丙型肝炎病毒NS5A反式激活蛋白7的重组表达及生物信息学分析

Prokaryotic expression and bioinformatics analysis of NS5A transactivating protein 7 of hepatitis C virus

目的构建丙型肝炎病毒NS5A反式激活蛋白7(NS5ATP7)的原核表达载体,诱导其在大肠埃希菌中的表达,并初步探讨其结构与功能。方法应用逆转录PCR(RT-PCR)技术,以HepG2细胞mRNA为模板,扩增获得NS5ATP7基因片段,连接到pGEM-T载体,酶切鉴定及测序正确后插入至原核表达载体pET-32a(+)中,转化大肠埃希菌BL21,IPTG诱导以获得NS5ATP7融合蛋白的表达,SDS-PAGE、Western blot免疫印迹分析和证实该融合蛋白表达的特异性,并应用生物信息学方法对该融合蛋白的结构和功能进行预测。结果利用RT-PCR扩增获得大小为891bp的NS5ATP7基因片段,插入pET-32a(+)表达载体,转化BL21宿主菌,经IPTG诱导,成功获得了大小为48kD的目的蛋白,Western blot进一步证实了该蛋白具有特异性免疫反应识别。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成功表达了NS5ATP7融合蛋白,结合生物信息学分析结果,为研究NS5ATP7蛋白的免疫原性和生物学特性奠定了基础。

Objective To construct prokaryotic expression vector of NS5A transactivating protein 7 of hepatitis C virus （NS5ATP7）, induce the expression of NS5ATP7 in E, coli, and to predict its structure and function by bioinformatics analysis. Methods NS5ATP7 gene was amplified by reverse transcription PCR （RT-PCR） using HepG2 cells mRNA as template, and was ligated into pGEM-T cloning vector. After verifying the sequence of the inserted fragraent by restriction enzyme digestion identification and sequencing, NS5ATP7 was cloned into inducible prokaryotic expression vector pET-32a（＋） and transfected into competent E. coli BL21. The protein expression was induced with IPTG and the product was analyzed by SDS-PAGE and Western blotting hybridization. The structure and function of NS5ATP7 were predicted using bioinformatics techniques. Results NS5ATP7 gene with about 891bp was amplified by RT- PCR, which was coincident with that we expected, and it was then inserted into expression vector pET-32a（＋）. Under the induction of IPTG, the recombinant NS5ATP7 was expressed successfully. SDS-PAGE and Western blotting assay showed that this recombinant protein possessed good immunogenieity. Bioinformatics method such as ExPASy, TMHMM and SignalP analysis indicated that NS5ATP 7 was full of helices, had neither transmembranous structure nor signal peptide. Conclusions The recombinant NS5ATP7 gene could be successfully expressed in prokaryotic expression system of E. coli, which might lay the foundation of studying the immunogenicity and biological charactersitics of the NS5ATP7. Bioinformatics analysis may provide a new method to analyze the structure and function of a new protein.