结核分枝杆菌RD区新功能性基因的发现及其免疫机制与应用的初步研究
发布时间：2019-06-26 21:15
【摘要】：背景：结核分枝杆菌(Mycobacterium tuberculosis, M.tb)感染引起的结核病(Tuberculosis, TB)是全球三大传染病杀手之一。据2015年最新流行病学统计,每年新增结核病例960万,导致150万死亡。全球约有1/3人口感染结核,其中90-98%为潜伏感染,其中2-10%会发展为活动性结核,加之耐药株的流行、与HIV或HCV共感染等情况使得该疾病严重威胁着人类健康。近年来,结核病在诊断、预防及致病机制的研究方面虽然已取得了一定进展,但仍存在许多不足,需要进一步地完善及深入。目前,结核病的诊断主要依赖胸片、痰涂片抗酸染色、TST、PCR、微生物培养等手段；但这些方法或灵敏度低、或检查周期长。近年来发展的IFN-γ释放检测试验(IGRAs)具有了较高的灵敏度及特异度,但该类型检测试剂盒是仅以ESAT-6、CFP10多肽为刺激抗原使诊断仍存在漏检情况。在结核病预防方面,唯一可用的疫苗是卡介苗(Bacillus Calmette-Guerin, BCG),但仅能为未成年儿童提供可靠的保护性,成人中免疫保护性非常低下。已有研究显示重组BCG为改良疫苗的最佳方法,但难点在于寻找合适的靶标抗原。筛选优势靶标抗原不仅可用于提高诊断率还可应用于疫苗研究。巨噬细胞是结核分枝杆菌寄生的主要宿主细胞,部分结核分枝杆菌可逃避宿主免疫系统、尤其是巨噬细胞的追杀。但具体机制尚不清楚,深入了解结核分枝杆菌与宿主巨噬细胞相互作用的机制和途径,对于解释结核分枝杆菌致病机理特别是它在巨噬细胞中长期寄生的原因有着重要的作用。BCG基因组中的某些片段相对于牛型结核分枝杆菌或结核分枝杆菌H37Rv来说发生缺失,这些缺失片段被定义为RD区(Region of Differences, RD)。基因芯片技术研究发现BCG较H37Rv丢失了RD1-16区,近年来许多研究报道显示,许多该丢失区域所表达的蛋白中有很多功能性蛋白,包括具有很强的免疫原性的基因、介导免疫逃逸的基因等。目的：筛选RD区中优势免疫原性抗原,评估其在结核诊断中的用途；利用该RD抗原构建能增强BCG免疫原性的新型重组BCG疫苗(rBCG::Rv2645);筛选RD区中可能存在的抗巨噬细胞杀伤基因,探讨结核分枝杆菌的逃逸机制。方法：本研究通过构建RD10-14区基因的31种原核表达质粒、表达纯化相应RD蛋白、ELISPOT等方法筛选出优势免疫原性抗原。以该蛋白作为结核特异性抗原评估其诊断用途。同时,利用电转技术将该RD基因导入BCG中构建重组疫苗rBCG::Rv2645,通过免疫Balb/c、鼠、恒河猴后,评估其免疫原性及H37Rv攻毒后的抗结核免疫保护能力。另外,利用表达RD蛋白建立的BL21-RDs菌种库,筛查这些RD蛋白对巨噬细胞的影响；通过巨噬细胞吞噬试验及菌落计数、LDH释放实验等筛选出能抗巨噬细胞杀伤的RDs基因,从而进一步探讨该蛋白与巨噬细胞的相互作用机制。结果：本研究首次从RD10-14区中筛选出来源于RD13区的分泌型蛋白Rv2645能刺激T淋巴细胞产生高水平特异性的IFN-γ,Rv2645特异性1FN-y水平在结核病人PBMCs中明显高于健康人(包括BCG接种者)；在结核病人与健康人的鉴别诊断中,Rv2645的检测灵敏度与特异度高达90.0%、98.2%,其中结核病人的诊断结果与临床上使用CFP10-ESAT6为刺激抗原的T-SPOT.TB诊断结果相比,相符率可达98%。但Rv2645与CFP10-ESAT6联合使用后比单独使用CFP10-ESAT6或Rv2645抗原效果更好,能将灵敏度从88.0%左右提升至96.0%,且具有类似高特异度98.2%；且Rv2645特异性皮肤试验可区分结核感染及BCG接种人群。我们还成功构建了重组疫苗rBCG::Rv2645;在Balb/c小鼠及恒河猴的动物实验中发现,相对于BCG而言,rBCG::Rv2645能提升更高的免疫原性及抗结核免疫保护作用。另外,本研究发现来源于RD14区的Rv1768 (PE-PGRS31)基因能逃逸巨噬细胞的杀伤。进一步的研究发现Rv1768是通过与巨噬细胞的Annexin A2结合从而降低胞内钙离子浓度,进一步抑制了吞噬溶酶体的成熟。结论：本研究首次发现的Rv2645蛋白是结核病T细胞免疫的特异性识别抗原,在结核诊断试剂开发及结核疫苗设计方面具有一定的潜力；另外,所筛选的Rv1768抗原为结核杆菌逃逸机制的探讨提供了新方向。
[Abstract]:BACKGROUND: The tuberculosis (TB) caused by the infection of Mycobacterium tuberculosis (M. tb) is one of the three major infectious diseases in the world. According to the latest epidemiological statistics in 2015, a total of 9.6 million tuberculosis was added to each year, resulting in a total of 1.5 million deaths. Approximately 1/3 of the world's population is infected with tuberculosis, of which 90-98 per cent are latent infections, of which 2-10 per cent will develop to active tuberculosis, and the prevalence of drug-resistant strains, in combination with HIV or HCV co-infection, poses a serious threat to human health. In recent years, although some progress has been made in the research of the diagnosis, prevention and pathogenesis of tuberculosis, there are still many shortcomings, which need to be further improved and in-depth. At present, the diagnosis of tuberculosis mainly depends on chest film, sputum smear acid-fast staining, TST, PCR and microorganism culture, but these methods have low sensitivity or long check period. The development of IGRAs in recent years has high sensitivity and specificity, but this type of detection kit is only ESAT-6, CFP10 polypeptide as the stimulation antigen to make the diagnosis still have a leak detection condition. In the area of tuberculosis prevention, the only available vaccine is the Bacillus Calmette-Guerin (BCG), but only can provide reliable protection for the minor children, and the immune protection in the adult is very low. The research has shown that the recombinant BCG is the best method for improving the vaccine, but the difficulty is to find the appropriate target antigen. Screening the advantage target antigen not only can be used for improving the diagnosis rate, but also can be used for vaccine research. Macrophages are the main host cells of the parasitic Mycobacterium tuberculosis, and some of the M. tuberculosis can escape the host immune system, in particular the collection of macrophages. But the mechanism and approach of the interaction between the Mycobacterium tuberculosis and the host macrophage are not clear, and it is important to explain the pathogenic mechanism of the Mycobacterium tuberculosis, especially the cause of the long-term and long-term parasitism of the macrophage. Some of the fragments in the BCG genome are missing relative to Mycobacterium tuberculosis or Mycobacterium tuberculosis H37Rv, which are defined as the Region of Differential, RD. Gene chip technology has found that BCG has lost RD1-16 region with H37Rv. In recent years, many studies have shown that many functional proteins in the protein expressed in many of the lost regions include genes with strong immunogenicity, genes that mediate immune escape, and the like. Objective: To screen the advantage immunogenic antigen in the RD area and to evaluate its use in the diagnosis of tuberculosis, and to construct a new recombinant BCG vaccine (rBCG: Rv2645) which can enhance the immunogenicity of BCG by using the RD antigen, and to screen the anti-macrophage killing genes that may be present in the RD area. To explore the mechanism of the escape of Mycobacterium tuberculosis. Methods: This study was used to construct 31 prokaryotic expression plasmids of RD10-14 region gene, to express and purify the corresponding RD protein, ELISPOT and other methods to screen out the advantage immunogenic antigen. The protein is used as the specific antigen of the tuberculosis to evaluate the diagnostic use thereof. At the same time, the RD gene was introduced into BCG to construct the recombinant vaccine rBCG: Rv2645, and the immunogenicity and the anti-tuberculosis immune protection ability after challenge of H37Rv were assessed by immunization of Balb/ c, murine and rhesus monkeys. In addition, the influence of the RD proteins on the macrophages is screened by using the BL21-RDs strain library established by the expression of the RD protein, and the RDs gene capable of anti-macrophage killing can be screened through the phagocytosis test of the macrophage and the colony count, the LDH release experiment and the like, So as to further explore the interaction mechanism between the protein and the macrophage. Results: The first time in the study, the secreted protein Rv2645 from the RD13 region can stimulate T-lymphocytes to produce high-level-specific IFN-1, and the specific 1FN-y level of Rv2645 is significantly higher in the PBMCs than in healthy people (including BCG vaccination). In the differential diagnosis of tuberculosis and healthy people, the detection sensitivity and specificity of Rv2645 were as high as 90.0% and 98.2%, among which, the diagnostic results of TB patients and the clinical use of CFP10-ESAT6 as the stimulating antigen. However, the combined use of Rv2645 and CFP10-ESAT6 is better than that of CFP10-ESAT6 or Rv2645 alone, and the sensitivity can be increased from about 88.0% to 96.0%, with a similar high specificity of 98.2%; and the Rv2645 specific skin test can distinguish the tuberculosis infection and the BCG vaccination population. We have also successfully constructed the recombinant vaccine rBCG: Rv2645; in Balb/ c mice and in the animals of the rhesus monkey, rBCG: Rv2645 can promote higher immunogenicity and anti-tuberculosis immune protection against BCG. In addition, this study found that the Rv1768 (PE-PGR31) gene from the RD14 region can escape the killing of macrophages. Further studies have found that Rv1768 is to reduce the intracellular calcium ion concentration by binding to the Annexin A2 of the macrophages, which further inhibits the maturation of the phagocytic lysosomes. Conclusion: The Rv2645 protein, which was first found in this study, is a specific identification antigen of T cell immunity, and has potential in the development of tuberculosis diagnosis reagent and the design of the tuberculosis vaccine. In addition, the screened Rv1768 antigen provides a new direction for the study of the escape mechanism of the tubercle bacillus.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R378.911
本文编号：2506497
[Abstract]:BACKGROUND: The tuberculosis (TB) caused by the infection of Mycobacterium tuberculosis (M. tb) is one of the three major infectious diseases in the world. According to the latest epidemiological statistics in 2015, a total of 9.6 million tuberculosis was added to each year, resulting in a total of 1.5 million deaths. Approximately 1/3 of the world's population is infected with tuberculosis, of which 90-98 per cent are latent infections, of which 2-10 per cent will develop to active tuberculosis, and the prevalence of drug-resistant strains, in combination with HIV or HCV co-infection, poses a serious threat to human health. In recent years, although some progress has been made in the research of the diagnosis, prevention and pathogenesis of tuberculosis, there are still many shortcomings, which need to be further improved and in-depth. At present, the diagnosis of tuberculosis mainly depends on chest film, sputum smear acid-fast staining, TST, PCR and microorganism culture, but these methods have low sensitivity or long check period. The development of IGRAs in recent years has high sensitivity and specificity, but this type of detection kit is only ESAT-6, CFP10 polypeptide as the stimulation antigen to make the diagnosis still have a leak detection condition. In the area of tuberculosis prevention, the only available vaccine is the Bacillus Calmette-Guerin (BCG), but only can provide reliable protection for the minor children, and the immune protection in the adult is very low. The research has shown that the recombinant BCG is the best method for improving the vaccine, but the difficulty is to find the appropriate target antigen. Screening the advantage target antigen not only can be used for improving the diagnosis rate, but also can be used for vaccine research. Macrophages are the main host cells of the parasitic Mycobacterium tuberculosis, and some of the M. tuberculosis can escape the host immune system, in particular the collection of macrophages. But the mechanism and approach of the interaction between the Mycobacterium tuberculosis and the host macrophage are not clear, and it is important to explain the pathogenic mechanism of the Mycobacterium tuberculosis, especially the cause of the long-term and long-term parasitism of the macrophage. Some of the fragments in the BCG genome are missing relative to Mycobacterium tuberculosis or Mycobacterium tuberculosis H37Rv, which are defined as the Region of Differential, RD. Gene chip technology has found that BCG has lost RD1-16 region with H37Rv. In recent years, many studies have shown that many functional proteins in the protein expressed in many of the lost regions include genes with strong immunogenicity, genes that mediate immune escape, and the like. Objective: To screen the advantage immunogenic antigen in the RD area and to evaluate its use in the diagnosis of tuberculosis, and to construct a new recombinant BCG vaccine (rBCG: Rv2645) which can enhance the immunogenicity of BCG by using the RD antigen, and to screen the anti-macrophage killing genes that may be present in the RD area. To explore the mechanism of the escape of Mycobacterium tuberculosis. Methods: This study was used to construct 31 prokaryotic expression plasmids of RD10-14 region gene, to express and purify the corresponding RD protein, ELISPOT and other methods to screen out the advantage immunogenic antigen. The protein is used as the specific antigen of the tuberculosis to evaluate the diagnostic use thereof. At the same time, the RD gene was introduced into BCG to construct the recombinant vaccine rBCG: Rv2645, and the immunogenicity and the anti-tuberculosis immune protection ability after challenge of H37Rv were assessed by immunization of Balb/ c, murine and rhesus monkeys. In addition, the influence of the RD proteins on the macrophages is screened by using the BL21-RDs strain library established by the expression of the RD protein, and the RDs gene capable of anti-macrophage killing can be screened through the phagocytosis test of the macrophage and the colony count, the LDH release experiment and the like, So as to further explore the interaction mechanism between the protein and the macrophage. Results: The first time in the study, the secreted protein Rv2645 from the RD13 region can stimulate T-lymphocytes to produce high-level-specific IFN-1, and the specific 1FN-y level of Rv2645 is significantly higher in the PBMCs than in healthy people (including BCG vaccination). In the differential diagnosis of tuberculosis and healthy people, the detection sensitivity and specificity of Rv2645 were as high as 90.0% and 98.2%, among which, the diagnostic results of TB patients and the clinical use of CFP10-ESAT6 as the stimulating antigen. However, the combined use of Rv2645 and CFP10-ESAT6 is better than that of CFP10-ESAT6 or Rv2645 alone, and the sensitivity can be increased from about 88.0% to 96.0%, with a similar high specificity of 98.2%; and the Rv2645 specific skin test can distinguish the tuberculosis infection and the BCG vaccination population. We have also successfully constructed the recombinant vaccine rBCG: Rv2645; in Balb/ c mice and in the animals of the rhesus monkey, rBCG: Rv2645 can promote higher immunogenicity and anti-tuberculosis immune protection against BCG. In addition, this study found that the Rv1768 (PE-PGR31) gene from the RD14 region can escape the killing of macrophages. Further studies have found that Rv1768 is to reduce the intracellular calcium ion concentration by binding to the Annexin A2 of the macrophages, which further inhibits the maturation of the phagocytic lysosomes. Conclusion: The Rv2645 protein, which was first found in this study, is a specific identification antigen of T cell immunity, and has potential in the development of tuberculosis diagnosis reagent and the design of the tuberculosis vaccine. In addition, the screened Rv1768 antigen provides a new direction for the study of the escape mechanism of the tubercle bacillus.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R378.911
【参考文献】
相关期刊论文 前2条
1 P.D.O.Davies;胡远莲;;结核病的诊断与误诊[J];国际结核病与肺部疾病杂志(中文版);2009年01期
2 刘晓清;;新的结核分枝杆菌RD1基因编码产物介导的T细胞反应在结核感染诊断中的应用[J];中国医学科学院学报;2008年03期
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