Randomization of P2/NC protease cleavage site of HIV-1 Gag protein and and its phage display / 第二军医大学学报

ABSTRACT

Objective:To construct the phage display library of randomized P2/NC protease cleavage sequences in HIV-1 Gag protein,so as to provide evidences for studying the effect of drug resistance-associated mutations on susceptible cleavage sequences of HIV-1 PR.Methods: CAP2 fragments and NC fragments of HIV-1 gag were generated by PCR.StuⅠ restriction site was introduced to the 5′ terminal of CAP2 fragments and the protease cleavage sequences of 3′ terminal of CAP2 fragments were randomized.The overlapping CAP2 sequence was introduced to the 5′ terminal of NC fragments and Sal Ⅰ restriction site was introduced to the 3′ terminal.CAP2 and NC fragments were linked by overlapping PCR method and the fused CAP2 and NC fragments were cloned into pCANTAB5S-LD3 to construct the phage display library of the P2/NC cleavage site in HIV-1 Gag protein.Results: As much as 2.1?10~6 clones were obtained in the phage library and the titer was 3.0?10~(12)TU/ml.There were 52.1% clones containing inserted CAP2/NC fragments.Sequence analysis of 12 samples showed that nucleotide acids and amino acids were randomly distributed at randomized PR cleavage sites.Nine of 10 positive monoclonal phages specifically bound to human IgG.Conclusion: CAP2/NC protease cleavage sequences of HIV-1 Gag protein have been successfully randomized and its phage display library has been constructed,which may help to screen the phages containing susceptible cleavage sequences of PR with drug resistance-associated mutations and establish the phage model for in vitro screening of PR inhibitor.