大肠杆力不耐热肠毒素突变体LTR72基因的克隆及序列分析

利用PCR和点突变PCR技术，制备了大肠杆菌不耐热肠毒素（LT）突变体LTR72和LT的基因片段，经酶切连接后构建了突变体重组质粒pLTR72，经酶切和序列测定证明重组子构建正确，目的突变点第72位的丙氨酸密码子已被突变成为精氨酸密码子。同时，试验构建了可以表达天然LT的表达质粒pLT作对照，二级结构分析表明，LTR72及LT之间没有显著差异。     

Expression of Putative Escherichia coli Heat-labile Enterotoxin (LT) Receptors on Intestinal Brush Borders from Pigs of Different Ages

Many strains of enterotoxigenic Escherichia coli (ETEC) that cause diarrhoea in young piglets secrete a heat-labile enterotoxin (LTp) that binds to specific glycoconjugates on porcine intestinal epithelial cells. Binding of LTp to an appropriate glycoconjugate facilitates the uptake and trafficking of the toxin into the cell, where it stimulates intracellular changes that promote fluid secretion and diarrhoea. The objective of the current study was to identify the LTp-binding glycoconjugates on porcine intestinal epithelial cells, the natural target cells for LTp. We found that LTp binds, in an age-correlated manner, to an acidic glycosphingolipid (GSL) that co-migrated with GM1 on thin-layer chromatography (TLC), a small acidic GSL that appears to be a sulphatide, a neutral GSL that co-migrated with neolactotetraglycosylceramide (nLc4) on TLC, and two glycoproteins (36 and 205 kDa). Of these potential LTp receptors, the GM1-co-migrating GSL was detected most intensely in young animals, while the other four LTp-binding glycoconjugates were detected most intensely in older pigs (54 weeks). Since ETEC primarily cause disease in young piglets, the GM1-co-migrating GSL is the most likely candidate for a functional LTp receptor.     

大肠杆菌热敏感肠毒素的检测与制备

为探索猪源大肠杆菌热敏感肠毒素的简易检测方法和纯化方法,拟采用兔回肠结扎试验、兔皮肤蓝斑试验和平板免疫溶血试验比较检测热敏感肠毒素的方法。初步试验表明:平板免疫溶血试验能够快速灵敏地检测出热敏感肠毒素,并具有很强的特异性,比肠结扎试验灵敏度高,而且简便易行;采用SephadexG 100纯化热敏感肠毒素LT并采用SDS PAGE电泳检测蛋白纯度,表明该方法可以除去杂蛋白,肠毒素蛋白分子量为12 5KD和25KD     

Unique features of bovine lymphocytes exposed to a staphylococcal enterotoxin

We previously demonstrated that stimulation of bovine peripheral blood mononuclear cells (PBMCs) with staphylococcal enterotoxin C (SEC), led to an inversion of the CD4⁺:CD8⁺ T cell ratio and generation of an atypical CD8⁺ T cell subpopulation expressing CD26. In the present study, we examined T cell apoptosis and proliferation profiles of PBMC subpopulations in cultures stimulated with SEC. Unlike when stimulated with concanavalin A, nucleic acid synthesis in bovine PBMC cultures stimulated with SEC was low during the first four days but increased greatly on day 5. In contrast, nucleic acid synthesis in human PBMC cultures stimulated with SEC increased continuously. To investigate the mechanism of delayed bovine T cell proliferation, various cell phenotypes were monitored. The inversion of the bovine CD4⁺:CD8⁺ T cell ratio in PBMC cultures stimulated by SEC was associated with higher proliferation and lower apoptosis of CD8⁺ T cells compared to CD4⁺ T cells. The mRNA levels for interleukin (IL)-4 and IL-13 were sustained over 4 days but IL-12 mRNA levels dropped to background on day 2. These data suggest that SEC induces a prolonged Th-2-biased microenvironment, and together with the inversion of the bovine CD4⁺:CD8⁺ T cell ratios in bovine PBMC cultures with SEC, may in part explain the inability of the mammary immune system to establish an effective response to Staphylococcus aureus infections.     

表达Ⅰ型耐热肠毒素的重组大肠杆菌对7日龄仔猪小肠功能的影响

试验旨在研究表达Ⅰ型耐热肠毒素(STa)的重组大肠杆菌对7日龄仔猪小肠功能的影响。选取24头7日龄仔猪,随机分为4个处理组,分别为对照组、STa组、LMG194组和K88组。整个试验期间均以人工乳饲喂,预饲期4d,试验第5天进行攻毒,STa组、LMG194组及K88组分别在早晚两次口服灌喂2×109 CFU大肠杆菌LMG194-pBAD-STa、LMG194和K88,对照组灌服等量生理盐水,试验第7天早上各组灌服D-木糖并采血,屠宰并取空肠、回肠组织样品,测量仔猪血常规指标、血浆D-木糖含量及二胺氧化酶(DAO)活力,以及吸收与转运相关基因与蛋白表达量。结果显示,与对照组相比,STa组仔猪血液中中性粒细胞、中性粒细胞比率显著降低(P<0.05),淋巴细胞比率显著升高(P<0.05),LMG194组与K88组仔猪血液中中性粒细胞比率显著降低(P<0.05),淋巴细胞比率显著升高(P<0.05);STa组与K88组仔猪血浆D-木糖含量显著降低(P<0.05);STa组、LMG194组及K88组血浆DAO活力显著升高(P<0.05);STa组与LMG194组空肠AQP8、AQP10、KCNJ13和b0,+AT基因表达量均显著降低(P<0.05),K88组空肠AQP8、AQP10基因表达量显著升高(P<0.05);STa组回肠AQP8基因表达量显著降低(P<0.05),AQP10、SGLT-1基因表达量显著升高(P<0.05),LMG194组、K88组AQP8基因表达量显著升高(P<0.05),K88组AQP10、KCNJ13、b0,+AT、SGLT-1基因表达量显著降低(P<0.05);STa组、LMG194组及K88组空肠HSP70蛋白的表达量显著升高(P<0.05)。以上结果表明,表达Ⅰ型耐热肠毒素的重组大肠杆菌可导致7日龄仔猪小肠产生炎症,吸收与转运能力下降。     

Preparation,Identification and Purification of Polyclonal Antibody against STa

Enterotoxic Escherichia coli (ETEC) can cause acute diarrhea in human and animals, and its key virulent factor is heat-stable enterotoxin (ST).ST is a peptide with small molecular weight and great toxicity but without immunogenicity, so how to keep its immunogenicity and reduce its toxicity has become a hot research topic for preparing ST toxoid vaccine.In this study, rabbit serum albumin (RSA) was purified by rivanol method and activated in 0.2% glutaraldehyde solution before RSA was conjugated to synthetic methanol-soluble STa, SDS-PAGE result showed that an approximate 108.5 ku complete antigen was obtained.New Zealand White rabbits were immunized with the conjugate four times, the serum was then collected and used to purify immunoglobulin by immunoprecipitation with Protein A/G Plus-Agarose.Dot-ELISA and Western blotting result showed that the titer of the antiserum both reached 1:400.These results indicated that anti-STa polyclonal antibody was successfully prepared, which provided the way for screening of ST structure analogues.     

spa typing and enterotoxin gene profile of Staphylococcus aureus isolated from bovine raw milk in Korea

Staphylococcus aureus is a major etiological pathogen of bovine mastitis, which triggers significant economic losses in dairy herds worldwide. In this study, S. aureus strains isolated from the milk of cows suffering from mastitis in Korea were investigated by spa typing and staphylococcal enterotoxin (SE) gene profiling. Forty-four S. aureus strains were isolated from 26 farms in five provinces. All isolates grouped into five clusters and two singletons based on 14 spa types. Cluster 1 and 2 isolates comprised 38.6% and 36.4% of total isolates, respectively, which were distributed in more than four provinces. SE and SE-like toxin genes were detected in 34 (77.3%) isolates and the most frequently detected SE gene profile was seg, sei, selm, seln, and selo genes (16 isolates, 36.3%), which was comparable to one of the genomic islands, Type I νSaβ. This is a first report of spa types and the prevalence of the recently described SE and SE-like toxin genes among S. aureus isolates from bovine raw milk in Korea. Two predominant spa groups were distributed widely and recently described SE and SE-like toxin genes were detected frequently.     

Experimental model of enterotoxigenic Escherichia coli infection in pigs: potential for an early recognition of colibacillosis by monitoring of behavior

The hypothesis that altered behavior is a sign for an early recognition of disease was tested. The experiment was conducted to evaluate the behavioral patterns of pigs in a model of postweaning colibacillosis. Twenty-five weaned pigs (from a herd that was previously found to be highly susceptible to F4⁺ Escherichia coli strains) were randomly assigned into 5 groups, kept in isolated pens under the controlled ambiental conditions. One day after weaning, the pigs from three groups were intragastrically inoculated (via orogastric tube) with either F4ac⁺ (1466 or 2407) or F4⁻ (1467) nonenterotoxigenic E. coli (non-ETEC) strains, respectively. The pigs from the fourth group were inoculated with F4ac⁺ ETEC strain M1823 and the remaining 5 pigs that received broth containing 1.2% sodium bicarbonate were kept as noninoculated controls. The pigs were examined daily and the frequency and duration of their behavioral patterns, such as eating, drinking, lying, standing, urinating, defecating, rooting and playing were monitored for 300 h during a period of 10 days. In this model, three conditions were also observed in F4-susceptible pigs: (1) acute fatal diarrheal disease; (2) moderate diarrhea and weight loss and (3) no diarrhea and weight loss. The incidence (both frequency and duration) of defecating was significantly higher (P<0.05) in pigs inoculated with F4ac⁺ ETEC strain M1823 as compared to that of noninoculated (control) pigs. Pigs inoculated with F4ac⁺ non-ETEC strain 1466 had a significantly lower frequency of eating (P<0.05) and frequency/duration of drinking (P<0.05) than did the controls. The 1466-inoculated pigs, had an increased diarrhea score, but frequency/duration of defecating was not significantly different. Pigs inoculated with F4ac⁺ non-ETEC strain 2407 spent more time in lying (P<0.05) than did noninoculated pigs. Conversely, the pigs that received F4⁻ non-ETEC strain 1467 laid shorter (P<0.05) and ate/drank less frequently (P<0.05) than the controls. It was concluded that the changed occurrence of defecating and eating in pigs that were inoculated with either F4ac⁺ ETEC (M1823) or non-ETEC (1466) strain, respectively, was consistent with the pending clinical disease, i.e. postweaning colibacillosis.     

空肠弯曲菌肠毒素间接ELISA检测法的建立

通过对ＣＥ３３种检测方法的比较研究,证实间接ＥＬＩＳＡ是检测ＣＥ最为敏感,简便,快速的方法,在２４ｈ内即可得到结果。ＣＨＯ法也较敏感,但要求较高。大鼠肠袢试验虽然操作繁杂,却是传统的肠毒素检测法,结果更具说服力。     

抗STa多克隆抗体的制备、鉴定和纯化

产肠毒素大肠杆菌(ETEC)可引起人及动物急性腹泻,其关键毒力因子为耐热肠毒素(ST)。由于ST是一种具有强烈毒性但缺乏免疫原性的小分子肽,如何在保持其免疫原性的同时减弱其毒性已成为制备ST类毒素疫苗的研究热点。本试验以利凡诺法提纯兔血清白蛋白(RSA),经0.2%戊二醛活化,再与人工合成的甲醇可溶性STa偶联,十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示获得分子质量约108.5 ku的完全抗原。用该偶联物免疫新西兰白兔4次,采集抗血清并用Protein A/G 琼脂糖小珠进行纯化,斑点酶联免疫吸附试验(dot-ELISA)和免疫印迹(Western blotting)结果显示抗血清效价均为1:400。试验结果表明抗STa多克隆抗体成功制备,这为筛选ST结构类似物做有益的铺垫。