毒素源性大肠杆菌定居因子抗原CS6基因的克隆

通过构建人源毒素源性大肠杆菌野生株E519/66A大质粒的基因文库,成功地筛选出能表达定居因子抗原CS6菌毛的阳性克隆,初步确定了克隆DNA片段的限制性内切酶图谱。CS6抗原的编码和调控基因集中在一大小为4.6kb的DNA区段中,该片段能产生两种分子量大小不同、但抗原反应交叉的菌毛蛋白。本研究获得的CS6抗原阳性的重组菌株可用于人源ETEC多价疫苗的研制,克隆的基因片段亦可作为研究CS6菌毛蛋白基因表达及调控的基础。     

肠毒素大肠杆菌定居因子CFA/Ⅰ和CS6在减毒福氏志贺氏菌中的共表达

定居因子CFA/I和CS6是肠毒素大肠杆菌 (ETEC)中重要的两种优势抗原 ,是ETEC疫苗研制的首选组分。采用基因重组技术将二者构建在以asd基因为选择标记的重组质粒上 ,与asd基因缺失突变型减毒福氏志贺氏菌FWL0 1构成宿主 载体平衡致死系统。实验结果表明 ,重组疫苗候选株能够稳定表达CFA/I和CS6抗原 ,并可在菌体表面形成相应菌毛。重组菌口服免疫BALB/c小鼠后 ,可诱生相应的抗CFA/I和CS6的特异性血清抗体IgG和分泌型抗体sIgA ,说明以志贺氏菌为载体 ,可以构建同时表达多个定居因子抗原的ETEC多价菌苗     

CS3表面抗原基因的表达和纤毛装配元件的研究

在肠毒素大肠杆菌(ETEC)的已知定居因子抗原(colonization factor antigen,CFA)中,CFA Ⅰ、CFA Ⅱ和CFA Ⅳ分布较广,是优势血清型。在上述的CFAs中,CFA Ⅰ为单一抗原组分,而CFA Ⅱ则有3种抗原组分,分别称为CS1、CS2和CS3。临床分离株中常以CS1/CS3、CS2/CS3或单独以CS3的形式出现,可见CS3是CFA Ⅱ阳性菌株的共同抗原成分。已知CS3是由分子质量60Mu的质粒编码的。从CS3基     

肠毒素大肠杆菌定居因子CFA／I和CS6在减毒福氏志贺氏菌中的共表达

定居因子CFA／I和CS6是肠毒素大肠杆菌(ETEC)中重要的两种优势抗原,是ETEC疫苗研制的首选组分。采用基因重组技术将二者构建在asd基因为选择标记的重组质粒上,与asd基因缺失突变型减毒福氏志贺氏菌FWL01构成宿主．载体平衡致死系统,实验结果表明,重组疫苗候选株能够稳定表达CFA／I和CS6抗原,并可在菌体表面形成相应菌毛。重组菌口服免疫BALB／c小鼠后,可诱生相应的抗CFA／I和CS6的特异性血清抗体IsG和分泌型抗体sIgA．说明以志贺氏菌为载体．可以构建同时表达多个定居因子抗原的ETEC多价菌苗。     

CS3纤毛抗原表达调控机理的研究

定居因子抗原(CFA)是肠毒素大肠杆菌(ETEC)的重要毒性因子和保护性抗原,大多以菌毛的形式存在。此类抗原的表达及菌毛的装配需要2类基因的共同作用,即亚基结构基因和辅助蛋白结构基因。亚基是纤毛抗原的基本组成单位,而辅助蛋白则参与亚基蛋白分子的输送及装配过程。CS3亚基结构基因位于辅助蛋白结构基因的下游,处于CS3操纵子的3′端。CS3亚基结构基因的核苷酸序列分析揭示,在其翻译起始位点的上游存在着rbs位点及原核     

产肠毒素原性E.coli菌苗的研究进展

<正> 产肠毒素原性大肠杆菌(ETEC)是当前世界上的一个重要病原,虽然确切的调查资料尚难以得到,但据近来的研究估计,全世界每年大约有4亿腹泻病例,五岁以下儿童因之死亡的有70万。ETEC也是旅游者腹泻的一个重要病因。由于人们对这种病原的认识相当晚,因而近十年来才注意其菌苗的研制。 关于ETEC腹泻的致病机理常有评述,但简要说来,ETEC引起腹泻需要有两类毒力因子,即定居因子和肠毒素。病原体必须首先粘附到小肠的近测,这一过程是由称为定居因子抗原(CFA)的纤毛蛋白粘附素(adhesins)介导的。已经识别出许多这样的因子如CFA/I,CS1,CS2,CS3,CS4,CS5,CS6以及PCFO159:H4。早期的报     

肠毒素大肠杆菌CS3定居因子抗原和融合肠毒素基因在减毒痢疾杆菌中的共表达

肠毒素和定居因子抗原 (CFAs)是肠毒素大肠杆菌 (ETEC)两种主要的致病因素。有效的ETEC疫苗应包括这两种成分。采用基因重组技术 ,将定居因子CFA/II的共有抗原成分CS3菌毛抗原和LT B/ST融合肠毒素基因克隆至以asd基因为选择标记的重组质粒上 ,与asd基因剔除的弗氏志贺氏菌Fwl0 1构成了宿主 载体平衡致死系统。结果表明 ,在无抗生素条件选择的情况下 ,该重组菌可稳定表达CS3菌毛抗原和LT B/ST融合肠毒素抗原。通过口服和鼻饲方式免疫小鼠 ,可诱生相应的血清IgG抗体 ,同时能够检测到分泌型IgA的产生 ,表明该重组菌可以有效的诱导产生粘膜免疫。     

肠毒素大肠杆菌定居因子抗原基因CFA-I在痢疾杆菌中的表达

通过体外重组的方法 ,构建了包含asd基因的重组表达质粒 pZHY2 1,与福氏志贺氏菌Fwl0 1构成了宿主 载体平衡致死系统 ,Western印迹结果表明 ,在没有抗生素条件选择的情况下 ,稳定表达肠毒素大肠杆菌定居因子抗原CFA I。电镜结果显示 ,在重组菌株的菌体表面 ,表达产物能够装配成菌毛。重组菌通过口服和鼻饲免疫小鼠后 ,可以诱生CFA I的抗体 ;同时可以检测到分泌型IgA产生 ,表明重组菌可以诱导相应的粘膜免疫反应     

肠毒素大肠杆菌定居因子抗原基因CFA—1在痢疾杆菌中的表达

通过体外重组的方法,构建了包含asd基因的重组表达质粒pZHY21,与福氏志贺氏菌Fwl101构成了宿主－载体平衡致死系统,Westem印迹结果表明,在没有抗生素条件选择的情况下,稳定表达肠毒素大肠杆菌定居因子抗原CFA－I。电镜结果显示,在重组菌株的菌体表面,表达产物能够装配成菌毛。重组菌通过口服和鼻饲免疫小鼠后,可以诱生CFA－I的抗体;同时可以检测到分泌型IgA产生,表明重组菌可以诱导相应的粘膜免疫反应。     

肠毒素大肠杆菌定居因子CS6蛋白的纯化和抗体制备

将大肠杆菌E519／66A在CFA固体培养基上培养,收菌后经热水浴脱蛋白、硫酸铵分级盐析、超速离心及S-200柱纯化,获得了相对分子量约14600的高纯度CS6蛋白。用其免疫家兔,然后颈动脉采血。ELSIA法检测血清,测得效价为1：32000。Westem印迹证实CS6抗原蛋白可特异性结合抗CS6多克隆抗血清。本研究建立了分离纯化肠毒素大肠杆菌定居因子CS6抗原蛋白的有效方法;获得了高纯度的野生型定居因子抗原CS6蛋白及相应的多克隆抗血清。对于进一步研究肠毒素大肠杆菌腹泻疫苗具有重要意义。     

Positive regulation of colonization factor antigen I (CFA/I) production by enterotoxigenic Escherichia coli producing the colonization factors CS5, CS6, CS7, CS17, PCFO9, PCFO159:H4 and PCFO166

Entertoxigenic Escherichia coli (ETEC) strains of nineteen serogroups which produced colonization factors (coli-surface-associated antigens CS5, CS6, CS7 and CS17, colonization factor antigen CFA/III and putative colonization factors PCFO159:H4, PCFO166 and PCFO9) were tested for hybridization with a DNA probe containing the cfaD sequence that regulates expression of CFA/I. Strong colony hybridization, similar to that with the CFA/I-positive control strain H10407, occurred with ETEC strains of serogroups O27, O159 and O169 which produced CS6 antigen, and with all the strains which produced PCFO166 fimbriae. Weak colony hybridization, compared to the control strain, was found with ETEC producing CS5 fimbriae with CS6 antigen, CFA/III fimbriae with CS6 antigen, CS7 fimbriae or PCFO159:H4 fimbriae. CS6-antigen-positive strains of serogroups O79, O89 and O148 and all the CS17-antigen-positive and PCFO9-fimbriae-positive strains were negative in colony hybridization tests with the cfaD probe. Plasmid DNA of nine ETEC strains and their colonization-factor-negative derivatives was tested for hybridization with the cfaD probe and with ST and LT oligonucleotide probes. The sequences that hybridized with the cfaD probe were on the plasmids which coded for enterotoxin production. Fifteen strains were transformed with NTP513, a recombinant plasmid which contains the CFA/I region 1 fimbrial subunit operon but lacks a functional cfaD sequence, in order to determine whether DNA in any of these strains could substitute for the cfaD sequence in the regulation of production of CFA/I fimbriae.(ABSTRACT TRUNCATED AT 250 WORDS)     

CS3抗原基因的表达及纤毛装配元件的研究

在肠毒素大肠杆菌(ETEC)的已知定居因子中,CS3是临床分离株中最常见的抗原之一。为了研究CS3纤毛装配的基本元件,绘制了CS3亚基结构基因和辅助蛋白编码区的限制酶谱。通过亚克隆的亚基基因和不同辅助蛋白基因之间的互补性表达结果,确定了CS3纤毛装配所需要的辅助蛋白的DNA功能片段。微细胞分析结果显示,CS3基因的有效表达和纤毛装配至少需要6条蛋白多肽,分子量分别为(×10~3)15、17、24、27、48和90。除了15×10~3/17×10~3的蛋白多肽为CS3亚基外,其余的蛋白多肽参与CS3亚基的转运及纤毛的装配。根据以上结果初步确定了上述相关基因的相对位置。     

The fimbriae of human enterotoxigenic Escherichia coli strain 334 are related to CS5 fimbriae

Escherichia coli strain 334 is a human enterotoxigenic strain of serotype O15:H11 which had previously been shown to produce 'attachment pili'. These fimbriae were compared with other colonization factors. From strain 334 a mannose-resistant haemagglutination positive colony 334A and a mannose-resistant haemagglutination negative variant 334C were isolated. By electron microscopy the fimbriae of strain 334A were shown to have a helical structure resembling coli-surface-associated antigen (CS5) fimbriae. An antiserum was raised to strain 334A and absorbed with a fimbriae-negative variant of that strain, 334C. By immuno-electron microscopy this antiserum was shown to coat fimbriae of strain 334A but not CS5 fimbriae produced by strain E17018A. Conversely, CS5 antiserum did not coat the fimbriae produced by strain 334A. No antigenic cross-reaction was detected between these intact fimbriae when anti-strain 334A serum and CS5 antiserum were used in immunodiffusion tests. By enzyme-linked immunosorbent assays (ELISAs) the fimbriae of strain 334A were shown to be antigenically unrelated to most other human ETEC adhesins, namely colonization factor antigens (CFA/I, CFA/III and CFA/IV), coli-surface-associated antigens (CS1, CS2, CS3, CS4, CS6 and CS17) and putative colonization factors (PCFO159:H4 and PCFO166). However, a heated suspension of strain 334A reacted weakly with CS5 antiserum in an ELISA. By SDS-PAGE the fimbriae of strain 334A were shown to consist of subunits of similar size to CS5 subunits, that is about 21.5 kDa. Western immunoblotting revealed that the subunits of 334A and CS5 fimbriae shared common epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)     

CS3 fimbriae of enterotoxigenicEscherichia coli as chimeric expression carriers of heterologous antigenic determinants

CS3 fimbriae, which are the strong immunogen, are produced by human enterotoxigenicEscherichia coli (ETEC). The possibility of using CS3 as a carrier of foreign antigenic determinants was investigated. A SacII site sequence was inserted into the structural gene of CS3 subunit by site-specific mutagenesis based on analyzing and predicting the properties of the proteins. A recombinant strain expressing CS3/CTP3 hybrid fimbriae was constructed by inserting the sequence encoding CTP3 into the SacII site created by directed mutagenesis in the structural gene of CS3 subunit and transforming the recombinant plasmid into the host of DH5α. The result of SDS-PAGE showed that the hybrid CS3/CTP3 molecules were the fusion proteins with molecular masses of about 18.5 ku. Inoculating mice orally and intraperitoneally showed that both antibodies against CS3 and CTP3 were elicited. These results indicate that CS3 pili could be an exposure vector for heterologous antigenic determinants and become a powerful tool for the development of oral vaccines directed against mucosal pathogens.     

CS3 fimbriae of enterotoxigenic Escherichia coli as chimeric expression carriers of heterologous antigenic determinants

CS3 fimbriae, which are the strong immunogen, are produced by human enterotoxigenic Escherichia coli (ETEC). The possibility of using CS3 as a carrier of foreign antigenic determinants was investigated. A SacⅡ site sequence was inserted into the structural gene of CS3 subunit by site-specific mutagenesis based on analyzing and predicting the properties of the proteins. A recombinant strain expressing CS3/CTP3 hybrid fimbriae was constructed by inserting the sequence encoding CTP3 into the SacII site created by directed mutagenesis in the structural gene of CS3 subunit and transforming the recombinant plasmid into the host of DH5α. The result of SDS-PAGE showed that the hybrid CS3/CTP3 molecules were the fusion proteins with molecular masses of about 18.5 ku. Inoculating mice orally and intraperitoneally showed that both antibodies against CS3 and CTP3 were elicited. These results indicate that CS3 pili could be an exposure vector for heterologous antigenic determinants and become a powerful tool for the development of oral vaccines directed against mucosal pathogens.     

Assessing the expression of enterotoxigenic Escherichia coli-specific surface antigens in recombinant strains by transmission electron microscopy and immunolabeling.

Infections with enterotoxigenic Escherichia coli (ETEC) are a major cause of travelers' diarrhea worldwide. Colonization of the small intestine mucosa is dependent on specific colonization factor antigens (CFA) and coli surface (CS) antigens. CFA/1, CS3, and CS6 are the most prevalent fimbrial antigens found in clinical isolates. The goal of our study was to visualize the morphology of CS3 and CS6 fimbriae in wild-type and recombinant E. coli strains by means of transmission electron microscopy in conjunction with negative staining and immunolabeling. Corresponding ETEC genes were cloned into E. coli K12 strain DH10B. Expression of fimbriae was dependent on culture conditions and sample handling. Specific immunolabeling of fimbriae unequivocally demonstrated the presence of all types of surface antigens investigated. Negative staining was effective in revealing CS3 but not CS6. In addition, this technique clearly demonstrated differences in the morphology of genetically and immunologically identical CS3 surface antigens in wild-type and recombinant strains. This paper provides a basis for the assessment of recombinant vaccines.     

Stability of the Encoding Plasmids and Surface Expression of CS6 Differs in Enterotoxigenic Escherichia coli (ETEC) Encoding Different Heat-Stable (ST) Enterotoxins (STh and STp)

Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC isolates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located.