噬菌体展示耐药突变HIV-1蛋白酶敏感切割位点六肽随机文库的构建

目的构建耐药性突变HIV-1蛋白酶(protease PR)敏感切割位点序列体外噬菌体筛选模型,研究HIV-1蛋白酶耐药性突变与敏感切割序列的关系。方法用含随机核苷酸序列的引物PCR方法对HIV-1gag基因上的CAP2片段的PR切割位点处的氨基酸序列进行随机化,再将重组CAP2片段和NC片段拼接克隆于噬菌体展示载体LD3-pCANT-AB5S上,建立HIV-1蛋白酶靶蛋白切割位点随机化的噬菌体展示文库。结果该噬菌体展示文库库容量为2.6×106,滴度为4.1×1015TU·L-1,CAP2片段插入率为47.8%;序列分析显示切割位点中随机化的核苷酸与氨基酸均呈随机性分布。结论成功地构建HIV-1蛋白酶的敏感切割序列噬菌体筛选文库,为筛选到突变PR敏感切割序列噬菌体及研究耐药HIV-1蛋白酶抑制剂与突变PR的关系打下基础。

Construction six peptide phage library for in vitro screening of susceptible cleavage sequences of HIV-1 protease with drug resistance-associated mutation

Aim To construct the phage library for in vitro screening of susceptible cleavage sequences of HIV-1 protease with drug resistance-associated mutation so as to explore the relationship of drug resistance-associated mutation with susceptible cleavage sequences of HIV-1 protease.Methods CAP2 fragment which was introduced randomized sequences at P2/NC cleavage site of HIV-1 protease was generated by PCR with the random nucleotide primers. The recombined CAP2 fragment and NC fragment were linked and cloned into phage display vector LD3-pCANTAB5S. Results The phage library with the size of 2.6×106 was obtained and the titer was 4.1×1015TU·L-1. About 47.8% clones contained inserted CAP2 fragments. Sequence analysis of 10 samples showed that nucleotide acids and amino acids at randomized PR cleavage site distributed randomly.Conclusions The phage library for screening susceptible cleavage sequences at P2/NC cleavage site of HIV-1 PR has been constructed successfully, which is helpful for in vitro screening the phages containing susceptible cleavage sequences of PR with drug resistance mutations and laying the foundation of screening of PR inhibitor in vitro.