Prokaryotic expression and bioinformatics analysis of NS5A transactivating protein 7 of hepatitis C virus / 解放军医学杂志

ABSTRACT

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 fragment 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 immunogenicity. Bioinformatics method such as ExPASy, TMHMM and SignalP analysis indicated that NS5ATP7 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.