HIV-1感染者抗p24和gp41体液免疫应答的特征及流行病学意义

发布时间：2018-02-26 23:27

本文关键词： HIV-1 p24 gp41 体液免疫近期感染长期感染　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:人类免疫缺陷病毒(Human Immunodeficiency Virus,HIV)是导致获得性免疫缺陷综合征(Acquired Immunodeficiency Syndrome,AIDS)的病原体。HIV-1 的跨膜糖蛋白gp41在HIV-1众多蛋白中最为保守,能诱导产生多个中和抗体,是免疫检测试剂盒中最为常见的检测抗原及疫苗研发的目标。衣壳蛋白p24在病毒蛋白中含量最为丰富,其氨基酸序列在多种亚型中高度保守,也是感染后最早能检测到的抗原,使其也成为重要的生物标志物。研究发现在HIV-1感染者体内存在体液免疫应答模式的转变,提示我们p24和gp41的免疫优势表位或能成为区分不同感染模式的生物标志物,对建立实验室检测方法有利用价值。研究目的:本课题通过重组表达的p24、gp41蛋白,合成p24、gp41的系列肽段作为包被抗原,探究与HIV-1感染人群外周血与病毒蛋白或肽段的免疫反应特征,探讨HIV-1感染者的体液免疫应答模式及其特点,分析HIV-1感染者体液免疫应答模式与感染者病程进展指标的关联,发现有使用价值的特殊生物标志物,建立能区分不同感染时期的实验室检测方法。研究方法:构建p24的重组表达质粒,转化至大肠杆菌感受态细菌。经过诱导表达可溶性重组蛋白并对重组蛋白进行纯化。表达产物用SDS-PAGE电泳和间接ELISA分析鉴定其活性。收集不同人群来源的HIV-1阳性血浆样本,利用商品化试剂盒将样本区分为近期感染和长期感染,观察不同人群在感染时期分布上的差异。分别合成3条覆盖p24和gp41不同区域的肽段。分析不同人群,不同感染时期的样本与重组蛋白及肽段的免疫反应特点。分析不同感染时期的样本与肽段的免疫应答情况,筛选出具有区分HIV-1不同感染时期能力的肽段。优化反应条件,初步建立区分HIV-1不同感染时期的实验室检测方法。通过与两种商品化试剂盒鉴定结果的比较,评价方法的敏感性和特异性。研究结果:成功构建p24的原核表达质粒并诱导表达,蛋白电泳与预期相符,间接ELISA分析显示p24具有抗原特异性。88.3%(233/264)的样本不与三个覆盖p24主要免疫优势表位的三个肽段反应,与此形成对比的是,87.7%(236/269)的样本至少与一个gp41的肽段反应。随着感染时间的延长,HIV-1感染者体内gp41-p1特异性抗体滴度逐步上升,感染时间与gp41-p1的酶免实验OD值之间的相关系数为0.78。将gp41和gp41-p1作为捕捉抗原,gp41抗体阳性和gp41-p1抗体阴性定义为近期感染,gp41抗体阳性和gp41-p1抗体阳性定义为长期感染,初步建立了一种能区分HIV-1新近和长期感染的实验室检测方法。研究结论:HIV-1感染者针对p24和gp41的体液免疫应答模式存在较大差异,此为依据,初步建立了一种区分HIV-1新近感染和长期感染的实验室检测方法。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91

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