結(jié)核分枝桿菌RD區(qū)新功能性基因的發(fā)現(xiàn)及其免疫機制與應用的初步研究
發(fā)布時間：2019-06-26 21:15
【摘要】：背景：結(jié)核分枝桿菌(Mycobacterium tuberculosis, M.tb)感染引起的結(jié)核病(Tuberculosis, TB)是全球三大傳染病殺手之一。據(jù)2015年最新流行病學統(tǒng)計,每年新增結(jié)核病例960萬,導致150萬死亡。全球約有1/3人口感染結(jié)核,其中90-98%為潛伏感染,其中2-10%會發(fā)展為活動性結(jié)核,加之耐藥株的流行、與HIV或HCV共感染等情況使得該疾病嚴重威脅著人類健康。近年來,結(jié)核病在診斷、預防及致病機制的研究方面雖然已取得了一定進展,但仍存在許多不足,需要進一步地完善及深入。目前,結(jié)核病的診斷主要依賴胸片、痰涂片抗酸染色、TST、PCR、微生物培養(yǎng)等手段；但這些方法或靈敏度低、或檢查周期長。近年來發(fā)展的IFN-γ釋放檢測試驗(IGRAs)具有了較高的靈敏度及特異度,但該類型檢測試劑盒是僅以ESAT-6、CFP10多肽為刺激抗原使診斷仍存在漏檢情況。在結(jié)核病預防方面,唯一可用的疫苗是卡介苗(Bacillus Calmette-Guerin, BCG),但僅能為未成年兒童提供可靠的保護性,成人中免疫保護性非常低下。已有研究顯示重組BCG為改良疫苗的最佳方法,但難點在于尋找合適的靶標抗原。篩選優(yōu)勢靶標抗原不僅可用于提高診斷率還可應用于疫苗研究。巨噬細胞是結(jié)核分枝桿菌寄生的主要宿主細胞,部分結(jié)核分枝桿菌可逃避宿主免疫系統(tǒng)、尤其是巨噬細胞的追殺。但具體機制尚不清楚,深入了解結(jié)核分枝桿菌與宿主巨噬細胞相互作用的機制和途徑,對于解釋結(jié)核分枝桿菌致病機理特別是它在巨噬細胞中長期寄生的原因有著重要的作用。BCG基因組中的某些片段相對于牛型結(jié)核分枝桿菌或結(jié)核分枝桿菌H37Rv來說發(fā)生缺失,這些缺失片段被定義為RD區(qū)(Region of Differences, RD)�；蛐酒夹g(shù)研究發(fā)現(xiàn)BCG較H37Rv丟失了RD1-16區(qū),近年來許多研究報道顯示,許多該丟失區(qū)域所表達的蛋白中有很多功能性蛋白,包括具有很強的免疫原性的基因、介導免疫逃逸的基因等。目的：篩選RD區(qū)中優(yōu)勢免疫原性抗原,評估其在結(jié)核診斷中的用途；利用該RD抗原構(gòu)建能增強BCG免疫原性的新型重組BCG疫苗(rBCG::Rv2645);篩選RD區(qū)中可能存在的抗巨噬細胞殺傷基因,探討結(jié)核分枝桿菌的逃逸機制。方法：本研究通過構(gòu)建RD10-14區(qū)基因的31種原核表達質(zhì)粒、表達純化相應RD蛋白、ELISPOT等方法篩選出優(yōu)勢免疫原性抗原。以該蛋白作為結(jié)核特異性抗原評估其診斷用途。同時,利用電轉(zhuǎn)技術(shù)將該RD基因?qū)隑CG中構(gòu)建重組疫苗rBCG::Rv2645,通過免疫Balb/c、鼠、恒河猴后,評估其免疫原性及H37Rv攻毒后的抗結(jié)核免疫保護能力。另外,利用表達RD蛋白建立的BL21-RDs菌種庫,篩查這些RD蛋白對巨噬細胞的影響；通過巨噬細胞吞噬試驗及菌落計數(shù)、LDH釋放實驗等篩選出能抗巨噬細胞殺傷的RDs基因,從而進一步探討該蛋白與巨噬細胞的相互作用機制。結(jié)果：本研究首次從RD10-14區(qū)中篩選出來源于RD13區(qū)的分泌型蛋白Rv2645能刺激T淋巴細胞產(chǎn)生高水平特異性的IFN-γ,Rv2645特異性1FN-y水平在結(jié)核病人PBMCs中明顯高于健康人(包括BCG接種者)；在結(jié)核病人與健康人的鑒別診斷中,Rv2645的檢測靈敏度與特異度高達90.0%、98.2%,其中結(jié)核病人的診斷結(jié)果與臨床上使用CFP10-ESAT6為刺激抗原的T-SPOT.TB診斷結(jié)果相比,相符率可達98%。但Rv2645與CFP10-ESAT6聯(lián)合使用后比單獨使用CFP10-ESAT6或Rv2645抗原效果更好,能將靈敏度從88.0%左右提升至96.0%,且具有類似高特異度98.2%；且Rv2645特異性皮膚試驗可區(qū)分結(jié)核感染及BCG接種人群。我們還成功構(gòu)建了重組疫苗rBCG::Rv2645;在Balb/c小鼠及恒河猴的動物實驗中發(fā)現(xiàn),相對于BCG而言,rBCG::Rv2645能提升更高的免疫原性及抗結(jié)核免疫保護作用。另外,本研究發(fā)現(xiàn)來源于RD14區(qū)的Rv1768 (PE-PGRS31)基因能逃逸巨噬細胞的殺傷。進一步的研究發(fā)現(xiàn)Rv1768是通過與巨噬細胞的Annexin A2結(jié)合從而降低胞內(nèi)鈣離子濃度,進一步抑制了吞噬溶酶體的成熟。結(jié)論：本研究首次發(fā)現(xiàn)的Rv2645蛋白是結(jié)核病T細胞免疫的特異性識別抗原,在結(jié)核診斷試劑開發(fā)及結(jié)核疫苗設(shè)計方面具有一定的潛力；另外,所篩選的Rv1768抗原為結(jié)核桿菌逃逸機制的探討提供了新方向。
[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.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R378.911
本文編號：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.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R378.911
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相關(guān)期刊論文 前2條
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