本文選題：人類免疫缺陷病毒 + 整合酶�。� 參考：《福建醫(yī)科大學(xué)》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(zhuǎn)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(xì)胞的基因組DNA中,另外它亦在病毒RNA基因的逆轉(zhuǎn)錄及整合前復(fù)合物的進(jìn)核轉(zhuǎn)運(yùn)過程中起作用。在對(duì)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(xiàn),漸漸顯示出它們的局限性。因此, IN及其與宿主細(xì)胞因素間相互作用成為AIDS治療研究的新熱點(diǎn)。在對(duì)HIV-1 C端功能的研究中發(fā)現(xiàn),它可以與DNA非特異性結(jié)合,與宿主細(xì)胞轉(zhuǎn)運(yùn)因子importin7相互作用,在病毒RNA基因的逆轉(zhuǎn)錄、整合等HIV-1復(fù)制中的多個(gè)階段起作用。目前對(duì)IN的研究多采用“突變分析”的方法,但此方法可能會(huì)改變IN的構(gòu)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(jié)構(gòu)與功能,所以具有獨(dú)特的優(yōu)勢(shì)。目前的研究已經(jīng)發(fā)現(xiàn)IN與importin7的特異性作用位點(diǎn)位于IN的CTD,但是未見關(guān)于importin7與IN作用位點(diǎn)的研究報(bào)道�；诖�,本研究第一部分應(yīng)用雜交瘤技術(shù)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(yīng)用細(xì)胞基礎(chǔ)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(jié)構(gòu)、功能及開發(fā)抗HIV-1 IN的藥物或疫苗打下基礎(chǔ)。 本研究第一部分,構(gòu)建了HIV-1 IN第142-288位氨基酸的谷胱甘肽S轉(zhuǎn)移酶(glutathione S-transferase ,GST)標(biāo)簽的融合蛋白表達(dá)質(zhì)粒pGEX-4T1-GST- IN142-288,在原核表達(dá)系統(tǒng)中進(jìn)行表達(dá)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(yīng)用雜交瘤技術(shù)制備了表達(dá)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(yàn)(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(duì)mAbs進(jìn)行檢測(cè)。結(jié)果顯示,共制備了45個(gè)ELISA陽性的雜交瘤克隆,其中有12個(gè)克隆WB與IP陽性, 3個(gè)克隆免疫熒光染色陽性。對(duì)12個(gè)WB與IP陽性的克隆分泌的mAb進(jìn)行亞型測(cè)定,結(jié)果顯示重鏈亞型中3個(gè)為IgG1,6個(gè)為IgG2a, 2個(gè)為IgG2b, 1個(gè)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(gòu)建了表達(dá)importin7第207-836位氨基酸及第442-836位氨基酸融合蛋白的質(zhì)粒sVCMVin-T7-Imp7 207-836和sVCMVin-T7-Imp7 442-836。將上述兩個(gè)質(zhì)粒分別與表達(dá)野生型HIV-1 IN的質(zhì)粒CMV-YFP-IN共轉(zhuǎn)染HEK-293T細(xì)胞,采用細(xì)胞基礎(chǔ)上共免疫沉淀(cell-based co-IP)的方法對(duì)兩種importin7融合蛋白與YFP-IN融合蛋白間的相互作用進(jìn)行研究。結(jié)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(diǎn)可能位于importin7的中間區(qū)域,至少一個(gè)位點(diǎn)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(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【共引文獻(xiàn)】

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