HIV-1整合酶的生物活性及酶動(dòng)力學(xué)分析
發(fā)布時(shí)間：2018-10-10 07:49
【摘要】：[目的] 整合酶(integrase)是人類免疫缺陷病毒(human immunodeficiency virus,HIV)基因編碼的蛋白質(zhì),由前期融合蛋白Pr160gag—pol經(jīng)病毒自身編碼的蛋白酶作用形成的產(chǎn)物。整合酶催化HIV逆轉(zhuǎn)錄合成的雙鏈cDNA整合至宿主DNA上,在HIV復(fù)制中起著關(guān)鍵的作用;由于正常宿主細(xì)胞沒有整合酶,因此整合酶是設(shè)計(jì)HIV病毒抑制劑的良好靶點(diǎn)。在整合反應(yīng)過(guò)程中,整合酶表現(xiàn)出四種活性:DNA結(jié)合、DNA 3'末端加工、末端轉(zhuǎn)移(病毒DNA整合進(jìn)宿主DNA)、去整合。在結(jié)合到雙鏈平末端病毒DNA(供體DNA)后,整合酶切去3'端二個(gè)核苷酸—GT,然后切割宿主DNA的5'末端,并將病毒DNA整合進(jìn)宿主DNA(受體DNA)。整合過(guò)程是可逆的,當(dāng)整合DNA產(chǎn)物的類似物存在時(shí),整合酶可將原整合產(chǎn)物重新分解為病毒DNA和宿主DNA部分。晶體結(jié)構(gòu)研究顯示:整合酶由三個(gè)結(jié)構(gòu)域組成:1、N-末端結(jié)構(gòu)域,包含一個(gè)鋅指結(jié)構(gòu)。2、中心區(qū),包含酶的催化中心。3、C-末端結(jié)構(gòu)域,非特異性結(jié)合DNA。因?yàn)橐郧捌毡椴捎玫姆椒ㄊ褂脦в蟹派湫詷?biāo)記的病毒DNA底物,和整合酶共育后,經(jīng)過(guò)變性膠電泳以及放射自顯影,顯示整合酶反應(yīng)的產(chǎn)物。但這種實(shí)驗(yàn)都有耗時(shí)較長(zhǎng)及放射線污染的缺點(diǎn)。我們旨在建立一種快速簡(jiǎn)便安全的體外篩選HIV-1整合酶抑制劑的方法。 [方法] 包含HIV-1整合酶基因的重組質(zhì)粒PT7-His-TX-WT-IN在E.coli BL21(DE3)中以包含體形式高效表達(dá)HIV-1整合酶(IN)。所獲包含體經(jīng)過(guò)洗滌,稀釋復(fù)性,并通過(guò)鎳柱純化,獲得高純度及可溶的整合酶蛋白(IN)。我們采用一種基于Biotin-Avidin EILSA(BA-ELISA)的方法,測(cè)定分析整合酶的3'端酶切活性以及病毒DNA整合活性,并據(jù)此得到酶的動(dòng)力學(xué)參數(shù)及整合酶的比活性,我們還分析了核糖體失活蛋白Ⅰ型(RIP Ⅰ)luffin-a對(duì)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，
本文編號(hào)：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，
本文編號(hào)：2261222
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