结核分枝杆菌的分子免疫学进展

结核分枝杆菌(H37Rv)全基因组测序的完成有力地支持了人们对其毒力基因和保护性抗原的寻求与研究。本文就结核杆菌的致病和在体内巨噬细胞中长期存活的机制、参与免疫应答的几类T细胞的作用及其调节机制等方面的研究进展作一综述，以期为抗结核新型疫苗的设计提供参考。     

结核分枝杆菌重组MPT64蛋白抗原诱发豚鼠迟发型超敏反应的研究

结核菌素纯蛋白衍生物 (ＰＰＤ)含多种蛋白成分 ,与多种分枝杆菌存在交叉 ,尤其在鉴别结核菌感染和卡介苗接种阳转者上有一定的困难 ,所以ＰＰＤ作为结核病诊断试剂有其局限性。现已发现结核分枝杆菌Ａｇ85、ＭＰＴ64、38ｋＤ抗原、ＭＰＴ63、ＥＳＡＴ6等 ,均能诱发豚鼠发生迟发性超敏反应[1] 。具有高特异性的新的诊断试剂或许就存在于他们或他们的组合中。ＭＰＴ64蛋白是结核分枝杆菌生长过程中的分泌蛋白 ,它能诱发结核分枝杆菌感染的豚鼠发生很强的ＤＴＨ反应 ,部分ＢＣＧ在减毒过程中丢失了编码该蛋白的基因 ,特异性较强 ,用于皮肤试验…     

MPT64--卡介苗重组疫苗的构建

全球有三分之一的人口约2 0亿感染过结核分枝杆菌,其中16 0 0万人处于活跃期,每年有约10 0 0万新感染者。由于用药的不规范,耐药和耐多药结核病显著增多,增加了治疗的难度;流动人口的增加使结核病的传播机会增加;而艾滋病与结核病合并感染,使问题变的更为严峻。当前预防结核病的卡介苗效果在不同地区有很大差异,改进疫苗的要求提到日程。MPT6 4是结核分枝杆菌分泌蛋白中含量较多的一种,具有免疫保护力,是亚单位疫苗和核酸疫苗的候选者之一。本研究通过穿梭质粒将基因导入卡介苗,希望能在卡介苗中表达MPT6 4蛋白,增强其免疫原性和保护力。…     

结核分枝杆菌的分子免疫学进展

结核分枝杆菌 (H37Rv)全基因组测序的完成有力地支持了人们对其毒力基因和保护性抗原的寻求与研究。本文就结核杆菌的致病和在体内巨噬细胞中长期存活的机制、参与免疫应答的几类T细胞的作用及其调节机制等方面的研究进展作一综述 ,以期为抗结核新型疫苗的设计提供参考     

SELEX技术筛选结核分枝杆菌MPT64蛋白DNA适配子方法学研究

目的 利用指数富集配体系统进化(SELEX)技术筛选能与结核分枝杆菌MPT64蛋白特异结合的寡核苷酸适配子.方法 体外合成长度为78个核苷酸的随机ssDNA文库,利用SEL-EX技术筛选,以MPT64蛋白为靶物质进行12轮筛选,利用生物素-亲和素显色系统检测寡核苷酸与蛋白的结合性.结果 SELEX技术筛选MPT64蛋白适配子的技术体系:PCR扩增的最佳退火温度为65℃;文库优化时,最佳Mg2+的浓度为1.5 mmol/L;不对称PCR法制备ssDNA时,Mg2+的浓度为0.75 mmol/L;以酶联板为介质筛选时,在10轮后筛选达到饱和,且随着筛选轮数的增加,PCR扩增产物的电泳条带逐渐单一、致密,亲和性检测显示第10轮获得的适配子库,比初始的文库亲和性吸光度(A)值增加了9.18倍.将适配子库克隆,随机挑取10个单克隆子,利用混合夹心法检测其与MPT64的亲和性,亲和性范围分布在0.572～1.606之间.结论 已初步筛选到与MPT64蛋白高亲和性结合的DNA适配子.     

结核分枝杆菌Rv3245 蛋白抗原免疫学特性的研究

目的:研究结核分枝杆菌Rv3425 蛋白免疫学特性,评估其诊断应用价值及在结核致病性中的作用。方法:将Rv3425 基因克隆至pET28a 载体中,诱导表达Rv3425 融合蛋白并进行纯化;利用ELISA、Western blot 分析其抗原性与特异性;流式检测该抗原对巨噬细胞凋亡的影响。结果:获得了可溶性原核表达融合蛋白Rv3425,Rv3425 蛋白能刺激灭活结核分枝杆菌免疫小鼠脾细胞产生高水平的特异性IFN鄄酌、纯化的Rv3425 蛋白能与结核感染小鼠血清特异性结合,其特异性血清抗体IgG 及IgM 在结核病人中的水平明显高于健康人,发现该蛋白能诱导巨噬细胞的凋亡。结论:本研究发现结核分枝杆菌Rv3425 蛋白具有较强的抗原性且能促进巨噬细胞的凋亡,这些发现对于结核病的诊断及致病机制的研究具有重要价值。     

结核分枝杆菌Ag85A质粒DNA疫苗治疗小鼠耐药结核病的效果研究

目的 研究结核分枝杆菌Ag85A质粒DNA疫苗治疗小鼠耐药结核病的效果.方法 用结核分枝杆菌高耐利福平低耐异烟肼临床分离株HB361尾静脉注射17～19 g的6～8周龄雌性BALB/c小鼠后,将小鼠随机均匀地分为6组,感染后第3天开始,分别用生理盐水(A组)、pVAX1空载体(B组)、利福平(C组)、微卡菌苗(D组)、Ag85A质粒DNA疫苗(E组)、利福平和Ag85A质粒DNA疫苗(F组)治疗60d,每组10只小鼠.治疗结束后3周,分别取肺和脾观察病理改变,称取重量做菌落计数.结果 治疗结束后3周,与对照组比较,D组、E组和F组肺脏病变有不同程度减轻,病变局限,病变范围分别为50%、20%、20%,2/3区域可见正常的肺泡结构,肺泡轮廓相对清晰,细胞分布均匀.与A组相比,D组、E组和F组肺脏菌落数分别减少了52%、68%、78%;脾脏菌落数依次减少了48%、65%、79%.结论 与对照组相比,Ag85A质粒DNA疫苗单独应用或与利福平联合应用治疗小鼠耐药结核病均显示疗效.     

结核分枝杆菌Rv0901基因重组耻垢分枝杆菌的构建及其细胞作用

目的对结核分枝杆菌Rv0901基因的功能进行研究。方法以PCR扩增Rv0901基因编码序列,定向克隆人穿梭表达质粒pMV261获得重组穿梭表达质粒,将重组质粒电穿孔进入耻垢分枝杆菌,构建重组Rv0901基因的耻垢分枝杆菌,对重组耻垢分枝杆菌进行诱导表达,用SDS-PAGE检测表达结果。比较耻垢分枝杆菌和重组耻垢分枝杆菌对THP-1细胞的不同作用。结果成功构建重组穿梭表达质粒及重组Rv0901基因的耻垢分枝杆菌,重组菌诱导THP-1细胞的凋亡率高于耻垢分枝杆菌,其感染THP-1细胞后细胞的存活率低于耻垢分枝杆菌的感染,重组菌感染THP-1细胞后细胞培养液中NO(一氧化氮)的产生高于耻垢分枝杆菌的感染。结论Rv0901基因可能与结核毒力存在一定关系。     

结核分枝杆菌Ag85B-MPT64融合基因疫苗的免疫效果观察

目的:研究并比较结核分枝杆菌(H37Rv)Ag85B、MPT64DNA.以及两者的融合基因(AM)的免疫原性。方法:雌性C57BL/6小鼠25只,随机分为5组,即A组(PBS)、B组(peDNA3.1)、C组(pcDNA/Ag85B)、D组(pcDNA/MPT64)和E组(pcDNA/AM)。分别于胫前肌注射7.5 g/L利多卡因和质粒混合物(1:4,100μL,含质粒70μg/次),间隔2 wk免疫 1次,共3次。末次免疫后4 wk取血分离血清测定总IgG,同时分离脾淋巴细胞,用PPD刺激后分别做脾淋巴细胞增殖实验(MTT比色法)和测定脾淋巴细胞培养上清中IFN-γ的水平。结果:Ag85B、MPT64和AM质粒DNA,均能诱导小鼠产生较高水平的PPD特异性IgG。免疫小鼠脾淋巴细胞体外经PPD刺激后,能产生特异性淋巴细胞增殖和分泌IFN-γ。peDNA/AM组IFN-γ的分泌水平明显高于pcDNA/Ag85B和pcDNA/MPT64免疫组(P<0.05)。结论:结核分枝杆菌Ag85B-MPT64融合基因疫苗,能在小鼠体内诱导特异性细胞和体液免疫。     

外泌体与结核分枝杆菌相互作用的研究进展

结核病是目前世界各国重点防控传染性疾病之一。外泌体(exosomes)是多种细胞分泌的一种含有特异性与非特异性蛋白成分的囊泡结构,是机体免疫细胞之间重要的信息传递介质,其在机体免疫反应中发挥着重要作用。外泌体能呈递表面抗原,激活多种细胞免疫因子,介导巨噬细胞杀伤结核杆菌的细胞免疫反应。外泌体与结核分枝杆菌相互作用研究已经成为当前重大研究热点,有望开发成预防结核病的新型疫苗。     

Rapid diagnosis of tuberculosis in aspirate, effusions, and cerebrospinal fluid by immunocytochemical detection of Mycobacterium tuberculosis complex specific antigen MPT64

The aim of the study was to evaluate the diagnostic potential of immunocytochemical staining for the detection of Mycobacterium tuberculosis complex-specific antigen MPT64, in tuberculous lymph node aspirates, cerebrospinal fluid, and effusions from pleura and abdomen. One hundred ninety patients with a diagnosis of tuberculosis (cases) and 80 patients with nontuberculous lesions (controls) were enrolled and differentiated on the basis of clinical features, histology, cytology, clinical biochemistry, Ziehl-Neelsen staining, Lowenstein-Jensen culture, and response to antituberculous therapy. Cervical lymph nodes fine-needle aspirate (n = 150), cerebrospinal fluid (n = 27), pleural fluid (n = 41), and peritoneal fluid (n = 52) were collected and stained with anti-MPT64 and anti-BCG antibodies using immunocytochemistry. Nested-PCR for IS6110 was done for comparison and to calculate the diagnostic indices of the ICC staining. ICC staining with anti-MPT64 was positive in 128/190 (67.4%) tuberculous smears and in 4/80 (5%) control smears thus giving sensitivity of 67.4% and the specificity of 95%, while anti-BCG was positive in 112 (58.9%) tuberculous smears and in 16/80 (20%) control smears with sensitivity of 58.9% and specificity of 80%. When diagnostic validation of ICC was done using PCR as the gold standard, the overall sensitivity, specificity, positive- and negative-predictive values for ICC staining in smears with anti-MPT64 was 96, 96, 95, and 97%, respectively, while the corresponding values for anti-BCG were 87, 88, 86, and 88%. ICC staining using anti-MPT64 represents a robust and simple method for establishing an early etiological diagnosis of M. tuberculosis complex infection in extrapulmonary tuberculosis.     

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

Purpose

Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed.

Materials and Methods

The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains.

Results

The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%).

Conclusion

The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.     

Mapping of the delayed-type hypersensitivity-inducing epitope of secreted protein MPT64 from Mycobacterium tuberculosis.

The gene encoding the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was expressed in Escherichia coli K-12 and purified as a recombinant protein. The purified recombinant MPT64 elicited delayed-type hypersensitivity (DTH) in outbred guinea pigs sensitized with Mycobacterium bovis BCG Tokyo. The skin reactions were comparable to those obtained with native MPT64. No skin reactions were observed when either recombinant MPT64 or native MPT64 was used in guinea pigs sensitized with M. bovis BCG Danish 1331. Amino- and carboxy-terminal deletion mutants of MPT64 were purified as fusion proteins for the mapping of DTH-inducing epitopes on recombinant MPT64 by use of the guinea pig skin test model. The part of the molecule responsible for the biological activity was located at the carboxy-terminal end. Further studies with overlapping synthetic peptides have pinpointed the biological activity at a single DTH-inducing epitope consisting of 15 residues between amino acids Gly-173 and Ala-187. Screening by PCR of 56 clinical isolates of M. tuberculosis from Danish and Tanzanian patients demonstrated the presence of mpt64 in all of the strains. These results point to MPT64 as a possible candidate for a skin test reagent specific for diagnosis of human tuberculosis.     

Characterization of the secreted MPT53 antigen of Mycobacterium tuberculosis

MPT53 is a secreted protein of Mycobacterium tuberculosis. Southern transfer and hybridization showed mpt53 to be conserved in the M. tuberculosis complex and to have homology with DNA from Mycobacterium avium and other nontuberculous mycobacteria. However, anti-MPT53 polyclonal antibodies detected no antigen in the culture filtrates of M. avium and other nontuberculous mycobacteria. MPT53 of M. tuberculosis induced strong, tuberculosis-specific antibody responses in guinea pigs but induced no delayed-type hypersensitivity. Involvement in immune responses during human tuberculosis was very modest.     

Human T-Cell Epitopes on the Mycobacterium tuberculosis Secreted Protein MPT64

Mycobacterium tuberculosis secretes a number of proteins into the extracellular envirornment, some of which are restricted to the M. tuberculosis complex. These proteins are targets for T- and B-cell immune responses in tuberculosis (TB) patients and their contacts. The authors have mapped the immunogenic regions of the MPT64 protein of M. tuberculosis using peripheral blood mononuclear cells (PBMC) from TB patients and a set of overlapping peptides encompassing the complete sequence of the protein. T-cell epitopes which induced proliferation or interferon-gamma (IFN-γ) release were distributed over the full length of the protein. A C-terminal region of the protein, however, contains sequences recognized in the context of multiple HLA-DR phenotypes by more than 80% of the subjects tested. The nature of the T-cell response was further investigated by generating MPT64-specific T-cell lines. These lines also identified the T-cell epitopes in the C-terminal region of the protein. On stimulation with antigen the lines secreted IFN-γ, but not interleukin 4 (IL-4). A minority of TB patients (6/32) mounted an antibody response to MPT64. Sera from half (3/6) of these identified two linear antibody binding sites. These results confirm the significance of this protein in the immune response to tuberculosis infection.     

Reduced apoptosis and increased inflammatory cytokines in granulomas caused by tuberculous compared to non-tuberculous mycobacteria: role of MPT64 antigen in apoptosis and immune response

Inhibition of apoptosis of infected macrophages by pathogenic mycobacteria is suggested to be an important virulence mechanism, but little is known about the mycobacterial proteins involved in the inhibition of apoptosis. In this study we investigated differences in apoptosis and immune response and their correlation with the expression of Mycobacterium tuberculosis complex-specific secretory protein MPT64 in lesions caused by tuberculous or non-tuberculous mycobacteria by analysing the in situ expression of apoptosis-related proteins (FasL, Fas, Bax, Bcl-2), apoptotic cells, inflammatory cytokines [tumour necrosis factor (TNF)-alpha, interleukin (IL)-10, transforming growth factor (TGF)-beta, interferon (IFN)-gamma] and MPT64 antigen. The discrimination of mycobacteria was made by nested polymerase chain reaction (PCR) amplification of IS6110, which is specific for M. tuberculosis complex organisms. Forty-seven cases of lymphadenitis with necrotic granulomas were evaluated. With nested PCR, 30/47 cases were positive for M. tuberculosis. MPT64 antigen was detected specifically in the PCR-positive cases. Granulomas caused by tuberculous mycobacteria had fewer apoptotic cells, higher numbers of cells expressing TNF-alpha and TGF-beta and less extensive necrosis than granulomas caused by non-tuberculous mycobacteria. There was a significant negative correlation between apoptotic cells and the number of cells expressing MPT64 antigens, suggesting a role for MPT64 protein in the inhibition of apoptosis. Granulomas with higher amounts of MPT64 also showed a greater number of cells expressing TGF-beta than those with lower amounts of MPT64. In conclusion, this study supports the hypothesis that inhibition of apoptosis is a virulence mechanism for tuberculous mycobacteria. Correlation of MPT64 antigen with expression of macrophage deactivating cytokines and reduced apoptosis suggests its role in pathogenesis and bacillary persistence.     

Characterization of a Mycobacterium leprae antigen related to the secreted Mycobacterium tuberculosis protein MPT32.

Secreted proteins may serve as major targets in the immune response to mycobacteria. To identify potentially secreted Mycobacterium leprae antigens, antisera specific for culture filtrate proteins of Mycobacterium tuberculosis were used to screen a panel of recombinant antigens selected previously by leprosy patient sera. Four potentially secreted antigens were identified by this approach, and one was recognized by antibodies specific for MPT32, a secreted M. tuberculosis protein. The DNA coding for the M. leprae antigen, which we have designated 43L, was isolated and characterized and found to encode a 25.5-kDa protein that is preceded by a consensus signal peptide of 39 amino acids. The N-terminal amino acid sequence of 43L shows 50% homology with the 20 known N-terminal amino acids of MPT32, and 47% homology was found with the N terminus of a 45/47-kDa antigen complex identified in Mycobacterium bovis BCG. These findings indicate that 43L represents an antigen related to MPT32 and the M. bovis BCG 45/47-kDa complex and that 43L is likely to be a protein secreted by M. leprae. Purified recombinant 43L protein is recognized by antibodies and T cells from healthy contacts and leprosy patients, illustrating that secreted proteins are of importance in the immune response to M. leprae.