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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。
Objective To construct prokaryotic expression vector of disulfide stabilized anti-HIV-1 gp41 single chain antibody (dsFv) gene and express and identify it. Methods The site-directed mutagenesis was used to construct the recombinant plasmid pUC57-d41 containing the disulfide bond-stable anti-HIV-1 gp41 gene. The recombinant plasmid was digested with BamHⅠand HindⅢ and inserted into pET-28a (+ BL21 (DE3), induced by IPTG, and expressed by SDS-PAGE and Western blot. The target protein was purified and refolded, and the antigen binding activity and relative stability were tested. Results The recombinant plasmid pET-d41 was confirmed by restriction enzyme digestion. The relative molecular mass of the expressed product was about 28000, which was completely consistent with the expected value of the theory. The highest expression of the target protein may account for 45.48% of the total bacterial protein. After purified by Ni-NTA affinity chromatography and renaturation, the purity of the protein reached more than 95%. The anti-HIV-1 gp41 dsFv had antigen-binding activity and the stability was better than scFv. Conclusion The disulfide-stabilized prokaryotic expression vector of the anti-HIV-1 gp41 single chain antibody (dsFv) gene was successfully constructed and expressed, which laid the foundation for further study of its biological function.