结核分枝杆菌Mtb9.9家族蛋白免疫功能的研究

【作者】 彭超；

【导师】 王洪海；

【作者基本信息】 复旦大学 ， 微生物学， 2011， 博士

【摘要】 结核病是由结核分枝杆菌(Mycobacterium tuberculosis,简称为M.tb)引起的一种慢性传染病。据世界卫生组织(WHO)统计,当前全球约有1/3的人感染了结核杆菌,每年死于结核病的人数达到300万。随着耐药结核的增加和HIV共感染,结核病成了危害人类健康的重要传染病之一。要控制结核病的流行与蔓延的关键在于弄清楚结核的耐药机制、有效地疫苗保护、早期诊断以及规范的多药物联合的化学治疗。目前广泛使用的唯一结核疫苗BCG的保护效果不理想,迫切需要新型结核疫苗的开发,另外改良的诊断试剂对于检测结核分枝杆菌感染是必须的,研究新的血清学诊断方法是对现有诊断方法的一个有力补充。定量蛋白质组学(quantitative proteomics)是研究结核耐药机制、找到新的疫苗和诊断候选蛋白的有力工具。我们利用定量蛋白质组学的相对和绝对定量的等量异位标签技术(Isobaric Tag for Relative and Absolute Quantitation,iTRAQ)研究了两株临床分离的广泛耐药结核(extensively drug resistant tuberculosis, XDR-TB)和标准株H37Rv之间的差异蛋白表达。发现Mtb9.9c蛋白在两株XDR-TB中分别上调表达了1.7和21.3倍。Mtb9.9c蛋白是Mtb9.9家族中的一员。这是一组由94个氨基酸组成的相对分子质量为9.9kDa的蛋白家族,H37Rv株的Mtb9.9家族主要由Mtb9.9a (Rvl793)、 Mtb9.9c (Rvll98)、 Mtb9.9d (Rv1037c)和Mtb9.9e (Rv2346c)基因编码,其序列相互间具有高度同源性,本论文以这一家族蛋白为研究对象,探索了Mtb9.9家族蛋白作为亚单位疫苗和诊断抗原的潜力。本文成功地在大肠杆菌(Escherichia coli, E.coli中克隆、表达和纯化了Mtb9.9家族蛋白。将重组蛋白和弗氏不完全佐剂(IFA)联合免疫C57BL/6小、鼠,在第三次免疫后的第三周,收集小鼠血清和脾细胞进行Western Blot、细胞免疫和体液免疫检测。结果显示Mtb9.9家族蛋白之间有相似和特异的抗原表位。我们用Elispot发现了免疫小鼠产生IFN-γ的脾细胞数量增多。Elisa检测到细胞因子IFN-γ、 TNF-a的分泌量比对照多,但IL-4几乎没有变化。上述结果说明了Mtb9.9家族蛋白主要诱导Thl型细胞免疫。体液免疫检测了小鼠免疫后血清中IgG、 IgG1、 IgG2a、 IgG2b和IgG2c的抗体水平,发现Mtb9.9家族蛋白能诱导小鼠产生强烈的体液免疫应答,说明这是一组免疫原性抗原。另外IgG2c和IgG2b的产生量都比IgGl多,进一步证实了Mtb9.9家族蛋白诱导以Th1型为主的细胞免疫。为了进一步评估Mtb9.9家族蛋白的诊断潜力,我们克隆、表达、纯化了CFP-10蛋白做为血清学诊断的对照蛋白,来检测Mtb9.9家族蛋白针对结核病人血清以及接种BCG的健康对照者(n=61)血清的抗体反应。结核病人血清包括全敏肺结核病人的血清(n=101)、肺外结核病人(n=10)和多耐药病人血清(n=60)。结果显示在诊断耐多药病人血清方面,Mtb9.9a蛋白的敏感性可以达到75%,比对照CFP-10蛋白的敏感性53.3%要高,但是它的特异性为83.6%,略低于对照CFP-10蛋白的88.5%。研究中还发现,重组Mtb9.9a蛋白针对接种BCG的健康对照者和耐多药结核病人的血清学反应具有显著的差异(P<0.0001)。证明了Mtb9.9a蛋白具有用于耐多药结核病人的血清学诊断的潜力。这一结果在文献检索中未见报道。综上所述,Mtb9.9家族蛋白主要介导Thl型细胞免疫,具有作为亚单位疫苗的应用潜力。另外Mtb9.9a蛋白可以区别耐多药病人和健康对照,有希望作为耐多药结核病人的血清学诊断的候选抗原。更多还原

【Abstract】 Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (M.tb). According to the World Health Organization (WHO) statistics, one-third of the world’s population was infected with mycobacterium tuberculosis, causing over3million deaths annually. Moreover, with the emergence of multidrug-resistant TB and the spread of HIV, tuberculosis became one of the main infectious diseases which destroyed people’s health. Understanding the drug resistant mechanism、 good vaccine development and early diagnosis followed by chemotherapy are the major control strategy. The only available vaccine BCG shows disputable protection efficacy against pulmonary tuberculosis, therefore it is urgently needed to develop more effective vaccines against TB. Furthermore, improved diagnostic reagents are needed for the detection of Mycobacterium tuberculosis infections, and the development of a serodiagnostic test would complement the diagnostic methods presently available.Quantitative proteomics is a strong tool to study M.tb drug-resistant mechanism, find new vaccine and diagnostic proteins. We use isobaric tag for relative and absolute quantitation (iTRAQ) to study the differently expressed protein between two extensively drug resistant tuberculosis (XDR-TB) with H37Rv. We found that Mtb9.9c up express1.7and21.3fold in two XDR-TBs, respectively. Mtb9.9c belongs to Mtb9.9protein family. Mtb9.9protein family consisting of five members Mtb9.9A (Rv1793), Mtb9.9B (Rv3619c), Mtb9.9C (Rv1198), Mtb9.9D (Rv1037c) and Mtb9.9E (Rv2346c), shows>90%aa sequence identity.Mtb9.9proteins were highly homologous and were predicted to encode94amino acids with molecular mass of9.9kD. Because of they are highly homologous and antigenic proteins, we study whether they have potential to be subunit vaccine and diagnostic antigen.First we cloned, expressed and purified Mtb9.9family proteins in E.coli. And then we immunize mouse with Mtb9.9family proteins and IFA, at the third week after three immunization, we gather mouse serum and spleen cells to do western blot, cellular immunity and humoral immunity tests. The result of western blot showed that Mtb9.9proteins have same and typical antigenic epitope. The Mtb9.9proteins induced an increased Th1type cellular and humoral immune response in mice, characterized by an elevated concentration of IFN-y and TNF-a in antigen stimulated splenocyte culture and a strong lgG2c antibody response.In order to evaluate whether Mtb9.9can be diagnostic antigen or not, we cloned, expressed and purified CFP-10protein as control. When tested in a conventional ELISA, the Mtb9.9a protein revealed statistically significant antigenic distinction between healthy BCG-vaccinated controls (n=61) and MDR tuberculosis patients. The panel of patient sera comprised sera from pulmonary tuberculosis patients (n=101) and extra-pulmonary tuberculosis patients (n=10) and MDR patients (n=60). It was shown that serum immunoglobulin G antibodies positive rate of recombinant protein antigen Mtb9.9a was75.0%which was higher than CFP-10in MDR pulmonary tuberculosis patients, the specificity of these proteins were83.6%and88.5%, respectively. This study demonstrates that Mtb9.9a protein can be a useful diagnostic marker in MDR pulmonary tuberculosis. In conclusion, Mtb9.9family proteins have the potential to be subunit vaccine because they have the Th1type cellular immunization. Besides Mtb9.9a can distinguish MDR patients and health control, so it also can be use as candidate antigen as serological test.更多还原