丙型肝炎病毒NS5A反式激活蛋白2在大肠埃希菌中表达及生物信息学分析  

Prokaryotic expression and bioinformatics of NS5A transactivating protein 2 of hepatitis C virus

作　　者：李晓光[1,2] 洪源[1] 王琦[1] 张锦前[1] 成军[1] 

机构地区：[1]北京地坛医院传染病研究所,北京100011 [2]哈尔滨医科大学附属第二临床医院感染病科

出　　处：《中西医结合肝病杂志》2009年第4期217-220,共4页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

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。Objective： Constructing prokaryotic expression vector of NSSATP2 gene, and inducing the expression of recombinant protein in E. coli. Methods： NSSATP2 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, NSSATP2 was cloned into inducible prokaryotie expression vector pET-32a （ ＋ ） and transformed into E. coli BL21 （DF-3） . The expression of recombinant NSSATP2 was induced by IPTG, then sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blotting hybridization were done; Predicting structure and function of NSSATP2 with bioinformaties methods. Results： NSSATP2 gene was amplified by RT-PCR successfully. The recombinant expression vector was constructed and confirmed by restriction enzyme digestion. The expression of NSSATB2 was verified by SDS-PAGE and Western blot; Bioinformatics predicting results showed that NSSATP2 was full of helices, no transmembrane structure and no signal peptide. Conclusion： The recombinant NSSATP2 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 NSSATP2.

关 键 词：原核表达 病毒非结构蛋白质类 反式激活蛋白 肝炎病毒 丙型 

分 类 号：R373[医药卫生—病原生物学]