丙型肝炎病毒1bDY株ns5a基因的克隆及其所表达融合蛋白的免疫原性分析

目的:克隆和表达丙型肝炎病毒(HCV)1b型地方株DY株ns5a基因,为进一步研究和应用该基因及其表达产物奠定基础。方法:采用基因重组技术完成实验。设计特异的引物,采用巢式PCR法,从含HCV 1b DY株全长cDNA的质粒HCV17中扩增出目的片段,约480bp;将其插入克隆载体pMD18-Tvector中,再与原核表达载体pET-28a重组;经酶切、PCR及测序鉴定后转化入BL21菌株,在IPTG诱导下进行融合蛋白的表达。采用SDS-PAGE电泳及Western-blot检测NS5A蛋白表达水平。结果:含有HCV 1b DY株ns5a基因的重组体构建成功,并得以表达蛋白,并且NS5A蛋白具有与HCV患者阳性血清中的多克隆的结合活性,具有很好的免疫原性。结论:运用原核细胞基因工程技术成功构建和表达了HCV 1b DY株ns5a基因,为进一步研究HCVns5a的基因型及探讨该基因编码的NS5A蛋白的性质和生物学活性创造条件。

Cloning and expressing of ns5a gene of hepatitis C virus 1b DY strain and the study of its immunogenicity

Objective:To clone and express the ns5a gene of HCV 1b DY strain and to make a base to study and utilize the gene and the expressed product. Methods: HCV ns5a gene was amplified with nested PCR from the plasmid HCV17 of HCV 1b DY strain and inserted into the plasmid PMD18-T vector. The cloned HCV ns5a gene was separated and subcloned into expression vector pET-28a and induced by IPTG in E.coli BL21.The expressed product was identified by SDS-PAGE and Western-blot methods. Results:Recombinant expression plasmid for HCV 1b ns5a and pET-28a-ns5a has been successfully constructed and expressed successfully. The recombinant protein well maintained the ability of native NS5A to combine with the serum from patient infected by HCV. Conclusion:HCV ns5a gene was cloned and expressed by using the prokaryotic cell gene engineering. This might be helpful for further studies of characterizations of the ns5a gene.