丙型肝炎病毒1b DY株ns5a基因的克隆及其在大肠杆菌中的表达

Cloning and expression of ns5a gene of hepatitis C virus 1b strain DY in Escherichia coli

目的克隆和表达丙型肝炎病毒(HCV)1b型地方株DY株ns5a基因。方法运用原核细胞基因工程技术。设计目的基因的特异引物,采用巢式PCR法,从含HCV 1b DY株全长cDNA的质粒HCV17中扩增出约480 bp的目的片段,将其插入克隆载体pMD18-Tvector中,再亚克隆到原核表达载体pET-28a中;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蛋白的表达水平。结果成功构建了含有HCV1b DY株ns5a基因的重组体,并得以表达。结论成功构建和表达了HCV1b 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了条件。

To clone and express the ns5a gene of hepatitis C virus （HCV） 1 b strain DY. Methods By using the prokaryotic cell gene engineering, HCV ns5a gene was amplified with nested PCR from the plasmid HCV17 of HCV lb strain DY full-length gene and inserted into the cloning 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 pet-28a-ns5a was constructed and expressed successfully. Conclusion HCV ns5a gene was cloned and expressed. This might be helpful for further studies on the nature and biological properties of the ns5a gene.