Establishing A Phage Model For In Vitro Screenning Of HIV PR Inhibitors And Primary Research

The primers were designed according to the CAP2 amino acid sequence of HIV-1 HXB2 subtype and the E.coli preferred codon. The CAP2 DNA fragment with E.coli preferred codon was synthesized in vitro by overlapping PCR and sequenced after inserted into pMD18-T vector by T/A method. Then the PR DNA sequence was cloned into PET32a(+) vector which is used as expression vector in E.coli. Expression of HIV PR was induced by IPTG in E.coli BL21-TRXB and the expressed PR protein was purified by the Ni-NTA affinity column. The purified CAP2 protein was analyzed by SDS-PAGE and ELISA. The HIV CAP2 DNA fragment with E.coli preferred codon was successfully synthesized and its corresponding amino acid sequence is identical with the primary amino acid sequence of HIV-1 HXB2 subtype. The expression vector PET32a(+)-CAP2 was constructed successfully, The HIV-1 CAP2 was expressed in E.coli BL21-TRXB after the induction by IPTG with a relative molecular weight of 26000. The purifyed CAP2 protein has a concentration of 6.65mg/ml and showed specific reaction to the anti-HIV positive blood sera.HIV gag protein CAP2NC were cleavaged by SF2 PR in viro,it showed that PR substrate which we had expressed in E.coli has favorable activer function of biology.Simultaneity, substrate of HIV PR displayed on surface of phages were also cleavaged by SF2 PR,subsequenly protease inhibitor indinavir can be used to inhibit cleavage the function of SF2 PR.HIV-1 gag CAP2 fragment with induced randomized protease cleavage signal sequences at the 3' terminal and Stu I restriction site at the 5' terminal was generated by PCR amplication. HIV-1 gag NC fragment with induced 5' terminal sequense overlapping CAP2 3' terminal region and Sal I restriction site at the 3' terminal was also generated by PCR. The CAP2/NC fragment with randomized protease cleavage...