丙型肝炎病毒非结构蛋白3的体外表达与纯化

In vitro expression and purification of hepatitis C virus non-structural protein 3

目的构建丙型肝炎病毒非结构蛋白3(HCV NS3)表达质粒并在大肠杆菌中表达其全长蛋白。方法克隆HCV NS3的核酸序列3420-5312位点,插入pQE-11质粒的BamH1限制性酶切位点,将此重组质粒转入大肠杆菌表达,用Westernblot方法筛选阳性克隆,将含有额外拷贝的argU、ileY和leuW的tRNA基因的pACYA质粒引入HCV NS3表达系统以提高HCV NS3蛋白的表达量。收集培养扩增的大肠杆菌菌体,用卵白溶菌酶裂解,收集含有HCV NS3重组蛋白的不溶性包涵体,将包涵体充分洗涤后,溶于含10mmol/L二硫苏糖醇的缓冲液中。释放的可溶性蛋白用SDS-PAGE电泳分离、洗脱,用离心过滤方法浓缩,乙醇沉淀,重复洗涤去除内毒素。结果用此方法克隆的HCVNS3全长基因片段表达的重组蛋白分子量为69kD。含有额外拷贝的argU、ileY和leuW的tRNA基因的pACYA质粒引入HCV NS3表达系统后,HCV NS3蛋白的产出率可提高10倍以上。此方法生产的NS3纯化蛋白质产量,每升培养物为40mg。内毒素含量低于20EU/mg NS3蛋白。结论 HCV NS3与HCV的免疫逃逸及感染的慢性化有密切关系,是HCV的抗病毒治疗和疫苗研究的重要靶点,全长HCV NS3蛋白的体外合成为HCV研究提供了一个有用的工具。

Objective This study was designed to construct the Hepatitis C virus non-structural protein 3(HCV NS3)expression vector and introduce this vector into Escherichia Coli(E.coli)to produce the full-length HCV NS3 protein.Methods A full-length HCV NS3 gene encoding amino acids 1027-1657(nucleotides 3420-5312)was inserted into the BamHI restriction enzyme site of plasmid pQE-11 and expressed in E.coli cells.Positive expressing clones were selected by Western blot.A pACYA plasmid with tRNA genes containing extra copies of argU,ileY and leuW was introduced into the HCV NS3 expression clone by transformation to increase the expression level of HCV NS3 recombinant protein.The expanded E.coli cells were collected and lysed by hen egg-white lysozyme.The insoluble inclusion bodies containing HCV NS3 recombinant protein were collected by centrifugation and dissolved in Tris-HCl buffer containing 10 mmol/L dithiothreitol to release the recombinant protein.The soluble recombinant protein released was purified by SDS-PAGE and concentrated by centrifugal filtration and ethanol precipitation.The contaminated endotoxin was removed by repeated washing with 0.9% NaCl,10 mmol/L sodium deoxycholic acid.Results The molecule weight of this full-length recombinant HCV NS3 protein was 69 kD.After introduction of pACYC-based plasmid containing extra copies of argU,ileY and leuW tRNA genes,the production of HCV NS3 recombinant protein by HCV NS3 expression clone was increased 10 times more than that by its mother clone.The final production of purified HCV NS3 recombinant protein was 40 mg per liter of culture with the endotoxin level was less than 20 EU/mg NS3 protein.Conclusions HCV NS3,in cooperation with HCV NS4A,plays an important role in the immune evasion and chronic infection of HCV,and therefore,is one of the most important targets in the studies for anti-viral therapy and vaccine development.Thus,the production of full-length HCV NS3 recombinant protein provided a useful tool in HCV research.