【作者】 李华；

【导师】 张先恩； 王殿冰；

【作者基本信息】 华中农业大学 ， 微生物学， 2017， 博士

【摘要】 就发病率和死亡率而言,结核病是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)引起的世界第二大传染病。长期以来,Mtb的感染机制一直是结核病研究领域中悬而未决的基本科学问题之一。为此,本研究利用TMT(Tandem Mass Tag)定量蛋白质组学技术,通过对巨噬细胞感染Mtb后蛋白质组的系统研究及后续生物学验证,发现了一批在感染过程中受Mtb毒力调控且影响宿主细胞命运的候选蛋白,为进一步解析巨噬细胞应对Mtb侵染的免疫机制提供了丰富依据。具体结果如下:1)在巨噬细胞分别感染H37Rv(强毒株)和H37Ra(弱毒株)的差异蛋白质组研究中,共鉴定到6,702个蛋白,其中235个为差异表达蛋白,并通过Western blot实验验证了部分蛋白的差异表达。这些蛋白涉及细胞凋亡、凝血和氧化磷酸化等生物学过程,并初步揭示了宿主细胞分别感染两种M.tb后的四种不同机制。2)在上述差异表达蛋白中,AHSG蛋白为抗炎性因子,基因敲降实验证明AHSG蛋白通过凋亡途径帮助Mtb逃避免疫。凝血因子10(F10)是凝血酶通路中的一员,可以代替补体结合到病毒表面激活免疫反应,通过ELISA实验和免疫荧光实验证明F10蛋白可以与分枝杆菌结合。本研究首次初步揭示AHSG蛋白和F10蛋白在巨噬细胞感染Mtb后免疫反应中的作用,其具体机制还需后续深入研究。3)γ-干扰素是激活巨噬细胞免疫应答的关键因子。M.tb分别侵染γ-干扰素激活前后的巨噬细胞,共鉴定到257个差异表达蛋白,并通过Western blot实验验证了部分蛋白的差异表达。这些差异蛋白涉及细胞凋亡、抗原递呈和凝血等生物学过程。分析结果表明,γ-干扰素通过增强感染后巨噬细胞对抗原的呈递能力提高机体对M.tb的免疫。由于M.tb的慢生长特性(大约是大肠杆菌生长速率的1/50-1/70)极大地制约了结核病的诊断、药物研发和基础研究的进程,除了上述工作之外,本研究同时尝试了M tb快速生长菌的构建工作。本课题组成功的构建了与M.tb近缘且生长较快的M smegatis基因组Cosmid文库并转化至M.tb,期望通过Mtb中大量M.smegmatis基因的异源表达,改变Mtb原有代谢网络,进而加快M.tb的生长速度。目前的结果表明Mtb中cosmid质粒片段丢失,后期将对cosmid骨架质粒的复制子进行优化,并通过构建低DNA重组酶活性、弱DNA限制修饰系统的M.tb优化菌种来实现该课题目标。更多还原

【Abstract】 Tuberculosis(TB),caused by Mycobacterium tuberculosis(M.tb),is the second infectious disease in the world,because of its morbidity and mortality.However,the infection mechanism of M.tb remains to be elucidated now.In this study,we used TMT(Tandem Mass Tag)quantitative proteomics to investigate the proteome of M.tb infected macrophages.We identified some macrophage proteins that are regulated by the virulent factor of M.tb..These proteins possibly have great effect on the fate of macrophages.These findings would contribute to further illustrate how macrophages reponse to the infection of M.tb.The main results are as follows:1)In the proteomic studies of M.tb infected macrophages,6,702 proteins were identified;H37Ra and H37Rv caused 235 differentially expressed proteins in macrophages.These proteins are related to the biological processes of apoptosis,coagulation and oxidative phosphorylation and indicate four possible different mechanisms of macrophages infected by two M.tb strains respectively.2)Among the above differential expressed proteins,AHSG was negative acute-phase reactant.AHSG may help M.tb to escape from macrophage killing by knockdown experiment.The coagulation factor F10 was one factor of blood coagulation cascade,which could bind to virus surface and active immune response.F10 would take part in the M.tb infection by binding to the surface of M.tb by ELISA and immunofluorescence.This research revealed the function of AHSG and F10 during M.tb infecting macrophages for the first time.3)Interferon-y(IFN-y)is a key factor in macrophage immune response.After M.tb infection,IFN-y acitivated macrophages produced 257 differentially expressed proteins comparing with those not treated macrophages.These proteins are related to such biological processes as apoptosis,antigen presentation and coagulation.The results show that IFN-y obviously enhances the host immune response against M.tb by improving the ability of macrophage antigen presentation after infection.Additionally,as the slow growth of M.tb(about 1/50-1/70 of E.coli growth rate)significantly hinders the diagnosis,treatment and scientific research of tuberculosis,we try to construct fast-growing M.tb strains in this study.M.smegmatis is one of Mycobacterium sp with rapid growth.Therefore,we firstly constructed the cosmid library of M.smegmatis.Then transformed the library into M.tb.We considered the original metabolic network of M.tb would be changed by the heterologous expression of M.smegmatis genes in M.tb and thus create fast growing M.tb.Large amount of M.smegmatis derived genes,were lost in M.tb.We believe this project would be finished by constructing of M.tb strain,the DNA recombinant enzyme activity and DNA limited modification system of which were weak.更多还原