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目的探讨脑脊液来源人类免疫缺陷病毒(HIV)tat真核表达载体的构建,并研究TAT蛋白在真核细胞的表达。方法从脑脊液中扩增出HIV tat基因,构建pcDNA-tat真核表达载体,所构建载体经酶切和测序鉴定。将构建的表达载体转染293T细胞系,通过免疫荧光检测TAT蛋白的表达。结果成功扩增tat基因,酶切和测序结果证实正确构建了表达载体pcDNA-tat,免疫荧光证实所构建载体能在293T细胞中有效表达HIV TAT目的蛋白。结论成功构建真核表达载体pcDNA-tat,为了解HIV在中枢神经系统的潜伏机制奠定实验基础。
Objective To investigate the construction of eukaryotic expression vector of human immunodeficiency virus (HIV) tat from cerebrospinal fluid and to study the expression of TAT protein in eukaryotic cells. Methods HIV tat gene was amplified from cerebrospinal fluid and eukaryotic expression vector pcDNA-tat was constructed. The constructed vector was identified by restriction enzyme digestion and sequencing. The constructed expression vector was transfected into 293T cell line and the expression of TAT protein was detected by immunofluorescence. Results The tat gene was successfully amplified. The recombinant plasmid pcDNA-tat was successfully constructed by restriction enzyme digestion and sequencing. Immunofluorescence confirmed that the constructed recombinant plasmid efficiently expressed HIV TAT protein in 293T cells. Conclusion The eukaryotic expression vector pcDNA-tat was successfully constructed, which laid the experimental foundation for understanding the latent mechanism of HIV in the central nervous system.