本文選題：人類免疫缺陷病毒 + 整合酶　； 參考：《福建醫(yī)科大學》2008年博士論文

【摘要】： 人類免疫缺陷病毒I型(human immunodeficiency virus type I, HIV-1)的pol基因編碼的三種酶:逆轉錄酶(reverse transcriptase, RT),蛋白酶(protease,PR)和整合酶(integrase, IN )。IN的主要功能是將病毒cDNA整合到宿主細胞的基因組DNA中,另外它亦在病毒RNA基因的逆轉錄及整合前復合物的進核轉運過程中起作用。在對艾滋病(acquired immune deficiency syndrome, AIDS)的治療過程中,由于傳統(tǒng)抗RT及抗PR等藥物的高毒性,易引起代謝紊亂以及抗藥病毒株的出現,漸漸顯示出它們的局限性。因此, IN及其與宿主細胞因素間相互作用成為AIDS治療研究的新熱點。在對HIV-1 C端功能的研究中發(fā)現,它可以與DNA非特異性結合,與宿主細胞轉運因子importin7相互作用,在病毒RNA基因的逆轉錄、整合等HIV-1復制中的多個階段起作用。目前對IN的研究多采用“突變分析”的方法,但此方法可能會改變IN的構象。通過“單克隆抗體”的方法來研究IN,可以在不改變IN的前提下,通過其與IN相互作用來反映IN的結構與功能,所以具有獨特的優(yōu)勢。目前的研究已經發(fā)現IN與importin7的特異性作用位點位于IN的CTD,但是未見關于importin7與IN作用位點的研究報道�；诖�,本研究第一部分應用雜交瘤技術制備了抗IN第142-288位氨基酸蛋白的單克隆抗體;第二部分應用細胞基礎上共免疫沉淀的方法探討了IN與importin7片段蛋白間的相互作用。本研究旨在為HIV-1 IN的結構、功能及開發(fā)抗HIV-1 IN的藥物或疫苗打下基礎。 本研究第一部分,構建了HIV-1 IN第142-288位氨基酸的谷胱甘肽S轉移酶(glutathione S-transferase ,GST)標簽的融合蛋白表達質粒pGEX-4T1-GST- IN142-288,在原核表達系統(tǒng)中進行表達后純化了融合蛋白GST-IN142-288。純化了兩種用于篩選雜交瘤克隆的野生型IN融合蛋白:His-IN與T7-IN。以GST-IN142-288作為抗原免疫BALB/c小鼠,應用雜交瘤技術制備了表達單克隆抗體(monoclonal antibody, mAb)的雜交瘤克隆。采用酶聯(lián)免疫吸附試驗(enzyme-linked immuno-sorbent Assay , ELISA ) , Western-blot ( WB ) ,免疫沉淀(Immuno-precipitation ,IP)及免疫熒光染色的方法對mAbs進行檢測。結果顯示,共制備了45個ELISA陽性的雜交瘤克隆,其中有12個克隆WB與IP陽性, 3個克隆免疫熒光染色陽性。對12個WB與IP陽性的克隆分泌的mAb進行亞型測定,結果顯示重鏈亞型中3個為IgG1,6個為IgG2a, 2個為IgG2b, 1個為IgM;所有的輕鏈亞型均為κ。 本研究的第二部分探討IN與importin7的相互作用。構建了表達importin7第207-836位氨基酸及第442-836位氨基酸融合蛋白的質粒sVCMVin-T7-Imp7 207-836和sVCMVin-T7-Imp7 442-836。將上述兩個質粒分別與表達野生型HIV-1 IN的質粒CMV-YFP-IN共轉染HEK-293T細胞,采用細胞基礎上共免疫沉淀(cell-based co-IP)的方法對兩種importin7融合蛋白與YFP-IN融合蛋白間的相互作用進行研究。結果顯示T7-importin7 207-836與T7-imp7 442-836均能與YFP-IN相互作用。提示IN與importin7相互作用的位點可能位于importin7的中間區(qū)域,至少一個位點位于第442-836位氨基酸之間。
[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.

【學位授予單位】：福建醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R392

【共引文獻】

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