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
发布时间：2018-08-06 21:36
【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)。
[Abstract]:Part one
Polymorphism analysis of T cell epitope coding gene in Mycobacterium tuberculosis
The infection, occurrence, development and outcome of tuberculosis are related to the cellular immune response of the body. Among them, T lymphocytes play an important role in the immune response of the organism to Mycobacterium tuberculosis..T lymphocyte recognition is dependent on the short peptide fragment (the epitopes), which is produced by the hydrolysis of exogenous proteins and is compatible with the major tissue (MH). C) binding. Studies on certain viruses, bacteria and protists (such as HIV-1, hepatitis C virus, Plasmodium falciparum and meningococcal) suggest that the genes encoding the antigen are highly variable in order to escape the host's immune performance. However, the changes in the antigen of Mycobacterium tuberculosis under the host immune pressure and the resulting evolutionary machine Research on the T cell antigen epitopes of Mycobacterium tuberculosis can further understand the interaction mechanism between Mycobacterium tuberculosis and host and the immune response induced by Mycobacterium tuberculosis, further reveal the immunological pathogenesis of Mycobacterium tuberculosis, the methods for improving immunological diagnosis and new vaccines. The research is of great significance.
In this study, we selected 180 representative strains of Chinese strains, and amplified 480 T cell antigen epitopes, compared the difference in gene and amino acid levels, screened out the potential antigen epitopes and proteins that might have immune escape, and used mega5.0 software to apply these epitope differences to genotyping and entry. Chemical analysis revealed the genetic evolution relationship between Mycobacterium tuberculosis and organism.
The results showed that in the 480 epitopes, 415 epitopes were highly conserved and 65 epitopes were mutated at the gene level, accounting for 13.54%. At the amino acid level, there were 60 epitopes of amino acids, which accounted for 18 of 12.5%. proteins at the gene level, and a few epitopes in the PstS1 gene and esxL base. Because of the MPT64, esxO, lppX, and Hypothetical protein MT0322 genes, they all changed two and more amino acids. In the 480 epitopes of this study, the value of dN/dS is more than 1 of the dN/dS of 12 genes of 1.38., which suggests that these genes are likely to have the role of pressure selection in genetics. Immune escape.
180 strains could be divided into 9 large clusters by coding epitope gene polymorphism, and some Spoligotyping types (such as T family, U family, CAS family, H37Rvfamily and BCG) showed a certain aggregation. But the other Spoligotyping strains were distributed among the clusters, especially in the Beijing family, which showed no obvious aggregation. The strains of the Ming Beijing family are different in the immune response to the interaction of the T cells of the body, and there are obvious genetic heterogeneity. 180 strains can be divided into 3 large clusters by removing the synonymous mutation and random mutation sites. The strains are typed from this angle, which reflects the difference of the interaction between Mycobacterium tuberculosis and the body T cells. It helps us to develop different immunization strategies for different types of bacteria.
The second part
Evaluation of 4 VNTR loci typing for clinical isolates of Mycobacterium tuberculosis in China
The results of the genotyping of Mycobacterium tuberculosis show that the epidemic of tuberculosis in the world is mainly caused by several Mycobacterium tuberculosis families, and the different gene families have unique molecular characteristics, regional distribution and pathogenicity. Mycobacterium is scattered in the repeat unit variable number tandem repeat sequence (mycobacterial interspers). Ed repetitive Units-Variable number tandem repeat, MIRU-VNTR) classification method is one of the most commonly used techniques for the study of Mycobacterium tuberculosis genotyping. It has been widely used in the epidemiological study of tuberculosis in all parts of the world. Different resolution of different VNTR loci can be used in different VNTR loci combinations to obtain different genotypes. At present, the 12 site VNTR typing method is widely used and is recommended as a routine method in tuberculosis control. After that, the 15 site VNTR and 24 site VNTR are recommended as the standard method for the VNTR typing of Mycobacterium tuberculosis.
In this study, two strains of CCDC5079 (CP002884) and CCDC5180 (CP002885) have been sequenced, and 7 strains of NCBI (H37Rv, H37Ra, F11, Bovis, BCG-Pasteur, BCG-Tokyo and CDCl551) were sequenced, and 4 strains were screened by the total genome alignment of 9 strains. The candidate VNTR loci BJ1, BJ2, BJ3, and BJ4. were genotyping and evaluating the genotyping effect of 225 strains of Mycobacterium tumefaciens isolated from China.
The results show that 225 strains of bacteria can be divided into 6 gene clusters, 165 genotypes and the largest gene cluster (cluster V) including 139 strains, 111 of which are Beijing family strains. The HGI, BJ2, BJ3 and BJ4 of 225 strains of HGI are 0.995 in 0.634,0.917,0.697 and 0.910. four loci respectively, indicating a good score. In addition, 126 Beijing families were divided into 15 subclusters with the 4 loci VNTR, and the HGI of 126 Beijing families with the four loci of 0.447,0.878,0.315 and 0.850., respectively, reached 0.988., respectively.
The repetitions of Mycobacterium bovis and BCG strains in BJ1 (1) and BJ2 (5.5) are different from those of other mycobacteria. It may be used for identification of Mycobacterium tuberculosis and Mycobacterium bovis and BCG strains. In addition, FJ06057 is a Mycobacterium African, and its site BJ1 also shows a unique copy number (8).
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R378
本文编号：2169059
[Abstract]:Part one
Polymorphism analysis of T cell epitope coding gene in Mycobacterium tuberculosis
The infection, occurrence, development and outcome of tuberculosis are related to the cellular immune response of the body. Among them, T lymphocytes play an important role in the immune response of the organism to Mycobacterium tuberculosis..T lymphocyte recognition is dependent on the short peptide fragment (the epitopes), which is produced by the hydrolysis of exogenous proteins and is compatible with the major tissue (MH). C) binding. Studies on certain viruses, bacteria and protists (such as HIV-1, hepatitis C virus, Plasmodium falciparum and meningococcal) suggest that the genes encoding the antigen are highly variable in order to escape the host's immune performance. However, the changes in the antigen of Mycobacterium tuberculosis under the host immune pressure and the resulting evolutionary machine Research on the T cell antigen epitopes of Mycobacterium tuberculosis can further understand the interaction mechanism between Mycobacterium tuberculosis and host and the immune response induced by Mycobacterium tuberculosis, further reveal the immunological pathogenesis of Mycobacterium tuberculosis, the methods for improving immunological diagnosis and new vaccines. The research is of great significance.
In this study, we selected 180 representative strains of Chinese strains, and amplified 480 T cell antigen epitopes, compared the difference in gene and amino acid levels, screened out the potential antigen epitopes and proteins that might have immune escape, and used mega5.0 software to apply these epitope differences to genotyping and entry. Chemical analysis revealed the genetic evolution relationship between Mycobacterium tuberculosis and organism.
The results showed that in the 480 epitopes, 415 epitopes were highly conserved and 65 epitopes were mutated at the gene level, accounting for 13.54%. At the amino acid level, there were 60 epitopes of amino acids, which accounted for 18 of 12.5%. proteins at the gene level, and a few epitopes in the PstS1 gene and esxL base. Because of the MPT64, esxO, lppX, and Hypothetical protein MT0322 genes, they all changed two and more amino acids. In the 480 epitopes of this study, the value of dN/dS is more than 1 of the dN/dS of 12 genes of 1.38., which suggests that these genes are likely to have the role of pressure selection in genetics. Immune escape.
180 strains could be divided into 9 large clusters by coding epitope gene polymorphism, and some Spoligotyping types (such as T family, U family, CAS family, H37Rvfamily and BCG) showed a certain aggregation. But the other Spoligotyping strains were distributed among the clusters, especially in the Beijing family, which showed no obvious aggregation. The strains of the Ming Beijing family are different in the immune response to the interaction of the T cells of the body, and there are obvious genetic heterogeneity. 180 strains can be divided into 3 large clusters by removing the synonymous mutation and random mutation sites. The strains are typed from this angle, which reflects the difference of the interaction between Mycobacterium tuberculosis and the body T cells. It helps us to develop different immunization strategies for different types of bacteria.
The second part
Evaluation of 4 VNTR loci typing for clinical isolates of Mycobacterium tuberculosis in China
The results of the genotyping of Mycobacterium tuberculosis show that the epidemic of tuberculosis in the world is mainly caused by several Mycobacterium tuberculosis families, and the different gene families have unique molecular characteristics, regional distribution and pathogenicity. Mycobacterium is scattered in the repeat unit variable number tandem repeat sequence (mycobacterial interspers). Ed repetitive Units-Variable number tandem repeat, MIRU-VNTR) classification method is one of the most commonly used techniques for the study of Mycobacterium tuberculosis genotyping. It has been widely used in the epidemiological study of tuberculosis in all parts of the world. Different resolution of different VNTR loci can be used in different VNTR loci combinations to obtain different genotypes. At present, the 12 site VNTR typing method is widely used and is recommended as a routine method in tuberculosis control. After that, the 15 site VNTR and 24 site VNTR are recommended as the standard method for the VNTR typing of Mycobacterium tuberculosis.
In this study, two strains of CCDC5079 (CP002884) and CCDC5180 (CP002885) have been sequenced, and 7 strains of NCBI (H37Rv, H37Ra, F11, Bovis, BCG-Pasteur, BCG-Tokyo and CDCl551) were sequenced, and 4 strains were screened by the total genome alignment of 9 strains. The candidate VNTR loci BJ1, BJ2, BJ3, and BJ4. were genotyping and evaluating the genotyping effect of 225 strains of Mycobacterium tumefaciens isolated from China.
The results show that 225 strains of bacteria can be divided into 6 gene clusters, 165 genotypes and the largest gene cluster (cluster V) including 139 strains, 111 of which are Beijing family strains. The HGI, BJ2, BJ3 and BJ4 of 225 strains of HGI are 0.995 in 0.634,0.917,0.697 and 0.910. four loci respectively, indicating a good score. In addition, 126 Beijing families were divided into 15 subclusters with the 4 loci VNTR, and the HGI of 126 Beijing families with the four loci of 0.447,0.878,0.315 and 0.850., respectively, reached 0.988., respectively.
The repetitions of Mycobacterium bovis and BCG strains in BJ1 (1) and BJ2 (5.5) are different from those of other mycobacteria. It may be used for identification of Mycobacterium tuberculosis and Mycobacterium bovis and BCG strains. In addition, FJ06057 is a Mycobacterium African, and its site BJ1 also shows a unique copy number (8).
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R378
【参考文献】
相关期刊论文 前1条
1 全国结核病流行病学抽样调查技术指导组;第四次全国结核病流行病学抽样调查报告[J];中华结核和呼吸杂志;2002年01期
，本文编号：2169059
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