结核分枝杆菌信号转导调控网络研究及新型免疫抗原的筛选鉴定和运用初探
发布时间：2018-08-27 19:08
【摘要】：结核病是由结核分枝杆菌(简称结核菌)感染引起的慢性传染病,长期以来严重影响人类的健康。目前全世界的结核病疫情依然十分严峻,是全球仅次于艾滋病感染导致死亡的第二大传染性疾病。全球近1/3的人口被结核菌感染,大部分处于潜伏性感染状态。当人体免疫力由于各种原因有所下降的时候,结核菌便开始生长,逐渐发展成活动性结核。这在艾滋病患者中,比率会更高。据世界卫生组织2011年全球结核病防控报告：在2010年,全球新增850-920万结核病例,120-150万病例死于结核病(其中不包括HIV阳性患者)。长期以来,由于结核病治疗周期长,导致治疗不彻底,抗生素在某种程度上的不合理使用及结核菌自身的进化,世界各地出现越来越多的耐多药结核菌(Multidrug-resistant TB, MDR-TB)和广泛耐药结核菌(Extensively drug-resistant TB, EDR-TB),使得抗结核药物面临巨大困境。而目前作为防止结核病的唯一疫苗牛分枝杆菌BCG (Mycobacterium bovis Bacillus Calmette-Guerin, BCG)的效果也不尽人意。该疫苗能对婴儿和儿童提供很好的保护,但是对成年人的保护效果降低且存在较大的差异。这也使得社会中的绝大多数群体成为结核菌潜在的感染对象。结核病的防治仍然是全球面临的一个艰巨任务,特别是在在贫困地区及发展中国家。 我国仍然是全球22个结核病高负担国家之一。结核病年发病人数约为130万,占全球发病人数的14%,位居全球第二位。近年来,我国每年报告肺结核发病人数始终位居全国甲乙类传染病的前列；耐多药肺结核危害日益凸显,每年新发患者人数约12万,未来数年内可能出现以耐药菌为主的结核病流行态势；结核菌／艾滋病病毒双重感染患者人数持续增加,防治工作急待加强；中西部地区、农村地区结核病防治形势严峻。因此,新型药物,疫苗及诊断技术都必须不断发展和进步,以应对和控制目前和未来严峻的结核病疫情。 首先,诊断是治疗的前提。目前结核病的诊断方法主要包括影像学、微生物学、免疫学和分子生物学诊断。基于特异性抗原-抗体反应的血清学免疫诊断方法因其耗时短,方法简单,样品容易获得及适应人群广泛等优点而备受青睐。目前存在的很多基于胶体金试纸条检测技术的快速结核病检测试盒由于灵敏性和特异性差异较大而不能满足临床使用要求。开发高特异性和高灵敏性的诊断方法主要依赖于特异性抗原的鉴定及检测方法的选择。电化学免疫传感器是将免疫测定法与高灵敏的电化学传感技术相结合的一类新型生物传感器,被广泛应用于痕量免疫原性物质的分析研究。因此,本实验结合蛋白质凝胶双向电泳与Western blot技术,将结核菌溶菌蛋白与结核病患者血清进行免疫杂交,鉴定杂交点,利用ELISA技术进行抗原性验证,再与免疫电化学传感器结合并探索在血清学检测中的运用潜力。免疫杂交发现4个信号较强的杂交点,LC-MS/MS鉴定产生最强信号的抗原蛋白为rv2175c基因编码的保守假设性的调节蛋白Rv2175c。克隆,表达并纯化Rv2175c后,利用该蛋白免疫兔子成功制备多克隆抗体血清。利用酶联免疫检测法(enzyme linked immunosorbent assay, ELISA)技术对几组健康人血清及结核病患者血清中抗Rv2175c特异性IgG进行检测,发现这些血清中对应抗体浓度非常低,且在健康人血清与结核病患者血清之间无明显差异。进一步利用包被了Rv2175c蛋白的免疫电化学传感器检测到结核病患者血清中存在较强信号,暗示结核病患者血清中存在一些能与Rv2175c蛋白发生特异性相互作用的抗体或其它蛋白。本文对结果进行了深入分析,并提出ELISA与免疫电化学传感器之间结果的差异,为后续相关实验提供经验。 此外,结核菌在传播,胞内持留性感染等过程中都面临着各种苛刻的生存环境。因此,强大的信号转导系统是保存其能够感应信号并进行下游调控的重要基础,也是结核菌生存的关键。结核菌的信号转导系统主要由双组份调控系统(Two-component systems, TCS),真核样丝氨酸／苏氨酸蛋白激酶(Ser/Thr protein kinases, STPKs)和丝氨酸／苏氨酸磷酸酶系统,酪氨酸蛋白激酶(Protein tyrosine kinase, PTK)和酪氨酸磷酸酶系统,σ因子系统组成。PtkA是目前结核菌中发现的唯一的酪氨酸蛋白激酶,其自磷酸化和底物蛋白磷酸化均发生在酪氨酸残疾上。编码酪氨酸的密码子为GC含量很低的UAU或UAC,而分枝杆菌属细菌均为高CG含量,均在65%左右。酪氨酸在这样的情况下并没有优势,但PtkA仍然结核菌的漫长进化中保存了下来,因此它必然在结核菌中承担者比较重要的功能。而目前对PtkA仅有的了解是它能够磷酸化酪氨酸磷酸酶PtpA。 为了探索PtkA在结核菌中的更多生理功能,本研究首先从野生型结核菌H37Rv及结核菌H37Rv ΔptkA缺失突变菌株,牛分枝杆菌BCG和牛分枝杆菌BCGΔptkA缺失突变株入手,用蛋白质凝胶双向电泳方法对比野生型菌株与缺失突变菌株之间胞质蛋白表达谱上的差异。结果显示ptkA基因的缺失对结核菌的胞质蛋白表达谱影响较大,而对牛分枝杆菌则没有明显的影响。进一步的质谱分析鉴定了结核菌H37Rv ΔptkA菌株与结核菌H37Rv相比,表达量明显下降的三个蛋白点为低氧反应蛋白1,热休克蛋白HspX和普遍胁迫蛋白Rv2623。这些蛋白涉及多方面的环境压力应急及免疫原性等,且都同属于结核菌休眠调控蛋白DosR调控子,暗示了PtkA可能涉及结核菌的压力反应,休眠与结核菌免疫原性等方面。而在ptkA基因及其临近基因在序列和基因组上的排布与结核菌完全一样的牛分枝杆菌中,ptkA的缺失对胞质蛋白表达谱几乎没有影响,进一步暗示ptkA可能在结核菌中发挥了重要功能。本实验为后续的工作提供了一个大体的研究方向。 另外,PtkA作为一个蛋白酪氨酸激酶,其发挥生理功能的方式是通过磷酸化其底物蛋白,改变底物蛋白的功能活性,进而调控相关的代谢。而PtpA是其已知的唯一磷酸化底物。PtpA通过结合巨噬细胞空泡膜上的H+-ATPase中的H亚基并去磷酸化HOPS (homotypic vacuole fusion and vacuole protein sorting)复合体上VPS33B(human class C Vacuolar Protein Sorting VPS33B)蛋白来阻止吞噬体与溶酶体的结合,在结核菌的持留性感染中发挥至关重要的作用。对如此重要的一个生理过程,PtkA是如何感应细胞中的环境,在什么样的条件下磷酸化和调控PtpA的功能呢?它自身又有没有受到其它蛋白的调控呢?除了PtpA以外,PtkA还调控着哪些重要的生理功能呢?为此,我们首先将PtkA与结核菌的胞质蛋白在体外进行孵育,以期能够发现更多的磷酸化底物蛋白,却意外的发现了PtkA蛋白受到结核菌中内源性蛋白激酶的磷酸化。结合已有知识,推定磷酸化PtkA的内源性蛋白激酶应该为STPKs。我们首先克隆,表达和纯化了PknB, PknD, PknG和PknH。体外蛋白激酶反应发现这四个蛋白激酶均能对PtkA进行不同程度的磷酸化,其中以PknD对PtkA的磷酸化活性最强,PknG次之。进一步的实验证实PknD以时间及浓度依赖的方式对PtkA进行磷酸化,证明了PtkA是PknD的特异性磷酸化底物。利用LC-MS/MS分析发现PknD对PtkA的磷酸化位点为T119和另外一个靠近其自磷酸化位点y262的基序,暗示其可能影响自磷酸化位点附近的结构,进而改变其自磷酸化活性,间接改变其底物磷酸化活性。因此,STPKs可能通过调控PtkA而间接调控PtpA的功能,影响结核菌与宿主之间的相互作用和持留性感染。本实验结果又一次揭示了结核菌中信号转导系统之间的相互调控关系,展示了结核菌信号传导系统的复杂性和调控的多层次性。
[Abstract]:Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (tuberculosis) infection, which has seriously affected human health for a long time. Nowadays, the epidemic of tuberculosis in the world is still very serious, and it is the second largest infectious disease in the world that causes death after HIV infection. When the immunity of the human body declines for various reasons, the tuberculosis bacteria begin to grow and gradually develop into active tuberculosis. This is even higher in AIDS patients. According to the World Health Organization's Global Tuberculosis Prevention and Control Report 2011, 85-92 million new cases of tuberculosis worldwide in 2010, 1.2-1.5 million new cases of tuberculosis. One died of tuberculosis (excluding HIV-positive patients). Over the years, more and more multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have emerged around the world due to long treatment cycles leading to incomplete treatment, unreasonable use of antibiotics to a certain extent, and the evolution of the tuberculosis bacterium itself. Extensive drug-resistant TB (EDR-TB) makes antituberculosis drugs in dire straits. At present, Mycobacterium bovis Bacillus Calmette-Guerin (BCG), the only vaccine to prevent tuberculosis, is not satisfactory. The vaccine provides good protection for infants and children, but for adults. This also makes the vast majority of the community a potential target for TB infection. TB control remains a global challenge, especially in poor areas and developing countries.
China is still one of the 22 countries with high burden of tuberculosis in the world. The annual incidence of tuberculosis is about 1.3 million, accounting for 14% of the global incidence, ranking second in the world. About 120,000 people will be infected with drug-resistant bacteria in the next few years, the number of patients with TB / HIV double infection will continue to increase, and the prevention and control work needs to be strengthened urgently. Steps to tackle and control the current and future severe tuberculosis epidemic.
Diagnosis is the premise of treatment. Currently, the diagnostic methods of tuberculosis mainly include imaging, microbiology, immunology and molecular biology. Serological immunodiagnosis based on specific antigen-antibody reaction is favored for its advantages of short time-consuming, simple method, easy sample availability and wide population adaptation. Many rapid tuberculosis test kits based on colloidal gold strips can not meet the clinical requirements because of their high sensitivity and specificity. The development of high specificity and sensitivity diagnostic methods mainly depends on the identification of specific antigens and the selection of detection methods. A new type of biosensor, which combines the determination method with highly sensitive electrochemical sensing technology, has been widely used in the analysis of trace immunogenic substances. ELISA was used to verify the antigenicity, and then combined with immunoelectrochemical sensors to explore the potential application in serological detection. Immunohybridization revealed four strong signal hybridization sites. LC-MS/MS identified the antigen protein producing the strongest signal as the conserved hypothetical regulatory protein Rv2175c encoded by rv2175c gene. Polyclonal antibody serum was successfully prepared by immunizing rabbits with Rv2175c. Anti-Rv2175c specific IgG was detected by enzyme linked immunosorbent assay (ELISA) in several groups of healthy human serum and tuberculosis serum. The results showed that the concentration of anti-Rv2175c specific IgG in these sera was very low and healthy. There was no significant difference between human serum and tuberculosis serum. Further, strong signals were detected in the serum of tuberculosis patients by immunoelectrochemical sensors coated with Rv2175c protein, suggesting that there were antibodies or other proteins in the serum of tuberculosis patients that could interact specifically with Rv2175c protein. The differences between ELISA and immunoelectrochemical sensors were analyzed in detail, which provided experience for the follow-up experiments.
In addition, Mycobacterium tuberculosis is confronted with a variety of harsh living environments in the process of transmission and intracellular persistent infection. Therefore, a powerful signal transduction system is an important basis for preserving its ability to sense signals and regulate downstream. It is also the key to the survival of Mycobacterium tuberculosis. Poent systems, TCS, Ser/Thr protein kinases (STPKs) and serine/threonine phosphatase systems, protein tyrosine kinase (PTK) and tyrosine phosphatase systems, and_factor systems are the only tyrosine protein kinases found in tuberculosis at present. Both autophosphorylation and substrate protein phosphorylation occur on tyrosine disability. The codon encoding tyrosine is UAU or UAC with very low GC content, whereas Mycobacterium bacteria are both high CG content at about 65%. PtkA is now known only to be able to phosphotyrosine phosphatase PtpA.
In order to explore more physiological functions of PtkA in tuberculosis, we compared the cytoplasmic eggs of wild-type strains H37Rv and tuberculosis H37Rv ptkA deletion mutants, Mycobacterium bovis BCG and Mycobacterium bovis BCG ptkA deletion mutants by protein gel two-dimensional electrophoresis. The results showed that the deletion of ptkA gene had a great influence on the cytoplasmic protein expression profile of Mycobacterium bovis, but had no obvious effect on Mycobacterium bovis. Heat shock protein HspX and universal stress protein Rv2623. These proteins are involved in various environmental stress emergencies and immunogenicity, and both belong to tuberculosis dormancy regulatory protein DosR regulator, suggesting that PtkA may be involved in stress response, dormancy and tuberculosis immunogenicity. In Mycobacterium bovis, the deletion of ptkA has little effect on the expression profile of cytoplasmic proteins, suggesting that ptkA may play an important role in the pathogenesis of tuberculosis.
PtkA, as a protein tyrosine kinase, plays a physiological role by phosphorylating its substrate proteins, altering the functional activity of substrate proteins, and then regulating related metabolism. PtpA is the only phosphorylated substrate known to be PtkA. PtpA dephosphorylates HOPS by binding to H subunits in H + - ATPase on macrophage vacuoles. VPS33B (human class C Vacuolar Protein in Sorting VPS33B) proteins on the homotypic vacuole fusion and vacuole protein sorting complex prevent phage-lysosome binding and play a crucial role in tuberculosis retention infection. For such an important physiological process, how PtkA senses the ring in cells Under what conditions are PtkA phosphorylated and regulated? Is PtkA itself regulated by other proteins? Besides PtpA, what other important physiological functions does PtkA regulate? To this end, we first incubate PtkA with the cytoplasmic proteins of Mycobacterium tuberculosis in vitro in order to discover more phosphorylated substrate eggs. We first cloned, expressed and purified PknB, PknD, PknG and PknH. In vitro protein kinase reactions found that these four protein kinases can not carry out PtkA. The phosphorylation of PtkA by PknD was the strongest, followed by PknG. Further experiments confirmed that PtkA was phosphorylated by PknD in a time-and concentration-dependent manner, which proved that PtkA was a specific phosphorylation substrate of PknD. LC-MS/MS analysis showed that the phosphorylation site of PknD to PtkA was T119 and another one was close to its self-phosphorylation site. The sequence of the phosphorylated site y262 suggests that it may affect the structure near the site of autophosphorylation, thereby altering its autophosphorylation activity and indirectly altering its substrate phosphorylation activity. Therefore, STPKs may indirectly regulate the function of PtpA by regulating PtkA, and affect the interaction between tuberculosis and host and persistent infection. The relationship between the signal transduction systems in tuberculosis was revealed again, and the complexity and multi-level regulation of the signal transduction system in tuberculosis were demonstrated.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.11
本文编号：2208170
[Abstract]:Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (tuberculosis) infection, which has seriously affected human health for a long time. Nowadays, the epidemic of tuberculosis in the world is still very serious, and it is the second largest infectious disease in the world that causes death after HIV infection. When the immunity of the human body declines for various reasons, the tuberculosis bacteria begin to grow and gradually develop into active tuberculosis. This is even higher in AIDS patients. According to the World Health Organization's Global Tuberculosis Prevention and Control Report 2011, 85-92 million new cases of tuberculosis worldwide in 2010, 1.2-1.5 million new cases of tuberculosis. One died of tuberculosis (excluding HIV-positive patients). Over the years, more and more multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have emerged around the world due to long treatment cycles leading to incomplete treatment, unreasonable use of antibiotics to a certain extent, and the evolution of the tuberculosis bacterium itself. Extensive drug-resistant TB (EDR-TB) makes antituberculosis drugs in dire straits. At present, Mycobacterium bovis Bacillus Calmette-Guerin (BCG), the only vaccine to prevent tuberculosis, is not satisfactory. The vaccine provides good protection for infants and children, but for adults. This also makes the vast majority of the community a potential target for TB infection. TB control remains a global challenge, especially in poor areas and developing countries.
China is still one of the 22 countries with high burden of tuberculosis in the world. The annual incidence of tuberculosis is about 1.3 million, accounting for 14% of the global incidence, ranking second in the world. About 120,000 people will be infected with drug-resistant bacteria in the next few years, the number of patients with TB / HIV double infection will continue to increase, and the prevention and control work needs to be strengthened urgently. Steps to tackle and control the current and future severe tuberculosis epidemic.
Diagnosis is the premise of treatment. Currently, the diagnostic methods of tuberculosis mainly include imaging, microbiology, immunology and molecular biology. Serological immunodiagnosis based on specific antigen-antibody reaction is favored for its advantages of short time-consuming, simple method, easy sample availability and wide population adaptation. Many rapid tuberculosis test kits based on colloidal gold strips can not meet the clinical requirements because of their high sensitivity and specificity. The development of high specificity and sensitivity diagnostic methods mainly depends on the identification of specific antigens and the selection of detection methods. A new type of biosensor, which combines the determination method with highly sensitive electrochemical sensing technology, has been widely used in the analysis of trace immunogenic substances. ELISA was used to verify the antigenicity, and then combined with immunoelectrochemical sensors to explore the potential application in serological detection. Immunohybridization revealed four strong signal hybridization sites. LC-MS/MS identified the antigen protein producing the strongest signal as the conserved hypothetical regulatory protein Rv2175c encoded by rv2175c gene. Polyclonal antibody serum was successfully prepared by immunizing rabbits with Rv2175c. Anti-Rv2175c specific IgG was detected by enzyme linked immunosorbent assay (ELISA) in several groups of healthy human serum and tuberculosis serum. The results showed that the concentration of anti-Rv2175c specific IgG in these sera was very low and healthy. There was no significant difference between human serum and tuberculosis serum. Further, strong signals were detected in the serum of tuberculosis patients by immunoelectrochemical sensors coated with Rv2175c protein, suggesting that there were antibodies or other proteins in the serum of tuberculosis patients that could interact specifically with Rv2175c protein. The differences between ELISA and immunoelectrochemical sensors were analyzed in detail, which provided experience for the follow-up experiments.
In addition, Mycobacterium tuberculosis is confronted with a variety of harsh living environments in the process of transmission and intracellular persistent infection. Therefore, a powerful signal transduction system is an important basis for preserving its ability to sense signals and regulate downstream. It is also the key to the survival of Mycobacterium tuberculosis. Poent systems, TCS, Ser/Thr protein kinases (STPKs) and serine/threonine phosphatase systems, protein tyrosine kinase (PTK) and tyrosine phosphatase systems, and_factor systems are the only tyrosine protein kinases found in tuberculosis at present. Both autophosphorylation and substrate protein phosphorylation occur on tyrosine disability. The codon encoding tyrosine is UAU or UAC with very low GC content, whereas Mycobacterium bacteria are both high CG content at about 65%. PtkA is now known only to be able to phosphotyrosine phosphatase PtpA.
In order to explore more physiological functions of PtkA in tuberculosis, we compared the cytoplasmic eggs of wild-type strains H37Rv and tuberculosis H37Rv ptkA deletion mutants, Mycobacterium bovis BCG and Mycobacterium bovis BCG ptkA deletion mutants by protein gel two-dimensional electrophoresis. The results showed that the deletion of ptkA gene had a great influence on the cytoplasmic protein expression profile of Mycobacterium bovis, but had no obvious effect on Mycobacterium bovis. Heat shock protein HspX and universal stress protein Rv2623. These proteins are involved in various environmental stress emergencies and immunogenicity, and both belong to tuberculosis dormancy regulatory protein DosR regulator, suggesting that PtkA may be involved in stress response, dormancy and tuberculosis immunogenicity. In Mycobacterium bovis, the deletion of ptkA has little effect on the expression profile of cytoplasmic proteins, suggesting that ptkA may play an important role in the pathogenesis of tuberculosis.
PtkA, as a protein tyrosine kinase, plays a physiological role by phosphorylating its substrate proteins, altering the functional activity of substrate proteins, and then regulating related metabolism. PtpA is the only phosphorylated substrate known to be PtkA. PtpA dephosphorylates HOPS by binding to H subunits in H + - ATPase on macrophage vacuoles. VPS33B (human class C Vacuolar Protein in Sorting VPS33B) proteins on the homotypic vacuole fusion and vacuole protein sorting complex prevent phage-lysosome binding and play a crucial role in tuberculosis retention infection. For such an important physiological process, how PtkA senses the ring in cells Under what conditions are PtkA phosphorylated and regulated? Is PtkA itself regulated by other proteins? Besides PtpA, what other important physiological functions does PtkA regulate? To this end, we first incubate PtkA with the cytoplasmic proteins of Mycobacterium tuberculosis in vitro in order to discover more phosphorylated substrate eggs. We first cloned, expressed and purified PknB, PknD, PknG and PknH. In vitro protein kinase reactions found that these four protein kinases can not carry out PtkA. The phosphorylation of PtkA by PknD was the strongest, followed by PknG. Further experiments confirmed that PtkA was phosphorylated by PknD in a time-and concentration-dependent manner, which proved that PtkA was a specific phosphorylation substrate of PknD. LC-MS/MS analysis showed that the phosphorylation site of PknD to PtkA was T119 and another one was close to its self-phosphorylation site. The sequence of the phosphorylated site y262 suggests that it may affect the structure near the site of autophosphorylation, thereby altering its autophosphorylation activity and indirectly altering its substrate phosphorylation activity. Therefore, STPKs may indirectly regulate the function of PtpA by regulating PtkA, and affect the interaction between tuberculosis and host and persistent infection. The relationship between the signal transduction systems in tuberculosis was revealed again, and the complexity and multi-level regulation of the signal transduction system in tuberculosis were demonstrated.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.11
【参考文献】
相关期刊论文 前1条
1 戴小锋;刘仲明;;乙肝电化学免疫传感器阵列电极的研制[J];生物医学工程学杂志;2008年02期
，本文编号：2208170
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