人APOBEC3H hap Ⅱ在昆虫表达系统中的表达及其纯化  

Expression of human apolipoprotein B m RNA-editing catalytic polypeptide-like protein 3H hapⅡ in insect expression system and purification of expressed product

作　　者：李婷[1] 王虹[1] 张晓红[1] 董燕[1] 王涛 于晓方[1] 韩雪[1] 

机构地区：[1]天津大学生命科学学院,天津300072

出　　处：《中国生物制品学杂志》2016年第3期262-265,269,共5页Chinese Journal of Biologicals

摘　　要：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 recombinant baculovirus vector expressing human apolipoprotein B m RNA-editing catalytic polypeptide-like protein 3H（APOBEC3H） hap II（A3H hap Ⅱ）, express in insect cell expression system, and purify the expressed product by Ni-NTA affinity chromatography. Methods A3 H hapⅡ gene was amplified by PCR and tagged with His, then inserted into baculovirus transfer vector p Fast Bac1. The constructed recombinant plasmid was transformed to competent E. coli DH10 Bac cells, and the obtained recombinant plasmid Bacmid-A3 H hap Ⅱ-His was transfect to sf9 cells. The generated recombinant baculovirus was amplified, of which the passage 3（P3） was infected to sf9 cells. The expressed target protein was identified by Western blot, purified by Ni-NTA affinity chromatography, and determined for specific binding to HIV-1 Vif by co-immunoprecipitation assay. Results Recombinant baculovirus vector Bacmid-A3 H hapⅡ-His was constructed successfully. The target protein with a relative molecular mass of about 20 000 was expressed in sf9 cells infected with P3 of recombinant baculovirus, which showed specific binding to HIV-1 Vif.Conclusion The insect cell expression system for A3 H hap II was constructed successfully, which laid a foundation of further study on the mechanism of interaction between A3 H hap Ⅱ and HIV-1 Vif and design of anit-HIV drugs targeting the interaction site.

关 键 词：APOBEC3H hapⅡ Vif基因 昆虫表达系统 基因表达 纯化 

分 类 号：R978.7[医药卫生—药品] Q786[医药卫生—药学]