脑脊液来源人类免疫缺陷病毒tat真核表达载体的构建及鉴定

Construction and in vitro expression of an eukaryotic vector encoding HIV tat from cerebrospinal fluid samples

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 construct an eukaryotic plasmid which contains HIV-1 tat from cerebrospinal fluid samples, and to study the TAT protein expression in the eukaryotic cell in vitro. Methods HIV-1 tat gene was amplified from cerebrospinal fluid samples by PCR. The tat gene was inserted into the eukaryotic plasmid pcDNA3.1, and the resultant recombinant plasmid was confirmed by restriction endo-nuclease and sequencing. The pcDNA3.1-tat was transfected into 293 T cell, and the expression of TAT protein was analyzed by immunoflurescence method. Results Tat gene was amplified successfully, the recombinant plasmid pcDNA3.1-tat was correctly constructed, and TAT protein could be expressed in 293T cells. Conclusion pcDNA3.1-tat plasmid has been constructed successfully, which has established experiment basis for research of HIV latency mechanism in the central nervous system.