| Background and Objective:Acquired immunodeficiency syndrome (AIDS) is an immune deficiency disease caused by human immunodeficiency virus (HIV) infection. HIV-I can not only regulate by exploiting host regulatory mechanisms but also exert self-regulation by encoding some regulatory proteins. In HIV-1 genome, there are sites on which transcription factors can bind in 5'long terminal repeats (LTR), The interactions between regulatory proteins and DNA or other proteins form a complex network, which regulate the expressions of structural and non-structural proteins of virus. The study on LTR areas and regulatory proteins of HIV-1 will pave a new way for development of anti-AIDS drugs.The tradition Chinese medicine (TCM) play a unique role in anti-AIDS, however, lack of scientific and effective drug screening system let application of TCM in anti-AIDS lag behind western medicine.Thus, how to screen out effective anti-AIDS drugs from rich TCM resources is aproblem needed to be solved urgently. In this study, we constructed a new method, which used reporter gene techniques, to screen potential anti-AIDS drugs targeting at HIV-I promoters. At same time, pseudotyped virus was used to validate the efficacy of this system.Methods:The synthetical HIV-I core promoters were inserted into vectors pGL4.17 and positive recombinant vectors were confirmed by double restriction digestion and DNA sequencing. pGL4.17-HIV1P vectors were transfected into HT-H9 cells with liposome. The stable tranfected cell line was selected with G418. The cells were divided into corresponding groups after expanding. The adult Wistar rats were administered intragastrically with 4 kinds of Chinese traditional medicine decoction (Cortex Eucommiae, Radix Scutellariae, Radix Sophorae Flavescentis and Shuanghuanglian Oral Liquid). Seven days later, blood was obtained and the separated serum was termed as medicated serum. After treated by the 4 kinds of medicated and control serums, the HIV-I promoter activity was determined through luciferase reporter assay and a screening system which can select drug targeting at HIV-1 promoter was constructed.293T cells were transfected by pseudotyped virus pNL4-3.luc.R-E-and treated with corresponding medicated serum. And then ELISA assay was applied to quantify p24 protein expression and validate the reliability of this screening system. Experiment data were analyzed with SPSS 13.0 software pack.Results:1 pGL4.17 vector was inserted by synthetical HIV-1 core promoter. The engineered vector, which was confirmed with double restriction digestion and DNA sequencing, was accordance with purpose of experimental design. Thus, a positive recombinant pGL4.17-HIV1P was obtained.2 The stable transfected HT-H9 cell line with pGL4.17-HIV1P was obtained by G418 screening.3 Data of luciferase assay indicated that the values of relative light unit (RLU) increased dramatically in RSF-(1110780.5±15076.6), CE-(864557.5±15441.9) and RS-(1054500.5±26279.0) medicated serum-treated groups, compared with control (291089.6±10457.7, P<0.01), while RLU value of Shuanghuanglian Oral Liquid group was reduced remarkably versus control (P<0.01). 4 Expression of p24 protein was quantified with ELISA assay as according optical density (OD) value. Results were:RSF, OD=2.267±0.159; CE, OD=1.851±0.076 and RS, OD=2.044±0.122. They were all increased remarkably compared with control group (1.403±0.024, P<0.01). While OD value of Shuanghuanglian Oral Liquid medicated serum-treated group(1.042±0.023) was decreased evidently (P<0.01)Conclusions:1 Vectors in which luciferase reporter gene was controlled by HIV-1 promoter was constructed successfully. Thus, a screening system is set up, through which drugs targeting at virus promoter can be selected. The feasibility of this system is confirmed using pseudotyped virus.2 Shuanghuanglian Oral Liquid, which can significantly suppress the activity of HIV-1 promoter in vitro, may have potential anti-AIDS effect. |
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