多聚TAR-CoreRNA诱饵对人免疫缺陷病毒1型Tat蛋白活性的抑制作用

Inhibition of Human Immunodeficiency Virus Type 1(HIV 1)Tat Transactivation By Multimerized TAR Core RNA Decoy

为了抑制Ｔａｔ蛋白的反式激活作用，在细胞内大量表达外源ＴＡＲＲＮＡ使其与Ｔａｔ蛋白结合，从而竞争性抑制其与ＨＩＶ－１ＬＴＲ的ＴＡＲＲＮＡ元件结合．构建了以ＨＩＶ－１ＬＴＲＹＬ１５８（－１５８～＋１８０）为启动子，分别含有４，８和１５个拷贝的ＴＡＲ－ＣｏｒｅＲＮＡ诱饵（ｄｅｃｏｙ）表达质粒；以荧光酶基因为报告基因，检测了瞬时共转染体系中含不同拷贝数的ＴＡＲ－ＣｏｒｅＤＮＡ转录产物对Ｔａｔ蛋白反式激活作用的影响．结果证明，ＴＡＲ－ＣｏｒｅＲＮＡ诱饵对Ｔａｔ蛋白活性具有很强的抑制作用，其抑制程度与ＴＡＲ－ＣｏｒｅＤＮＡ串联体的拷贝数有关．

The human immunodeficiency virus type 1(HIV 1)Tat protein transactivates HIV 1 gene expression at the transcriptional level by interacting with its response element(TAR)of the promoter region in the viral long terminal repeat(LTR).Therefore,Tat and TAR are desirable target for recombinant gene therapy against HIV 1 infection.To inhibit trans activity of Tat protein,TAR RNA was overexpressed to make Tat protein bind TAR decoy competitively with TAR RNA of HIV 1 LTR.By using HIV 1 YL 158 as an autoregulated promoter,three eucaryotic expression vectors,which expressed 4,8 and 15 copies tandem TAR Core decoies,were constructed.After transfecting Jurkat cell and measuring the relative activities of luciferase,inhibition rates of different copies of TAR core RNA were recorded.The results showed that all the products of these three expression plasmids could repress Tatmediated gene expression significantly in a transiently transfection assay.The results also demonstrated that the inhibition was dependent upon the numbers of transcribed TAR Core elements contained in the expression plasmids.