APOBEC3G蛋白的原核表达及纯化

目的：原核表达重组APOBEC3G蛋白，为其功能及免疫原性研究奠定基础。方法：提取H9细胞全细胞基因组RNA，通过RT-PCR获得目的基因，经纯化、酶切后克隆到原核表达载体pET32a中，转化大肠杆菌BL21（DE3）菌株获得表达工程菌株，并对表达条件和纯化条件进行优化；利用Western Blot分析鉴定目的蛋白。结果：构建了APOBEC3G蛋白的原核表达载体Apo-His-pET32a，并在大肠杆菌中获得高表达，目的蛋白以可溶性蛋白形式存在；经Ni-NTA亲和层析柱一步纯化，获得了高纯度的重组APOBEC3G蛋白，蛋白浓度可达1．2mg／mL；Westem Blot显示获得了目的蛋白。结论：在原核表达系统中表达、纯化了可溶性APOBEC3G蛋白，为进一步对其进行免疫原性和功能研究奠定了基础。

Prokaryotic Expression and Purification of the APOBEC3G Protein

Objective： To clone, prokaryotic express and purify APOBEC3G protein in vitro. Methods： The gene fragment coding APOBEC3G protein was amplified by RT-PCR and inserted into plasmid pET32a to construct a recombinant prokaryotic expression vector Apo-His-pET32a. The vector was transformed into E.coli BL21（DE3）. The expression and purification condition was optimized. The purified recombinant protein was identified with Western Blot. Results： The recombinant APOBEC3G protein expressing vector was constructed and transformed into E.coli BL21 （DE3）, and the soluble APOBEC3G protein was expressed under induction with 0.1 mmoL/L IPTG for 3 hours. High-purity naive APOBEC3G protein was purified with Ni-NTA affinity chromatograph, and the concentration of proteins was 1.2 mg/mL. Western Blot showed that the recombinant protein could be identified by specific antibody. Conclusion： The recombinant APOBEC3G protein was successfully expressed and purified in E.coli. This lays foundation for the study of the structure and functions of APOBEC3G protein in vitro.