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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。
AIM: To express Tat protein efficiently in E. coli BL21 (DE3). Methods: The complete sequence of HIV-1Tat gene was constructed by PCR, and the Tat gene was mutated by site-directed mutagenesis (AAG28 was replaced by CAG and AAG50 was replaced by CAG) to eliminate the transcriptional activity of native Tat protein. The mutated Tat gene was ligated to the chaperone 10 (chap10) gene, subcloned into the expression vector pET28a, and expressed in E. coli. The expressed product was identified by Western blot. Results: The complete Tat gene sequence was successfully constructed by three rounds of PCR respectively. The constructed recombinant plasmid pET28a- chap10 Tat was highly expressed in E. coli BL21 (DE3). Western blot analysis showed that there was one specific band at a relative molecular mass (Mr) of 24,000. Conclusion: The fusion of chap10 gene and HIV-1Tat gene can make Tat protein highly expressed in E. coli, which lays a foundation for its role in the pathogenesis of AIDS.