预防幼畜腹泻双价工程菌苗株的构建及表达

Bam HI部分酶切表达K99抗原的重组质粒pMGK99,将其与相同酶切的含有F41菌毛基因的片段相连接,构建了载有两种抗原基因的质粒pMG611。通过转化大肠杆菌K12HB101、RRI和c600,得到HBlOl(pMG611)、RRl(pMG611)和C600(pMG611)菌株。经甘露糖抗性血凝试验、玻片凝集试验和western blot分析证明K99和F4l两种菌毛抗原在每株菌中均获表达。sDs—PAGE结果表明所表达K99和F4l菌毛亚单位的分子量与其各自野生株所产生的相同,分别是17200和29800Da,ELIsA测定HB101(pMG611)菌株表达K99和K41菌毛抗原的水平与相应出发菌株相同,而高于其野生株。本实验对质粒pMG611表达K99和F41菌毛抗原的影响因素也进行了研究。     

K_(99)—F_(41)双纤毛菌的构建

产肠毒素大肠杆菌能引起仔猪、犊牛、羔羊等新生幼畜发生急性腹泻、K_(99)和F_(41)是这类产肠毒素大肠杆菌所产生的两种在免疫性质上不同的纤毛抗原。 提取K_(99)质粒,用HindⅢ酶消化,再用低熔点琼脂糖电泳回收分子量约为11Md的K_(99)的HindⅢ片段,并将该片段与经过碱性磷酸酶处理的PBR322载体重组,转化大肠杆菌C_(600)。用ELISA筛选K_(99)抗原阳性克隆。 E.coli83919F_(41)~+是非致病的产生F_(41)纤毛抗原的野生菌株。且F_(41)抗原基因是由染色体编码的。采用转化的方法将K99人工重组质粒转化到E.coli83919F_(41)~+中构建成K_(99)—F_(41)双纤毛基因工程菌。双纤毛菌中K_(99)抗原表达达到给体菌的水平,而F_(41)抗原的表达未受到明显的干扰。     

大肠杆菌F18菌毛及其亚型的PCR鉴定

F18菌毛是产肠毒素大肠杆菌 (ETEC)与产vero细胞毒素大肠杆菌 (VTEC)的重要致病因子 ,可介导细菌对小肠细胞的黏附 ,并具有F18ab和F18ac 2个抗原亚型。根据已发表的F18ab菌毛A亚单位 (FedA ab)的基因 (fedA ab)设计 3条引物 ,建立了 2种聚合酶链式反应 (PCR)扩增方法。通过对F18ab 大肠杆菌、F18ac 大肠杆菌、K88 大肠杆菌、K99 大肠杆菌、987P 大肠杆菌、F4 1 大肠杆菌的试验 ,结果表明所建立的PCR方法可特异性鉴定F18 大肠杆菌并区别其亚型F18ab与F18ac     

大肠杆菌K99菌毛fan操纵子的克隆、表达及活性

[目的]在体外克隆和表达猪产肠毒素大肠杆菌(ETEC) K99菌毛操纵子fan结构基因,并检测重组菌毛的相关生物学活性.[方法]以猪源分离的表达K99菌毛ETEC C83907株制取模板,成功PCR扩增出编码K99菌毛的fan操纵子,约5.7 kb.将fan操纵子克隆人表达质粒载体pBR322,筛选出含正确阳性质粒的重组菌.进一步将上述的重组质粒DNA转化至不含任何菌毛的大肠杆菌SE5000株,同时将空载体pBR322质粒转化入SE5000构建阴性对照菌株.[结果]该重组菌能与鼠抗K99菌毛单克隆抗体发生明显的凝集反应,与新生仔猪小肠上皮细胞刷状缘BBV分子有强烈凝集反应.电镜观察到上述重组菌表面大量表达K99菌毛,用热抽提法提纯其表达的K99菌毛,并经SDS-PAGE电泳和考马斯亮蓝染色,可以得到分子量约为18.5kDa的主要蛋白条带.纯化菌毛免疫小鼠后制备出高效价的鼠抗血清,能与携带K99菌毛的C83907、C83914、C83260野生株发生强烈的凝集反应,而与携带其他菌毛的ETEC不反应.玻板凝集试验和Western blot结果表明:体外表达的K99菌毛具有和野生K99菌毛相同的抗原性.用表达K99菌毛的重组菌进行HeLa细胞体外黏附试验和黏附抑制实验,结果表明:重组菌和野生菌株一样具有较强的粘附性,而且用重组菌毛制备的鼠抗血清能有效地抑制上述重组菌或野生菌株对细胞系的黏附结合.[讨论]本研究为进一步研究K99菌毛生物学作用建立了良好的实验平台.     

毒素源性大肠杆菌K88ac抗原基因的次级克隆和重组质粒的限制图

从我国引起仔猪腹泻的野生株E. coli 79-1454克隆了K88ac抗原基因,获得了重组体E. coli RR1(pNZ8801),分子量为10 Md。为了得到分子量更小的重组体,用E．coRI消化重组质粒,除去中间约3.2 Md的片段,得到次级克隆株E. coli RR1(pNZ8802),质粒分子量为6.8 Md。测定其K 88ac抗原为阳性,而且其产生的K88at抗原量略高于初级克隆株。电镜照片表明重组体表面长有菌毛。萤光抗体染色和猪刷状缘细胞粘附试验亦表明有良好的粘附生物活性。此重组体可以与本人克隆的K99抗原基因构建成复合重组体。本文还作了重组体的限制性酶切图。     

带有K88与K99两种伞毛抗原基因的重组质粒的构建

产肠毒素大肠杆菌能引起仔猪、犊牛、羔羊等新生幼畜发生急性腹泻,K88与K99是这类产肠毒素大肠杆菌所产生的两种在免疫性质上不同的伞毛抗原。质粒pTK8899—6和pTK8899—8是由来自K99质粒DNA的4．5×10⁶道尔顿大小的BamHI片段与带有K88伞毛抗原基因的质粒pTK90DNA重组而构建成的;带有pTK889g一6或pTK8899—8的大肠杆菌c600菌株均能同时产生K88与K99两种伞毛抗原。限制性酶切图谱的研究表明,pTK88g9—6与pTK8899—8 DNA的分子量均为11．85×10⁶道尔顿,但这两种重组DNA中所带Kg9伞毛抗原基因的片段连接在载体DNA上的方向彼此相反,带有pTK8899—6或pTK8899—8的大肠杆菌c600菌株在产生K88或K99伞毛抗原的能力上没有明显的差别。带有K88与K99两种伞毛抗原基因重组质粒的大肠杆菌菌株有可能用作预防仔猪,犊牛和羔羊急性腹泻的菌苗。     

抗大肠埃希氏菌F41粘附素特异单克隆抗体的研制及其初步应用

应用杂交瘤技术获得7株能稳定分泌F41特异单克隆抗体的杂交瘤细胞系,分别命名为Ll0、B10、C32、B1、E7、E40和B49。在直接凝集试验、酶联免疫吸附试验和间接荧光试验中,这7株单克隆抗体对所试的31株肠道菌中所有F41阳性菌株都发生反应,与F41阴性菌株则无反应性。抗原竞争ELISA试验结果发现,这些单克隆抗体是针对F41粘附素上相同或十分相近的抗原决定簇。采用肢体金标记技术和免疫电镜证实,这些抗原决定簇在F41粘附素的每条纤毛上多次重复出现。体外肠吸附抑制试验表明,7株F41l特异单克隆抗体对B41M菌株具有很强的抑制能力,对B41菌株则必须用F41和K99单克隆抗体同时作用才具有完全的肠吸附抑制效果。本文用直接酶标单克隆抗体建立的酶联免疫斑点试验具有高度的特异性和灵敏性,可广泛用于现场快速诊断。     

大肠杆菌K88ac菌毛操纵子fae全基因的克隆、表达及生物学活性初步研究

目的：在体外克隆和表达猪肠产毒性大肠杆菌（ETEC）K88ae菌毛操纵子,触结构基因,并检测重组菌毛的相关生物学活性。方法：利用长PCR技术以猪ETECK88ae株C83902基因组DNA为模板扩增编码K88菌毛操纵子触基因,克隆入表达质粒载体pBR322,构建和筛选重组质粒pBR322-fae,转化至不含任何菌毛的大肠杆菌EP株;电镜观察重组菌表面菌毛表达情况;用热抽提法提纯表达的重组菌毛;用纯化菌毛免疫小鼠制备高效价抗血清;用SDS-PAGE和Western blot检测重组菌毛的抗原性,用细胞黏附和黏附抑制试验检测其生物学活性。结果和结论：在电镜下观察到重组菌表面大量表达K88ae菌毛,该重组菌与兔抗K88ae菌毛单因子阳性血清、鼠抗K88ac菌毛单克隆抗体均产生凝集反应;纯化菌毛经SDS-PAGE,结构单位菌毛呈单一的相对分子质量约26×10^3的蛋白条带;纯化菌毛免疫小鼠后可制备出高效价的鼠抗血清,玻板凝集试验和Western blot结果表明体外表达的K88ae菌毛具有与K88ae野生菌毛相同的抗原性;猪小肠上皮细胞系黏附和黏附抑制实验结果表明重组EP菌和野生菌株一样具有较强的黏附猪小肠上皮细胞系的能力,而且提纯重组菌毛制备出的鼠抗血清能有效抑制上述重组菌或野生菌株对猪小肠上皮细胞系的黏附结合。     

大肠杆菌F18菌毛操纵子全基因克隆、表达及生物学活性

利用PCR技术以猪产肠毒素大肠杆菌F18标准菌株107/86和2134P基因组DNA为模板成功地扩增出编码F18ab和F18ac完整菌毛操纵子fed基因。将它们分别克隆入表达质粒载体pET-22b( ),结合酶切和核苷酸序列分析证明了PCR预期扩增产物的正确性。然后将克隆的重组载体DNA转化至大肠杆菌BL21(DE3),构建和筛选出分别含F18ab和F18ac完整fed基因的重组菌,经过IPTG诱导表达,在电镜下观察到上述两种重组菌能分别大量表达F18ab和F18ac菌毛。用热抽提法提纯其诱导表达的F18ab和F18ac菌毛,经SDS-PAGE电泳和考马斯亮蓝染色发现提纯后菌毛获单一分子量约为15kDa蛋白条带,免疫家兔后制备出高效价的兔抗血清,玻板凝集试验和Western blot结果表明:体外诱导表达的F18ab和F18ac菌毛具有和野生F18菌毛相同的抗原性。用表达F18ab和F18ac菌毛的上述2株重组菌分别进行小肠上皮细胞体外吸附试验和吸附抑制试验,结果表明:2株重组菌和野生菌株一样具有较强的粘附易感仔猪小肠上皮细胞的能力,而用表达F18ab和F18ac重组菌提纯的菌毛制备出兔抗血清都能有效地抑制上述重组菌或野生菌株对易感仔猪小肠上皮细胞的吸附结合。     

动物源产肠毒素大肠杆菌(ETEC)黏附素研究进展

动物源产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)是引起动物(尤其是幼龄动物)腹泻的主要病原菌。已知黏附素和肠毒素是ETEC中两种重要的毒力因子,在致病性中两者缺一不可。其中黏附素结合到宿主易感肠上皮细胞是ETEC感染的第一步,也是最重要的关键步骤。动物源ETEC的菌毛黏附素主要包括K88、K99、987P、F18、F17和F41等。人们从20世纪60年代就开始了ETEC菌毛黏附素的相关研究,包括菌毛的基因、结构组成、生物合成、菌毛表达的调控机制以及黏附素和宿主受体相互作用等,这些研究基础有助于我们深入了解ETEC病原菌的感染机理;并且在疾病诊断和新疫苗的开发中具有重大意义。     

Morphological description of surface structures on strain B41 of bovine enterotoxigenic Escherichia coli bearing both K99 and F41 antigens

In order to describe morphologically the structures on the cell surface of bovine enterotoxigenic Escherichia coli, variants of reference strain B41 (K99+F41+) either negative for K99 and positive for F41 antigens (variants B41A, B41*C), or phenotypically negative for both antigens (variants B41B1, B41B2, B41*CB), and a transconjugant harbouring the K99 plasmid and expressing the K99 adhesin [transconjugant B41 x H510a:H510(2)] were examined by transmission electron microscopy using negative staining. Several negative staining procedures were tested for strain B41 and variant B41A: direct harvesting of strains into ammonium molybdate (2%, w/v), with bacitracin (50 micrograms ml-1) as wetting agent, gave the best results. Three morphologically distinct structures on the cell surface could be identified in cultures grown on Minca medium. Firstly, thin, filamentous, flexible fibrillar structures, presenting a helical structure and a mean diameter of approximately 3 nm, were recognized as K99 fimbriae, since they were present on strain B41 and on transconjugant H510(2), but not on K99-negative variants nor on the recipient strain H510a. Secondly, coil-like structures with a diameter of about 17-20 nm were observed on strain B41 and on variants B41A and B41*C. These structures appeared to consist of two or more curled filaments (diameter 3 nm) joined to coil on themselves into dense spirals. They were very rare in variants B41B1 and B41B2 and were absent on variant B41*CB and on a transconjugant B41* x B41*CB, which had re-acquired the K99 plasmid and which again exhibited K99 fimbriae. Strains B41 and variant B41A gown at 37 degrees C for 24 h on sheep-blood agar exhibited coiled structures like those seen on Minca medium. In contrast, after growth at 18 degrees C for 48 h (which inhibits the synthesis of F41 antigen), coiled structures were no longer expressed on the cell surface of strain B41 and variants B41A and B41*C. Thus the presence of coiled structures correlated with the expression of F41 antigen in strains and variants, which suggests that F41 had a coiled morphology. Finally, straight fimbriae (diameter 6.5-7 nm) were observed on the cell surface of every strain and variant. Their expression on the cell surface was enhanced by several subcultures in th e static broth, and it was inhibited by subculture on agar, but not by culture at 18 degrees C after serial subcultures in static broth. These facts indicated that the straight fimbriae could be common fimbriae, and excluded their being F41 structures.     

The production of F41 fimbriae by piglet strains of enterotoxigenic Escherichia coli that lack K88, K99 and 987P fimbriae

The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique. Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E. coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain. The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen. The MR haemagglutinating properties of an antigen extract containing material B from E. coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E. coli strains that produce both F41 and K99 fimbriae. These sera also gave an anionic precipitation line with the MR haemagglutinin from E. coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae. OK antisera to K99+ F41- bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin. Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E. coli strain 1706 showed irregular, poorly defined filamentous material surrounding some,though not all, bacteria but regular fimbrial structures were not visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequences related to the major subunit gene fedA of F107 fimbriae in porcine Escherichia coli strains that express adhesive fimbriae

Abstract Porcine Escherichia coli strains isolated from cases fo postweaning diarrhea or edema disease were analysed for the presence of fedA , the major subunit gene of F107 fimbriae. The E. coli isolates were known to contain colonisation factor '8813', or to express F107, 2134P or other fimbriae, different from F4, F5, F6, and F41. PCR with fedA -specific primers, restriction enzyme digestion of the PCR product, and nucleotide sequence analysis demonstrated that 2134P pili, colonisation factor '8813' and fimbriae identified on Australian strains of the O141 serotype belong to one family of F107 fimbrial antigens.     

Molecular cloning of the F8 fimbrial antigen from Escherichia coli

Abstract The genetic determinant coding for the P-specific F8 fimbriae was cloned from the chromosome of the Escherichia coli wild-type strain 2980 (O18:K5:H5:F1C, F8). The F8 determinant was further subcloned into the Pst I site of pBR322 and a restriction map was established. In a Southern hybridization experiment identity between the chromosomally encoded F8 determinant of 2980 and its cloned counterpart was demonstrated. The cloned F8 fimbriae and those of the wild type strain consist of a protein subunit of nearly 20 kDa. F8 fimbriated strains were agglutinated by an F8 polyclonal antiserum, caused mannose-resistant hemagglutination and attached to human uroepithelial cells. The cloned F8 determinant was well expressed in a variety of host strains.     

Lactobacillus acidophilus expressing recombinant K99 adhesive fimbriae has an inhibitory effect on adhesion of enterotoxigenic Escherichia coli

The most common enteric colibacillosis in neonatal and newborns is caused by enterotoxigenic Escherichia coli(ETEC). Colonization of ETEC in the small intestine is associated with adhesions using fimbriae, which is known as a specific adhesion factor and provides highly specific means for anchoring and prerequisite for an infectious agent. In the present study we have engineered Lactobacillus acidophilus to produce recombinant K99 fimbriae, which is used for the colonization to the intestine of pigs. The expression of K99 fimbrial protein was confirmed using SDS-PAGE, immunoblot and agglutination analyses. To evaluate a function of the K99 fimbrial protein, inhibition and competition tests were performed on pre-screened intestinal brush border from pigs. The tests showed that recombinant L. acidophilus, not control L. acidophilus, had a significant inhibitory effect to and competition against K99+ E. coli in a dose dependent manner. In conclusion, we demonstrated that recombinant K99 fimbriae producing L. acidophilus was able to prevent E. coli binding to intestinal brush border.     

Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88.

The genetic determinant for production of the adhesive antigen F41 was isolated from a porcine enterotoxigenic Escherichia coli strain by cosmid cloning. The cloned DNA included sequences homologous to those of hybridization probes prepared from the K88 adhesive antigen operon. Transposon insertions which inactivated F41 production mapped to the same region of DNA showing homology with the K88 genes, demonstrating the genetic relatedness of F41 and K88. Hybridization of a K88 gene probe to plasmid and total DNA from the porcine E. coli isolate from which the F41 gene was cloned indicated that F41 is chromosomally encoded by this strain. This observation was extended to other F41-producing animal isolates. A large number of animal E. coli isolates were examined with K88, F41, and K99 gene probes and for mannose-resistant hemagglutination of human group O erythrocytes and K88 and F41 antigen production. All K88 and F41 antigen producers possessed genetic homology with the K88 and F41 gene probes. Most, but not all, F41-producing strains possessed homology to the K99 gene probe, reflecting the previously observed association of F41 and K99 antigen production. In the strains examined, homology with the K99 gene probe was plasmid associated, whereas homology with the F41 gene probe was chromosomal. The K88 antigen-producing strains showed no homology with the K99 probe. A number of strains possessed homology with the K88 and F41 gene probes and were mannose-resistant hemagglutination positive, but did not produce K88 or F41 antigens. This suggests that there are adhesins among animal isolates of E. coli which are genetically related to but antigenically distinct from K88 and F41.     

Comparison between Enterotoxigenic Escherichia coli Strains Expressing “F42,” F41 and K99 Colonization Factors

Two enterotoxigenic Escherichia coli (ETEC) strains (coded 567/7 and 103) isolated from piglets with neonatal diarrhea were described as producers of a new adhesin (F42). With the use of molecular biology and immunology techniques such as DNA hybridization with probes for F41 and K99 genes and Western-blotting of the superficial proteins of these strains and standard E. coli strains carrying genes for F41 and K99 adhesins, it was demonstrated that this new adhesin either shares extensive genetic and immunological determinants with F41 adhesin or they are the same fimbriae.     

Role of type 1 fimbriae and mannose in the development of Escherichia coli K12 biofilm: from initial cell adhesion to biofilm formation

The influence of type 1 fimbriae, mannose-sensitive structures, on biofilm development and maturation has been examined by the use of three isogenic Escherichia coli K12 strains: wild type, fimbriated, and non-fimbriated. Experiments with the three strains were done in minimal medium or Luria–Bertani broth supplemented with different concentrations of d-mannose. The investigation consisted of: (1) characterizing the bacterial surface of the three strains with respect to hydrophilicity and surface charge, (2) investigating the effect of type 1 fimbriae on bacterial adhesion rate and reversibility of initial adhesion on glass surfaces, and (3) verifying the role of type 1 fimbriae and exopolysaccharides (EPS) in biofilm maturation. The results suggest that type 1 fimbriae are not required for the initial bacterial adhesion on glass surfaces as the non-fimbriated cells had higher adhesion rates and irreversible deposition. Type 1 fimbriae, however, are critical for subsequent biofilm development. It was hypothesized that in the biofilm maturation step, the cells synthesize mannose-rich EPS, which functions as a ‘conditioning film’ that can be recognized by the type 1 fimbriae.