【摘要】 目的:构建丙型肝炎病毒NS5A反式激活蛋白2(NS5ATP2)原核表达载体并诱导其在大肠埃希菌中表达。方法:应用逆转录聚合酶链反应(RT-PCR)技术,以从HepG2细胞提取的mRNA为模板,扩增NS5ATP2目的基因片段,T-A克隆连接到pGEM-T载体,测序正确后将目的片段插入原核表达载体pET-32a(+)中,转化大肠埃希菌BL21(DE3),IPTG诱导NS5ATP2融合蛋白的表达,并通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western blot免疫印迹分析和证实融合蛋白的表达;以生物信息学方法对蛋白结构和功能进行预测。结果:利用RT-PCR扩增获得NS5ATP2基因片段,IPTG诱导BL21宿主菌成功表达了NS5ATP2,经SDS-PAGE和Western blot分析得到证实;生物信息学预测结果显示此蛋白富含螺旋结构,为非跨膜蛋白,无信号肽。结论:利用大肠埃希菌BL21(DE3)能够成功表达NS5ATP2蛋白,为今后研究NS5ATP2蛋白的免疫原性和生物学特性奠定了的基础。更多还原

【Abstract】 Objective:Constructing prokaryotic expression vector of NS5ATP2 gene,and inducing the expression of recombinant protein in E.coli.Methods:NS5ATP2 gene which was amplified with reverse transcription polymerase chain reaction(RT-PCR)from HepG2 cells mRNA,was ligated into pGEM-T cloning vector.After verified by sequencing,NS5ATP2 was cloned into inducible prokaryotic expression vector pET-32a(+)and transformed into E.coli BL21(DE3).The expression of recombinant NS5ATP2 was induced by IPTG,then sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting hybridization were done;Predicting structure and function of NS5ATP2 with bioinformatics methods.Results:NS5ATP2 gene was amplified by RT-PCR successfully.The recombinant expression vector was constructed and confirmed by restriction enzyme digestion.The expression of NS5ATB2 was verified by SDS-PAGE and Western blot;Bioinformatics predicting results showed that NS5ATP2 was full of helices,no transmembrane structure and no signal peptide.Conclusion:The recombinant NS5ATP2 gene can be expressed in prokaryotic expression system of E.coli successfully,which will set a foundation for studying the immunogenicity and bioinformatics of the NS5ATP2.更多还原