霍乱弧菌O139和El Tor菌株毒力岛区域分子特征比较

目的:比较O139霍乱弧菌(VC O139)菌株MO45基因组与O1群El Tor霍乱弧菌(EVC)菌株N16961基因组中毒力岛(VPI)区域分子特征.方法:制备霍乱弧菌VPI上游VC0815基因探针.从VC O139菌株MO45基因组文库中筛选一个既含有VC0815基因又含有tcpA基因的克隆,命名为pCOSVC081545,绘制pCOSVC081545 VPI上游片段的限制性酶切图谱,并与N16961同段序列的限制性酶切图谱进行比较.结果与结论:pCOSVC081545和N16961中VPI上游约1 500 bp片段的限制性酶切图谱基本相同,支持O139霍乱弧菌是EVC O抗原突变而来的观点.     

霍乱弧菌HutX蛋白的晶体结构

目的 获取纯化的霍乱弧菌HutX蛋白晶体和衍射数据.方法 通过基因克隆和蛋白表达得到HutX蛋白,通过镍柱亲和层析、Source Q阴离子交换柱层析和Superdex 200分子筛柱层析等纯化所得蛋白,并用蛋白质印迹法进行验证.将所获蛋白进行结晶条件筛选及悬滴优化,X线衍射分析所得晶体的结构.结果 蛋白质印迹分析间接证明所得到的蛋白为HutX蛋白,并得到HutX蛋白的晶体及衍射数据.结论 为进一步研究HutX的蛋白结构及其在霍乱弧菌亚铁血红素利用机制中发挥的作用提供了参考.     

霍乱弧菌O1群菌株的群体遗传学特征

霍乱的病原菌是Ｏ１群霍乱弧菌，但在Ｏ１群霍乱弧菌中，仅有部分可以引起霍乱流行，因此不同菌株在流行病学上的意义差异很大。为探讨Ｏ１群菌不同型别和亚群之间的遗传关系和进化变异规律，应用多位点酶电泳法对Ｏ１群霍乱弧菌进行研究。结果：Ｏ１群霍乱弧菌的大多基因位点等位基因频率较低，尤其是古典型菌株和埃尔托型流行株，大多被检测基因为非多态位点；Ｏ１群霍乱弧菌的平均遗传多态值（ＨＯ１）为０．１８３，平均杂合度（ｈＯ１）为５．６７；其中古典型菌、埃尔托型流行株、埃尔托型非流行株的平均遗传多态值和平均杂合度分别为：ＨＣＶＣ＝０．１０６，ｈＣＶＣ＝１．５；ＨＥＳＥＶＣ＝０．０５６，ｈＥＳＥＶＣ＝２．３；ＨＮＳＥＶＣ＝０．４２５，ｈＮＳＥＶＣ＝５。结果显示：古典型和埃尔托型流行株作为霍乱的病原体是一个遗传关系紧密的群体，而埃尔托型非流行株遗传关系较为离散，与流行株之间的关系也较远，可能是其长期作为环境菌株进化变异的结果     

A case of Vibrio vulnificus septicaemia acquired in Victoria

The clinical features and laboratory findings of a case of Vibrio vulnificus septicaemia are reported. The illness occurred in a previously well 68-year-old man who was accidentally spiked in the buttock by the dorsal spine of a flathead caught in Tamboon Inlet, near Mallacoota, Victoria. The clinical picture of an acute septicaemic illness with shock, associated with metastatic cellulitic lesions on the lower limbs progressing to bulla formation, skin necrosis, necrotising fasciitis and myositis, is characteristic of V. vulnificus septicaemia. It would appear that tetracyclines are the drugs of choice. Surgical debridement may be necessary. Clinical recognition of this rare but characteristic illness will facilitate early effective chemotherapy. To our knowledge this is the southern-most case reported in Australia.     

重庆市霍乱弧菌的分子流行病学研究

目的 分析重庆市1992-2006年霍乱弧菌菌株的相关性.方法 94株霍乱弧菌基因组DNA经NotⅠ酶切,通过脉冲场凝胶电泳获得电泳图谱,利用BioNumerics软件对图谱进行聚类分析.结果 检测94株霍乱弧菌毒力基因,ctxA阳性92株,其中有2株水产品市场霍乱监测中分离的菌株ctxA为阴性:tcpA阳性91株,其中有2株水产品市场霍乱监测中发现菌株,1株从病人分离的0139群霍乱弧菌tcpA为阴性.94株霍乱弧菌被分为44种脉冲场凝胶电泳图谱,聚类分析将大部分菌株分为三群.从甲鱼分离的0139群霍乱弧菌与同期流行的0139群霍乱弧菌优势PFGE型别一致,并且也为产毒株.结论 重庆市霍乱弧菌存在紧密相关的流行克隆群.甲鱼中分离的0139群霍乱弧菌与霍乱疫情分离菌株之间高度同源,被污染的甲鱼可能是食源性霍乱暴发的主要传染来源之一,海、水产品的监测是霍乱防控的重点.霍乱弧菌的脉冲场凝胶电泳分析,有助于霍乱的主动监测和传染来源的追踪.     

A molecular epidemiological study of Vibrio cholerae in Australia

Two hundred and eight strains of Vibrio cholerae serovar O1 and 454 non-O1 strains, which were isolated within or imported into Australia, were tested for the production of cholera enterotoxin and for hybridization with gene probes for cholera enterotoxin. Genetic relationships among representative isolates were studied by a comparison of the restriction-endonuclease digest patterns of the chromosomal DNA and by Southern-blot analysis of toxigenic strains with the cholera-toxin gene probe. V. cholerae O1 isolates from patients and from the environment were predominantly toxigenic in contrast with the non-O1 isolates. The local O1 isolates were found to be diverse genetically and were unrelated to the imported O1 isolates, to those from other endemic areas and to non-O1 isolates.     

Detection of Vibrio cholerae 01 from aquatic environment in Sarawak

The detection of Vibrio cholerae 01 from the aquatic environment of Daro and Bintulu in Sarawak was carried out following an outbreak of cholera. Conventional culture methods and detection of ctx gene by polymerase chain reaction technique were carried out on 80 water samples. Only one sample was positive by culture methods while 8 were positive by PCR. DNA finger printing by pulsed-field gel electrophoresis showed that the clinical isolates in Daro and Bintulu were genetically identical while the environmental isolate was closely related. Recovery of Vibrio cholerae by culture method is poor and newer methods of detection should be developed.     

霍乱弧菌非O1群菌株的群体遗传结构

近年来非Ｏ１群霍乱弧菌在人类疾病上的流行病学意义也日益受到人们的关注。应用多位点酶电泳法对９８株非Ｏ１群霍乱弧菌１７个酶基因位点进行检测分析。结果：在被检测的基因位点中，大多位点的等位基因数较多，等位基因频率相对较低，但多数位点具有１～２个优势等位基因；平均遗传多态值和杂合度分析表明，检测的１７个酶基因位点都是多态的，平均遗传多态值ＨＮＯ１为０．６２７，平均杂合度ｈＮＯ１为６．５，说明非Ｏ１群霍乱弧菌群体遗传结构复杂，群体中的变异程度很高；这与非Ｏ１群霍乱弧菌广泛分布于自然环境水体中是一致的。结果提示：在被检测地区，非Ｏ１群霍乱弧菌中尚不具有遗传性状稳定的较大优势群体存在；同时也说明环境的自然选择在细菌进化变异中的重要作用。文中还对非Ｏ１群霍乱弧菌群体遗传结构的明显变化可能导致的流行病学意义进行了讨论     

霍乱弧菌O139菌株的分子生物学特征

为阐明Ｏ１３９霍乱弧菌的主要分子生物学特征，并为探讨其来源提供科学依据，应用核酸脉冲场凝胶电泳、基因探针Ｓｏｕｔｈｅｒｎ杂交等方法，对国内外分离的２３株Ｏ１３９菌和一些Ｏ１群菌株进行分析。结果表明，所有Ｏ１３９菌株均表现为一致的且具有特征性的酶切图谱；它们与埃尔托型流行株的图谱较与古典型菌株图谱更为相似，与埃尔托非流行株和其他非Ｏ１菌株的遗传差异较大。在ＳｍａＩ图谱中，新疆分离的Ｏ１３９菌株显示出与当地１９８９年分离的埃尔托型流行株较接近的图谱；Ｓｏｕｔｈｅｒｎｂｌｏｔ结果显示在不同的酶切图谱分子杂交（ＳｍａＩ、ＳａｌＩ、ＸｂａＩｃｔｘ，ＢｇｌＩ１６ｓｒＲＮＡ）中，Ｏ１３９菌株与Ｏ１流行株之间有一定的差异；我国分离的Ｏ１３９菌株具有一致的图谱，与印度和孟加拉国分离的Ｏ１３９菌株之间有较小差异；新疆分离的Ｏ１３９菌株与该地１９８９年分离的某些ＥＶＣ流行株的图谱相似。基因检测表明，Ｏ１３９菌株都具有ｃｔｘ、ｚｏｔ、ａｃｅ和ＲＳ１；ＭＥＥ分析表明，Ｏ１３９菌株的ＥＴ型与ＥＶＣ流行株不可区分。     

LAMP方法检测霍乱弧菌的研究

目的建立一种快速检测霍乱弧菌的方法。方法利用霍乱弧菌的hlyA基因设计特异性引物,建立LAMP检测方法,以及优化反应体系,并进行LAMP的特异性和灵敏度实验,同时与普通PCR和荧光定量PCR进行比较。结果成功建立霍乱弧菌的LAMP检测方法,得到最优的反应体系,在特异性实验中,霍乱弧菌均呈阳性,而副溶血弧菌、溶藻孤菌、最小孤菌、1梅氏孤菌等均为阴性;在灵敏度实验中,霍乱弧菌的最低检测限为6.2pg／tube,是普通PCR的1000倍。比荧光定量PCR低一个数量级。结论LAMP方法具有很高的特异性和灵敏度,可用于霍乱孤菌的检测。     

霍乱弧菌毒力强弱及菌株同源性的快速检测

目的建立一套快速检测霍乱弧菌毒力强弱及不同菌株同源性的实验方法。方法设计4对引物进行多重PCR检测菌株毒力基因特征;采用随机扩增多态性DNA(RAPD)结合SPSS软件分析不同菌株的同源性。结果通过多重PCR同时检测菌株CtxA、TcpA、Ace、Zot4种主要的毒力基因,并快速判定其毒力强弱;RAPD产生的指纹图谱条带清晰、数目较多、片段大小分布均匀,结合SPSS软件分析可判断菌株间的同源性关系。结论多重PCR和RAPD结合SPSS软件分析可在4h内完成对霍乱弧菌毒力强弱及不同菌株间同源性的检测,有助于在疫情处理中判断病菌来源和及时采取适当的处理措施。     

霍乱弧菌菌体蛋白双向电泳技术的建立

目的 建立霍乱弧菌全菌体蛋白的双向电泳技术,获得分辨度高、重复性好的双向电泳图谱.方法 利用适当的裂解液处理霍乱弧菌,提取全菌蛋白;采用pH梯度等电聚焦对全菌蛋白进行双向电泳;考马斯亮蓝染色后获得的双向电泳图谱,并利用ImageMaster 2D Elite 5.0图象分析软件进行分析,所得的数据用SPSS15.0进行统计分析.结果 得到了(1081±16)个蛋白斑点,蛋白主要集中在pI 4.24～7.20之间,重复胶的匹配点数为(1057±28),匹配率为97.85%.结论 建立了霍乱弧菌全菌双向电泳分析方法,2-DE图谱中蛋白位点的分辨率和重复性非常高,获得了较为理想、清晰的双向电泳图谱,为进一步研究其蛋白质组学奠定了基础.     

霍乱毒素ctxA基因的克隆及原核表达

目的 构建霍乱毒素A亚基基因（ctxA）的融合表达载体。并在原核细胞中表达,为霍乱弧菌肠毒素A亚基（CTA）免疫原性的研究及其作为免疫佐剂的研究提供基础。方法 以霍乱弧菌DNA为模板．PCR扩增获得霍乱弧菌ctxA基因,与带有硫氧还蛋白（Trx）基因的高效原核表达质粒pET32a（＋）定向重组,构建重组质粒．转化大肠杆菌BL21（DE3）,并经限制性核酸内切酶酶切鉴定、PCR和核酸序列分析后,以IPTG诱导表达Trx—CTA融合蛋白,用SDS—PAGE及Western blot进行鉴定。结果 限制性核酸内切酶酶切鉴定、PCR和核酸序列分析表明,我们扩增出了霍乱弧菌787bp的ctxA基因,成功构建了重组质粒pET—ctxA,经SDS—PAGE及Western blot分析显示重组质粒pET—ctxA在原核细胞中得到了高效融合表达。结论 霍乱弧菌ctxA基因在大肠杆菌中得到了高效表达。     

Toxin-coregulated pilus-loaded microparticles as a vaccine against Vibrio cholerae O139

The cholera epidemics is an important public health problem in many developing countries. Highly effective and preventive vaccines against cholera are under investigation as alternatives to the one available presently. Much of the vaccine research focuses on colonization factors. Colonization of a human by the Vibrio cholerae (V. cholerae Ol strain is mediated by toxin-coregulated pilus (TCP), 1 which was shown to play a role in the infant mouse cholera model and subsequently in human volunteers. 2 TCP-loaded vaccines could potentially provide cross-protection among experimental strains. Data have indicated that poly (D,L-lactide)-polyethylene glycol copolymer (PELA)microparticles loaded antigens were strongly immunogenic, 3 and that these microparticles served as an effective delivery system for a single dose of vaccine. 4Microparticle formulation could represent the next generation of vaccines, as they are highly effective at delivery of vaccines, thus requiring fewer doses. 5 We prepared PELA microparticles loaded with TCP for testing as a vaccine; their targeting distributions were identified and related immune responses were analyzed.     

Toxin-coregulated pilus-loaded microparticles as a vaccine against Vibrio cholerae O139

The cholera epidemics is an important public health problem in many developing countries. Highly effective and preventive vaccines against cholera are under investigation as alternatives to the one available presently. Much of the vaccine research focuses on colonization factors. Colonization of a human by the     

霍乱肠毒素基因DNA疫苗的构建和体外表达

目的构建霍乱弧菌ctxAB融合基因分子佐剂DNA疫苗,并在NIH3T3细胞中进行表达。方法用限制性核酸内切酶从原核表达重组质粒pET32a-ctxAB上切下ctxAB基因,导入真核表达载体pcDNA3.1（＋）,重组子经限制性酶切分析、PCR鉴定正确后,命名为pcDNA3.1-ctxAB。用脂质体法将重组质粒pcDNA3.1-ctxAB转染NIH3T3细胞,采用免疫荧光法和Western blot对pcDNA3.1-ctxAB的瞬时表达产物和稳定表达产物进行鉴定。结果真核表达重组质粒pcDNA3.1-ctxAB成功转入NIH3T3细胞,利用免疫荧光技术检测到其在细胞膜和细胞浆中获得瞬时表达,对稳定转染细胞用Western blot分析,发现在约40.5kDa处有阳性杂交信号。结论成功构建霍乱弧菌ctxAB基因分子佐剂DNA疫苗,并在NIH3T3细胞中获得表达。