【作者】 徐皓亮；

【导师】 韩伟； 黄永平；

【作者基本信息】 华东理工大学 ， 中药学， 2011， 硕士

【摘要】 HIV-1是一种人类免疫缺陷病毒,感染人体后导致艾滋病的发生。目前艾滋病的治疗药物主要为反转录酶以及蛋白酶抑制剂等,副作用多,不能达到根治目的。因此,研究开发新的抗HIV-1药物十分重要。根据HIV-1病毒转录复制过程中,需要HIV-1编码的调节蛋白Tat和LTR启动子中-TAR RNA序列特异性结合,以激活转录。因此,阻断Tat-TAR的结合有可能抑制病毒的转录复制。本课题通过构建两种实验模型,筛选具有能有阻断Tat-TAR结合并具有转录抑制活性的化合物。通过构建Tat的真核表达载体pCMV-Tat以及LTR荧光素酶报告基因载体pGL3-LTR,将两者共转染HeLa细胞后,检测荧光素酶活性,以观察Tat蛋白对LTR启动子的转录激活作用,建立转录活化细胞模型,用于筛选转录抑制性化合物。将HIV-1 Tat (1-86aa)编码序列插入原核表达质粒pET28a中,转化E.coli BL21(DE3), IPTG诱导表达,并用Ni+金属螯合层析介质纯化获得Tat蛋白；把编码TARRNA的DNA序列插入含有T7启动子的载体pBluescript II SK(+)中,用T7 RNA多聚酶体外转录合成TAR RNA。用毛细管电泳法分析Tat与TAR的结合活性,以观察活性化合物对Tat-TAR的阻断作用。,结果显示：通过对68个化合物的筛选,并结合细胞毒性试验,总共筛选出了10个能够有效抑制转录的化合物；制备的Tat和TAR能相互结合,此结合能被转录抑制性化合物AO081/15571098阻断,说明此模型可用于筛选阻断Tat-TAR结合的化合物。更多还原

【Abstract】 Human immunodeficiency virus type 1(HIV-1) is the cause of AIDS(Acquired Immune Deficiency Syndrome). There are no drugs can cure AIDS completely up to now. So search for new drugs that can cure AIDS has become very important. Because of the transcription of HIV-1 requires specific interaction of Tat with TAR RNA, disruption of Tat-TAR interaction could inhibit HIV-1 transcription and replication. So we construct two models for screening compounds which can inhibit the Tat binding to TAR and the Tat activated transcription of HIV-1.HIV-1 Tat (1-86aa) coding sequence and LTR sequence was inserted into eukaryotic expression plasmid pCMV-HA and luciferase report gene vector pGL3-Basic, named pCMV-Tat and pGL3-LTR. The two vectors were transfected into HeLa cells, the activity of luciferase were detected with illuminator. This Tat activated transcription experiments was used as cell model for screening transcription inhibitory compounds.HIV-1 Tat coding sequence was inserted into the prokaryotic expression plasmid pET28a and transformed into E. coli BL21(DE3). Tat protein expression was induced by IPTG and purified with Ni-NTA column affinity chromatography. Then the TAR sequence was inserted into pBluescript II SK(+),which contain T7 promoter. The TAR RNA was synthesized by T7 RNA polymerase transcription in vitro. The binding activity of Tat with TAR was analyzed by Capillary Electrophoresis. This experiment was used as Tat-TAR binding model in vitro to test the binding blocking activity of compounds.The results show that:we screened 10 active compounds which can inhibit Tat activated transcription through cell model; Tat protein can combine TAR effectively and the compound AO081/15571098 can block the binding of Tat and TAR.更多还原