抗结核分枝杆菌感染的免疫机制

对宿主利用体内各种细胞与结核分支杆菌的相互作用进行了综述.机体抵抗结核分枝杆菌感染的机制包括特异性免疫和非特异性免疫,参与非特异性免疫的主要有巨噬细胞和γδT细胞,另外,树突状细胞在引发T细胞免疫方面起着关键的作用.由于结核分支杆菌是胞内寄生菌,特异性免疫以细胞免疫为主,主要包括CD4+T细胞免疫和CD8+T细胞免疫.     

C-di-GMP signaling and implications for pathogenesis of Mycobacterium tuberculosis

C-di-GMP is a ubiquitous bacterial second messenger that regulates a wide range of bacterial physiological processes including biofilm formation, virulence, motility and cell differentiation. Here, we have summarized our current knowledge on the upstream signaling factors and downstream effectors of c-di-GMP in addition to the interaction between c-di-GMP and eukaryotic organisms. New discoveries in these areas have enriched our understanding of the diversity of c-di-GMP signaling pathways and provide important clues for us to explore the roles of c-di-GMP signaling in human pathogens such as Mycobacterium tuberculosis.     

小鼠结核分枝杆菌感染模型用于评价重组HBHA疫苗免疫效果的实验研究

目的通过复制小鼠结核病模型评价重组HBHA疫苗的免疫效果。方法将重组肝素结合血凝素(HBHA)疫苗接种BALB/c小鼠,观察其诱导的细胞免疫应答水平,并以MTB H37Rv毒株攻击免疫小鼠,研究重组疫苗诱导的保护力。结果重组HBHA免疫BALB/c小鼠后可诱发细胞毒作用,将体内MTB裂解。重组疫苗不仅能刺激CD4+T,CD8+T增殖、活化,还可诱导脾细胞分泌IFN-γ、IL-2等细胞因子。H37Rv毒株攻击后,被免疫小鼠的组织病理学症状减轻。结论本研究通过检测小鼠体内细胞免疫应答和肺脏组织病理学改变等指标,客观的反映了重组疫苗免疫小鼠结核模型的保护效果。     

The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis.

Tuberculosis is responsible for one in four of all avoidable adult deaths in developing countries. Increased frequency and accelerated fatality of the disease among individuals infected with human immunodeficiency virus has raised worldwide concern that control programmes may be inadequate, and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in several recent fatal outbreaks in the United States. Isonicotinic acid hydrazide (isoniazid, INH) forms the core of antituberculosis regimens; however, clinical isolates that are resistant to INH show reduced catalase activity and a relative lack of virulence in guinea-pigs. Here we use mycobacterial genetics to study the molecular basis of INH resistance. A single M. tuberculosis gene, katG, encoding both catalase and peroxidase, restored sensitivity to INH in a resistant mutant of Mycobacterium smegmatis, and conferred INH susceptibility in some strains of Escherichia coli. Deletion of katG from the chromosome was associated with INH resistance in two patient isolates of M. tuberculosis.     

Thrombin-stimulated cell division involves proteolysis of its cell surface receptor.

A cell-surface component of molecular weight 43,000 is cleaved by thrombin on cells that divide after thrombin treatment, but is not cleaved on cells that are unresponsive to its mitogenic action. Studies with a photoreactive derivative of thrombin showed that its cell surface receptor has a molecular weight of 43,000. This indicates that thrombin must cleave its receptor to stimulate cell division.     

Neuronal cell killing by the envelope protein of HIV and its prevention by vasoactive intestinal peptide

The clinical manifestations of AIDS (acquired immune deficiency syndrome) often include neuropsychiatric and neurological deficits, including early memory loss and progressive dementia. HIV (human immunodeficiency virus), the aetiological agent of AIDS, is probably carried by infected macrophages in the central nervous system. The virus enters cells by binding its envelope glycoprotein gp120 to the CD4 antigen present on brain and immune cells. From the data reported in this paper, we now suggest that the neuronal deficits associated with HIV may not be entirely a result of infectivity, but that gp120 shed from HIV could directly produce the neuropathology as a result of its interference with endogenous neurotrophic substances. It is known that an analogue of a sequence contained in vasoactive intestinal peptide (VIP) occurs in all known sequenced gp120 isolates and that VIP is important for neuronal survival in cell culture. Here we show that purified gp120 from two diverse HIV isolates and a recombinant gp120 from a third isolate were all potent in specifically producing significant neuronal cell death in dissociated hippocampal cultures derived from fetal mice, and that this could be reduced by monoclonal antibodies against the murine CD4 antigen and completely antagonized by VIP.     

Regulation of cancer cell migration and bone metastasis by RANKL

Bone metastases are a frequent complication of many cancers that result in severe disease burden and pain. Since the late nineteenth century, it has been thought that the microenvironment of the local host tissue actively participates in the propensity of certain cancers to metastasize to specific organs, and that bone provides an especially fertile 'soil'. In the case of breast cancers, the local chemokine milieu is now emerging as an explanation for why these tumours preferentially metastasize to certain organs. However, as the inhibition of chemokine receptors in vivo only partially blocks metastatic behaviour, other factors must exist that regulate the preferential metastasis of breast cancer cells. Here we show that the cytokine RANKL (receptor activator of NF-kappaB ligand) triggers migration of human epithelial cancer cells and melanoma cells that express the receptor RANK. RANK is expressed on cancer cell lines and breast cancer cells in patients. In a mouse model of melanoma metastasis, in vivo neutralization of RANKL by osteoprotegerin results in complete protection from paralysis and a marked reduction in tumour burden in bones but not in other organs. Our data show that local differentiation factors such as RANKL have an important role in cell migration and the tissue-specific metastatic behaviour of cancer cells.     

结核分枝杆菌膜蛋白Rv0849免疫学特性的初步检测

探讨了结核分枝杆菌膜蛋白Rv0849作为药泵蛋白的转运功能及其作为外源蛋白免疫诱导C57BL/6小鼠产生免疫应答的能力.从H37Rv基因组中扩增获得Rv0849基因,酶切后连接到分枝杆菌穿梭质粒pMV261上构建重组质粒pMV0849,并以耻垢分枝杆菌为载体构建获得重组菌株SM(0849).检测药物对重组菌株SM(0849)的最小抑菌浓度(MIC)变化,用重组菌株免疫C57BL/6小鼠检测其作为外源蛋白免疫诱导小鼠产生的免疫应答反应.结果表明,重组菌株SM(0849)对药物的耐受性没有明显提高,但能够较早、较好地诱导小鼠体液免疫应答水平,并能通过刺激小鼠脾脏淋巴细胞促使其分泌高水平的肿瘤坏死因子-α和干扰素-γ.Rv0849所编码的蛋白具有较弱的药泵功能,但有潜力成为一种新的免疫抗原.     

结核分枝杆菌RipB基因的克隆、表达及对藤黄微球菌生长的影响

提取肺结核分枝杆菌基因组DNA,PCR扩增RipB基因并将其连接到表达载体pET-21a上,重组质粒转化大肠杆菌BL21(DE3)感受态细胞后,用IPTG诱导表达重组蛋白.SDSPAGE表明:重组RipB表达蛋白量约占菌体总蛋白量的40％,而其在37℃表达时主要为包涵体,在15℃表达时主要在可溶上清内;Ni柱一步亲和层析获得重组RipB;100 pmol/L重组RipB可显著促进藤黄微球菌生长.     

结核分枝杆菌ESX-1分泌系统的研究进展

目的:综述结核分枝杆菌ESX-1分泌系统的组成及其组分的作用。方法:检索并查阅结核分枝杆菌ESX-1分泌系统相关的文献,并进行归纳总结。结果:ESX-1系统是以分泌ESAT-6及CFP-10为特征的分泌系统,包括编码基因及十余种分泌调节基因。结论:结核分枝杆菌毒力分子ESAT-6及CFP-10的分泌是多种相关编码基因共同作用的结果。     

结核分枝杆菌ICL抑制CTBP1表达并促进细胞凋亡

异柠檬酸裂合酶(Isocitrate Lyase,ICL)参与乙醛酸循环,是结核分枝杆菌能在宿主体内潜伏的关键因素之一.为了探索ICL在结核菌与宿主相互作用中的地位,首先通过酵母双杂交技术在人淋巴细胞cDNA文库中筛选得到一个新的ICL相互作用蛋白CTBP1(C Terminal Binding Protein 1).然后分别采用GST pull-down和Co-IP实验证实了这两个蛋白在体外和体内与ICL相互作用的特异性.通过使ICL在HEK293T细胞中过表达,发现内源性CTBP1在转录水平和蛋白水平的表达都受到了抑制,同时导致细胞增殖减缓、凋亡加快,并出现G2/M期阻滞等改变.这些结果表明,结核菌ICL在参与介导细胞凋亡、影响宿主细胞活性的过程中起着重要的作用.     

Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice

Tuberculosis is the leading cause of death in the world resulting from a single bacterial infection. Despite its enormous burden on world health, little is known about the molecular mechanisms of pathogenesis of Mycobacterium tuberculosis. Bacterial multiplication and concomitant tissue damage within an infected host, including experimentally infected mice, occurs primarily in the lungs-the favoured niche of M. tuberculosis. Although it has been proposed that the distinctive cell wall of M. tuberculosis is important for virulence, rigorous genetic proof has been lacking. Here, using signature-tagged mutagenesis, we isolated three attenuated M. tuberculosis mutants that cannot synthesize or transport a complex, cell wall-associated lipid called phthiocerol dimycocerosate (PDIM) which is found only in pathogenic mycobacteria. Two mutants have transposon insertions affecting genes implicated in PDIM synthesis; the third has a disruption in a gene encoding a large transmembrane protein required for proper subcellular localization of PDIM. Synthesis and transport of this complex lipid is only required for growth in the lung; all three mutants are unaffected for growth in the liver and spleen. This clearly shows that a lipid is required for M. tuberculosis virulence.     

表达结核杆菌抗原的重组酿酒酵母免疫小鼠研究

近年来研究显示全细胞重组酿酒酵母疫苗具有治疗性疫苗潜能.为了研究重组酿酒酵母结核病候选疫苗的免疫效果,将IFN-γ基因与结核杆菌抗原蛋白Ag85B、ESAT6的基因融合,利用pHR酿酒酵母表达系统和同源重组方法,成功构建了以酿酒酵母Y16为宿主的表达融合蛋白IF-γ-Ag85B和IF-γ-ESAT6-Ag85B的重组酿...     

结核分枝杆菌Rv2626c基因的表达、纯化和鉴定

克隆结核分枝杆菌Rv2626c基因,构建其原核表达载体,并进行表达、纯化及鉴定.采用聚合酶链反应(PCR)方法从结核分枝杆菌H37Rv基因组中扩增出Rv2626c基因片段,克隆入pMD18-T载体,序列测定正确后亚克隆入原核表达载体pPro-EXHTb,转化入E.Coli DH5α,经IPTG诱导表达,SDS-PAGE分析后与抗His单抗进行Western-blot,以Ni-NTA亲和层析纯化目的蛋白.PCR扩增获得的目的基因为432bp,与预期大小一致,测序与Genebank公布的基因序列完全一致.成功构建原核表达载体pPro-EXHTb-2626c,转化E.Coli DH5α后能特异性表达目的蛋白,大小约16KD,与理论值相一致.蛋白可溶性分析表明该蛋白主要以可溶性方式存在,纯化蛋白经Western-blot鉴定有特异性反应条带.成功构建结核分枝杆菌Rv2626c原核表达载体pPro-EXHTb-2626c,并获得纯化的目的蛋白,为研究新型结核疫苗的靶抗原奠定了基础.     

Heterologous Expression of Mycobacterium tuberculosis Ag85B in Pichia pastoris

Tuberculosisisaworldwidehealthproblemandmaybethemostcommoncauseofdeathfromanysingleinfectiousagent.Theannualnumberofdeathscausedbythisdiseaseisanticipatedtoreach3.5×106by2005.Theserioussituationhascreatedanurgentneedfora newvaccinetopreventTB[1].TheMycob…     

结核分枝杆菌Hsp16.3单克隆抗体的制备及其特性鉴定

制备抗结核分枝杆菌Hsp16.3的单克隆抗体,并对其生物学特性进行鉴定。将含目的基因的表达载体pProEXHTb-Hsp16.3,通过E.coli DH5α诱导表达,获得含有6譎is的Hsp16.3蛋白,采用Ni-NTA纯化试剂盒进行目的蛋白的纯化,并用透析方法进行蛋白复性。将复性的蛋白免疫BALB/c小鼠,利用杂交瘤技术进行细胞融合,间接ELISA筛选阳性杂交瘤细胞株。将获得的Hsp16.3阳性杂交瘤细胞株分别利用间接ELISA法和Western blot方法进行效价、相对亲和力及特异性的测定。获得了三株Hsp16.3的单克隆抗体,分别命名为3H6F9、1D5E1和4H8G6,其效价分别为1:1?07、1:1?06和1:1?06,相对亲和力分别为0.0001 mg/mL、0.001 mg/mL和0.001 mg/mL,并且无交叉反应性。所获得的结核分枝杆菌Hsp16.3的单克隆抗体效价高、特异性强,为进一步研究Hsp16.3在结核分枝杆菌潜伏感染中的作用提供了有效的工具。     

四川省结核分枝杆菌基因分型中MIRU-VNTR位点稳定性评价

摘要: 本文收集四川省2009-2013年184株结核分枝杆菌菌株,用标准12 MIRU-VNTR位点组对四川省结核分枝杆菌菌株进行基因分型研究,评估四川地区一线VNTR分型位点的稳定性,确定四川地区保守位点组合及主要MIT型。184株结核分枝杆菌基因分型后得到51个基因型,12个基因型成簇。本研究确定当前四川地区12高分辨力位点VNTR分型方法中的7个MIRU位点依然具有较高的分辩能力（h > 0.55）。MIRU02, MIRU20, MIRU23, MIRU24为多态性最低的4个位点（0 ≤ h < 0.4）,保守位点组的主要MIT型为“2251”。四川省结核分枝杆菌VNTR位点的多态性保持稳定,目前采用的12高分辨力VNTR位点依然稳定可靠,本研究首次确定了四川地区保守位点组及主要MIT型,为下一步结核分枝杆菌进化研究奠定了基础。