發(fā)布時間：2018-02-03 17:56

  本文關(guān)鍵詞： 結(jié)核分枝桿菌 卡介苗 B細(xì)胞抗原表位 dN/dS　出處：《中國疾病預(yù)防控制中心》2016年博士論文　論文類型：學(xué)位論文

【摘要】：結(jié)核病是一種主要由結(jié)核分枝桿菌感染導(dǎo)致的慢性傳性疾病。傳統(tǒng)的觀點認(rèn)為結(jié)核病的感染免疫以細(xì)胞免疫為主,但越來越多的研究表明B細(xì)胞主導(dǎo)的體液免疫反應(yīng)在抗原呈遞、刺激機體產(chǎn)生保護(hù)性抗體、調(diào)節(jié)宿主免疫應(yīng)答等方面起到重要作用。B細(xì)胞抗原表位是抗原表面與B細(xì)胞相互識別、刺激B細(xì)胞產(chǎn)生抗體或分泌細(xì)胞因子、調(diào)節(jié)免疫應(yīng)答的特殊結(jié)構(gòu)�？ń槊�(Bacille Calmette-Guerin, BCG),是牛結(jié)核分枝桿菌連續(xù)傳代培養(yǎng)而得到減毒活疫苗。盡管一直以來有關(guān)卡介苗在人群中接種產(chǎn)生的保護(hù)效果存在較大爭議,但BCG仍是迄今為止唯一獲準(zhǔn)在全球廣泛使用的預(yù)防結(jié)核的疫苗。卡介苗刺激機體產(chǎn)生免疫保護(hù)的機制仍然不是十分清楚；且卡介苗在全球范圍內(nèi)的廣泛使用,產(chǎn)生了多個不同的克隆,每個克隆所具有的保護(hù)性抗原、理化性質(zhì)、免疫特征等不同,所產(chǎn)生的免疫保護(hù)效果也存在差異。為了對機體的體液免疫系統(tǒng)進(jìn)行深入的認(rèn)識、為新型疫苗的研發(fā)提供必要的數(shù)據(jù)支持,我們從結(jié)核分枝桿菌的人B細(xì)胞抗原表位入手,對來自全球不同地區(qū)的卡介苗基因組以及180株結(jié)核分枝桿菌國內(nèi)臨床分離菌株中B細(xì)胞抗原表位編碼基因采用多種生物分析軟件進(jìn)行比較分析,研究其在不同卡介苗菌株及結(jié)核分枝桿菌菌株中的分布差異、以及可能對不同卡介苗所產(chǎn)生的保護(hù)力和不同結(jié)核分枝桿菌菌株的致病能力所產(chǎn)生的潛在影響。結(jié)果表明,目前已知的399個結(jié)核分枝桿菌人B細(xì)胞原表位分別由81個基因編碼；無論是在卡介苗菌株中,還是在結(jié)核分枝桿菌臨床分離菌株中,絕大多數(shù)B細(xì)胞抗原表位均高度保守。在13株卡介苗菌株的基因組中321個B細(xì)胞抗原表位的編碼序列未發(fā)生任何變化；全部表位根據(jù)變化情況可分為5個Group, Group1包含321個B細(xì)胞抗原表位、在全部卡介苗中均高度保守；Group2包含15個B細(xì)胞抗原表位、在全部卡介苗中存在相同的點突變；Group3包含26個B細(xì)胞抗原表位、在全部卡介苗中缺失；Group4包含13個B細(xì)胞抗原表位、在部分卡介苗中缺失；Group5包含23個B細(xì)胞抗原表位、在不同的卡介苗中發(fā)生特異性的變化。BCG-Tokyo 172和BCG-China菌株中擁有357個完整的結(jié)核分枝桿菌人B細(xì)胞抗原表位,為13株卡介苗中擁有B細(xì)胞抗原表位數(shù)量最多的疫苗株,從B細(xì)胞抗原表位分布角度考慮BCG-Tokyo 172和BCG-China株在接種人群后可能刺激機體產(chǎn)生較強的抗結(jié)核體液免疫反應(yīng)。最初的卡介苗菌株在擴散階段的無限制傳代中,由于面臨不同的環(huán)境壓力、逐漸丟失部分人B細(xì)胞抗原表位,進(jìn)而演變?yōu)楸Ｗo(hù)力不同的BCG菌株；在對卡介苗進(jìn)行改造時,可以考慮采用恢復(fù)現(xiàn)有卡介苗中丟失的B細(xì)胞抗原表位的策略來增強疫苗的接種保護(hù)力。在180株結(jié)核分枝桿菌臨床分離菌株中,293個結(jié)核分枝桿菌人B細(xì)胞抗原表位未發(fā)生任何堿基序列變化,104個結(jié)核分枝桿菌人B細(xì)胞抗原表位的編碼序列發(fā)生了不同程度的變化,78個表位的多肽序列發(fā)生了變異；在150株擁有完整測序結(jié)果的菌株中,未檢測到表位缺失現(xiàn)象,1株菌中發(fā)生了12個B細(xì)胞抗原表位編碼序列的變化、為發(fā)生B細(xì)胞抗原表位編碼序列變化最多的菌株,10株菌中397個B細(xì)胞抗原表位編碼序列未發(fā)生任何變化,99.33%臨床分離菌株(149/150)中結(jié)核分枝桿菌人B細(xì)胞抗原表位的變化數(shù)量不超過10個；結(jié)核分枝桿菌臨床菌株中B細(xì)胞抗原表位的分布高度保守。在臨床菌株中,通過對人B細(xì)胞抗原表位編碼基因突變頻率的分析,發(fā)現(xiàn)結(jié)核分枝桿菌的表位編基因較為保守；在80個B細(xì)胞抗原表位編碼基因中,27.5%B細(xì)胞抗原表位編碼基因(22/80)的dN/dS比值大于1、受正向選擇作用傾向于發(fā)生免疫逃避現(xiàn)象,其余基因較為保守；核酸變異大多位于基因中非B細(xì)胞抗原表位區(qū)。在全部B細(xì)胞抗原表位編碼基因中,4個基因中發(fā)生多個菌株的基因片段插入與缺失,必將會對編碼蛋白的功能產(chǎn)生重要影響。B細(xì)胞抗原表位及編碼基因的高度保守使得結(jié)核分枝桿菌更容易被宿主識別、在人群中廣泛傳播；在全部編碼序列中,無論是B細(xì)胞抗原表位的編碼基因、B細(xì)胞抗原表位的編碼區(qū)還是非表位的編碼區(qū),大多數(shù)情況下其dN/dS值均小于1、傾向于受純化選擇壓力的作用,這也提示我們在疫苗設(shè)計的時候應(yīng)充分考慮采用添加可變抗原成分來提高疫苗的保護(hù)力。
[Abstract]:Tuberculosis is a major infection by Mycobacterium tuberculosis caused by chronic disease transmission. The traditional view is that the infection of tuberculosis in immune cells, but more and more studies show that B cells dominated the humoral immune response in antigen presentation, stimulate the body to produce protective antibody, regulating host immune response plays an important role.B cell epitope is mutual recognition with B cell surface antigen, stimulate B cells to produce antibodies or cytokine secretion, regulating immune response. The special structure of BCG (Bacille Calmette-Guerin, BCG), Mycobacterium tuberculosis is continuously subcultured and attenuated live vaccine. Although there is considerable controversy has been about the protective effect of BCG in the crowd were produced, but BCG is so far the only widely used in the world to prevent tuberculosis vaccine BCG stimulation machine. The mechanism of body immune protection is still not very clear; and the widespread use of BCG in the global scope, produced a number of different clones, protective antigen of each clone has the physicochemical properties, immune characteristics, immune protective effect generated by the differences of the humoral immune system of the body in order to. The in-depth understanding, to provide necessary data support for the research and development of new vaccines, we start from Mycobacterium tuberculosis B cell epitopes from different regions of the world and 180 strains of Mycobacterium tuberculosis BCG genome biological analysis software were analyzed by a gene encoding surface antigen of B cells of domestic clinical isolates of Mycobacterium strain, study its distribution in different strains of BCG and Mycobacterium tuberculosis strain differences, and force protection are likely to produce different BCG and different The potential impact of the pathogenicity of Mycobacterium tuberculosis strains produced. The results show that the currently known 399 Mycobacterium tuberculosis B cell epitope respectively by 81 genes encoding; both in BCG strains, or in Mycobacterium tuberculosis clinical isolates, the vast majority of B cell epitopes are highly conserved in 13 strains of BCG strain genome encoding a sequence of 321 B cell epitopes did not change any; all epitopes according to the changes can be divided into 5 Group, Group1 contains 321 B cell epitopes in all BCG vaccine are highly conserved; Group2 contains 15 B cell antigen epitope has the same mutation in all BCG; Group3 contains 26 B cell epitopes, deletion in all BCG; Group4 contains 13 B cell epitopes, deletion in part of BCG; Group5 package Containing 23 B cell epitopes, changes of specific.BCG-Tokyo in different BCG in 172 and BCG-China strains have 357 complete Mycobacterium tuberculosis B cell epitope, 13 strains of BCG have B cell epitope of the largest number of vaccine strains, considering the distribution angle of 172 and BCG-Tokyo BCG-China strain in vaccinated population may induce strong humoral immune responses of anti tuberculosis tables from the B cell antigen. The initial BCG strain in the diffusion phase unrestricted passage, in the face of pressure to the environment, gradually lost the part of human B cell epitopes, and the evolution of BCG strains of different protection in; the transformation of the BCG, you can consider using recovery strategy table B cell antigen loss of BCG vaccination to enhance existing protective vaccines. In 180 isolates of Mycobacterium tuberculosis Isolates of Mycobacterium tuberculosis, 293 human B cell epitope without any base sequence changes, changes of 104 Mycobacterium tuberculosis B cell epitope encoding sequence of the mutated peptide sequence of 78 tables; has a complete sequence of the 150 strains. Not detected, epitope deletion phenomenon, 1 strains had 12 B cell epitope encoding sequence changes for B cell epitope encoding sequence changes in most strains, 10 strains in 397 B cell epitope encoding sequence without any change in clinical isolates (99.33% 149/150) number no more than 10 changes of Mycobacterium tuberculosis in human B cell epitope; Mycobacterium tuberculosis clinical strains in the distribution of B cell epitopes are highly conserved. In clinical strains, the epitope encoding based on human B cell antigen By the analysis of mutation frequency, found Mycobacterium tuberculosis epitope encoding gene is conservative; in the 80 B cell epitope encoding gene, 27.5%B cell epitope encoding gene (22/80) dN/dS ratio was greater than 1, the positive selection is prone to immune escape phenomenon, the more conservative gene; genetic variation are located in the B cell antigen epitope gene. In all non B cell epitope encoding gene, gene fragments of multiple strains occurred in 4 genes insertion and deletion, will produce important influence of highly conservative.B cell epitopes and the gene encoding for encoding protein function to Mycobacterium tuberculosis more likely to be the host recognition, widely spread in the crowd; all in the encoding sequence, either B cell epitope encoding gene, B cell epitope encoding area or non epitope encoding region, mostly Its dN/dS value is less than 1 under a number of cases, and tends to be affected by the purification selective pressure. This suggests that we should consider adding variable antigen components to improve vaccine protection when designing vaccines.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R378.911
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本文編號：1487996