Cloning and prokaryotic expression of binding protein 4 spliced variant HBeBP4A of hepatitis B virus e antigen

Objective:To clone a new gene,binding protein 4 spliced variant HBeBP4A of hepatitis B virus e antigen,and construct its recombinant prokaryotic expression vector,and induce the expression of recombinant protein in Escherichia coli.Methods:Reverse transcription-polymerase chain reaction(RT-PCR) and bioinformatic technique were used to amplify HBeBP4A from HepG2 cDNA template.The novel DNA fragment was ligated into pGEM-T easy 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,and then cultured and induced with IPTG.The expressed HBeBP4A was analyzed and confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting hybridization.Results:The spliced DNA fragment of HBeBP4A was successfully amplified by RT-PCR,and its expression vector was constructed.After transformation with pET-32a(+)-HBeBP4A and induction with IPTG,recombinant HBeBP4A was expressed and confirmed by SDS-PAGE and Western blotting.Conclusion:A novel gene-binding protein 4 pliced variant HBeBP4A of hepatitis B virus e antigen has been recognized,and its recombinant prokaryotic expression vector pET-32a(+)-HBeBP4A has been constructed.The recombinant HBeBP4A gene can be expressed in prokaryotic expression system of E.coli.