本文關(guān)鍵詞： HIV-1 p24 gp41 體液免疫 近期感染 長期感染　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景:人類免疫缺陷病毒(Human Immunodeficiency Virus,HIV)是導(dǎo)致獲得性免疫缺陷綜合征(Acquired Immunodeficiency Syndrome,AIDS)的病原體。HIV-1 的跨膜糖蛋白gp41在HIV-1眾多蛋白中最為保守,能誘導(dǎo)產(chǎn)生多個中和抗體,是免疫檢測試劑盒中最為常見的檢測抗原及疫苗研發(fā)的目標(biāo)。衣殼蛋白p24在病毒蛋白中含量最為豐富,其氨基酸序列在多種亞型中高度保守,也是感染后最早能檢測到的抗原,使其也成為重要的生物標(biāo)志物。研究發(fā)現(xiàn)在HIV-1感染者體內(nèi)存在體液免疫應(yīng)答模式的轉(zhuǎn)變,提示我們p24和gp41的免疫優(yōu)勢表位或能成為區(qū)分不同感染模式的生物標(biāo)志物,對建立實驗室檢測方法有利用價值。研究目的:本課題通過重組表達的p24、gp41蛋白,合成p24、gp41的系列肽段作為包被抗原,探究與HIV-1感染人群外周血與病毒蛋白或肽段的免疫反應(yīng)特征,探討HIV-1感染者的體液免疫應(yīng)答模式及其特點,分析HIV-1感染者體液免疫應(yīng)答模式與感染者病程進展指標(biāo)的關(guān)聯(lián),發(fā)現(xiàn)有使用價值的特殊生物標(biāo)志物,建立能區(qū)分不同感染時期的實驗室檢測方法。研究方法:構(gòu)建p24的重組表達質(zhì)粒,轉(zhuǎn)化至大腸桿菌感受態(tài)細菌。經(jīng)過誘導(dǎo)表達可溶性重組蛋白并對重組蛋白進行純化。表達產(chǎn)物用SDS-PAGE電泳和間接ELISA分析鑒定其活性。收集不同人群來源的HIV-1陽性血漿樣本,利用商品化試劑盒將樣本區(qū)分為近期感染和長期感染,觀察不同人群在感染時期分布上的差異。分別合成3條覆蓋p24和gp41不同區(qū)域的肽段。分析不同人群,不同感染時期的樣本與重組蛋白及肽段的免疫反應(yīng)特點。分析不同感染時期的樣本與肽段的免疫應(yīng)答情況,篩選出具有區(qū)分HIV-1不同感染時期能力的肽段。優(yōu)化反應(yīng)條件,初步建立區(qū)分HIV-1不同感染時期的實驗室檢測方法。通過與兩種商品化試劑盒鑒定結(jié)果的比較,評價方法的敏感性和特異性。研究結(jié)果:成功構(gòu)建p24的原核表達質(zhì)粒并誘導(dǎo)表達,蛋白電泳與預(yù)期相符,間接ELISA分析顯示p24具有抗原特異性。88.3%(233/264)的樣本不與三個覆蓋p24主要免疫優(yōu)勢表位的三個肽段反應(yīng),與此形成對比的是,87.7%(236/269)的樣本至少與一個gp41的肽段反應(yīng)。隨著感染時間的延長,HIV-1感染者體內(nèi)gp41-p1特異性抗體滴度逐步上升,感染時間與gp41-p1的酶免實驗OD值之間的相關(guān)系數(shù)為0.78。將gp41和gp41-p1作為捕捉抗原,gp41抗體陽性和gp41-p1抗體陰性定義為近期感染,gp41抗體陽性和gp41-p1抗體陽性定義為長期感染,初步建立了一種能區(qū)分HIV-1新近和長期感染的實驗室檢測方法。研究結(jié)論:HIV-1感染者針對p24和gp41的體液免疫應(yīng)答模式存在較大差異,此為依據(jù),初步建立了一種區(qū)分HIV-1新近感染和長期感染的實驗室檢測方法。
[Abstract]:Background: human immunodeficiency virus (Human Immunodeficiency Virus, HIV) is the leading cause of acquired immunodeficiency syndrome (Acquired Immunodeficiency, Syndrome, AIDS) of the pathogen.HIV-1 transmembrane glycoprotein gp41 in HIV-1 protein in many of the most conservative, can induce a neutralizing antibody, and antigen detection is a common target for vaccine development the immune detection kit. P24 virus capsid protein in the protein content in the most abundant, its amino acid sequence is highly conserved in various subtypes, but also infected the earliest detectable antigen, which has also become important biomarkers. The study found that the HIV-1 infection exists in the change of humoral immune response model the p24 and gp41 suggests that the immunodominant epitopes can be biomarkers to distinguish different patterns of infection, to establish a laboratory testing method with favorable value. Research Research objective: this topic through recombinant p24, gp41 protein synthesis, p24, gp41 series of peptides as antigen on immune responses and HIV-1 infection in peripheral blood and viral proteins or peptides, humoral immune response model of HIV-1 infection and its characteristics, correlation analysis of HIV-1 infected body fluids the immune response pattern and infection progression index, found that the use value of the special biological markers, establish a detection method can distinguish the different infection stages in laboratory. Methods: to construct p24 recombinant expression plasmid, transformed into E.coli competent bacteria. After induced expression of soluble recombinant protein and the recombinant proteins were purified. The product by SDS-PAGE electrophoresis and indirect ELISA analysis identified the activity expression. HIV-1 positive plasma samples were collected from different groups, using the commercial kit divides the sample into the recent Infection and long-term infection, infection in different populations in different observation distributions. 3 were synthesized covering p24 and gp41 in different regions of the peptide. Analysis of different populations, the immune response characteristics of different infection period samples with recombinant protein and peptide analysis. The immune response of different infection period and the peptide and screened with peptide HIV-1 distinguish different infection stages. The ability to optimize the reaction conditions, the initial establishment of detection method to distinguish different HIV-1 infection during the laboratory. By comparing the identification results of two kinds of commercial kit and the evaluation method of sensitivity and specificity. Results: the successful construction of p24 prokaryotic expression plasmid and induce the expression of protein electrophoresis, in line with expectations, indirect ELISA analysis showed that p24 with antigen specific.88.3% (233/264) samples and three of three peptides covering p24 major immunodominant epitopes of anti We should, by contrast, 87.7% (236/269) samples with at least one gp41 peptide reaction. With prolonged infection, HIV-1 infection in vivo gp41-p1 specific antibody titers gradually increased, the correlation coefficient between the OD value for 0.78. gp41 and free gp41-p1 as capture antigen and gp41-p1 infection the enzyme, gp41 antibody and gp41-p1 antibody negative defined as infection, gp41 antibody and gp41-p1 antibody positive is defined as a long-term infection, we established a new HIV-1 and can distinguish the long-term infection of laboratory testing method. Conclusion: HIV-1 infection in p24 and gp41 of the humoral immune response model has obvious difference, this is according to preliminary set up a distinction between newly infected HIV-1 and long-term infection of laboratory testing method.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.91

【參考文獻】

相關(guān)期刊論文 前1條

1 ;Immune Responses to Six Synthetic Peptides of Capsid Protein with Sera from HIV-1 Infected Individuals[J];Cellular & Molecular Immunology;2005年04期

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本文編號：1540161