结核分枝杆菌T细胞抗原表位编码基因多态性分析及4个VNTR位点的筛选与评估

【作者】 蒋毅；

【导师】 阚飙； 万康林；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2012， 博士

【摘要】 第一部分结核分枝杆菌T细胞抗原表位编码基因多态性分析结核病的感染、发生、发展及转归都与机体细胞免疫反应相关,其中,T淋巴细胞在机体对结核分枝杆菌的免疫应答中起重要作用。T淋巴细胞识别外来抗原依赖于短肽片段(即表位),它是由外源蛋白水解产生的,并与主要组织相容复合体(MHC)结合。在对某些病毒、细菌及原生生物(如HIV-1、丙型肝炎病毒、恶性疟原虫及脑膜炎球菌)的研究表明编码抗原的基因为了逃避宿主的免疫表现为高度可变。然而,在对结核分枝杆菌在宿主免疫压力下的抗原的变化及由此产生的进化机制的研究较少。对结核分枝杆菌的T细胞抗原表位的研究可以进一步理解结核分枝杆菌与宿主之间的相互作用机制和结核菌抗原分子引起的免疫应答反应,进一步揭示结核菌的免疫学致病机制,对改进免疫学诊断的方法及进行新疫苗的研究具有重要意义。在本次研究中,我们选取了有代表性的180株中国菌株,并对其480个T细胞抗原表位基因进行PCR扩增,比较其在基因及氨基酸水平的差异,筛选出可能发生免疫逃逸的抗原表位及蛋白,同时采用mega5.0软件将这些表位差异用于基因分型及进化分析,揭示结核分枝杆菌在与机体相互作用方面的遗传进化关系。结果表明,在480个抗原表位中,有415个表位序列高度保守,65个表位在基因水平发生了变异,占13.54%；在氨基酸水平上,共有60个表位发生了氨基酸的改变,占12.5%。有18个蛋白在基因水平发生了变化,变化较大的几个表位位于PstS1基因、esxL基因、MPT64基因、esxO基因、lppX基因和Hypothetical protein MT0322基因中,它们都发生了两个及以上氨基酸的改变。在本次研究中的480个表位中,dN/dS的值为1.38。有12个基因的dN/dS的值大于1,这说明这些基因在遗传学上是有压力选择的作用而可能发生免疫逃逸。以编码表位基因多态性对菌株进行分型,可将180株菌株分为9大簇,有些Spoligotyping型(如T家族、U家族、CAS家族、H37Rvfamily及BCG)表现为一定的聚集性。但是其余Spoligotyping型的菌株在各簇中分散分布,尤其是北京家族没有表现明显的聚集性,说明北京家族的菌株在与机体的T细胞相互作用发生免疫反应方面是不同的,存在着明显的遗传异质性。去掉同义突变和随机突变位点可将180株菌株分为3大簇。从该角度对菌株进行分型,体现了结核分枝杆菌与机体T细胞相互作用的差异,有助于我们对不同的菌型制定不同的免疫策略。第二部分4个VNTR位点用于中国结核分枝杆菌临床分离菌株分型的评价结核分枝杆菌的基因分型研究结果显示全世界的结核病的流行主要由几种结核分枝杆菌家族引起,并且不同的基因家族各具有独特的分子特征、地区性分布和致病性。分枝杆菌散在重复单元-可变数目串联重复序列(mycobacterial interspersed repetitive units-variable number tandem repeat, MIRU-VNTR)分型方法是研究结核分枝杆菌基因分型常用的技术方法之一,已广泛应用于世界各地的结核病流行病学研究中。不同VNTR位点的分辨率不同,用不同的VNTR位点组合可以得到不同的基因分型结果。目前12位点VNTR分型方法应用较为广泛并推荐作为结核控制中的常规方法。之后,15位点VNTR和24位点VNTR被推荐为结核分枝杆菌VNTR分型的标准方法。本研究用中国疾病预防控制中心传染病所结核室CCDC5079(CP002884)及CCDC5180(CP002885)两株已经完成全基因组测序的菌株序列,以及NCBI发布的7株(H37Rv,H37Ra,F11,Bovis, BCG-Pasteur, BCG-Tokyo和CDCl551)全基因组序列,经9株菌株全基因组比对,筛选出4个候选VNTR位点BJ1、BJ2、BJ3和BJ4。采用此4个位点对225株中国临床分离的分枝杆菌复合群菌株进行基因分型并对其分型效果进行评估。结果表明,在用此4位点可将225株菌分成6个基因簇,165个基因型。最大的基因簇(cluster V)包括139株菌,其中111株是北京家族菌株。位点BJl,BJ2,BJ3和BJ4对225株菌分型的HGI分别为0.634,0.917,0.697和0.910。四个位点组合的HGI达到0.995,说明了很好的分辨率和基因分型能力。此外,采用此4位点VNTR对126株北京家族分成15亚簇。对126株北京家族分型的HGI分别为0.447,0.878,0.315和0.850。四个位点组合的HGI达到0.988。牛分枝杆菌和BCG菌株在BJ1(1.0)和BJ2(5.5)的重复次数与其它分枝杆菌不同,可能用于结核分枝杆菌和牛分枝杆菌及BCG菌株的鉴定。此外,FJ06057为非洲分枝杆菌,它在位点BJ1也表现了独特的拷贝数(8.0)。更多还原

【Abstract】 Part ⅠDiversity in the encoding genes of T cell epitopes in Mycobacterium tuberculosisInfection, occurrence, development and prognosis of tuberculosis (TB) are dependent on the different mechanisms of the cell immune response of the body. T lymphocytes play an important role in immunity to tuberculosis in humans. Recognition of foreign antigens by T lymphocytes depends on binding of short peptide fragments (termed epitopes), derived by proteolysis of foreign proteins, to the major histocompatibility complex (MHC) proteins on the surfaces of macrophages and dendritic cells. Studies in human pathogenic viruses, bacteria and protozoa have revealed that genes encoding antigens tend to be highly variable as a consequence of diversifying selection to evade host immunity. However, there are few studies on whether similar evolutionary mechanisms operate in TB and whether the bacteria undergo antigenic variation in response to host immune pressure. Studies on T cell epitopes of Mycobacterium tuberculosis (M. tuberculosis) can help us further understand the mechanism of interaction between TB and host, immune response caused by TB antigen molecules and pathogenesis of TB. In addition, it benefits to improve the methods of immune diagnosis and develop new vaccines.In this study, we chose180representative strains in China for amplifying the gene sequences of480T cell epitopes in M.tuberculosis genome, comparing the differences of these epitopes in gene and amino acids levels and finding out epitopes and even proteins which probably undergo antigenic variation in response to host immune pressure. Mega5.0software was used to genotype and analyze these strains phylogeneticly. It was revealed the phylogenetic and evolutionary relationship between TB strains and human.The results showed that415epitopes were hyperconserved and65epitopes (13.54%) changes in gene level among all480epitopes. There were60epitopes changes in amino acid level, which accounted for12.5%.18proteins changed in gene sequences and6proteins (PstS1, esxL, MPT64, esxO, lppx and MT0322) had the greater changes with at least2amino acid changes. Among480epitopes in this study, the value of dN/dS is1.38. dN/dS values of12genes were above one, suggesting that these genes may undergo antigenic variation in response to host immune pressure.180strains were genotyped into9major clusters based on polymorphism in480epitopes. Some strains with certain spoligotypes like T, U, CAS, H37Rv family and BCG presented a certain degree of aggregation. However, other stains were dispersed in different clusters. Beijing family strains displayed no significant aggregation. This indicated that Beijing strains presented diversify in interaction between human T cell and pathogen. After synonymous mutations and random mutations were removed,180strains were divided into3major clusters. Genotypes based on this method reveal the diversify interaction between TB strains and human T cell. Part IIEvaluation of Four Candidate VNTR Loci for Genotyping225Chinese Clinical Mycobacterium Tuberculosis Complex StrainsThe genotyping results of Mycobacterium tuberculosis (M tuberculosis) showed that the prevalence of tuberculosis in the world mainly caused by several special M. tuberculosis genetic lineage (family) strains. Each family strains have unique molecular characteristics, regional distribution and pathogenicity. The method with variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) is one of the common methods for the genotyping M. tuberculosis, and has been widely used in epidemiological research of tuberculosis around the world. Combinations of different multiple VNTR loci can get different genotype clusters and has different discriminatory power. Currently,12-locus VNTR is the most widely used and has been integrated in TB control systems on a national scale. Later after that, the15-locus and24-locus sets have been proposed as a basis for standardized MIRU-VNTR typing of M. tuberculosis.Here, by sequencing2Chinese M. tuberculosis strains CCDC5079(CP002884) and CCDC5180(CP002885) and using published sequence data,4promising VNTR loci were identified and applied to genotype225Chinese clinical M. tuberculosis complex strains, and then the discriminatory power was evaluated.The results showed that225strains had165distinct profiles and were divided into6clusters by these4-VNTRs. The predominant cluster (cluster V) included139strains. Most of them were Beijing family (111). Hunter-Gaston Index (HGI) of BJ1, BJ2, BJ3and BJ4was0.634,0.917,0.697and0.910respectively. The HGI of combination of4loci was0.995suggested a good discriminatory power. In addition,126Beijing strains could be divided into15subculsters by these4new VNTR loci, HGI of BJ1, BJ2, BJ3and BJ4in genotyping Beijing family strains were0.447,0.878,0.315and0.850respectively. The HGI of combination of4loci in genotyping Beijing family strains was0.988.It seemed that the M. bovis strains and M. bovis-BCG family strains have the unique copy number in BJ1and BJ2of which the repeat number was1.0and5.5respectively, which may be applicable to separate M. bovis and M. bovis-BCG family strains from clinical M. tuberculosis complex strains. In addition, BJ1pattern of FJ06057presents unique high copy number8.0in225strains, which indicate a special characteristic in M. africanum strain.更多还原