致病性钩端螺旋体限制性内切酶酶切分析分型研究

为了建立一种简便、稳定的钩体分型方法,我们采用限制性内切酶ＥｃｏＲＩ对致病性钩端螺旋体八个血清群５４个血清型６４株国内外钩体参考株及２７株野生株进行限制性内切酶酶切分析（ＲＥＡ）。结果表明：９１株菌中共有５９种不同的限制性内切酶图谱（ＲＥＰｓ）,根据前５条高分子酶切片段可以区分不同的ＲＥＰｓ;除部分血清群中个别不同的血清型有相同的ＲＥＰ型外,大部份血清型都有其独特的ＲＥＰ型,同一血清群往往拥有共同的酶切片段;所研究的同一血清型的国内和国际参考株的ＲＥＰｓ不同;大多数野生株的ＲＥＡ型和参考株相同,差异仅表现为个别高分子带的缺少和增加,ＲＥＡ分型和血清分型吻合较好,通过ＲＥＡ分型基本可区分不同的血清型。     

贵州省3株钩端螺旋体分离株PFGE分型与基因种鉴定

目的应用脉冲场电泳（PFGE）分型技术和16S rRNA基因测序分析技术分别对贵州省3株动物宿主钩 端螺旋体分离菌株进行分子分型和基因种鉴定,了解贵州省钩端螺旋体的分子流行病学特征。方法应用 DNA限制性内切酶Not I对钩端螺旋体染色体DNA酶切后,用PFGE将DNA片段分离,采用BionumericsV4.0将3 株钩体菌株PFGE图谱与中国15群15型参考菌株进行聚类分析;同时,应用PCR扩增几乎全长的钩体16S rRNA基因片段,并将扩增产物进行双向序列测定,并与GenBank数据库已注册的核酸序列进行同源性比对 、确定基因种、分析亲缘进化关系。结果来自贵州省的3株动物宿主分离钩体菌株PFGE带型命名为LepNot I 002和LepNot I 003,经聚类分析,3株菌株与黄疸出血群黄疸出血型赖株的相似性大于95%。16S rRNA 基因测序和分析表明,3株贵州分离钩体菌株之间的同源性为100%,与致病性钩体问号钩端螺旋体种（L. interrogans）不同血清型参考菌株的同源性达100%。结论3株贵州动物分离钩体菌株经PFGE分型鉴定与黄 疸出血群赖型赖株的相似性大于95%,经16S rRNA基因测序分析鉴定为L. interrogans种,上述两种方法 对贵州省钩体分离菌株的鉴定结果一致,有助于贵州省钩端螺旋体病的主动监测、暴发调查和传染源追踪 。     

问号钩端螺旋体赖株基因组中一个新溶血基因的表达及功能鉴定

目的对问号钩端螺旋体赖株基因组中LA0202基因进行克隆表达并对重组蛋白的溶血活性进行初步鉴定。方法以问号钩端螺旋体赖株基因组DNA为模板,PCR扩增LA0202基因并重组到原核表达载体pET28b(+),重组质粒经限制性核酸内切酶酶切并经测序鉴定后,在大肠埃希菌BL21中诱导表达重组蛋白。制备绵羊血平板对表达的重组蛋白进行溶血活性鉴定。RT-PCR检测钩端螺旋体赖株体外培养时LA0202基因的转录。结果在大肠埃希菌中成功表达LA0202基因,表达的重组蛋白具有溶血活性。RT-PCR结果显示钩端螺旋体赖株体外培养时LA0202基因发生了转录。结论问号钩端螺旋体赖株体外培养时LA0202基因发生了转录,LA0202可能为一个新的溶血素基因。     

钩端螺旋体核酸分子生物学研究进展

随着分子生物学的兴起,钩端螺旋体(简称钩体)的研究工作者相继利用DNA限制性内切酶图谱分析、分子杂交、分子克隆与PCR等分子生物学技术,对钩体DNA代谢基因的调控与抗原基因的表达均获得了新的认识,并为该病的检测、传染源和传播途径的追踪以及监测提供了一些快速、简便、可靠的方法。 一、钩体基因组大小及其G C%含量 肖建国等用脉冲凝胶电泳技术(PFGE)对不同群型的5株钩体基因组DNA分子量进行了研究,表明钩体基因     

问号赖型钩端螺旋体重组质粒pDL121的初步研究

目的　探讨问号赖型钩端螺旋体抗原基因的特点。方法　应用６ 种常见内切酶（ＢａｍＨＩ,Ｂｇｌ Ⅱ,ＥｃｏＲⅠ,Ｈｉｎｄ Ⅲ,ＰｓｔⅠ,Ｐｖｕ Ⅱ）对从问号赖型钩端螺旋体０１７ 株的基因文库中筛选出来的重组质粒ｐＤＬ１２１ 外源基因进行酶谱分析,同时用Ｄｉｇｏｘｉｎ 标记的ｐＤＬ１２１ 外源基因片段作探针对不同种属的致病性钩端螺旋体和非致病性钩端螺旋体基因进行杂交分析。结果　显示问号赖型钩端螺旋体重组质粒ｐＤＬ１２１ 外源基因没有６ 种内切酶的酶切位点,重组探针与致病性钩体（ｓｅｒｏｖａｒｌａｉｓｔｒａｉｎ０１７,ｓｅｒｏｖａｒ ｈｅｂｄｏｍａｄｉｓｓｔｒａｉｎ ５６６１０ ,ｓｅｒｏｖａｒ ｐｏｍｏｎａｓｔｒａｉｎ ５６６０８）有杂交信号,与非致病性钩体（ｓｅｒｏｖａｒ ｐａｔｏｃ ｓｔｒａｉｎ Ｐａｔｏｃ Ⅰ,ｓｅｒｏｖａｒｉｌｌｉｎｉｓｔｒａｉｎ ３０５５）无杂交信号,亦不识别大肠杆菌。结论　重组质粒ｐＤＬ１２１ 外源基因可能是问号赖型钩端螺旋体的一个新基因,进一步测序分析将揭示该基因本质,同时因该重组探针能鉴别致病性钩体和非致病性钩体,提示其可用于钩端螺旋体病的早期诊断及钩端螺旋体的鉴定和分类。     

酶谱多态性分析对非典型布鲁氏菌鉴定

用 Hind Ⅲ、Bgl I、EcoR I 三种限制性核酸内切酶消化了8株非典型布鲁氏菌的染色体DNA,并在0.8％琼脂糖、4％和10％的聚丙烯酞胺凝胶上进行了电泳,各菌株的酶切图谱未显出明显的不同。     

2009-2015年贵州省致病性钩端螺旋体分离株PFGE分型分析北大核心CSCD

目的了解贵州省2009-2015年分离自鼠类宿主动物的钩端螺旋体(简称钩体)PFGE带型特征,为当地钩体病的预防和控制提供科学依据。方法对贵州省近年分离自鼠类宿主动物的56株钩体采用致病性钩体特异引物进行PCR扩增鉴定,应用血清分群PCR对其进行血清群鉴定,采用包埋法提取钩体染色体DNA,利用核酸内切酶NotI对DNA进行酶切,酶切片段经PFGE电泳后采用凝胶成像系统拍照获得DNA指纹图谱,采用国家病原体分子分型实验数据分析与传输系统进行处理和聚类分析。结果致病性钩体特异性PCR和血清分群PCR鉴定显示56株钩体均为黄疸出血群钩体株,PFGE分型将其分为38个带型,聚类分析显示多数钩体株与黄疸出血群赖型具有较高的相似度,为主要流行优势带型,少部分钩体株与爪哇群和波摩那群代表菌株具有较高相似性,聚类关系相对较近。结论黄疸出血群赖型是贵州省近年流行的优势型,钩体株PFGE带型具有多样性,可为当地钩体病的监测、暴发调查和传染源追踪提供参考依据。     

两株不同毒力株钩端螺旋体37℃培养条件表达谱的差异

目的 钩端螺旋体黄疸出血群赖型赖株毒力株56601（中赖）与钩端螺旋体黄疸出血群赖型赖株减毒株（法赖）在遗传背景上具有很大的相似性，但毒力却差别很大。本研究利用钩端螺旋体的cDNA芯片在全基因组水平上研究毒力差别的机制。方法 利用钩端螺旋体的cDNA芯片，在37℃培养条件下以钩端螺旋体黄疸出血群赖型赖株毒力株56601（中赖）为测试株，以钩端螺旋体黄疸出血群赖型赖株减毒株（法赖）为对照株，测试了芯片表达谱的变化。结果 在37℃培养条件下，毒力株和减毒株的表达谱具有明显的差别。毒力株上调表达的基因有101个，下调表达的有71个。这些差异表达的基因在功能上可分为12类。结论 这些差异表达的基因可能在钩端螺旋体毒力株致病力方面发挥一定的作用。     

10株鹦鹉热衣原体菌株主要外膜蛋白基因的比较性研究

目的　进行比较性研究了 10株鹦鹉热衣原体菌株 ,分析其主要外膜蛋白 (MOMP)基因的限制性内切酶图谱和基因同源性。方法　接种鸡胚卵黄囊对菌株进行培养 ,收集培养物 ,通过聚合酶链式反应 ,扩增了 10株Cps的主要外膜蛋白基因 ,获得 10 5 8bp的片段 ,根据限制性片段长度多态性 (RFLPs)技术 ,利用AluⅠ酶切此片段 ,获得其限制性图谱。将此片段连入 pGEMT载体 ,进行基因测序。 结果　有 5株菌的MOMP基因完全相同 ,将它们与其它 5株比较 ,仅有个别碱基不同。提示MOMP基因是鹦鹉热衣原体中高度保守的序列     

福建O：3型小肠结肠炎耶氏菌分子流行病学的研究

本研究应用限制性内切酶酶切图谱分析的方法．以福建小肠结肠炎耶氏菌（Y．e）流行较严重的莆田、建瓯等地的人、猪、鼠、鸡中所分离到的O：3毒力质粒DNA，用限制性内切酶BamHI、EcoRI、HindⅢ进行酶切分析，结果三种酶切图谱各具特征性，但不同菌株间无差异，表明从福建不同地区、不同动物中所分离到的Y．eO：342MDa的毒力质粒DNA具有同源性，它们可能来自共同的起源。本研究进一步阐明了猪、鼠、鸡作为人Y．e病传染源的意义。     

致病性钩端螺旋体限制性内切酶酶切分析分型研究

64 Leptospira intemational and domestic reference strains.which belong to 54 serovars.and 27 field strains were examined by using EcoR I restriction endonuclease patterns(REPs) can only be identified by the first five high moleular-segments.reference strains from China and other countries have differert REPs.Most field strains have the same or similar REPs with corresponding reference strains.only a few difference in non-matching REPs.A notable result was that the field strains of serovar pomona have the same REP with the intemational reference strain but differnce from the domestic reference strain.     

Restriction endonuclease analysis of Chlamydia trachomatis DNA]

Twelve serovar type strains (C, D, E, F, G, H, I, J, K, L1, L2 and L3) and eight isolates (D, 4; E, 2; K, 2) of C. trachomatis were examined by restriction endonuclease analysis (REA) of DNA extracted from the elementary bodies. No difference was observed in DNA patterns of three serotypes (L1, L2 and L3) of the biovar LGV when they were digested with EcoRI and analysed by electrophoresis in a 0.6% agarose gel. The genital strains of the biovar trachoma (serovars D-K) showed similar EcoRI patterns with or without detectable differences. Serovar C of the biovar trachoma differed from the biovar LGV and the genital strains. Comparative analysis of DNAs digested with EcoRI, BamHI, HindIII, SalI and NcoI revealed that C. trachomatis isolates belonging to serovars D and K, but not E, could be subdivided into different genome types. These results suggest that DNA cleavage pattern analysis is useful for epidemiological, clinical, and taxonomic studies of C. trachomatis.     

钩端螺旋体内毒素的实验研究 Ⅰ.钩端螺旋体内毒素的生物学活性

钩端螺旋体波摩那群波摩那型56608株和赛玛伦群帕托克型帕托克Ⅰ株的酚水提取物具有较大肠杆菌内毒素为弱的生物学活性。该提取物使鱟血液变形细胞溶解物凝胶化,注入家兔后能使动物出现发热、血糖升高和Shwartzman现象,但较大剂量时方能致死小鼠。小鼠内脏病理检查结果表明,各脏器均出现DIC,尤以肺组织更为明显。     

Epidemiological fingerprinting of Klebsiella pneumoniae by small-fragment-restriction-endonuclease-analysis (SF-REA).

Epidemiological fingerprinting of Klebsiella pneumoniae was performed by restriction endonuclease analysis (REA) of whole cell DNA. 11 isolates from 4 patients in an intensive care unit and 80 unrelated strains were examined in this study. DNA was cleaved with restriction endonuclease EcoR I, electrophoresed on 10% polyacrylamide gels, and restriction fragment patterns were visualized by silver staining. The analysis of small fragments within the cleavage patterns (SF-REA) yielded sufficient information for reliable strain identification. The gel patterns of unrelated strains exhibited marked differences by direct visual comparison. In contrast, the isolates from the ICU could only be subdivided into 2 types, supporting our suspicion of nosocomial infections in some of these patients. SF-REA was evaluated with regard to interstrain discriminatory ability, reproducibility, and practicability. Our results indicate that SF-REA may be used as a rapid, precise and reliable technique in typing K. pneumoniae strains.     

我国4个钩端螺旋体血清群lipL21基因序列分析及其表达产物的鉴定

目的本文采用高保真PCR从我国主要流行的问号钩体黄疸出血群赖型56601株、波摩那群波摩那型56608株、流感伤寒群临型56609株及腐生性双曲钩体参考标准株三宝垄群patoc型PatocⅠ株基因组DNA中扩增了全长lipL21基因片段。序列分析结果表明,所克隆的4株钩体lipL21基因与已报道的相应序列(GenBankNo.:AY187271)比较,其核苷酸和氨基酸序列相似性分别高达99.64～99.82%和99.46～100%。所构建的问号钩体黄疸出血群赖型56601株lipL21基因原核表达系统在IPTG诱导下,能有效地表达目的重组蛋白rLipL21,其产量约为细菌总蛋白的10%。Westernblot结果证实,兔抗钩体TR/patocⅠ属特异性抗原血清能识别rLipL21并与之结合。上述实验结果提示,lipL21基因序列非常保守,其表达产物有良好免疫原性,可作为研制通用型钩体基因工程疫苗的候选抗原。     

Identification of type II restriction and modification systems in Helicobacter pylori reveals their substantial diversity among strains

A total of 22 type II restriction endonucleases with 18 distinct specificities have been identified in six Helicobacter pylori strains. Among these 18 specificities are three completely new endonucleases, Hpy178III, Hpy99I, and Hpy188I, that specifically cleave DNA at TCNNGA, CGWCG, and TCNGA sites, respectively. The set of endonucleases identified in each strain varies, but all have four- or five-base recognition sequences. Among 16 H. pylori strains, examination of the DNA modification status at the recognition sites of 15 restriction endonucleases reveals that each strain has a substantially different complement of type II modification systems. We conclude that the type II restriction-modification systems in H. pylori are highly diverse between strains, a unique characteristic of H. pylori. The diverse methylation status of H. pylori chromosomal DNA may serve as a new typing system to discriminate H. pylori isolates for epidemiological and clinical purposes. This study also demonstrates that H. pylori is a rich source of type II restriction endonucleases.     

抗波摩那型109株钩端螺旋体外膜抗原单克隆抗体的研制

本文用波摩那型109株钩体外膜抗原免疫的BALB/c鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,获得3株分泌抗波摩那型钩体外膜抗原单克隆抗体的杂交瘤细胞株,定名为LS_(15)、LS_(22)和LS_(28)。经酶标(ELISA)和显微镜凝集试验(MAT)证实LS_(22)和LS_(28)McAbs为波摩那型109株钩体特异的抗体;LS_(15)McAb为针对存在于一定钩体群型中共同抗原的非凝集抗体。杂交瘤细胞注入同系小鼠腹腔可诱生含高效价McAbs的腹水。经鉴定LS_(15)、LS_(22)和LS_(28)McAbs均属IgG_2亚类。     

The serovars of Leptospira interrogans isolated from cases of human leptospirosis in S?o Paulo, Brazil]

Eighteen strains of L. interrogans isolated from human cases were serotyped by the agglutinin-absorption test at Instituto Adolfo Lutz in S?o Paulo, Brazil. Fourteen were identified as serovar copenhageni (icterohaemorrhagiae serogroup), 2 as canicola (canicola serogroup), 1 as castellonis (Ballum serogroup) and 1 as pomona serogroup (serovar not yet defined). The frequency of serovar copenhageni in 100% of the isolates in icterohaemorrhagiae serogroup is emphasized and more studies to verify the real serovars prevalence as subsidy to the epidemiology of this infection are suggested by the authors.