中文摘要：

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.方法根据HIV-1 HXB2株PR DNA序列设计引物,用重叠延伸PCR方法合成使用大肠杆菌偏好密码子的HIV-1 PR的DNA编码序列,用T/A克隆法将其插入pMD18-T载体进行测序.将HIV-1 PR DNA克隆至原核表达载体pQE30,在大肠杆菌M15中进行诱导表达,用Ni-NTA亲和柱对表达蛋白进行纯化,SDS-PAGE鉴定,并用HIV阳性血清进行免疫反应性的鉴定.结果合成的使用大肠杆菌偏好密码子的HIV-1 PR的DNA编码序列,经测序显示其编码氨基酸序列与原始序列完全一致.所构建的HIV-1 PR原核表达质粒pQE30-pr,经IPTG诱导后,表达出相对分子质量为14 000的HIV-1 PR蛋白,纯化的目的蛋白浓度为7.74 mg/ml。ELISA检测该蛋白与HIV阳性血清呈特异性反应.结论已成功合成并克隆了使用大肠杆菌偏好密码子的HIV-1 PR的DNA编码序列,经表达及纯化的HIV-1 PR蛋白具有免疫学特性.

英文摘要：

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. coil-preferred cedon,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 prokaryotie expression vector pQE30 and expressed in E.coli M15 under induction of IPTG. The expressed product was purified by Ni-NTA affinity coIttmn chromatography and identified by SDS-PAGE, then analyzed for immunoreactlvity 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 I-tlV-1 PR,with E. coil-preferred codon ,was successfully synthesized and cloned ,and the HIV-1 PR with immunological character was expressed in E. coil and purified.