体内诱导抗原技术筛选病原菌体内诱导抗原的研究进展

体内诱导基因是病原菌在宿主体内能够表达而体外培养时却不能表达的功能基因,其对病原体在宿主体内的生存和致病具有重要意义。体内诱导抗原技术(in vivo induced antigen technology, IVIAT)已广泛应用于筛选病原体体内诱导基因,相较于其他用于筛选体内诱导基因的技术,IVIAT具有无需动物模型、检测病原菌在不同感染阶段产生的抗原等独特优势。IVIAT鉴定出的体内诱导抗原对病原体在宿主中的毒力、代谢及存活具有重要意义。现就IVIAT的原理、IVIAT筛选出的人类疾病相关病原菌的体内诱导抗原及其功能研究等作一概述。     

致病菌体内诱导基因的功能分析—体内表达技术的应用

病原菌体内诱导的基因在致病过程中起重要作用,体内表达技术是一类很有前景的研究体内诱导基因的技术,本介绍了体内表达技术的基本原理,类型以及在致病菌体内诱导基因方面的研究进展和应用前景。     

伤寒沙门菌感染过程中体内诱导表达蛋白抗原的筛选

在感染过程中,病原体会表达一系列基因产物以适应体内环境.因此,病原体只在宿主体内表达或高表达的基因可能与致病机制密切相关.本研究用体内诱导抗原技术（in vivo induced antigen technology,IVIAT）来筛选伤寒沙门菌感染过程中体内诱导表达的抗原,共筛选到7个体内诱导（in vivo induced,IVI）抗原,分别是BcfD（菌毛结构亚单位）、GrxC（谷氧还蛋白3）、SapB（ABC转运系统）、T3663（ABC未知转运系统）、T3816（可能的有关硫氰酸酶的硫转移酶）、T1497（可能的TonB依赖受体）和T3689（功能未知）,其中BcfD,GrxC,SapB,T3663和T3689这5个抗原与吸附处理后的健康志愿者的血清无交叉免疫反应.这些筛选到的体内诱导抗原是新药和疫苗开发的潜在靶标,同时也有利于新诊断方法的研究.     

耐药结核分枝杆菌感染动物模型所用菌株的筛选

本研究通过小鼠体内实验检测我国部分地区结核分枝杆菌耐药菌株的毒力,以筛选耐药结核分枝杆菌感染动物模型所用菌株。收集从我国部分省份98例结核病患者痰培养液中分离出的结核分枝杆菌,用比例法药敏试验进行结核分枝杆菌一线和二线药物的药敏试验,筛选出对二线药物敏感而对一线药物利福平或异烟肼耐药或敏感的菌株,然后进行小鼠体内毒力实验,对异烟肼耐药相关基因katG和利福平耐药相关基因rpoB测序并进行基因突变分析。从98株菌中筛选出药物敏感谱清晰的40株,进行小鼠体内毒力实验。结果显示,共35株半数死亡时间≤H37Rv的半数死亡时间,其中18株耐利福平合并耐异烟肼、5株单耐利福平,7株单耐异烟肼、5株对利福平和异烟肼均敏感。通过小鼠毒力研究,分别筛选出基因背景清晰,半数死亡时间≤7d的耐利福平合并耐异烟肼的菌株1株、半数死亡时间≤7d单耐利福平和异烟肼的菌株各1株,作为耐药结核分枝杆菌感染小鼠模型所用菌株及进一步进行豚鼠等其他动物模型感染用候选菌株。     

结核分枝杆菌抗原检测研究进展

结核病仍然是一个严重的全球性公共卫生问题,有效控制结核病的障碍在于缺乏早期、准确的诊断方法。机体受到结核分枝杆菌感染后,体内首先出现的是结核分枝杆菌特异性抗原。因此,结核分枝杆菌抗原检测作为结核病早期诊断的方法可能具有很高的诊断价值。我们简要综述了结核分枝杆菌抗原检测的相关研究进展。     

结核分枝杆菌免疫优势抗原研究进展

结核病是当今影响人类健康、流行性最广、病死率最高的感染性疾病之一。结核病的诊断和疫苗的构建成为当前的研究热点,筛选出结核分枝杆菌免疫优势抗原是快速准确的诊断结核病及研制安全有效的疫苗的关键。拟对近年来国内外学者发现的结核分枝杆菌免疫优势抗原的分子生物学特性研究进展进行综述。     

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。     

结核分枝杆菌的热休克蛋白及其抗原

<正>由病原性分枝杆菌引起的疾病,仍然是人类发病率和死亡率引起的主要原因,在发展中国家的死亡率中仅结核病就达7％。目前预防和控制分枝杆菌感染的研究,已转向包括病原菌与其哺乳类宿主细胞间相互关系的分子机理的阐明。根据结核杆菌和麻风杆菌一些重要蛋白抗原的序列分析,已知它们是热休克蛋白谱系中的成员,和大肠杆菌蛋白的Dnak、GroEL和GroES相关。本文分析了热休克对结核杆菌蛋白合成的作用,以期为阐明分枝杆菌分子遗传学与宿主-寄生间相互关系提供重要资料。     

结核分枝杆菌毒力的研究进展

结核分枝杆菌可以在人体内缓慢增殖,释放代谢产物,造成细胞损伤,引起结核病。其致病机制可能与其复杂的细胞壁成分密切相关。结核分枝杆菌细胞壁中的脂质、糖类及蛋白类物质在构成屏障结构,保护并辅助结核分枝杆菌在细胞内的生长及迁移,调节宿主免疫应答,造成宿主组织细胞损伤等方面发挥重要生物学作用。其表达受众多基因的精细调控。本文将对结核分枝杆菌细胞壁中的脂质阿拉伯甘露聚糖和分枝菌酸在其毒力中的作用,以及sig基因对毒力的调节作用作一综述。     

Identification of in vivo induced protein antigens of Salmonella enterica serovar Typhi during human infection

During infectious disease episodes, pathogens express distinct subsets of virulence factors which allow them to adapt to different environments. Hence, genes that are expressed or upregulated in vivo are implicated in pathogenesis. We used in vivo induced antigen technology (IVIAT) to identify antigens which are expressed during infection with Salmonella enterica serovar Typhi. We identified 7 in vivo induced (IVI) antigens, which included BcfD (a fimbrial structural subunit), GrxC (a glutaredoxin 3), SapB (an ABC-type transport system), T3663 (an ABC-type uncharacterized transport system), T3816 (a putative rhodanese-related sulfurtransferase), T1497 (a probable TonB-dependent receptor) and T3689 (unknown function). Of the 7 identified antigens, 5 antigens had no cross-immunoreactivity in adsorbed control sera from healthy subjects. These 5 included BcfD, GrxC, SapB, T3663 and T3689. Antigens identified in this study are potential targets for drug and vaccine development and may be utilized as diagnostic agents.     

Direct detection of Mycobacterium tuberculosis lipid antigens by thin-layer chromatography

Abstract Free lipids were extracted from Mycobacterium tuberculosis H37Rv, and their antigenicity was assessed directly on thin-layer chromatograms (TLC) by an immunostaining technique. A family of glycolipids, composed of trehalose acylated with multimethyl branched long-chain fatty acids, was investigated. The most polar of these glycolipids was identified as a possible specific surface antigen. A pair of novel polar glycolipids also showed positive antigenic reactions.     

结核分枝杆菌对宿主巨噬细胞死亡方式的调控

结核分枝杆菌（Mycobacterium tuberculosis,Mtb）感染后能抑制宿主巨噬细胞（M西）的免疫反应,并在其中生存、复制。研究表明Mtb减毒株感染主要诱导宿主Mφ凋亡,凋亡能抑制胞内Mtb的活力;而Mtb毒力株感染能抑制凋亡的完成,诱导Mφ坏死,最终导致Mtb扩散、感染临近细胞。通过对Mtb感染诱导宿主Mφ不同死亡方式的讨论,进一步认识Mtb的致病机制。     

寡核苷酸芯片研究结核分枝杆菌临床株和无毒株诱导的巨噬细胞U937凋亡相关基因的差异表达

结核病仍然是人类健康的主要威胁,结核分枝杆菌诱导的巨噬细胞凋亡是宿主防御反应之一,研究凋亡相关基因的差异表达有助于认识结核分枝杆菌致病机理和发现新的药物靶标。利用包括19200个基因或基因片段的DNA芯片研究巨噬细胞株U937对临床和实验室菌株感染的差异表达,Northem blotting和RT—PCR验证了芯片研究结果。Mtb H37Rv感染下调bcl—2,vitaminD受体、干扰素调控因子3、细胞色素氧化酶C表达,幅度分别为2-,3-,3-,2．5-倍,临床菌株感染上调SOD2、SOD3、丝氨酸蛋白酶、toll—like受体2、signal transducer and activator(STAT1)、hypoxia—inducible factor22等表达,幅度分别为2．9-,2．5-,2．5-,22-,2．4-,5．9-倍。结果提示,临床菌株感染更多促进凋亡,限制宿主的杀灭机理。该研究为进一步研究导致这些差异表达的结核分枝杆菌成分提供了基础。     

敲除pckA基因的结核杆菌引起的免疫反应的研究

研究结核杆菌pckA基因编码的磷酸烯醇型丙酮酸羧激酶(PEPCK)诱导机体产生的保护性免疫反应。用敲除pckA基因的牛结核杆菌BCG和野生型BCG分别感染小鼠,取肝、肺、脾进行病理分析,并进行脾细胞培养,检测CD4 、CD4 /CD8 、细胞因子IFNI-γI、L-12和TNF等。用敲除pckA基因的BCG感染的小鼠比野生型BCG感染的小鼠体内产生的结核结节少且不典型,炎性程度低。野生型BCG感染的小鼠脾脏内的CD4 T细胞和CD4 /CD8 、细胞因子IFN-γ、IL-12、TNF均明显高于敲除pckA基因BCG感染的小鼠。pckA基因为结核杆菌生长所必需,其编码产物PEPCK能够刺激机体产生免疫反应,是一种很好的疫苗候选分子。     

Horizontal transfer of a virulence operon to the ancestor of Mycobacterium tuberculosis

The contribution of interspecies horizontal gene transfer (HGT) to the evolution and virulence of Mycobacterium tuberculosis, the agent of tuberculosis in humans, has been barely investigated. Here we have studied the evolutionary history of the M. tuberculosis Rv0986-8 virulence operon recently identified, through functional genomics approaches, as playing an important role in parasitism of host phagocytic cells. We showed that among actinobacteria, this operon is specific to the M. tuberculosis complex and to ancestral Mycobacterium prototuberculosis species. These data, together with phylogenetic reconstruction and other in silico analyses, provided strong evidence that this operon has been acquired horizontally by the ancestor of M. tuberculosis, before the recent evolutionary bottleneck that preceded the clonal-like evolution of the M. tuberculosis complex. Genomic signature profiling further suggested that the transfer was plasmid mediated and that the operon originated from a gamma-proteobacterium donor species. Our study points out for the first time the contribution of HGT to the emergence of M. tuberculosis and close relatives as major pathogens. In addition, our data underline the importance of deciphering gene transfer networks in M. tuberculosis in order to better understand the evolutionary mechanisms involved in mycobacterial virulence.     

High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis Phosphoserine Phosphatase

The emergence of drug-resistant strains of Mycobacterium tuberculosis makes identification and validation of newer drug targets a global priority. Phosphoserine phosphatase (PSP), a key essential metabolic enzyme involved in conversion of O-phospho-l-serine to l-serine, was characterized in this study. The M. tuberculosis genome harbors all enzymes involved in l-serine biosynthesis including two PSP homologs: Rv0505c (SerB1) and Rv3042c (SerB2). In the present study, we have biochemically characterized SerB2 enzyme and developed malachite green-based high throughput assay system to identify SerB2 inhibitors. We have identified 10 compounds that were structurally different from known PSP inhibitors, and few of these scaffolds were highly specific in their ability to inhibit SerB2 enzyme, were noncytotoxic against mammalian cell lines, and inhibited M. tuberculosis growth in vitro. Surface plasmon resonance experiments demonstrated the relative binding for these inhibitors. The two best hits identified in our screen, clorobiocin and rosaniline, were bactericidal in activity and killed intracellular bacteria in a dose-dependent manner. We have also identified amino acid residues critical for these SerB2-small molecule interactions. This is the first study where we validate that M. tuberculosis SerB2 is a druggable and suitable target to pursue for further high throughput assay system screening.     

Identification of the new T-cell-stimulating antigens from Mycobacterium tuberculosis culture filtrate

The proteins secreted by Mycobacterium tuberculosis are an important target for vaccine development. To identify the antigens from M. tuberculosis culture filtrate (CF) that strongly stimulate T-cells, the CF was fractionated by ion-exchange chromatography and then non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mini-whole gel elution. Each fraction was screened for its ability to induce interferon-gamma (IFN-gamma) production in peripheral blood mononuclear cells isolated from healthy tuberculin reactors. The protein bands that strongly induced IFN-gamma production were subjected to N-terminal sequencing. Two new proteins, a 17-kDa protein (Rv0164, MTSP17) and an 11-kDa (Rv3204, MTSP11) protein, were identified. The recombinant MTSP17 (rMTSP17) and rMTSP11 induced significant production of IFN-gamma and interleukin (IL)-12p40 in peripheral blood mononuclear cells from healthy tuberculin reactors. Interestingly, IL-12p40 production in response to rMTSP11 was significantly higher than that in response to rMTSP17 or the three components of the antigen 85 complex. These results suggest that MTSP11 antigen should be further evaluated as a component of a subunit vaccine.     

细菌毒力基因体内表达检测技术研究进展

病原菌入侵宿主是一个及其复杂的过程。为了深入了解病原菌的致病机理,人们需要鉴定那些在感染过程中特异表达的细菌毒力基因。为此,多种体内实验模型被建立起来分析细菌在宿主体内的基因表达,它们包括了体内表达技术、信号标签突变技术、差异荧光诱导、体外转座进行基因组分析和作图技术以及体内诱导抗原技术等。文章对目前运用的这些研究方法进展进行综述,并讨论了它们的优点与不足。