obtained gene fragment into plasmid pET-28a (+).Transform the constructed recombinant plasmid pET-d41 to E.coli BL21 (DE3) for expression under induction of IPTG.Identify the expressed product by SDS-PAGE and Western blot.The expressed protein was purified,refolded and determined for antigen binding activity and relative stability.Results Restriction analysis proved that recombinant plasmid pET-d41 was correctly constructed.The relative molecular mass of expressed product was about 28 000,which was completely identical to that expected.The expressed prouduct contained 45.48% of total somatic protein and reached a purity of more than 95% after purification by Ni-NTA affinity chromatography.The protein after refolding showed antigen binding activity,and its stability was higher than that of scFv.Conclusion The prokaryotic expression vector for disulfide-stabilized HIV-1 gp41 single chain antibody was successfully constructed,which laid a foundation of further study on biological function of dsFv.