【摘要】 目的: 在E.coliBL21(DE3)中高效表达Tat蛋白。方法: 用PCR方法构建HIV- 1Tat基因全序列, 同时将Tat基因进行定点突变(AAG28替换为CAG, AAG50替换为CAG),以消除天然Tat蛋白的转录活性。将突变的Tat基因与伴侣10(chap10)基因连接后, 共同亚克隆入表达载体pET28a中,并在E.coli中表达, 表达产物用Westernblot进行鉴定。结果: 分别通过 3轮PCR, 成功地构建了Tat基因全序列。构建的重组质粒pET28a- chap10 Tat在E.coliBL21(DE3)中得到高效表达。Westernblot分析表明, 在相对分子质量 (Mr)为24 000处有 1条特异性的带。结论: chap10基因与HIV- 1Tat全基因的融合构建, 使Tat蛋白在大肠杆菌中得到高效表达,为其在爱滋病发病中作用研究奠定了基础。更多还原

【Abstract】 AIM: To express high-level the Tat protein in E.coli. METHODS: Full-length HIV-1 Tat gene was amplified artificially by PCR and Tat gene was mutated site-specifically (substitution the codons AAG encoding the lysine at the 28th and the 50th site by the CAG encoding glutamine) in order to eliminate the transcriptional activity of Tat protein. The site-mutated Tat gene was fused with chaperone10 gene, and then was subcloned into vector pET28a. The recombinant plasmid was expressed in E.coli BL21(DE3). The expressed products were identified by Western blot. RESULTS: Full-length HIV-1 Tat gene was amplified successfully by three rounds of PCR. The recombinant plasmid pET28a-chaperone 10-Tat was expressed efficiently in E.coli BL21(DE3). Western blot analysis showed the expressed Tat fusion protein with relative molecular mass (M r) 24 000 could bind to anti-His-tag monoclonal antibody. CONCLUSION: Full-length HIV-1 Tat gene was cloned and chaperone 10-Tat fusion protein was expressed efficiently in E.coli BL21(DE3), which will lay the foundation for researching the pathogenic effect of HIV-1 Tat on AIDS.更多还原