溶藻弧菌毒力菌株SSH文库的构建

[目的]通过构建基因文库为溶藻弧菌毒力基因的鉴定、致病机理的阐明奠定基础。[方法]应用抑制性差减杂交技术(suppression subtrative hybridization,SSH),以毒力菌株HN08155为测试子(Tester),构建了溶藻弧菌毒力菌株HN08155的SSH差减文库。[结果]文库共含有2 873个克隆;通过菌落PCR鉴定,2 786个克隆能扩增出250~1 000bp大小的目标片段;通过Southern斑点杂交,确定毒力菌株特异基因的阳性克隆347个,经测序和同源性比对分析,其中135个克隆的序列含有已知的77个基因,其中46个基因主要为代谢途径的多种酶类的编码基因,23个基因为其它基因,8个为毒力相关功能基因。[结论]这些毒力相关功能基因包括丝氨酸蛋白酶基因prk A、热休克蛋白基因dna J、趋化因子基因che W、甲基受体趋化蛋白(methyl-accepting chemotaxis protein)基因、双组分感应子(two-component sensor)基因、Ⅳ型菌毛(typeⅣpilin Pil A)基因、生物膜相关的表面蛋白(biofilm-associated surface protein)基因和外膜蛋白(outer membrane protein)基因;该文库中还含有166个未知新基因,占文库中总基因数的68.44%,溶藻弧菌的毒力基因很可能存在于这个文库中的未知新基因中。     

VHH/TLH溶血素基因在海洋弧菌中分布的研究

哈维氏弧菌(V.harveyi)的VHH溶血素是对海水养殖鱼类的潜在致病因子。哈维氏弧菌的VHH溶血素基因与副溶血弧菌(V.parahaemolyticus)的TLH热不稳定性溶血素基因具有高度相似性,其氨基酸序列的相似性达到85.6 %。根据哈维氏弧菌vhhA溶血素基因序列,合成一个地高辛标记的VHH基因探针,利用其进行Southern Blot ,检测VHH溶血素基因在57株弧菌(包括26株国际标准菌株,20株哈维氏弧菌,11株副溶血弧菌)中的分布情况。结果显示,VHH基因探针与13株弧菌标准菌株有强杂交信号,包括2株溶藻胶弧菌(V.alginolyticus) ,2株哈维氏弧菌以及1株霍氏格里蒙菌(Grimontia hollisae) ,坎贝氏弧菌(V.campbellii) ,辛辛那提弧菌(V.cincinatiensis) ,费氏弧菌(V.fischeri) ,拟态弧菌(V.mimicus) ,飘浮弧菌(V.natriegens) ,副溶血弧菌,解蛋白弧菌(V.proteolyticus)和火神弧菌(V.logei)。与6株弧菌标准菌株有弱杂交信号,包括鳗弧菌(V.anguillarum) ,河口弧菌(V.aestuarianus) ,美人鱼发光杆菌(Photobacterium damselae subsp.damselae) ,河弧菌(V.fluvialis) ,弗尼斯弧菌(V.furnissii)和创伤弧菌(V.vulnificus) ,而另外7株弧菌标准菌株中无杂交信号。所有的哈维氏弧菌菌株至少含有一条杂交带,其中菌株VIB645 , VIB 648和SF-1分别含有2条杂交带。11株副溶血弧菌中均含有一条杂交带。上述数据表明,vhh/tlh溶血素基因广泛分布于弧菌中,尤其是哈维氏弧菌相关菌株和费氏弧菌相关菌株中。另外对鳗弧菌VIB 72 ,坎贝氏弧菌VIB 285 ,飘浮弧菌VIB 299和哈维氏弧菌VIB 647的vhh/tlh溶血素基因进行克隆并测序,其氨基酸序列与VHH溶血素和TLH溶血素氨基酸序列的同源性分别为67 %~99 %和69 %~91 %。对vhh/tlh溶血素基因在弧菌中的分布研究,将有助于进一步确定这类溶血素基因在病原弧菌致病性中的作用。     

溶藻弧菌中类霍乱弧菌毒力岛转座酶基因及侧翼序列分子生物学特征分析

[目的]调查类似霍乱弧菌毒力岛(VPI)转座酶(vpiT)的基因是否在溶藻弧菌中分布,并了解其全序列及侧翼序列的分子生物学特征.[方法]对94株溶藻弧菌是否携带类似VPI的vpiT基因进行PCR检测,对阳性株进行了PCR产物直接测序,根据获得的部分已知序列,设计引物,通过反向PCR扩增出全长类似vpiT的基因valT及部分侧翼片段,对反向PCR产物进行克隆测序,然后对获得的valT及侧翼序列进行生物信息学分析.[结果]发现94株溶藻弧菌中只有从粤东对虾池水分离的2个株E06011、E0612在PCR检测中产生了预期扩增片段.测序表明两者序列(valT-S1)完全一致.根据反向PCR及克隆最终获得的溶藻弧菌E0601全长valT基因及部分侧翼序列valT-S3.对valT-S3生物信息学分析表明:valT是一个高度类似于霍乱弧菌毒力岛vpiT的转座酶基因.[结论]根据上述结果及相关文献,有理由相信valT基因及其侧翼片段是异源获得,霍乱弧菌VPI元件或整体很可能在包括溶藻弧菌在内的弧菌种间转移.     

鳗弧菌flaE基因的克隆及蛋白结构分析

[目的]:克隆鳗弧菌flaE基因并分析其蛋白结构,为研究该蛋白的生物学功能及免疫原性奠定基础。[方法]通过PCR方法对鳗弧菌flaE基因进行扩增,利用生物学软件对其序列及蛋白质结构进行分析。[结果]所得flaE基因的大小为841bp,其开放阅读框长度为798 bp,编码262个氨基酸。蛋白分子质量为28 411.4,理论等电点p I为5.70,脂肪系数为81.60,不稳定系数为31.81;为疏水性蛋白;没有信号肽和跨膜螺旋结构;保守区结构域属于flagellin家族;二级结构中以α螺旋为主,其次是无规则卷曲和β片层,少量β转角;三级结构与3k8v.1.A的结构模型相似率为90%。[结论]成功克隆了鳗弧菌flaE基因,Gen Bank登录号为KM091934。     

松材线虫伴生菌中产纤维素酶细菌的筛选、鉴定及其相关基因的克隆

[目的]从松材线虫伴生菌中筛选出高效降解纤维素的细菌菌株,初步鉴定后,对其相应的纤维素酶基因尝试克隆.[方法]首先从河南南阳松材线虫病疫区采集到的木材样本中,分离获得松材线虫.采用刚果红平板初筛法,从松材线虫伴生菌中获得具有分泌较高活性纤维素酶的细菌菌株.基于该菌株的形态学、生理学及16 s rDNA序列特征等对高活性菌株进行分类鉴定.设计兼并引物,从高活性菌株中克隆该菌株的纤维素酶基因,并进行序列分析.[结果]获得7株具有分泌较高活性纤维素酶的细菌菌株,其中编号为C8的菌株酶活最高.经鉴定该菌株归为肠杆菌属,命名为Enterobacter sp.C8.进一步从C8菌株中成功克隆出该菌株的一个全长1104 bp的纤维素酶基因(GenBank JQ845065),在NCBI比对后发现该基因分别与产气肠杆菌( Enterobacter aerogenes) KCTC 2190的纤维素合成酶亚单位BcsC的核苷酸序列有97％的同源性,氨基酸序列有92％的同源性 ;与克雷白氏肺炎菌(Klebsiella pneumoniae)的endo-1,4-D-glucanase基因有82％的同源性,与不可培养的细菌内切纤维素酶基因有82％的同源性.[结论]本文首次从松材线虫伴生菌中筛选到了一株简单的产纤维素酶的细菌菌株并从中克隆出了一个新型纤维素酶基因,为下一步进行新能源的利用奠定了理论基础.     

溶原性噬菌体在猪链球菌2型分离株中的检出和鉴定

[目的]为了研究噬菌体整合酶基因在猪链球菌2型(Streptococcus suis type 2,SS2)中的分布情况.[方法]根据噬菌体整合酶基因设计引物,建立了PCR方法,并对扩增产物进行测序.[结果]结果显示,25株SS2致病菌株均扩增出目的片段,非毒力株T15、5株其它血清型猪链球菌及兰氏C群猪源链球菌未扩增出目的片段.经丝裂霉素C诱导后,SS2致病菌株出现完全的细胞溶解,而非毒力株T15未出现溶解.SS2致病株HA9801和ZY05719诱导均产生溶原性噬菌体,分别命名为SS2-HA和SS2-ZY,电镜观察,二者均头部呈正六边形,无尾部,其核酸类型为dsDNA,可鉴定为复层噬菌体科(Tectiviridae)的成员.噬菌体SS2-HA和SS2-ZY整合酶基因序列与已报道的SS2噬菌体整合酶基因序列高度同源,显示SS2噬菌体整合酶具有较高的特异性.[结论]从SS2致病株中检出溶原性噬菌体和噬菌体整合酶基因,且噬菌体整合酶基因与SS2溶菌酶释放蛋白(mrp)等7种毒力相关基因有相关性,表明SS2的溶原性噬菌体可能与其致病性有关.     

对粉纹夜蛾高毒力cry9Eα基因的克隆及表达

[目的]从本实验室分离的Bt4菌株中克隆cry9Eα基因,并研究其表达和杀虫活性.[方法]以PCR-RFLP方法鉴定Bt4菌株含有cry9基因,然后以菌株Bt4的质粒为模板,利用全长引物F9EA/R9EA进行PCR扩增全长基因.[结果]将目的片段插入到表达载体pET21b,得到大肠杆菌重组表达质粒pETcrygEa.转化E.coli BL21(DE3),诱导后表达130 kDa的蛋白,再将cry9Eα7基因连接到穿梭载体pSXY422b,电激转化HD73-(cry-),得到工程菌BioHD9Ea7,提取Cry9Ea7晶体蛋白,并进行生物活性测定.生物活性测定结果显示CrygEa7蛋白对粉纹夜蛾(Trichoplusia ni)初孵幼虫具有高毒力,LC_(50)为0.044 μg/mL,而对甜菜夜蛾(Spodoptera exigua)和棉铃虫(Helicoverpa armigera)初孵幼虫未显示活性.[结论]克隆和表达了一个对粉纹夜蛾高毒力的基因cry9Eα7,并成功构建了工程菌BioHD9Ea7.     

2006-2008年深圳市食源性病例和外环境的副溶血性弧菌分离株分型特征比较

[目的]比较深圳市食源性病例和外环境中分离的副溶血性弧菌在血清分布、毒力基因携带情况和分子分型方面的特征.[方法]血清凝集法检测菌株血清型,多重PCR检测毒力基因tdh和trh基因携带情况,脉冲场凝胶电泳(PFGE)分析基因分型特征.[结果]98株食源性病例分离株的主要血清型为O3∶K6(40.8%)、O1∶KUT(7...     

抑制差减杂交筛选禽致病性大肠杆菌基因组差异片段及其分析

采用抑制差减杂交技术(Suppression subtractive hybridization,SSH)对禽致病性大肠杆菌E037株(血清型O78)与非致病菌株K-12MG1655以及同一O2血清型高致病菌株E058与低致病菌株E526进行基因组差异片段克隆与分析。从E037株中共检出17个特异性差异片段,E058株中共检出32个特异性差异片段。经同源分析,这些序列可分为4类:质粒相关序列、噬菌体相关序列、已知功能序列、未知功能序列。这些差异片段包含许多重要的大肠杆菌毒力相关基因,如大肠杆菌素、气杆菌素受体、铁基因簇等。49个片段中,14个片段与其它微生物基因组同源性较高。结果表明,大肠杆菌高致病株与低致病菌株或非致病菌株基因组间存在较多差异基因,其中包括毒力、毒力相关基因、代谢以及噬菌体等基因成分。     

苏云金芽胞杆菌Rpp39杀虫晶体蛋白基因的鉴定及cry2Aa12基因的克隆表达

[目的]本研究的目的是分析从四川生态条件下分离的苏云金芽胞杆菌Rpp39菌株的特性,从分子水平上揭示该菌株对鳞翅目高毒力的原因;进一步从中分离克隆cry2Aa基因,并对其进行初步的表达研究.[方法]本研究主要采用扫描电镜观察、PCR-RFLP鉴定法和SDS-PAGE分析法研究菌株的特性;采用PCR直接克隆法克隆cry2Aa全长基因,并亚克隆到原核表达载体pET-30a中,构建重组表达质粒pET-2Aa,再转入受体菌E.coli.BL21(DE3)中进行诱导表达;采用室内生物测定法测定表达产物对小菜蛾和水稻二化螟的毒力.[结果]经扫描电镜观察菌株Rpp39主要产生菱形、方形和圆形3种伴胞晶体;SDS-PAGE分析表明主要产生130 kDa和60 kDa左右2种蛋白;经PCR-RFLP鉴定,该菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Ia和cry2Aa五类杀虫晶体蛋白基因;1种cry2Aa类杀虫晶体蛋白全长基因被克隆,序列分析显示该基因的开放阅读框(ORF)为1902 bps,编码由634个氨基酸组成的蛋白质,氨基酸序列与Cry2Aa1蛋白同源性为99.7%,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry2Aa12.重组表达质粒pET-2Aa在E.coli BL21(DE3)中,经IPTG诱导能正常表达,SDS-PAGE电泳验证含有65 kDa表达蛋白.生物活性测定表明表达的包涵体蛋白对小菜蛾和二化螟具有杀虫活性,LC50分别为5.4 μg/mL和22.3μg/mL.[结论]菌株Rpp39及从中分离克隆的cry2Aa12基因来自四川生态条件,丰富了菌株及基因的资源,在资源积累方面具有重要意义.     

Polypeptides p40, pOM2, and pAngR are required for iron uptake and for virulence of the marine fish pathogen Vibrio anguillarum 775.

Insertions were created in three iron uptake genes in plasmid pJM1 of Vibrio anguillarum 775 to assess their in vivo effects on virulence in fish. Insertions that blocked p40, pOM2, and pAngR expression resulted in iron uptake-negative strains and in 4.2 x 10(5)-, 8.8 x 10(5)-, and 2.5 x 10(5)-fold attenuations in virulence, respectively. A strain with an insertion in the pAngR coding region still synthesized significant constitutive levels of the outer membrane protein pOM2 and persisted in fish for at least 14 days postinjection. The results demonstrate a direct relationship between virulence and three pJM1-encoded gene products and also the feasibility of constructing live attenuated strains of V. anguillarum that might be useful in future vaccines.     

Relationships between plasmids and phenotypes of presumptive strains of Vibrio anguillarum isolated from different fish species

On the basis of plasmid composition as well as serological and biochemical properties, 26 strains identified as Vibrio anguillarum isolated from diseased fish could be assigned to two different groups. Except for three reference strains, these++ strains were isolated from Norwegian fish. The four strains isolated from rainbow trout (Salmo gairdneri), the only strain isolated from char (Salvelinus alpinus), and three of six strains isolated from Atlantic salmon (Salmo salar) harbored a plasmid of 47 megadaltons (MDa). Restriction endonuclease analysis showed that this plasmid and the virulence plasmid pJM1, carried by V. anguillarum strain 775, were very similar but not identical. Strains harboring the 47-MDa plasmid had nearly identical biochemical properties and were serotype O1. Strains isolated from reared coastal cod (Gadus morhua), turbot (Scophthalmus maximus), halibut (Hippoglossus hippoglossus), free-living saithe (Pollachius virens), and partly from reared Atlantic salmon differed from strains harboring the 47-MDa virulence plasmid by not containing this plasmid, by having different biochemical traits, and by being serotype O2. Rainbow trout which were experimentally infected with a strain isolated from cod suffering from vibriosis developed clinical symptoms similar to those in cod but quite different from those usually seen in rainbow trout.     

Relationships between plasmids and phenotypes of presumptive strains of Vibrio anguillarum isolated from different fish species.

On the basis of plasmid composition as well as serological and biochemical properties, 26 strains identified as Vibrio anguillarum isolated from diseased fish could be assigned to two different groups. Except for three reference strains, these++ strains were isolated from Norwegian fish. The four strains isolated from rainbow trout (Salmo gairdneri), the only strain isolated from char (Salvelinus alpinus), and three of six strains isolated from Atlantic salmon (Salmo salar) harbored a plasmid of 47 megadaltons (MDa). Restriction endonuclease analysis showed that this plasmid and the virulence plasmid pJM1, carried by V. anguillarum strain 775, were very similar but not identical. Strains harboring the 47-MDa plasmid had nearly identical biochemical properties and were serotype O1. Strains isolated from reared coastal cod (Gadus morhua), turbot (Scophthalmus maximus), halibut (Hippoglossus hippoglossus), free-living saithe (Pollachius virens), and partly from reared Atlantic salmon differed from strains harboring the 47-MDa virulence plasmid by not containing this plasmid, by having different biochemical traits, and by being serotype O2. Rainbow trout which were experimentally infected with a strain isolated from cod suffering from vibriosis developed clinical symptoms similar to those in cod but quite different from those usually seen in rainbow trout.     

Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum.

Genetic evidence has previously suggested that a zinc metalloprotease is involved in the invasive mechanism of the fish pathogen Vibrio anguillarum NB10. In this study, the metalloprotease gene was cloned and sequenced. The sequence encodes a polypeptide (611 amino acids) that contains a putative signal sequence followed by a large leader sequence and the mature protein (44.6 kDa). Since the purified protein has a molecular mass of 36 kDa instead of the predicted 44.6 kDa, the mature protein is most likely processed a third time. Comparative analyses of the protein sequence showed high homologies to other bacterial metalloproteases within the zinc-binding and active-site regions. The Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase were exceptions in that the homology extended throughout the entire putative preproprotein. A chromosomal metalloprotease mutant was made via the integration of foreign DNA into the protease gene. This mutant did not secrete the metalloprotease, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide protein analysis and by growth on gelatin agar. Transcomplementation of the chromosomal mutation revived the secretion of the metalloprotease and its activity on gelatin agar. Interestingly, when supernatant proteins were analyzed by gelatin-SDS-polyacrylamide electrophoresis, two different proteases (75 and 30 kDa) were detected in the mutant strain but not in the transcomplemented strain or the wild-type strain. Moreover, fish infection studies were done, and implications for the role of the metalloprotease in the virulence mechanism of V. anguillarum are discussed.     

Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain

Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross-reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components.     

Comparative Proteomics of two Vibrio anguillarum Serotype O1 Strains with Different Virulence Phenotypes

Vibrio anguillarum is part of the natural flora in the aquatic habitat, but under certain circumstances it can cause terminal hemorrhagic septicemia in marine fish due to the action of virulence-associated proteins. In our study, V. anguillarum MN and 3010 were identified as serotype O1 by AFLP analysis, and the virulence of V. anguillarum MN was shown 50-fold higher than that of the strain 3101 by LD(50) tests with Japanese flounder Paralichthys olivaceus. Nine spots were noted as differentially expressed proteins by comparing the cellular and extracellular protein profiles of V. anguillarum MN and 3101. Mass spectrometry results showed OmpU and PrtV were highly expressed in the virulent strain MN but lowly expressed in the less virulent strain 3101. Expression level confirmed by semiquantitative RT-PCR showed that ompU and prtV were indeed highly expressed in the virulent strain MN. Together with similar amino acid sequences of both OmpU and PrtV in V. anguillarum MN and 3101, our study indicated that the expression level of OmpU and PrtV may be associated with the virulence of V. anguillarum.     

Evidence for the existence of distinct populations of Vibrio anguillarum serogroup O1 based on plasmid contents and ribotypes.

A total of 103 Vibrio anguillarum serogroup O1 strains displaying 15 different plasmid profiles were characterized with respect to biochemical properties and ribotypes. The results confirmed that V. anguillarum O1 is a biochemically homogeneous group. The 103 strains could be allocated to three main clusters with high similarity coefficients. None of the biochemical properties were connected with the presence of plasmids. In total, 12 different ribotypes were demonstrated, with HindIII being used as the restriction enzyme. Forty of the strains were isolated from the same Danish fish farm, some from the kidneys of diseased fish and some from the environment, and some strains were isolated from the mucus, gills, and feces of healthy fish. Nineteen of these isolates possessed the 67-kb virulence plasmid alone or in combination with other plasmids, while 21 had no plasmids. All strains isolated from the kidneys of diseased fish on this farm had plasmids. Irrespective of their origin (kidneys, gills, or mucus), all 19 strains carrying the 67-kb virulence plasmid had the same ribotype, profile 1, while isolates without plasmids belonged to five different profiles, all different from profile 1. These results suggest that pathogenic V. anguillarum O1 strains possessing a virulence plasmid and nonpathogenic strains without plasmids from a small geographical area and even from the same fish may constitute two essentially distinct populations. Thus, it may be suggested that an exchange of virulence plasmids among strains is unlikely to occur in vivo.     

Use of suppressor subtraction hybridization for finding strain-specific sequences in bacterial genomes

Comparisons of bacterial genomes demonstrate that even strains of one species may strikingly differ in gene set. Strain-specific genes are of considerable interest, as they may be responsible for distinguishing features, such as virulence or drug resistance, of the strain and may be employed as markers in epidemiological or evolutionary studies. Suppression subtractive hybridization (SSH) was shown to be suitable for generating a set of DNA fragments differing between two closely related bacterial strains. More than 95% DNA fragments selected by SSH proved to be specific for Staphylococcus aureus strains ZW compared with strain 29213.