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目的构建重组人APOBEC3H hapⅡ(A3H hapⅡ)杆状病毒表达载体,在昆虫细胞表达系统中表达,表达产物经Ni-NTA亲和层析柱纯化。方法采用PCR方法扩增A3H hapⅡ基因并加入His标签后,克隆至杆状病毒转移载体p Fast Bac1中,转化E.coli DH10Bac感受态细胞进行重组,构建质粒Bacmid-A3H hapⅡ-His。将质粒转染昆虫细胞sf9获得P1病毒,扩增后获得P3病毒,感染sf9细胞表达目的蛋白,并进行Western blot鉴定。经Ni-NAT树脂亲和层析柱对表达的蛋白进行纯化,并通过免疫共沉淀试验检测A3H hapⅡ-His与其相互作用蛋白HIV-1 Vif之间的特异性结合。结果成功构建了质粒Bacmid-A3H hapⅡ-His;P3病毒感染sf9细胞后成功表达出相对分子质量约20 000的目的蛋白,且能够与HIV-1 Vif特异性结合。结论成功建立A3H hapⅡ昆虫表达体系,为进一步研究A3H hapⅡ与HIV-1 Vif之间的相互作用机制,并针对其相互作用位点设计抗HIV药物奠定了基础。
Objective To construct the recombinant baculovirus expression vector APOBEC3H hap Ⅱ (A3H hap Ⅱ) and express it in insect cell expression system. The expressed product was purified by Ni-NTA affinity chromatography. Methods The A3H hapⅡ gene was amplified by PCR and inserted into the His-tag. The recombinant plasmid was cloned into the baculovirus transfer vector p Fast Bac1 and transformed into E. coli DH10Bac competent cells for recombination to construct the plasmid Bacmid-A3H hapⅡ-His. The plasmid was transfected into insect cell sf9 to obtain P1 virus. After amplification, P3 virus was obtained and infected into sf9 cells to express the target protein. The recombinant protein was identified by Western blot. The expressed protein was purified by Ni-NAT resin affinity chromatography and the specific binding between A3H hapⅡ-His and its interacting protein HIV-1 Vif was detected by co-immunoprecipitation. Results Plasmid Bacmid-A3H hapⅡ-His was constructed successfully. The P3 virus successfully infected sf9 cells and successfully expressed the target protein with molecular weight of about 20,000, and could specifically bind HIV-1 Vif. Conclusion The A3H hap Ⅱ insect expression system was successfully established, which laid the foundation for the further study of the interaction mechanism between A3H hapⅡ and HIV-1 Vif and the design of anti-HIV drugs based on their interaction sites.