發(fā)布時(shí)間：2018-08-27 19:08

【摘要】：結(jié)核病是由結(jié)核分枝桿菌(簡(jiǎn)稱結(jié)核菌)感染引起的慢性傳染病,長(zhǎng)期以來(lái)嚴(yán)重影響人類的健康。目前全世界的結(jié)核病疫情依然十分嚴(yán)峻,是全球僅次于艾滋病感染導(dǎo)致死亡的第二大傳染性疾病。全球近1/3的人口被結(jié)核菌感染,大部分處于潛伏性感染狀態(tài)。當(dāng)人體免疫力由于各種原因有所下降的時(shí)候,結(jié)核菌便開始生長(zhǎng),逐漸發(fā)展成活動(dòng)性結(jié)核。這在艾滋病患者中,比率會(huì)更高。據(jù)世界衛(wèi)生組織2011年全球結(jié)核病防控報(bào)告：在2010年,全球新增850-920萬(wàn)結(jié)核病例,120-150萬(wàn)病例死于結(jié)核病(其中不包括HIV陽(yáng)性患者)。長(zhǎng)期以來(lái),由于結(jié)核病治療周期長(zhǎng),導(dǎo)致治療不徹底,抗生素在某種程度上的不合理使用及結(jié)核菌自身的進(jìn)化,世界各地出現(xiàn)越來(lái)越多的耐多藥結(jié)核菌(Multidrug-resistant TB, MDR-TB)和廣泛耐藥結(jié)核菌(Extensively drug-resistant TB, EDR-TB),使得抗結(jié)核藥物面臨巨大困境。而目前作為防止結(jié)核病的唯一疫苗牛分枝桿菌BCG (Mycobacterium bovis Bacillus Calmette-Guerin, BCG)的效果也不盡人意。該疫苗能對(duì)嬰兒和兒童提供很好的保護(hù),但是對(duì)成年人的保護(hù)效果降低且存在較大的差異。這也使得社會(huì)中的絕大多數(shù)群體成為結(jié)核菌潛在的感染對(duì)象。結(jié)核病的防治仍然是全球面臨的一個(gè)艱巨任務(wù),特別是在在貧困地區(qū)及發(fā)展中國(guó)家。 我國(guó)仍然是全球22個(gè)結(jié)核病高負(fù)擔(dān)國(guó)家之一。結(jié)核病年發(fā)病人數(shù)約為130萬(wàn),占全球發(fā)病人數(shù)的14%,位居全球第二位。近年來(lái),我國(guó)每年報(bào)告肺結(jié)核發(fā)病人數(shù)始終位居全國(guó)甲乙類傳染病的前列；耐多藥肺結(jié)核危害日益凸顯,每年新發(fā)患者人數(shù)約12萬(wàn),未來(lái)數(shù)年內(nèi)可能出現(xiàn)以耐藥菌為主的結(jié)核病流行態(tài)勢(shì)；結(jié)核菌／艾滋病病毒雙重感染患者人數(shù)持續(xù)增加,防治工作急待加強(qiáng)；中西部地區(qū)、農(nóng)村地區(qū)結(jié)核病防治形勢(shì)嚴(yán)峻。因此,新型藥物,疫苗及診斷技術(shù)都必須不斷發(fā)展和進(jìn)步,以應(yīng)對(duì)和控制目前和未來(lái)嚴(yán)峻的結(jié)核病疫情。 首先,診斷是治療的前提。目前結(jié)核病的診斷方法主要包括影像學(xué)、微生物學(xué)、免疫學(xué)和分子生物學(xué)診斷。基于特異性抗原-抗體反應(yīng)的血清學(xué)免疫診斷方法因其耗時(shí)短,方法簡(jiǎn)單,樣品容易獲得及適應(yīng)人群廣泛等優(yōu)點(diǎn)而備受青睞。目前存在的很多基于膠體金試紙條檢測(cè)技術(shù)的快速結(jié)核病檢測(cè)試盒由于靈敏性和特異性差異較大而不能滿足臨床使用要求。開發(fā)高特異性和高靈敏性的診斷方法主要依賴于特異性抗原的鑒定及檢測(cè)方法的選擇。電化學(xué)免疫傳感器是將免疫測(cè)定法與高靈敏的電化學(xué)傳感技術(shù)相結(jié)合的一類新型生物傳感器,被廣泛應(yīng)用于痕量免疫原性物質(zhì)的分析研究。因此,本實(shí)驗(yàn)結(jié)合蛋白質(zhì)凝膠雙向電泳與Western blot技術(shù),將結(jié)核菌溶菌蛋白與結(jié)核病患者血清進(jìn)行免疫雜交,鑒定雜交點(diǎn),利用ELISA技術(shù)進(jìn)行抗原性驗(yàn)證,再與免疫電化學(xué)傳感器結(jié)合并探索在血清學(xué)檢測(cè)中的運(yùn)用潛力。免疫雜交發(fā)現(xiàn)4個(gè)信號(hào)較強(qiáng)的雜交點(diǎn),LC-MS/MS鑒定產(chǎn)生最強(qiáng)信號(hào)的抗原蛋白為rv2175c基因編碼的保守假設(shè)性的調(diào)節(jié)蛋白R(shí)v2175c�？寺�,表達(dá)并純化Rv2175c后,利用該蛋白免疫兔子成功制備多克隆抗體血清。利用酶聯(lián)免疫檢測(cè)法(enzyme linked immunosorbent assay, ELISA)技術(shù)對(duì)幾組健康人血清及結(jié)核病患者血清中抗Rv2175c特異性IgG進(jìn)行檢測(cè),發(fā)現(xiàn)這些血清中對(duì)應(yīng)抗體濃度非常低,且在健康人血清與結(jié)核病患者血清之間無(wú)明顯差異。進(jìn)一步利用包被了Rv2175c蛋白的免疫電化學(xué)傳感器檢測(cè)到結(jié)核病患者血清中存在較強(qiáng)信號(hào),暗示結(jié)核病患者血清中存在一些能與Rv2175c蛋白發(fā)生特異性相互作用的抗體或其它蛋白。本文對(duì)結(jié)果進(jìn)行了深入分析,并提出ELISA與免疫電化學(xué)傳感器之間結(jié)果的差異,為后續(xù)相關(guān)實(shí)驗(yàn)提供經(jīng)驗(yàn)。 此外,結(jié)核菌在傳播,胞內(nèi)持留性感染等過程中都面臨著各種苛刻的生存環(huán)境。因此,強(qiáng)大的信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)是保存其能夠感應(yīng)信號(hào)并進(jìn)行下游調(diào)控的重要基礎(chǔ),也是結(jié)核菌生存的關(guān)鍵。結(jié)核菌的信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)主要由雙組份調(diào)控系統(tǒng)(Two-component systems, TCS),真核樣絲氨酸／蘇氨酸蛋白激酶(Ser/Thr protein kinases, STPKs)和絲氨酸／蘇氨酸磷酸酶系統(tǒng),酪氨酸蛋白激酶(Protein tyrosine kinase, PTK)和酪氨酸磷酸酶系統(tǒng),σ因子系統(tǒng)組成。PtkA是目前結(jié)核菌中發(fā)現(xiàn)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發(fā)生在酪氨酸殘疾上。編碼酪氨酸的密碼子為GC含量很低的UAU或UAC,而分枝桿菌屬細(xì)菌均為高CG含量,均在65%左右。酪氨酸在這樣的情況下并沒有優(yōu)勢(shì),但PtkA仍然結(jié)核菌的漫長(zhǎng)進(jìn)化中保存了下來(lái),因此它必然在結(jié)核菌中承擔(dān)者比較重要的功能。而目前對(duì)PtkA僅有的了解是它能夠磷酸化酪氨酸磷酸酶PtpA。 為了探索PtkA在結(jié)核菌中的更多生理功能,本研究首先從野生型結(jié)核菌H37Rv及結(jié)核菌H37Rv ΔptkA缺失突變菌株,牛分枝桿菌BCG和牛分枝桿菌BCGΔptkA缺失突變株入手,用蛋白質(zhì)凝膠雙向電泳方法對(duì)比野生型菌株與缺失突變菌株之間胞質(zhì)蛋白表達(dá)譜上的差異。結(jié)果顯示ptkA基因的缺失對(duì)結(jié)核菌的胞質(zhì)蛋白表達(dá)譜影響較大,而對(duì)牛分枝桿菌則沒有明顯的影響。進(jìn)一步的質(zhì)譜分析鑒定了結(jié)核菌H37Rv ΔptkA菌株與結(jié)核菌H37Rv相比,表達(dá)量明顯下降的三個(gè)蛋白點(diǎn)為低氧反應(yīng)蛋白1,熱休克蛋白HspX和普遍脅迫蛋白R(shí)v2623。這些蛋白涉及多方面的環(huán)境壓力應(yīng)急及免疫原性等,且都同屬于結(jié)核菌休眠調(diào)控蛋白DosR調(diào)控子,暗示了PtkA可能涉及結(jié)核菌的壓力反應(yīng),休眠與結(jié)核菌免疫原性等方面。而在ptkA基因及其臨近基因在序列和基因組上的排布與結(jié)核菌完全一樣的牛分枝桿菌中,ptkA的缺失對(duì)胞質(zhì)蛋白表達(dá)譜幾乎沒有影響,進(jìn)一步暗示ptkA可能在結(jié)核菌中發(fā)揮了重要功能。本實(shí)驗(yàn)為后續(xù)的工作提供了一個(gè)大體的研究方向。 另外,PtkA作為一個(gè)蛋白酪氨酸激酶,其發(fā)揮生理功能的方式是通過磷酸化其底物蛋白,改變底物蛋白的功能活性,進(jìn)而調(diào)控相關(guān)的代謝。而PtpA是其已知的唯一磷酸化底物。PtpA通過結(jié)合巨噬細(xì)胞空泡膜上的H+-ATPase中的H亞基并去磷酸化HOPS (homotypic vacuole fusion and vacuole protein sorting)復(fù)合體上VPS33B(human class C Vacuolar Protein Sorting VPS33B)蛋白來(lái)阻止吞噬體與溶酶體的結(jié)合,在結(jié)核菌的持留性感染中發(fā)揮至關(guān)重要的作用。對(duì)如此重要的一個(gè)生理過程,PtkA是如何感應(yīng)細(xì)胞中的環(huán)境,在什么樣的條件下磷酸化和調(diào)控PtpA的功能呢?它自身又有沒有受到其它蛋白的調(diào)控呢?除了PtpA以外,PtkA還調(diào)控著哪些重要的生理功能呢?為此,我們首先將PtkA與結(jié)核菌的胞質(zhì)蛋白在體外進(jìn)行孵育,以期能夠發(fā)現(xiàn)更多的磷酸化底物蛋白,卻意外的發(fā)現(xiàn)了PtkA蛋白受到結(jié)核菌中內(nèi)源性蛋白激酶的磷酸化。結(jié)合已有知識(shí),推定磷酸化PtkA的內(nèi)源性蛋白激酶應(yīng)該為STPKs。我們首先克隆,表達(dá)和純化了PknB, PknD, PknG和PknH。體外蛋白激酶反應(yīng)發(fā)現(xiàn)這四個(gè)蛋白激酶均能對(duì)PtkA進(jìn)行不同程度的磷酸化,其中以PknD對(duì)PtkA的磷酸化活性最強(qiáng),PknG次之。進(jìn)一步的實(shí)驗(yàn)證實(shí)PknD以時(shí)間及濃度依賴的方式對(duì)PtkA進(jìn)行磷酸化,證明了PtkA是PknD的特異性磷酸化底物。利用LC-MS/MS分析發(fā)現(xiàn)PknD對(duì)PtkA的磷酸化位點(diǎn)為T119和另外一個(gè)靠近其自磷酸化位點(diǎn)y262的基序,暗示其可能影響自磷酸化位點(diǎn)附近的結(jié)構(gòu),進(jìn)而改變其自磷酸化活性,間接改變其底物磷酸化活性。因此,STPKs可能通過調(diào)控PtkA而間接調(diào)控PtpA的功能,影響結(jié)核菌與宿主之間的相互作用和持留性感染。本實(shí)驗(yàn)結(jié)果又一次揭示了結(jié)核菌中信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)之間的相互調(diào)控關(guān)系,展示了結(jié)核菌信號(hào)傳導(dǎo)系統(tǒng)的復(fù)雜性和調(diào)控的多層次性。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.11

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相關(guān)期刊論文 前1條

1 戴小鋒;劉仲明;;乙肝電化學(xué)免疫傳感器陣列電極的研制[J];生物醫(yī)學(xué)工程學(xué)雜志;2008年02期

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本文編號(hào)：2208170