The screening and expression of Hepatitis B virus-S1 protein trans activating gene 5

Objective To clone and express hepatitis B virus pre-S1 protein activating gene PS1TP5 in Escherichia coli. Methods The mRNA from HepG2 cells transfected with pcDNA 3.1(-)-preS1 and pcDNA 3.1(-) empty vector was isolated respectively.SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. The novel coding gene transactivated by pre-S1 protein of hepatitis B virus was screened by bioinformatics method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify PS1TP5 from HepG2 cDNA template. The novel DNA fragment was ligated into pGEM-T cloning vector by TA cloning. After digestion with restrictive enzyme and sequencing, the correct DNA fragment was inserted into inducible proeukaryotic expression vector pET-32a(+). The competent BL21 (DE3) E.coli was transformed, then cultured and induced with IPTG. The expressed PS1TP5 was analyzed and confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization.Results The DNA fragment of PS1TP5 was successfully amplified by RT-PCR, and its expression vector was constructed. After transformation with pET-32a(+)-PS1TP5 and induction with IPTG, recombinant PS1TP5 was expressed and confirmed by SDS-PAGE and Western blotting. Conclusions Human novel gene PS1TP5 was cloned by the application of SSH, RT-PCR and bioinformatics technique. And its recombinant prokaryotic expression vector pET-32a(+)-PS1TP5 was constructed. The recombinant PS1TP5 gene can be expressed in prokaryotic expression system of E.coli. These results will certainly bring some new clues to study immunogenicity of PS1TP5 and pathogenesis of the chronic hepatitis B.