微量元素对结核分枝杆菌生长的影响

本文报道了25种微量元素在改良罗氏培养基上对分枝杆菌菌株H,,Ra和BcG生长的促进作用。试验结果表明：N,、B、Mo、Cr、Zn、Se、Be等元素能显著地促进菌株的生长。Sr、Cu,Al、Si等元素也在一定程度上促进菌株的生长。培养基的pH值对微量元素的这种生长促进作用有一定影响。Zn、M。与草酸铵组合,对分枝杆菌菌株H,,Ra和BoG在酸性培养基上的初生长和后生长,均有明显的促进作用。而Co、Cu和Mn组合能部分地消除高pH值对分枝杆菌生长的抑制作用o N,和se有使分枝杆菌斜面培养物保持细胞和菌落完整的作用。     

结核分枝杆菌分泌蛋白ESAT-6的表达、纯化和鉴定

目的:克隆结核分枝杆菌分泌蛋白ESAT-6基因,并在大肠杆菌中进行表达和纯化。方法:用PCR方法从结核分枝杆菌H37Rv基因组扩增出ESAT-6基因片段,克隆至pMD18-T载体中,序列测定正确后,将其亚克隆到表达载体pGEX-4T-1并在大肠杆菌DH5α中表达,表达蛋白经SDS-PAGE及Western-blot分析后,亲和层析法纯化蛋白。结果:成功克隆了ESAT-6基因,并对其在E.coli中进行了表达,SDS-PAGE及Western-blot分析表明表达产物正确。通过GST纯化系统获得34kD纯化蛋白,与文献报道相符。结论:成功获得了纯化的ESAT-6蛋白,为进一步研究ESAT-6蛋白的致病机理提供了实验依据。     

结核分枝杆菌分泌蛋白ESAT-6的表达、纯化和鉴定

目的：克隆结核分枝杆菌分泌蛋白ESAT-6基因,并在大肠杆菌中进行表达和纯化。方法：用PCR方法从结核分枝杆菌H37Rv基因组扩增出ESAT-6基因片段,克隆至pMD18一T载体中,序列测定正确后,将其亚克隆到表达载体pGEX-4T－1并在大肠杆菌DH5α中表达,表达蛋白经SDS—PAGE及Westem—blot分析后,亲和层析法纯化蛋白。结果：成功克隆了ESAT-6基因,并对其在E．coli中进行了表达,SDS—PAGE及Western—blot分析表明表达产物正确。通过GST纯化系统获得34kD纯化蛋白,与文献报道相符。结论：成功获得了纯化的ESAT-6蛋白,为进一步研究ESAT－6蛋白的致病机理提供了实验依据。     

结核分枝杆菌早期分泌蛋白ESAT-6家族的研究进展

ESAT-6家族蛋白是结核分枝杆菌早期分泌蛋白中具有较强免疫原性的成分之一。卡介苗中缺乏该家族的一些重要成分。近年对其分布。结构,免疫学特征以及生物学用途方面的研究较多。该蛋白在作抗结核分枝杆菌亚单位疫苗的候选抗原,DNA疫苗的候选基因以及作为结核分枝杆菌感染的免疫学诊断试剂等领域体现出良好的研究及应用前景。     

结核分枝杆菌ESX-1分泌蛋白EspB结构和功能分析

结核分枝杆菌作为肺结核病的病原菌,在人类中致死率远高于其他病原菌.结核分枝杆菌具有特殊的疏水性细胞壁结构,这种致密的细胞壁结构帮助结核分枝杆菌抵御外界环境压力和来自宿主细胞的毒素.同时,它利用特殊的分泌系统将体内的毒力蛋白输出体外,ESX-1分泌系统就是其中之一.结核分枝杆菌ESX-1系统在结核分枝杆菌进入宿主细胞吞噬小体、逃逸至细胞质以及杀死吞噬细胞这些过程中发挥重要作用.研究表明,在结核分枝杆菌内膜上存在一个由多亚基组成、旨在帮助结核分枝杆菌向外输送分泌蛋白的分泌装置.在这个分泌装置的帮助下,结核分枝杆菌重要的毒力蛋白ESAT-6跨内膜向外分泌,EspB也通过这个内膜上的分泌装置被转运至胞外.EspB存在于静置培养的结核分枝杆菌的胶囊层中,也可在振荡培养的结核分枝杆菌的培养液中被检测.通过X射线晶体衍射分析,我们解析了EspB的晶体结构,相比于其他同源结构,发现了EspB的不同构象,即EspB单体能够自组装成为七聚体的规则结构,联系其与毒力因子ESAT-6具有共分泌的特点,七聚体构象的发现为解释EspB在结核分枝杆菌向外分泌蛋白的过程中发挥的作用提供线索,即EspB具有锚定在结核分枝杆菌胶囊层中,作为运输ESAT-6的孔道而存在的可能.     

结核分枝杆菌ESX-1分泌蛋白ESAT-6增强巨噬细胞吞噬功能

【目的】研究结核分枝杆菌(Mycobacterium tuberculosis)分泌蛋白ESAT-6(early secreted antigenictarget of 6 kDa)对巨噬细胞吞噬功能的影响。【方法】用重组质粒pFLAG-ESAT-6和pFLAG-EGFP转染RAW264.7细胞,经G418筛选,PCR、RT-PCR和Western blot鉴定,获得稳定表达flag-ESAT-6和flag-EGFP的RAW细胞系,然后用流式细胞术观察各稳转细胞系吞噬荧光微球的能力,并用共聚焦显微镜和菌落计数法检测稳转细胞系吞噬大肠杆菌(Escherichia coli)的能力。【结果】获得了稳定表达flag-ESAT-6的RAW-E6细胞系和表达flag-EGFP的RAW-EGFP细胞系;流式细胞术检测结果表明RAW-E6吞噬荧光微球的能力显著强于野生型细胞系RAW264.7和对照细胞系RAW-EGFP;菌落计数和激光共聚焦分析表明RAW-E6细胞系吞噬E.coli的能力也显著强于RAW264.7和RAW-EGFP。【结论】通过胞内表达发现结核分枝杆菌分泌蛋白ESAT-6能够增强巨噬细胞的吞噬功能,这将为深入理解结核分枝杆菌的致病机制提供新的思路。     

结核分枝杆菌蛋白抗原的研究进展

结核病当今世界人类致死的主要疾病之一,早期诊断发现病人、选择敏感的抗结核药物进行有效治疗是控制结核病的关键。而临床上对结核病患者检出率低,漏诊率和误诊率高,结果导致结核耐药的情况越来越严重。简便、快速、准确的免疫学检测方法在诊断结核病中起到了重要的作用。本文对用于免疫学检测的蛋白抗原作一综述。     

结构分枝杆菌分泌蛋白研究进展

结构分枝杆菌（ＭＴＢ）的分泌蛋白是目前发现对ＭＴＢ感染保护性最好的一组蛋白,对结构病的预防和诊断具有重要意义。本文从其组成、生物学功能、免疫性及其在疫苗研制和ＭＴＢ诊断中的潜在价值作一概述。     

核酸、核酸前体对结核分枝杆菌H37Ra和BCG生长的影响

在罗氏培基中添加嘧啶类化合物或其组合物,对结核分枝杆菌H37Ra和BCG菌株有明显的生长促进作用。这种生长促进作用在不利于生长的PH条件下更加明显。 种子斜面在4℃冰箱贮存的时间越久,嘧啶类化合物的生长促进作用越明显。鸟嘌呤和鸟嘌呤核苷酸对结核分枝杆菌的早期生长有促进作用。在罗氏培基中添加0．3—5mg/L的RNA或DNA,对菌株H37Ra和BCG的生长无刺激作用。     

结核分枝杆菌RPF样蛋白家族及其功能

上世纪末,Mukamolova等从藤黄微球菌（Micrococcus luteus）发现一种分泌蛋白-复苏促进因子（Rpf）.该因子在皮摩尔浓度下能促进休眠期的藤黄微球菌及其他数种高G＋C含量的革兰氏阳性菌复苏和生长.结核分枝杆菌全基因组发现和该因子同源性较高的5个基因,预测产物都是分泌蛋白,一级结构都含有一个转糖基域.5个基因有一个共同的功能未知的域,3级结构预测该保守结构域和溶菌酶同源.重组表达的Rpf对不同种均有活性.5个rpf在不同生长时期均有表达,但调控有差异.     

抗结核分枝杆菌疫苗的研究进展

结核病是由结核分枝杆菌感染引起的传染病,细胞免疫中的CD4+T细胞、CD8+T细胞、Th17细胞在对抗结核分枝杆菌感染中发挥重要作用,新近研究显示抗体特定的糖链修饰有助于清除病原体,提示体液免疫也可能参与免疫保护。目前使用的疫苗——卡介苗对婴幼儿重症结核病具有良好的保护力,但是对成人肺结核保护力欠佳,所以需要研发新的疫苗。目前已有数个新型疫苗进入临床试验。本文就结核分枝杆菌的免疫保护机制作一简要介绍,主要阐述现用疫苗——卡介苗及新型疫苗的研究现状,让读者对上述知识的进展有所了解。     

结核杆菌融合蛋白DNA疫苗构建及免疫保护研究

目的:评价结核DNA疫苗免疫鼠产生细胞因子和抵抗结核分枝杆菌攻击的能力。方法:将结核菌Mtb8.4基因和谷胱甘肽S转移酶基因插入pVAX1载体,构建表达Mtb8.4和GST融合蛋白的DNA疫苗pVS8.4G。小鼠分成5组,用pVS8.4G、pVAX1、pIL2S 100μg和PBS 0.1mL各免疫3次,间隔2w。另一组用BCG免疫1次。每组10只鼠在加强后,无菌取脾培养。另外10只小鼠用H37Rv攻击,2w后取脾、肝和肺培养结核菌并计数。结果:pVS8.4G免疫鼠脾细胞培养上清mIL-2和mIFN-γ平均为380.9和422.1pg/mL,显著高于阴性对照组,与BCG组无显著差异。5个组的平均mIL-6和mIL-10无显著差异。pVS8.4G免疫小鼠脾、肝和肺的平均结核菌载量分别为42 093.2、43 264.1和37 264.8CFU/g,低于pVAX1、pIL2S和PBS组相应器官的载量。结论:DNA疫苗pVS8.4G能刺激产生Th1型免疫应答,免疫鼠抵抗H37Rv攻击的能力增强。     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non‐synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species‐specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia‐related genes between M. bovis and M. tuberculosis.     

Electrochemical-based biosensors for detection of Mycobacterium tuberculosis and tuberculosis biomarkers

Abstract

Early detection of tuberculosis (TB) reduces the interval between infection and the beginning of treatment. However, commercially available tests cannot discriminate between BCG-vaccinated healthy persons and patients. Also, they are not suitable to be used for immunocompromised persons. In recent years, biosensors have attracted great attention due to their simple utility, accessibility, and real-time outputs. These sensors are increasingly being considered as pioneering tools for point-of-care diagnostics in communities with a high burden of TB and limited accessibility to reference laboratories. Among other types of biosensors, the electrochemical sensors have the advantages of low-cost operation, fast processing, simultaneous multi-analyte analyzing, operating with turbid samples, comparable sensitivity and readily available miniaturization. Electrochemical biosensors are sub-divided into several categories including: amperometric, impedimetric, potentiometric, and conductometric biosensors. The biorecognition element in electrochemical biosensors is usually based on antibodies (immunosensors), DNAs or PNAs (genosensors), and aptamers (aptasensors). In either case, whether an interaction of the antigen–antibody/aptamer or the hybridization of probe with target mycobacterial DNA is detected, a change in the electrical current occurs that is recorded and displayed as a plot. Therefore, impedimetric-based methods evaluate resistance to electron transfer toward an electrode by a Nyquist plot and amperometric/voltammetric-based methods weigh the electrical current by means of cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Electrochemical biosensors provide a promising scope for the new era of diagnostics. As a consequence, they can improve detection of Mycobacterium tuberculosis traces even in attomolar scales.     

FasL regulatory B-cells during Mycobacterium tuberculosis infection and TB disease

Tuberculosis (TB) disease remains a major health crisis. Infection with Mycobacterium tuberculosis (M.tb) cause a range of diseases ranging from latent infection to active TB disease. This active state of the disease is characterised by the formation of granulomas (a physical barrier in the lung), a structure thought to protect the host by controlling the infection through preventing the growth of the bacilli. Subsequently, the surviving bacteria become inactive and in most cases, TB reactivation is prevented by the immune response of the host. B-cells perform numerous immunological functions beyond antibody production to positively regulate the response to pathogenic assault. A subgroup of B-cells with regulatory functions express death-inducing ligands, such as Fas ligand (FasL). Expression and interaction of the Fas receptor-ligand promotes the induction of apoptosis and the induction of T-cell tolerance. Here, we focus on the significance of B-cells by addressing their FasL phenotype and regulatory functions during TB, with reference to disease in humans, non-human primates and mice.     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

结核分枝杆菌抗原重组酿酒酵母免疫诱导小鼠特异性免疫应答

近年来基于重组酿酒酵母全细胞的新型疫苗研究报道不断出现。以结核杆菌重要保护抗原ESAT6和Ag85B为对象,采用pHR酿酒酵母表达系统,构建了两种分别表达ESAT6-Ag85B(EA)和IFN-γ-ESAT6-Ag85B(IEA)融合抗原的重组酿酒酵母Yeast-EA和Yeast-IEA。重组酵母以皮下注射方式免疫小鼠后,小鼠产生高水平Ag85B特异性抗体,淋巴细胞分泌IFN-γ、IL-2等细胞因子,无IL-4产生,发生Th1型细胞免疫应答,其中Yeast-IEA效应更强,优于传统的BCG疫苗。实验证实重组酵母能够刺激树突状细胞的成熟分化。研究结果显示结核分枝杆菌抗原重组酿酒酵母全细胞疫苗具有发展成为新型抗结核疫苗的潜力。     

Secretory proteins of Mycobacterium habana induce a protective immune response against experimental tuberculosis

The capacity of proteins of Mycobacterium habana TMC 5135 secreted into culture medium during the mid-exponential growth phase (secretory proteins, SPs) to induce protective immunity against Mycobacterium tuberculosis H37Rv was studied in the mouse model. Mice immunized with SPs followed by a challenge with M. tuberculosis H37Rv showed lesser M. tuberculosis bacilli in their lung and spleen and survived longer than unimmunized controls. The findings suggest that SP antigens of M. habana are protective against tuberculosis infection.     

结核分枝杆菌重要诊断用抗原研究进展

血清学试验是结核诊断的重要依据。随着科学技术的进步,新的结核诊断用抗原不断被发现。我们简要综述了结核菌素蛋白衍生物、抗原85复合体、38kDa磷酸盐转运蛋白、6kDa早期分泌性蛋白、10kDa培养滤液蛋白、免疫性蛋白MPT64、主要分泌性免疫蛋白MPB70、表面脂蛋白MPB83等8种结核分枝杆菌重要抗原作为结核诊断用抗原的研究进展。