結(jié)核分枝桿菌T細(xì)胞抗原表位編碼基因多態(tài)性分析及4個(gè)VNTR位點(diǎn)的篩選與評估
發(fā)布時(shí)間：2018-08-06 21:36
【摘要】：第一部分 結(jié)核分枝桿菌T細(xì)胞抗原表位編碼基因多態(tài)性分析 結(jié)核病的感染、發(fā)生、發(fā)展及轉(zhuǎn)歸都與機(jī)體細(xì)胞免疫反應(yīng)相關(guān),其中,T淋巴細(xì)胞在機(jī)體對結(jié)核分枝桿菌的免疫應(yīng)答中起重要作用。T淋巴細(xì)胞識(shí)別外來抗原依賴于短肽片段(即表位),它是由外源蛋白水解產(chǎn)生的,并與主要組織相容復(fù)合體(MHC)結(jié)合。在對某些病毒、細(xì)菌及原生生物(如HIV-1、丙型肝炎病毒、惡性瘧原蟲及腦膜炎球菌)的研究表明編碼抗原的基因?yàn)榱颂颖芩拗鞯拿庖弑憩F(xiàn)為高度可變。然而,在對結(jié)核分枝桿菌在宿主免疫壓力下的抗原的變化及由此產(chǎn)生的進(jìn)化機(jī)制的研究較少。對結(jié)核分枝桿菌的T細(xì)胞抗原表位的研究可以進(jìn)一步理解結(jié)核分枝桿菌與宿主之間的相互作用機(jī)制和結(jié)核菌抗原分子引起的免疫應(yīng)答反應(yīng),進(jìn)一步揭示結(jié)核菌的免疫學(xué)致病機(jī)制,對改進(jìn)免疫學(xué)診斷的方法及進(jìn)行新疫苗的研究具有重要意義。 在本次研究中,我們選取了有代表性的180株中國菌株,并對其480個(gè)T細(xì)胞抗原表位基因進(jìn)行PCR擴(kuò)增,比較其在基因及氨基酸水平的差異,篩選出可能發(fā)生免疫逃逸的抗原表位及蛋白,同時(shí)采用mega5.0軟件將這些表位差異用于基因分型及進(jìn)化分析,揭示結(jié)核分枝桿菌在與機(jī)體相互作用方面的遺傳進(jìn)化關(guān)系。 結(jié)果表明,在480個(gè)抗原表位中,有415個(gè)表位序列高度保守,65個(gè)表位在基因水平發(fā)生了變異,占13.54%；在氨基酸水平上,共有60個(gè)表位發(fā)生了氨基酸的改變,占12.5%。有18個(gè)蛋白在基因水平發(fā)生了變化,變化較大的幾個(gè)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,它們都發(fā)生了兩個(gè)及以上氨基酸的改變。在本次研究中的480個(gè)表位中,dN/dS的值為1.38。有12個(gè)基因的dN/dS的值大于1,這說明這些基因在遺傳學(xué)上是有壓力選擇的作用而可能發(fā)生免疫逃逸。 以編碼表位基因多態(tài)性對菌株進(jìn)行分型,可將180株菌株分為9大簇,有些Spoligotyping型(如T家族、U家族、CAS家族、H37Rvfamily及BCG)表現(xiàn)為一定的聚集性。但是其余Spoligotyping型的菌株在各簇中分散分布,尤其是北京家族沒有表現(xiàn)明顯的聚集性,說明北京家族的菌株在與機(jī)體的T細(xì)胞相互作用發(fā)生免疫反應(yīng)方面是不同的,存在著明顯的遺傳異質(zhì)性。去掉同義突變和隨機(jī)突變位點(diǎn)可將180株菌株分為3大簇。從該角度對菌株進(jìn)行分型,體現(xiàn)了結(jié)核分枝桿菌與機(jī)體T細(xì)胞相互作用的差異,有助于我們對不同的菌型制定不同的免疫策略。 第二部分 4個(gè)VNTR位點(diǎn)用于中國結(jié)核分枝桿菌臨床分離菌株分型的評價(jià) 結(jié)核分枝桿菌的基因分型研究結(jié)果顯示全世界的結(jié)核病的流行主要由幾種結(jié)核分枝桿菌家族引起,并且不同的基因家族各具有獨(dú)特的分子特征、地區(qū)性分布和致病性。分枝桿菌散在重復(fù)單元-可變數(shù)目串聯(lián)重復(fù)序列(mycobacterial interspersed repetitive units-variable number tandem repeat, MIRU-VNTR)分型方法是研究結(jié)核分枝桿菌基因分型常用的技術(shù)方法之一,已廣泛應(yīng)用于世界各地的結(jié)核病流行病學(xué)研究中。不同VNTR位點(diǎn)的分辨率不同,用不同的VNTR位點(diǎn)組合可以得到不同的基因分型結(jié)果。目前12位點(diǎn)VNTR分型方法應(yīng)用較為廣泛并推薦作為結(jié)核控制中的常規(guī)方法。之后,15位點(diǎn)VNTR和24位點(diǎn)VNTR被推薦為結(jié)核分枝桿菌VNTR分型的標(biāo)準(zhǔn)方法。 本研究用中國疾病預(yù)防控制中心傳染病所結(jié)核室CCDC5079(CP002884)及CCDC5180(CP002885)兩株已經(jīng)完成全基因組測序的菌株序列,以及NCBI發(fā)布的7株(H37Rv,H37Ra,F11,Bovis, BCG-Pasteur, BCG-Tokyo和CDCl551)全基因組序列,經(jīng)9株菌株全基因組比對,篩選出4個(gè)候選VNTR位點(diǎn)BJ1、BJ2、BJ3和BJ4。采用此4個(gè)位點(diǎn)對225株中國臨床分離的分枝桿菌復(fù)合群菌株進(jìn)行基因分型并對其分型效果進(jìn)行評估。 結(jié)果表明,在用此4位點(diǎn)可將225株菌分成6個(gè)基因簇,165個(gè)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。位點(diǎn)BJl,BJ2,BJ3和BJ4對225株菌分型的HGI分別為0.634,0.917,0.697和0.910。四個(gè)位點(diǎn)組合的HGI達(dá)到0.995,說明了很好的分辨率和基因分型能力。此外,采用此4位點(diǎn)VNTR對126株北京家族分成15亞簇。對126株北京家族分型的HGI分別為0.447,0.878,0.315和0.850。四個(gè)位點(diǎn)組合的HGI達(dá)到0.988。 牛分枝桿菌和BCG菌株在BJ1(1.0)和BJ2(5.5)的重復(fù)次數(shù)與其它分枝桿菌不同,可能用于結(jié)核分枝桿菌和牛分枝桿菌及BCG菌株的鑒定。此外,FJ06057為非洲分枝桿菌,它在位點(diǎn)BJ1也表現(xiàn)了獨(dú)特的拷貝數(shù)(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).
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378
本文編號(hào)：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).
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 全國結(jié)核病流行病學(xué)抽樣調(diào)查技術(shù)指導(dǎo)組;第四次全國結(jié)核病流行病學(xué)抽樣調(diào)查報(bào)告[J];中華結(jié)核和呼吸雜志;2002年01期
，本文編號(hào)：2169059
本文鏈接：http://sikaile.net/xiyixuelunwen/2169059.html
最近更新
教材專著
