HIV-1整合酶单克隆抗体的制备及整合酶与Importin7相互作用的探讨

发布时间：2018-04-13 17:15

本文选题：人类免疫缺陷病毒 + 整合酶　；参考：《福建医科大学》2008年博士论文

【摘要】： 人类免疫缺陷病毒I型(human immunodeficiency virus type I, HIV-1)的pol基因编码的三种酶:逆转录酶(reverse transcriptase, RT),蛋白酶(protease,PR)和整合酶(integrase, IN )。IN的主要功能是将病毒cDNA整合到宿主细胞的基因组DNA中,另外它亦在病毒RNA基因的逆转录及整合前复合物的进核转运过程中起作用。在对艾滋病(acquired immune deficiency syndrome, AIDS)的治疗过程中,由于传统抗RT及抗PR等药物的高毒性,易引起代谢紊乱以及抗药病毒株的出现,渐渐显示出它们的局限性。因此, IN及其与宿主细胞因素间相互作用成为AIDS治疗研究的新热点。在对HIV-1 C端功能的研究中发现,它可以与DNA非特异性结合,与宿主细胞转运因子importin7相互作用,在病毒RNA基因的逆转录、整合等HIV-1复制中的多个阶段起作用。目前对IN的研究多采用“突变分析”的方法,但此方法可能会改变IN的构象。通过“单克隆抗体”的方法来研究IN,可以在不改变IN的前提下,通过其与IN相互作用来反映IN的结构与功能,所以具有独特的优势。目前的研究已经发现IN与importin7的特异性作用位点位于IN的CTD,但是未见关于importin7与IN作用位点的研究报道。基于此,本研究第一部分应用杂交瘤技术制备了抗IN第142-288位氨基酸蛋白的单克隆抗体;第二部分应用细胞基础上共免疫沉淀的方法探讨了IN与importin7片段蛋白间的相互作用。本研究旨在为HIV-1 IN的结构、功能及开发抗HIV-1 IN的药物或疫苗打下基础。本研究第一部分,构建了HIV-1 IN第142-288位氨基酸的谷胱甘肽S转移酶(glutathione S-transferase ,GST)标签的融合蛋白表达质粒pGEX-4T1-GST- IN142-288,在原核表达系统中进行表达后纯化了融合蛋白GST-IN142-288。纯化了两种用于筛选杂交瘤克隆的野生型IN融合蛋白:His-IN与T7-IN。以GST-IN142-288作为抗原免疫BALB/c小鼠,应用杂交瘤技术制备了表达单克隆抗体(monoclonal antibody, mAb)的杂交瘤克隆。采用酶联免疫吸附试验(enzyme-linked immuno-sorbent Assay , ELISA ) , Western-blot ( WB ) ,免疫沉淀(Immuno-precipitation ,IP)及免疫荧光染色的方法对mAbs进行检测。结果显示,共制备了45个ELISA阳性的杂交瘤克隆,其中有12个克隆WB与IP阳性, 3个克隆免疫荧光染色阳性。对12个WB与IP阳性的克隆分泌的mAb进行亚型测定,结果显示重链亚型中3个为IgG1,6个为IgG2a, 2个为IgG2b, 1个为IgM;所有的轻链亚型均为κ。本研究的第二部分探讨IN与importin7的相互作用。构建了表达importin7第207-836位氨基酸及第442-836位氨基酸融合蛋白的质粒sVCMVin-T7-Imp7 207-836和sVCMVin-T7-Imp7 442-836。将上述两个质粒分别与表达野生型HIV-1 IN的质粒CMV-YFP-IN共转染HEK-293T细胞,采用细胞基础上共免疫沉淀(cell-based co-IP)的方法对两种importin7融合蛋白与YFP-IN融合蛋白间的相互作用进行研究。结果显示T7-importin7 207-836与T7-imp7 442-836均能与YFP-IN相互作用。提示IN与importin7相互作用的位点可能位于importin7的中间区域,至少一个位点位于第442-836位氨基酸之间。
[Abstract]:Human immunodeficiency virus type I (human immunodeficiency virus type I, HIV-1) of the three enzymes: reverse transcriptase gene encoding pol (reverse transcriptase RT), protease (protease, PR) and integrase (integrase, IN) the main function of.IN is to integrate viral cDNA into the host cell genome DNA in the play in addition it also integrate and effect of reverse transcriptase gene in viral RNA complexes into the nuclear transport before the process. On HIV / AIDS (acquired immune deficiency syndrome, AIDS) in the treatment process, due to the high toxicity of traditional anti RT and anti PR drugs, causing metabolic disorder and the emergence of drug resistant strains. Gradually show their limitations. Therefore, IN and its interaction with the host cell factors become a new hotspot in the research of the treatment of AIDS. In a study of HIV-1 C terminal function, it can be combined with DNA non-specific and host cells Extracellular factor importin7 interaction in virus RNA gene transcription, HIV-1 integration of multiple stages in replication work. At present the study on IN by mutational analysis, but this method may change the conformation of IN. Through the method of monoclonal antibodies to IN research, in the premise of don't change the IN, through its interaction with IN to reflect the structure and function of IN, so it has a unique advantage. The present study has found that specific binding sites IN and importin7 in IN CTD, but not on the importin7 and IN sites of action research report. Based on this, the first part of this study application hybridoma technique to prepare antibody to IN 142-288 amino acid protein monoclonal antibody method; CO immunoprecipitation of second cells was discussed on the basis of the interaction between IN and importin7 protein fragment. This research The aim is to lay the foundation for the structure, function of HIV-1 IN and the development of drugs or vaccines against HIV-1 IN.
The first part of this study, constructed the HIV-1 IN 142-288 amino acid glutathione S transferase (glutathione S-transferase GST) fusion protein expression plasmid pGEX-4T1-GST- IN142-288 tag, in the prokaryotic expression system for expression of purified fusion protein GST-IN142-288. two wild-type IN screening hybridoma clones for the purification of His-IN fusion protein T7-IN. and GST-IN142-288 as antigen to immune BALB/c mice, prepared by hybridoma technique the expression of monoclonal antibody (monoclonal antibody, mAb). The hybridoma clones using enzyme-linked immunosorbent assay (enzyme-linked immuno-sorbent, Assay, ELISA), Western-blot (WB), immunoprecipitation (Immuno-precipitation, IP) and immuno fluorescence stain for the detection of mAbs. The results showed that 45 ELISA positive hybridoma clones were obtained, including 12 clones of WB and I P positive, 3 clones immunofluorescence staining positive. 12 WB and IP positive clones secreted mAb subtype determination, the results showed that 3 heavy chain subtypes were IgG1,6 IgG2a, 2 IgG2b, 1 IgM, all light chain subtypes were kappa.
The second part of this study is to investigate the interaction between IN and importin7. The plasmid sVCMVin-T7-Imp7 importin7 207-836 amino acids and 442-836 amino acid fusion protein 207-836 and sVCMVin-T7-Imp7 442-836. these two plasmids and CMV-YFP-IN plasmid expressing wild-type HIV-1 IN were transfected into HEK-293T cells, the cells on the basis of the co immunoprecipitation (cell-based co-IP) the method of fusion protein and YFP-IN fusion protein interaction between research on two types of importin7. The results showed that T7-importin7 207-836 and T7-imp7 442-836 could interact with YFP-IN. It is suggested that the middle region of IN interaction with importin7 sites located in importin7, at least one of the 442-836 sites are located between amino acids.

【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

【共引文献】