人免疫缺陷病毒1型TAT蛋白点突变对其反式激活作用的影响

Effect of Site-directed Mutagenesis on the trans-Activation Activity of Human Immunodeficiency Virus Type 1 Tat trans-Activator

不同免疫缺陷病毒（ＨＩＶ－１，ＨＩＶ－２和ＳＩＶ）的Ｔａｔ均有３个高度保守的结构域：Ｃｙｓ富集域、核心域和碱性氨基酸富集域，用ＰＣＲ定点突变法在ＨＩＶ－１Ｔａｔ蛋白的这些区域引入单氨基酸或多氨基酸突变；构建了以ＨＩＶ－１ＬＴＲ－１５８到＋８Ｏ区域为启动子，含有不同突变点的突变Ｔａｔ基因表达质粒；以荧光酶基因为报告基因，瞬时共转染Ｊｕｒｋａｔ细胞：分析不同氨基酸突变对Ｔａｔ的反式激活作用的影响。结果发现，突变后的Ｔａｔ蛋白的活性均极大地降低或丧失，表明这些序列内的氨基酸的确定性对Ｔａｔ的活性至关重要。

The Tat trans-activators encoded by the primate immunodeficiency virus(HIV-1,HIV-2,and SIV) share three highly conserved domains:Cys-rich domain,core region and basic domain.Site-directed mutagenesis by PCR method of these regions in the Tat gene of human immunodeficiency virus type 1 is shown to greatly reduce or abolish the activity of Tat protein when PM1-6 was cotransfected transiently with an expression vector that the reporter gene for luciferase(Luc) was driven by HIV-1 LTR(from-158 to+80) promoter.The wild-type and mutant Tat gene were also directed by HIV-1 LTR(-158 to +80 fragment).The results demonstrated that the precise sequence of these conserved regions are crucial for the function of Tat protein.