具有野生起点的丙型肝炎病毒RNA依赖的RNA聚合酶表达质粒的构建

CONSTRUCTION OF THE PLASMID FOR EXPRESSION OF AUTHENTIC HEPATITIS C VIRUS (HCV) RNA-DEPENDENT POLYMERASE (RDRP)

目的构建具有野生起点的丙型肝炎病毒ＲＮＡ依赖的ＲＮＡ聚合酶表达载体。方法根据疏水性分析选定待表达的丙型肝炎病毒非结构基因５ｂ区大部分基因。以聚合酶链反应扩增泛素基因。逆转录－聚合酶链反应扩增丙型肝炎病毒ＲＮＡ依赖的ＲＮＡ聚合酶基因。限制性酶切长度多态性分析重组克隆，并以ＤＮＡ序列分析方法最终确认。结果分别获得长２４０个碱基对的泛素基因与长１７６１个碱基对的丙型肝炎病毒ＲＮＡ依赖的ＲＮＡ聚合酶基因片段，并分两步克隆于表达质粒ｐＥＴ２６ｂ，限制性酶切长度多态性分析与序列分析显示克隆成功。结论成功构建了新型表达载体ｐＥＴ２６－Ｕｂ并用于构建具有野生起点的丙型肝炎病毒ＲＮＡ依赖的ＲＮＡ聚合酶表达质粒。

Objective To construct the expression plasmid of authentic hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp). Methods Hydrophobicity profile was used to determine HCV RdRp gene to be expressed. Plymerase chain reaction (PCR) and reverse-transcription PCR (RT-PCR) were used to amplify ubiquitin gene and HCV RdRp gene respectively. Restriction fragment length polymorphism (RFLP) was employed to screen for the presence of insert. The final construct was confirmed by sequencing. Results The 240 base-pair (bp) ubiquitin gene and the 1761 bp HCV RdRp gene were obtained. The genes were then cloned into plasmid pET26b in two steps. RFLP and seqencing showed that pET26-Ub consisted of pET26b and ubiquitin gene, and plasmid pET26-Ub-HCV RdRp contained HCV RdRp gene downstream of ubiquitin gene. Conclusion New expression vector pET26-Ub was constructed and could be used to express recombinant authentic HCV RdRp gene.