论文部分内容阅读
目的:构建HIV-1 Nef基因的原核表达载体并在大肠杆菌中进行表达,初步优化其表达条件。方法:利用特异性引物PCR扩增获得HIV-1 Nef基因片段,PCR产物经限制性内切酶双酶切后连接载体pET24a(+),构建重组质粒pET24a(+)-Nef。经双酶切鉴定及序列测定后的重组质粒转化入E.coliBL21,IPTG诱导表达目的蛋白,优化表达条件,SDS-PAGE及Western blot分析鉴定表达产物。结果:PCR扩增获得618bpDNA片段,酶切鉴定结果表明HIV Nef基因已克隆入原核表达载体pET24a(+)中,测序证明序列正确。SDS-PAGE和Western blot分析证实重组菌诱导后可表达相对分子质量(Mr)符合预期的融合蛋白。0.1mmol/LIPTG在37℃诱导6h为最佳表达条件,重组蛋白表达量可由15.8%增加到43.9%。结论:在大肠杆菌中成功地表达了HIV Nef蛋白,并初步获得了最优化的表达条件。
OBJECTIVE: To construct prokaryotic expression vector for HIV-1 Nef gene and express it in E. coli. The expression conditions of the gene were preliminarily optimized. Methods: HIV-1 Nef gene fragment was amplified by PCR with specific primers. The PCR product was digested with restriction enzyme and ligated into vector pET24a (+) to construct recombinant plasmid pET24a (+) - Nef. The recombinant plasmid was transformed into E.coliBL21 by double enzyme digestion and sequence determination. The target protein was induced by IPTG, and the expression conditions were optimized. The expression products were identified by SDS-PAGE and Western blot. Results: The 618bp DNA fragment was obtained by PCR. The results of restriction enzyme digestion showed that the HIV Nef gene was cloned into the prokaryotic expression vector pET24a (+). The sequencing proved the correct sequence. SDS-PAGE and Western blot analysis confirmed that the recombinant protein can express the corresponding molecular weight (Mr) in line with the expected fusion protein. 0.1 mmol / L IPTG induced at 37 ℃ for 6h was the best expression conditions, the recombinant protein expression increased from 15.8% to 43.9%. Conclusion: The HIV Nef protein was successfully expressed in E. coli and the optimized expression conditions were obtained preliminarily.