卡介苗和结核分枝杆菌中B细胞抗原表位多态性研究

发布时间：2018-02-03 17:56

  本文关键词： 结核分枝杆菌 卡介苗 B细胞抗原表位 dN/dS　出处：《中国疾病预防控制中心》2016年博士论文　论文类型：学位论文

【摘要】：结核病是一种主要由结核分枝杆菌感染导致的慢性传性疾病。传统的观点认为结核病的感染免疫以细胞免疫为主,但越来越多的研究表明B细胞主导的体液免疫反应在抗原呈递、刺激机体产生保护性抗体、调节宿主免疫应答等方面起到重要作用。B细胞抗原表位是抗原表面与B细胞相互识别、刺激B细胞产生抗体或分泌细胞因子、调节免疫应答的特殊结构。卡介苗(Bacille Calmette-Guerin, BCG),是牛结核分枝杆菌连续传代培养而得到减毒活疫苗。尽管一直以来有关卡介苗在人群中接种产生的保护效果存在较大争议,但BCG仍是迄今为止唯一获准在全球广泛使用的预防结核的疫苗。卡介苗刺激机体产生免疫保护的机制仍然不是十分清楚；且卡介苗在全球范围内的广泛使用,产生了多个不同的克隆,每个克隆所具有的保护性抗原、理化性质、免疫特征等不同,所产生的免疫保护效果也存在差异。为了对机体的体液免疫系统进行深入的认识、为新型疫苗的研发提供必要的数据支持,我们从结核分枝杆菌的人B细胞抗原表位入手,对来自全球不同地区的卡介苗基因组以及180株结核分枝杆菌国内临床分离菌株中B细胞抗原表位编码基因采用多种生物分析软件进行比较分析,研究其在不同卡介苗菌株及结核分枝杆菌菌株中的分布差异、以及可能对不同卡介苗所产生的保护力和不同结核分枝杆菌菌株的致病能力所产生的潜在影响。结果表明,目前已知的399个结核分枝杆菌人B细胞原表位分别由81个基因编码；无论是在卡介苗菌株中,还是在结核分枝杆菌临床分离菌株中,绝大多数B细胞抗原表位均高度保守。在13株卡介苗菌株的基因组中321个B细胞抗原表位的编码序列未发生任何变化；全部表位根据变化情况可分为5个Group, Group1包含321个B细胞抗原表位、在全部卡介苗中均高度保守；Group2包含15个B细胞抗原表位、在全部卡介苗中存在相同的点突变；Group3包含26个B细胞抗原表位、在全部卡介苗中缺失；Group4包含13个B细胞抗原表位、在部分卡介苗中缺失；Group5包含23个B细胞抗原表位、在不同的卡介苗中发生特异性的变化。BCG-Tokyo 172和BCG-China菌株中拥有357个完整的结核分枝杆菌人B细胞抗原表位,为13株卡介苗中拥有B细胞抗原表位数量最多的疫苗株,从B细胞抗原表位分布角度考虑BCG-Tokyo 172和BCG-China株在接种人群后可能刺激机体产生较强的抗结核体液免疫反应。最初的卡介苗菌株在扩散阶段的无限制传代中,由于面临不同的环境压力、逐渐丢失部分人B细胞抗原表位,进而演变为保护力不同的BCG菌株；在对卡介苗进行改造时,可以考虑采用恢复现有卡介苗中丢失的B细胞抗原表位的策略来增强疫苗的接种保护力。在180株结核分枝杆菌临床分离菌株中,293个结核分枝杆菌人B细胞抗原表位未发生任何碱基序列变化,104个结核分枝杆菌人B细胞抗原表位的编码序列发生了不同程度的变化,78个表位的多肽序列发生了变异；在150株拥有完整测序结果的菌株中,未检测到表位缺失现象,1株菌中发生了12个B细胞抗原表位编码序列的变化、为发生B细胞抗原表位编码序列变化最多的菌株,10株菌中397个B细胞抗原表位编码序列未发生任何变化,99.33%临床分离菌株(149/150)中结核分枝杆菌人B细胞抗原表位的变化数量不超过10个；结核分枝杆菌临床菌株中B细胞抗原表位的分布高度保守。在临床菌株中,通过对人B细胞抗原表位编码基因突变频率的分析,发现结核分枝杆菌的表位编基因较为保守；在80个B细胞抗原表位编码基因中,27.5%B细胞抗原表位编码基因(22/80)的dN/dS比值大于1、受正向选择作用倾向于发生免疫逃避现象,其余基因较为保守；核酸变异大多位于基因中非B细胞抗原表位区。在全部B细胞抗原表位编码基因中,4个基因中发生多个菌株的基因片段插入与缺失,必将会对编码蛋白的功能产生重要影响。B细胞抗原表位及编码基因的高度保守使得结核分枝杆菌更容易被宿主识别、在人群中广泛传播；在全部编码序列中,无论是B细胞抗原表位的编码基因、B细胞抗原表位的编码区还是非表位的编码区,大多数情况下其dN/dS值均小于1、倾向于受纯化选择压力的作用,这也提示我们在疫苗设计的时候应充分考虑采用添加可变抗原成分来提高疫苗的保护力。
[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.

【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】：2016
【分类号】：R378.911
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本文编号：1487996