摘要
目的：构建人类免疫缺病毒Tat基因真核表达质粒，并研究该质粒在真核细胞中的有效表达。方法：以含有HIV-1全长基因的质粒pNL4—3为模板，通过PCR扩增HIV-1 Tat第一外显子，应用基因工程技术将扩增的基因片段插入真核表达质粒pCDNA3．1（＋），构建重组真核表达质粒pCDNA3．1-tat，经限制性内切酶酶切分析及测序鉴定正确后，应用脂质体转染技术转染入huh-7细胞。RT—PCR检测其基因转录情况，WesternBlot鉴定其是否能够表达相应的目的蛋白质。结果：成功扩增了Tat基因，酶切和测序证明正确构建了重组真核表达质粒pCDNA3．1-tat，RT—PCR证实该质粒能在huh-7真核细胞中有效表达Hiv-1Tat目的片段。结论：成功构建了HIV—ITat基因的真核表达质粒载体，并在huh-7中表达，为在huh-7细胞模型中研究Tat的功能奠定了基础。
Objective： To construct an eukaryotic plasmid expressing HIV- 1 Tat and test whether the plasmid can be expressed in the eukaryotic cell in vitro. Methods ：The first exon of HIV - 1 Tat was amplified by PCR from the plasmid pNL4 - 3. Then the Tat gene was in- serted into the eukaryotie expression plasmid pCDNA3. 1 （ ＋ ） , and the resultant recombinant plasmid was confirmed by restriction endonu- clease and sequencing , then was designated as pCDNA3.1 - tat. The recombinant plasmid pCDNA3.1 - tat was transfeeted into eukaryot- ie cell huh - 7 by lipo - fectamine. RT - PCR was used to certify whether the purpose protein was correctly expressed in huh - 7 cells. Results The 240 bp DNA fragment of tat was correctly amplified. The recombinant pCDNA3.1 - tat was successfully constructed , and it could be correctly expressed in the huh - 7 cells. Conclusions ： This recombinant plasmid pCDNA3.1 - tat and the resulting huh - 7 cell model pro- vide a basis for further study on the function of HIV - 1 Tat .
