丙型肝炎病毒NS5B全长基因及截短形式基因的克隆、表达及鉴定

目的构建丙型肝炎病毒(HCV)NS5B-FL、NS5B-C21和NS5B-C51重组原核表达载体,并进行目的蛋白的表达及鉴定。方法利用PCR技术扩增3种长度的NS5B基因,经BamHI和XhoI双酶切后连接到经同样酶切的原核表达载体pET-28a(+)上,转化大肠杆菌BL21(DE3),IPTG诱导表达重组蛋白,并进行纯化及鉴定。结果所构建的3种重组质粒pET-28a(+)-NS5B-FL、pET-28a(+)-NS5B-C21和pET-28a(+)-NS5B-C51,转化大肠杆菌BL21(DE3)后,经PCR及酶切鉴定,均能扩增或酶切出相应大小的目的基因片段,表达的6His-NS5B-FL融合蛋白主要以包涵体形式存在,而表达的6His-NS5B-C21和6His-NS5B-C51融合蛋白的可溶性明显增加。纯化的6His-NS5B-C21蛋白未发生降解,6His-NS5B-FL和6His-NS5B-C51蛋白发生了部分降解。纯化后的3种蛋白均能与丙肝患者血清发生反应。结论已成功构建了3种NS5B基因的重组表达载体,并获得了目的蛋白表达,为进一步抗HCV药物的筛选奠定基础。

Cloning and Expression of Full-length and Truncated Hepatitis C Virus NS5B Gene and Identification of Expressed Product

Objective To construct the prokaryotic expression vectors for hepatitis C virus (HCV) NS5B-FL, NS5B-C21 and NS5B-C51 genes and identify the expressed product. Methods Amplify the NS5B-FL,NS5B-C21 and NS5B-C51 genes of HCV by PCR, identify by digestion with BamH I and Xho I and insert into prokaryotic expression vector pET-28a (+) respectively. Transform the constructed recombinant plasmids pET-28a (+)-NS5B-FL, pET-28a (+)-NS5B-C21 and pET-28a (+)-NS5B-C51 to E.coli BL21(DE3) for expression under induction of IPTG.Purify and identify the expressed protein.Results The recombinant E.coli BL21(DE3) transformed with pET-28a (+)-NS5B-FL, pET-28a (+)-NS5B-C21 and pET-28a (+)-NS5B-C51 was identified by PCR and restriction analysis, and the results showed that the target genes at expected lengths were amplified. The expressed 6His-NS5B-FL fusion protein mainly existed in a form of inclusion body.However, the solubility of expressed 6His-NS5B-C21 and 6His-NS5B-C51 increased significantly. The purified 6His-NS5B-C21 showed no degradation, while purified 6His-NS5B-FL and 6His-NS5B-C51 were degraded partially. All the 3 kinds of purified fusion proteins showed specific reactions with the sera of patients with hepatitis C.Conclusion The prokaryotic expression vectors for hepatitis C virus (HCV) NS5B-FL,NS5B-C21 and NS5B-C51 genes were successfully constructed, and 3 kinds of fusion proteins were expressed, which laid a foundation of screening of drugs against HCV.