十六个结核分枝杆菌新抗原的抗原表位鉴定与评价

发布时间：2018-07-26 06:41

【摘要】：结核病是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)引起的一种慢性传染性疾病,一直危害着人类健康。全球有三分之一的人已经感染结核分枝杆菌,有10%的人将可能在以后发展成为结核病。2015年全球新发结核病例有1040万,死于结核病的人数有140万人。近年来,随着新发结核耐药株和HIV/AIDs合并感染病例的增多,导致结核病的发病率增高、全球结核病负担日益加重,尽管从2000年到2015年,结核病引起的死亡率已经下降了 22%,结核病仍然位居引起人类死亡的十大病因之首。目前,卡介苗(BCG)仍然是全球范围内唯一获准广泛使用的用于抵抗结核病的疫苗。BCG是牛结核分枝杆菌经连续传代培养而得到的一种减毒活疫苗。虽然BCG疫苗对预防儿童中栗粒型结核和结核性脑膜炎等有较佳的效果,但BCG对成人结核病的保护率存在有很大差别,在0～80%之间,且BCG只能有效地预防原发性结核病。研究发现不同BCG疫苗株之间随着保护性抗原或者表位的丢失而导致不同的BCG株保护性差异,我们需要研发新的疫苗为更好控制结核病提供新的手段,因此我们需要寻找新的免疫优势抗原或在表位,将其用于研制重组BCG或者用于BCG接种后的加强免疫型新型疫苗。在本研究中,我们结合基因组学和生物信息学技术对H37Rv蛋白质组上的蛋白抗原进行筛选,将筛选出的蛋白抗原进行T细胞表位预测,选取预测中得分值较高的表位,对这些表位多肽进行合成。收集肺结核患者、肺部其他疾病患者和健康志愿者的外周血淋巴细胞,用多肽作为刺激原,进行进行伽马干扰素释放试验(interferon gamma release assay,IGRA),从而筛选出结核分枝杆菌免疫优势抗原或者表位。选取阳性率较高的抗原表位,与佐剂DDA和polyI:C乳化后免疫BALB/c小鼠,检测相应抗原刺激后的脾细胞上清中的IFN-γ、IL-2、IL-4和IL-10的浓度,同时检测血清中的IgG、IgG1和IgG2a抗体滴度。用流式}0胞仪检测CD3+CD4+T细胞和CD3+CD8+T细胞的比例,对这些表位的免疫原性进行评价。利用seppa2.0对B细胞表位预测,合成表位B细胞表位多肽,并利用肺结核患者和健康志愿者的血清对这些B细胞表位进行血清学评价。另外,本实验制备的重组蛋白Rv2351c,本研究中利用IGRA实验和动物实验对Rv2351c的诊断性能和免疫效果进行了评价。最终,我们共对H37Rv基因组上273个抗原的2726个T细胞表位和B细胞表位进行了预测,从中选取了来自13个抗原的265个人T细胞表位多肽和来自3个抗原的22条人B细胞抗原表位多肽进行合成。我们通过IGRA鉴定出了具有抗原性30个人T细胞抗原表位多肽,分别来自于13个结核抗原:Rv3793、Rv05 85c、Rv1490、Rv0446c、Rv 1547、Rv2201、Rv3573、Rv0197、Rv0674、Rv1566c、Rv1798、Rv2318和Rv2941。这些多肽的反应灵敏度在1.9%～30%之间,特异性在96.81%～100%之间。位于同一个抗原的多肽联合诊断后,单个抗原的反应灵敏度可达到1.9%～50%。Rv2351c蛋白的灵敏度为61.7%,特异度为91.4%。在动物实验中,11条多肽和Rv2351c蛋白均能诱导产生不同程度的细胞免疫应答。其中 P1(Rv3793)、P24(Rv0585c)、P26(Rv0585c)、P55(Rv1490)、P120(Rv0446c)、P121(Rv0446c)和 Rv2351c 主要诱导产生 Th1 和 Th2 混合型细胞免疫应答,P123(Rv0446c)、P136(Rv2201)、P166(Rv0197)、P309(Rv1798)和P318(Rv2318)多肽主要产生Th1型细胞免疫应答。与对照组相比,多肽P1(Rv3793)、P26(Rv0585c)、P120(Rv0446c)和 P121(Rv0446c)能引起小鼠体内 CD3+CD4+T 细胞的比例升高,多肽有 P123(Rv0446c)、P166(Rv0197)、P309(Rv1798)、P318(Rv2318)和 Rv2351c 蛋白均能引起小鼠体内 CD3+CD8+T细胞的比例显著升高,P24(Rv0585c)、P55(Rv1490)和 P136(Rv2201)能够同时引起CD3+CD4+T细胞和CD3+CD8+T细胞的比例同时升高。这11条多肽诱导产生的IgG抗体的滴度从1:44～1:5079不等,其中P121(Rv0446c)和P166(Rv0197)能够引起1:1000以上的IgG抗体滴度。Rv2351c引起IgG的抗体滴度达到1:2.2×106。Rv0674的9条B细胞表位多肽的反应灵敏度在54.55%～79.55%,特异度在71.25%～95%,除P203、P204以外,其他多肽的ROC曲线下面积均大于0.8。Rv1411c的7条多肽的反应灵敏度在46.5%～88.6%,特异度在75%～92.5%。Rv1477的6条多肽反应灵敏度为60.23%～76.14%,特异度在86.25%～95%。除了 P220之外,且ROC曲线下面积均在0.8以上。本研究成功鉴定的来自14个新结核抗原30个人T细胞表位和Rv2351c蛋白具有一定的抗原性,动物实验结果证实其中11条T细胞表位多肽和Rv2351c蛋白在小鼠体内能引起细胞免疫应答和体液免疫应答,具有一定的免疫原性。本研究对3个新抗原的22条人B细胞表位进行了血清学评价,结果证实这22条B细胞表位具有一定的血清学诊断价值。综上所述,本研究鉴定出来16个结核分枝杆菌新抗原,为结核病的诊断和疫苗的研究提供了一定的基础。
[Abstract]:Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB), which has been harmful to human health. 1/3 of the people all over the world have infected Mycobacterium tuberculosis, and 10% of them will be likely to develop into TB.2015 for 10 million 400 thousand of the global new tuberculosis cases in the future and die from tuberculosis. There are 1 million 400 thousand people. In recent years, with the increase of new TB drug resistant strains and HIV/AIDs combined infection cases, the incidence of tuberculosis increases and the global tuberculosis burden is increasing. The death rate of tuberculosis has fallen by 22% from 2000 to 2015, and nuclear disease remains the ten major cause of human death. First. At present, BCG is still the only widely used vaccine to resist tuberculosis worldwide..BCG is a live attenuated vaccine obtained by continuous subculture of Mycobacterium tuberculosis. Although the BCG vaccine has a better effect on preventing chestnut tuberculosis and tuberculous meningitis in children, the BCG against adult The protection rate of nuclear disease is very different, between 0 and 80%, and BCG can only effectively prevent primary tuberculosis. The study found that different BCG vaccine strains with the loss of protective antigens or epitopes cause different protective differences of BCG strains. We need to develop new vaccine to provide new means to better control tuberculosis. We need to find new immune dominant antigens or in epitopes for the development of recombinant BCG or a new type of immunized vaccine after BCG inoculation. In this study, we screened the protein antigens on the H37Rv proteome by combining the genomics and bioinformatics techniques, and the selected protein antigens were used for the T cell table. Interferon gamma release assay (IGRA) was used to screen out tuberculosis scores. The immunodominant antigen or epitope of the Mycobacterium was selected. The antigen epitopes with higher positive rate were selected and the BALB/c mice were immunized with the adjuvant DDA and polyI:C. The concentrations of IFN- gamma, IL-2, IL-4 and IL-10 in the splenocytes supernatant after the corresponding antigen stimulation were detected, and the serum IgG, IgG1 and IgG2a antibody titers in the serum were detected. The CD3+CD4+T was detected by flow}0 cytometer. The immunogenicity of these epitopes was evaluated by the ratio of the cells to the CD3+CD8+T cells. The epitopes of the epitopes of the epitopes of the epitopes of the epitopes were synthesized by using seppa2.0 to predict the epitopes of the B cells, and the serological evaluation of these B cell epitopes was carried out with the serum of the patients with pulmonary tuberculosis and healthy volunteers. In addition, the recombinant protein Rv2351c, prepared in this experiment, was studied in this study. The diagnostic and immune effects of Rv2351c were evaluated by IGRA and animal experiments. Finally, we predicted the 2726 T cell epitopes and B cell epitopes of 273 antigens on the H37Rv genome, and selected 265 T cell epitopes from 13 antigens and 22 human B cell antigen tables from 3 antigens. We identified 30 T cell antigen epitopes with antigenicity by IGRA, which were derived from 13 TB antigens: Rv3793, Rv05 85C, Rv1490, Rv0446c, Rv 1547, Rv2201, Rv3573, Rv0197, and the sensitivity of these peptides was between 1.9% and 30%, and the specificity was 96.81% To 100%. After the combined diagnosis of the same antigen, the sensitivity of the single antigen can reach 1.9% ~ 50%.Rv2351c protein sensitivity 61.7%. The specificity is 91.4%. in animal experiments, 11 polypeptide and Rv2351c protein can induce different degree of cellular immune response. Among them, P1 (Rv3793), P24 (Rv0585c), P26 (Rv05) 85C), P55 (Rv1490), P120 (Rv0446c), P121 (Rv0446c) and Rv2351c are mainly induced to produce a mixed cell immune response of Th1 and Th2. 446c) can cause an increase in the proportion of CD3+CD4+T cells in mice. The peptide has P123 (Rv0446c), P166 (Rv0197), P309 (Rv1798), P318 (Rv2318) and Rv2351c protein, which can cause a significant increase in the ratio of CD3+CD8+T cells in mice. At the same time, the titers of the IgG antibodies induced by these 11 peptides ranged from 1:44 to 1:5079, of which P121 (Rv0446c) and P166 (Rv0197) could cause the IgG antibody titer above 1:1000 to cause the antibody titer of IgG to the 1:2.2 * 106.Rv0674 9 cell epitopes of 54.55% to 79.55% and the specificity of 71.25% To 95%, in addition to P203 and P204, the sensitivity of the 7 peptides with areas larger than 0.8.Rv1411c under ROC curves of other peptides is 46.5% to 88.6%, and the sensitivity of 6 peptides with specificity of 75% to 92.5%.Rv1477 is 60.23% to 76.14%, the specificity is 86.25% to 95%. except P220, and the area under ROC curve is more than 0.8. The success of this study is successful. 30 T cell epitopes and Rv2351c proteins from 14 new TB antigens have a certain antigenicity. Animal experimental results confirm that 11 T cell epitopes and Rv2351c proteins can cause cellular immune response and humoral immune response in mice, and have a certain immunogenicity. This study on 22 people of 3 new antigens B thin. The cell epitopes were evaluated by serology. The results showed that the 22 B cell epitopes had a certain value of serological diagnosis. To sum up, 16 new Mycobacterium tuberculosis antigens were identified in this study, which provided a basis for the diagnosis of tuberculosis and the study of 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】：2017
【分类号】：R378.911

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