HIV-1整合酶的生物活性及酶动力学分析
发布时间：2018-10-10 07:49
【摘要】：[目的] 整合酶(integrase)是人类免疫缺陷病毒(human immunodeficiency virus,HIV)基因编码的蛋白质,由前期融合蛋白Pr160gag—pol经病毒自身编码的蛋白酶作用形成的产物。整合酶催化HIV逆转录合成的双链cDNA整合至宿主DNA上,在HIV复制中起着关键的作用;由于正常宿主细胞没有整合酶,因此整合酶是设计HIV病毒抑制剂的良好靶点。在整合反应过程中,整合酶表现出四种活性:DNA结合、DNA 3'末端加工、末端转移(病毒DNA整合进宿主DNA)、去整合。在结合到双链平末端病毒DNA(供体DNA)后,整合酶切去3'端二个核苷酸—GT,然后切割宿主DNA的5'末端,并将病毒DNA整合进宿主DNA(受体DNA)。整合过程是可逆的,当整合DNA产物的类似物存在时,整合酶可将原整合产物重新分解为病毒DNA和宿主DNA部分。晶体结构研究显示:整合酶由三个结构域组成:1、N-末端结构域,包含一个锌指结构。2、中心区,包含酶的催化中心。3、C-末端结构域,非特异性结合DNA。因为以前普遍采用的方法使用带有放射性标记的病毒DNA底物,和整合酶共育后,经过变性胶电泳以及放射自显影,显示整合酶反应的产物。但这种实验都有耗时较长及放射线污染的缺点。我们旨在建立一种快速简便安全的体外筛选HIV-1整合酶抑制剂的方法。 [方法] 包含HIV-1整合酶基因的重组质粒PT7-His-TX-WT-IN在E.coli BL21(DE3)中以包含体形式高效表达HIV-1整合酶(IN)。所获包含体经过洗涤,稀释复性,并通过镍柱纯化,获得高纯度及可溶的整合酶蛋白(IN)。我们采用一种基于Biotin-Avidin EILSA(BA-ELISA)的方法,测定分析整合酶的3'端酶切活性以及病毒DNA整合活性,并据此得到酶的动力学参数及整合酶的比活性,我们还分析了核糖体失活蛋白Ⅰ型(RIP Ⅰ)luffin-a对整合酶的抑制作用。
[Abstract]:[objective] integrase (integrase) is a protein encoded by human immunodeficiency virus (human immunodeficiency virus,HIV) gene. The product formed by the protease action of the Prophase fusion protein Pr160gag-pol by the virus itself. The integration of double-stranded cDNA catalyzed by HIV reverse transcription into host DNA plays a key role in HIV replication. Since there is no integrase in normal host cells, integrase is a good target for the design of HIV virus inhibitors. In the process of integration, integrase showed four kinds of activity: DNA-binding DNA 3'terminal processing, terminal transfer (virus DNA integrated into host DNA), to be unintegrated). After binding to the double stranded flat terminal virus DNA (donor DNA), the integrase cleans 3 'terminal two nucleotides -GT, then cleans the 5'terminal of host DNA, and integrates the virus DNA into the host DNA (receptor DNA). The integration process is reversible. When the analogs of the integrated DNA products are present, the integrase can redivide the prointegration products into viral DNA and host DNA parts. Crystal structure study shows that the integrase consists of three domains: 1: 1 + N-terminal domain, which contains a zinc-finger structure of 0.2, a central region, a catalytic center of the enzyme, a C-terminal domain, and a non-specific binding to DNA.. This is because the commonly used method used in the past is to use the DNA substrate with radiolabeled virus. After co-breeding with integrase, the product of integrase reaction is shown by denaturing gel electrophoresis and autoradiography. But these experiments have the disadvantages of long time consuming and radiation pollution. We aim to establish a rapid and safe method for screening HIV-1 integrase inhibitors in vitro. [methods] Recombinant plasmid PT7-His-TX-WT-IN containing HIV-1 integrase gene was highly expressed in E.coli BL21 (DE3) in the form of HIV-1 integrase (IN). After washing, dilution, renaturation and purification by nickel column, a high purity and soluble integrase protein (IN). Was obtained. A method based on Biotin-Avidin EILSA (BA-ELISA) was used to determine and analyze the 3'-terminal digesting activity of integrase and the integration activity of virus DNA. The kinetic parameters and specific activity of integrase were obtained. We also analyzed the inhibitory effect of ribosomal inactivated protein type I (RIP 鈪，
本文编号：2261222
[Abstract]:[objective] integrase (integrase) is a protein encoded by human immunodeficiency virus (human immunodeficiency virus,HIV) gene. The product formed by the protease action of the Prophase fusion protein Pr160gag-pol by the virus itself. The integration of double-stranded cDNA catalyzed by HIV reverse transcription into host DNA plays a key role in HIV replication. Since there is no integrase in normal host cells, integrase is a good target for the design of HIV virus inhibitors. In the process of integration, integrase showed four kinds of activity: DNA-binding DNA 3'terminal processing, terminal transfer (virus DNA integrated into host DNA), to be unintegrated). After binding to the double stranded flat terminal virus DNA (donor DNA), the integrase cleans 3 'terminal two nucleotides -GT, then cleans the 5'terminal of host DNA, and integrates the virus DNA into the host DNA (receptor DNA). The integration process is reversible. When the analogs of the integrated DNA products are present, the integrase can redivide the prointegration products into viral DNA and host DNA parts. Crystal structure study shows that the integrase consists of three domains: 1: 1 + N-terminal domain, which contains a zinc-finger structure of 0.2, a central region, a catalytic center of the enzyme, a C-terminal domain, and a non-specific binding to DNA.. This is because the commonly used method used in the past is to use the DNA substrate with radiolabeled virus. After co-breeding with integrase, the product of integrase reaction is shown by denaturing gel electrophoresis and autoradiography. But these experiments have the disadvantages of long time consuming and radiation pollution. We aim to establish a rapid and safe method for screening HIV-1 integrase inhibitors in vitro. [methods] Recombinant plasmid PT7-His-TX-WT-IN containing HIV-1 integrase gene was highly expressed in E.coli BL21 (DE3) in the form of HIV-1 integrase (IN). After washing, dilution, renaturation and purification by nickel column, a high purity and soluble integrase protein (IN). Was obtained. A method based on Biotin-Avidin EILSA (BA-ELISA) was used to determine and analyze the 3'-terminal digesting activity of integrase and the integration activity of virus DNA. The kinetic parameters and specific activity of integrase were obtained. We also analyzed the inhibitory effect of ribosomal inactivated protein type I (RIP 鈪，
本文编号：2261222
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