Expression of Protease of HIV-1 HXB2 Subtype in E.coli and Purification and Identification of Expressed Product

Objective To express HIV-1 HXB2 subtype in prokaryotic cells,then purify and identify the expressed product for the screening of HIV-1 Gag CAP2/NC protein phage displayed library with randomized P2/NC protease cleavage site.Methods The primers were designed according to the PR DNA sequence of HIV-1 HXB2 subtype,and the DNA sequence encoding HIV-1 PR,with E.coli-preferred codon,was synthesized by overlapping PCR,then inserted into pMD18-T vector by T/A cloning.After identification by sequencing,the amplified HIV-1 PR DNA was cloned into prokaryotic expression vector pQE30 and expressed in E.coli M15 under induction of IPTG.The expressed product was purified by Ni-NTA affinity column chromatography and identified by SDS-PAGE,then analyzed for immunoreactivity with HIV-positive serum.Results The sequence of amino acids encoded by the synthesized DNA was completely identical to the original amino acid sequence of HIV-1 HXB2 subtype.The HIV-1 PR,with a relative molecular weight of 14 000 ,was expressed in the constructed prokaryotic expression vector pQE30.The purified target protein reached a purity of 7.74 mg/ml .ELISA proved specific reaction of the expressed protein with HIV-positive serum.Conclusion The DNA sequence encoding HIV-1 PR,with E.coli-preferred codon,was successfully synthesized and cloned,and the HIV-1 PR with immunological character was expressed in E.coli and purified.