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目的:原核表达、纯化HIV-1型HXB2株蛋白酶(protease,PR),用于对HIV-1 Gag P2/NC蛋白酶切割位点序列随机突变的噬菌体展示文库的切割筛选及构建新的蛋白酶抑制剂(protease inhibitor,PI)类药物体外筛选模型。方法:根据HIV-1 HXB2株PR DNA序列设计引物,在PR序列上游添加自切割位点MGTVSFNF 8个氨基酸编码获得PR107 DNA编码序列,将PR107 DNA克隆至原核表达载体pET-32a,序列测定后转化大肠杆菌BL21 DE3进行诱导表达。表达产物经用Ni-NTA亲和柱纯化,经复性后,进行靶蛋白CAP2NC切割试验,SDS-PAGE检测切割结果。结果:成功合成了使用大肠杆菌偏好密码子的HIV-1 PRl07的DNA编码序列,序列测定显示其编码氨基酸序列与原始序列完全一致;成功构建了HIV-1 PR原核表达质粒pETa2a—PR107,转化大肠杆菌BL21 DE3经IPTG诱导后,表达出相对分子质量为30 000的HIV-1 PR融合蛋白,纯化的目的蛋白浓度为2.54 mg/ml;纯化蛋白经复性后具有对底物蛋白CAP2NC的切割活性,该作用能被PI药物抑制。结论:成功构建大肠杆菌偏好密码子带自切割位点的HIV-1 PR原核表达载体pET32a—PR107,在大肠杆菌中表达,经纯化复性获得了具有切割活性的HIV-1 PR融合蛋白。
OBJECTIVE: To purify HIV-1 type HXB2 strain protease (PR) by prokaryotic expression and to screen for a phage display library of randomly mutated HIV-1 Gag P2 / NC protease cleavage site and to construct a novel protease inhibitor (protease inhibitor, PI) drugs in vitro screening model. Methods: Primers were designed according to the PR DNA sequence of HIV-1 HXB2 strain. The coding sequence of PR107 DNA was amplified by inserting 8 amino acids of MGTVSFNF from the upstream of PR sequence. The PR107 DNA was cloned into prokaryotic expression vector pET-32a. E. coli BL21 DE3 induced expression. The expressed product was purified by Ni-NTA affinity column, after refolding, the target protein CAP2NC cleavage test, SDS-PAGE detection of cleavage results. Results: The DNA coding sequence of HIV-1 PR107 using Escherichia coli preferred codons was successfully synthesized. Sequence analysis showed that the encoded amino acid sequence was identical with the original sequence. The prokaryotic expression vector pETa2a-PR107 was successfully constructed and transformed into the large intestine After induced by IPTG, the recombinant baculovirus BL21 DE3 expressed HIV-1 PR fusion protein with a relative molecular mass of 30 000 and the purified target protein concentration was 2.54 mg / ml. After purification, the purified protein had the cleavage activity of the substrate protein CAP2NC , This effect can be PI drug inhibition. CONCLUSION: The prokaryotic expression vector pET32a-PR107 of HIV-1 PR, which encodes the preferred codon usage region of Escherichia coli, was successfully constructed and expressed in E. coli. The cleaved HIV-1 PR fusion protein was obtained after purification.