产肠毒素性大肠杆菌肠毒素的研究概况

本文分析了大肠杆菌耐热肠毒素 (heat-stable,ST)和不耐热肠毒素 (heat- labile,LT)生物学特性、分子水平机制及其应用价值。一方面着重讲述 LT作为粘膜免疫佐剂的研究前景 ,利用体外定点突变方法构建不同的突变株并且均具有不同程度的佐剂活性 ,是霍乱毒素 (Choleratoxin,CT)很有希望的替代品。另一方面讲述ETEC基因工程亚单位疫苗的研究概况。ETEC只有 LT、ST两类肠毒素 ,通过不同方式构建融合基因探讨对 ETEC性腹泻防治效果。目前 ,这两方面均卓有成效 ,但还有许多的难点需进一步的研究     

应用酶联免疫吸附试验双夹心改良法检测毒素原性大肠杆菌

大肠杆菌是引起猪、牛、羊腹泻常见的病原菌之一。特别是由毒素原性大肠杆菌引起的腹泻,近年来引起了国内外的广泛重视。毒素原性大肠杆菌(ETEC)产生两种肠毒素:即耐热肠毒素(ST)和不耐热肠毒素(LT)。ST是小分子的无抗原性毒素。而LT除在理化性质上与霍乱毒素(CT)相似     

大肠杆菌热敏毒素LT克隆基因的PCR定点诱变和重组表达的构建

为使大肠杆菌热敏毒素LT的毒素活性丧失的同时仍保留其较强的免疫原性，实验依据LT基因的同源序列设计并合成5条引物，采用突出末端PCR法和重组PCR法，将克隆于质粒pEWD299上的LT基因，分别引入BamHI、Ndel和xhol等位点，扩增出带有上述RE位点且含有m7和m112突变点的约1100bp和约800bp的DNA片段和不含突变点的约300bp的DNA片段，使控制LT毒力活性中心的第7位和第112位氨基酸的碱基分别发生诱变，各DNA片段经分熟、纯化、RE酶切和DNA连接酶连接后，插入经BamHI和XhoI双酶切的线性化的高效表达载体pGEX-4T-1的多克隆位点区，使LTm基因置于pTac强启动子下且与Thrombin蛋白基因融合表达，将连接产物转化入JM105菌株，挑出可疑阳性菌落，提取质粒，经酶切鉴定、PCR鉴定和DNA序列分析，证明读框正确，序列正确，获得了pGEX-4T-1(LT7t GEX-4T-1(LTm112两个重组表达质粒。为运用基因工程手段大量生产人 动物大肠杆菌流行性腹泻的疫苗抗原和幽门螺杆菌疫苗的粘膜免疫佐剂，完成了基因水平的工作。     

大肠杆菌Ⅱ型不耐热肠毒素(LT-Ⅱ)的研究进展

1983年Green等首次报道,从泰国水牛粪便中分离的大肠杆菌SA53株能产生一种细胞结合的毒素,其不耐热性和生物学活性均与LT相似,而抗原性与LT无关,不被霍乱肠毒素(CT)、人株LT(LTh)或猪株LT(LTp)的抗血清所中和,故当时将其称为LT样毒素。尔后,Pickett等(1986)认为,该毒素应隶属于霍乱弧菌-大肠杆菌不耐热肠毒素族内,系LT的另一血清群。以往发现的大肠杆菌LT抗原性与     

大肠杆菌不耐热肠毒素B亚单位在原核细胞中的高效表达

从含有LT全毒素基因操纵子的质粒EWD299中扩增出LT的B亚单位基因后定向克隆于原核表达载体pET28a中，转化大肠杆菌BL21（DE3）plyss，用IPTG诱导表达后，将全茵裂解，用SDS—PAGE和Western blot检测重组茵中外源蛋白的表达情况。结果表明，在原核细胞中高效表达了LTB蛋白，表达的重组蛋白占菌体蛋白总量的38％。     

大肠杆菌不耐热肠毒素的分子生物学及粘膜免疫佐剂效应

大肠杆菌不耐热肠毒素(Heat-Labile,LT)是由产肠毒素大肠杆菌(enterotoxigenicEscherichiacoli,ETEC)分泌的一种外毒素。LT既有较强的免疫原性又有较强的粘膜佐剂效应,其毒力较霍乱毒素(choleratoxin,CT)小而被认为是替代CT的最有潜力的粘膜免疫佐剂。本文从LT的分子结构、分子生物学特性、免疫原性、粘膜佐剂效应及其作用机制以及LT的粘膜免疫佐剂的应用等方面进行了讨论。     

利用CRISPR/Cas9和λ-Red级联技术构建产肠毒素大肠杆菌LT敲除菌株

本研究旨在利用CRISPR/Cas9和λ-Red级联的技术对产肠毒素大肠杆菌（enterotoxigenic Escherichia coli，ETEC）K88的热不稳定性肠毒素（heat-labile toxin，LT）基因进行无痕敲除并获得K88 LT^-缺陷菌株。通过序列比对获取LT两端同源序列，并构建包含LT边界、氯霉素筛选标记、sgRNA和LT同源臂的供体片段；将供体片段转化至ETEC K88，同时分别利用λ-Red同源重组系统和CRISPR/Cas9基因编辑系统，对LT基因进行敲除；通过PCR验证获得了K88 LT^-缺陷菌株，并通过试验测定了敲除菌株的溶血能力和生长曲线。结果显示，λ-Red同源重组系统可成功地将LT基因替换为相应的供体片段，CRISPR/Cas9基因编辑系统可高效地对筛选标记进行删除，最终通过λ-Red和CRISPR/Cas9结合的基因编辑系统可成功对ETEC K88的LT基因进行无痕敲除。体外试验结果表明，K88 LT^-缺陷菌株的溶血能力丧失，并且生长速度比野生型菌株减缓，LT可能和ETEC K88的致病能力和生长性能有关。表明λ-Red和CRISPR/Cas9级联的基因敲除方法可用于LT毒素基因及其他一些大肠杆菌基因的敲除。K88 LT^-缺陷菌株的构建为下一步研究LT毒素的致病机制奠定基础。     

利用CRISPR/Cas9和λ-Red级联技术构建产肠毒素大肠杆菌LT敲除菌株

本研究旨在利用CRISPR/Cas9和λ-Red级联的技术对产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)K88的热不稳定性肠毒素(heat-labile toxin,LT)基因进行无痕敲除并获得K88 LT~-缺陷菌株。通过序列比对获取LT两端同源序列,并构建包含LT边界、氯霉素筛选标记、sgRNA和LT同源臂的供体片段;将供体片段转化至ETEC K88,同时分别利用λ-Red同源重组系统和CRISPR/Cas9基因编辑系统,对LT基因进行敲除;通过PCR验证获得了K88 LT~-缺陷菌株,并通过试验测定了敲除菌株的溶血能力和生长曲线。结果显示,λ-Red同源重组系统可成功地将LT基因替换为相应的供体片段,CRISPR/Cas9基因编辑系统可高效地对筛选标记进行删除,最终通过λ-Red和CRISPR/Cas9结合的基因编辑系统可成功对ETEC K88的LT基因进行无痕敲除。体外试验结果表明,K88 LT~-缺陷菌株的溶血能力丧失,并且生长速度比野生型菌株减缓,LT可能和ETEC K88的致病能力和生长性能有关。表明λ-Red和CRISPR/Cas9级联的基因敲除方法可用于LT毒素基因及其他一些大肠杆菌基因的敲除。K88 LT~-缺陷菌株的构建为下一步研究LT毒素的致病机制奠定基础。     

热敏肠毒素促进产肠毒素大肠杆菌对小肠上皮细胞系IPEC-J2的黏附的初步研究

本试验利用PCR技术,以K88ac标准株C83902基因组DNA为模板扩增出热敏肠毒素(heat-labile toxins,LT)基因,大小约1.1 kb。将其克隆入表达质粒载体pACYC184,构建和筛选出含正确插入LT基因的pACYC-LT重组质粒。采用同样方法,构建和筛选出两种含点突变LT基因的重组质粒(pACYC-LT72和pACYC-LT192)。进一步将上述重组质粒DNA转化入不表达任何毒素的大肠杆菌SE5000株。GM1-ELISA结果表明,上述重组菌均能在体外正常表达LT毒素蛋白。以猪小肠上皮细胞系IPEC-J2为模型,比较了表达和不表达LT的细菌对细胞黏附性能。数据表明,LT的表达使细菌对肠上皮细胞的黏附效应明显增加(12.3±3.4倍)。两种LT毒素蛋白的单氨基酸突变体的表达证明了LT毒素的ADP核糖基化作用对其增强致病菌对肠细胞的黏附作用是必要的。蛋白激酶A的抑制剂Rp-cAMP、腺苷酸环化酶的抑制剂DDA和LT毒素的受体GM1都可阻断LT毒素对细菌黏附性能的提升作用。     

猪源产肠毒素大肠杆菌三重PCR检测方法的建立及应用

为建立一种简单、快速、灵敏、准确的产肠毒素大肠杆菌(ETEC)检测方法,根据产肠毒素大肠杆菌菌毛(K88)和毒素(STa和LT)基因分别设计合成了1对引物,对K88、STa和LT基因扩增条件进行优化,建立了检测K88、STa和LT的三重PCR方法。该方法对K88、STa和LT基因的扩增产物分别为499 bp,190 bp和373 bp;此外,该方法具有良好的灵敏性和特异性。本实验建立的三重PCR方法为致幼畜腹泻ETEC的检测提供了快速准确方法。用所建立的三重PCR方法对实验室从临床腹泻样品中分离的120株大肠杆菌进行检测,结果 9株为K88/LT/STa阳性,14株为K88/LT阳性,21株K88/STa阳性,13株LT/STa阳性,8株K88阳性,2株LT阳性,12株STa阳性。     

大肠杆菌K_(88ac)与LT(A~-B~ )抗原基因质粒在猪霍乱沙门氏菌弱毒株中的表达

应用细菌质粒转化技术,将大肠杆菌K_(?)与LT(A~-B~ )抗原基因重组质粒转入猪霍乱沙门氏菌弱毒菌苗株中.对获得的其中8个转化子进行鉴定的结果表明,转化的细菌仍保持沙门氏菌的形态、生化及抗原特性,同时可稳定地表达K(?)和LT-B两种抗原.用微量间接血凝试验、抗甘露糖豚鼠红细胞凝集试验(MRHA)、ELISA等对转化菌表达的K(?)抗原进行了测定,用间接免疫溶血试验对其表达的LT-B抗原进行了测定.结果,这两种抗原在转化的细菌中均可高效表达.电镜下观察,转化的细菌在其表面形成菌毛样结构.这种转化细菌表现出猪霍乱沙门氏菌与产肠毒素性大肠杆菌的两种抗原特性,为这种双价工程菌苗的研制提供了有价值的候选菌株.本研究结果还表明,猪霍乱沙门氏菌弱毒菌苗株可作为基因转化的有效受体菌.     

Prevalence of virulence genes in Escherichia coli strains recently isolated from young pigs with diarrhea in the US

Enterotoxigenic Escherichia coli (ETEC)-associated post-weaning diarrhea (PWD) is economically one of the most important diseases for the swine industry. Porcine ETEC strains typically express K88 or F18 fimbria and heat-labile (LT) and/or heat-stable (STa, STb) enterotoxins. However, recent studies indicate that EAST1 toxin, adhesin involved in diffuse adherence (AIDA-I) and porcine attaching and effacing-associated factor (paa) may also be expressed by ETEC strains associated with diarrhea. To better understand the virulence factors of E. coli strains that cause PWD, we applied PCR to screen for K88, F18, F41, 987P and K99 fimbrial genes; LT, STa, STb, Stx2e and EAST1 toxic genes; and AIDA-I, paa and EAE adhesin genes in E. coli strains recently isolated from young pigs with PWD in the US. Of 304 E. coli isolates from diarrheic pigs submitted for testing, 175 (57.6%) strains possessed fimbrial genes: K88 (64.6%), F18 (34.3%), F41 (0.57%), K99 (0.57%), 987P (0); toxin genes: LT (57.7%), STb (72.6%), STa (27.4%), STx2e (17.4%), EAST1 (35%); and adhesin genes: AIDA-I (26.9%), paa (60%), EAE (1.1%). All toxin genes except the EAST1 toxin gene, were almost exclusively associated with K88+ or F18+ isolates, and most of these isolates carried multiple toxin genes. The non-fimbrial adhesin paa was found present in over half of the K88+ isolates. A total of 129 (42%) isolates carried no fimbrial genes, including 66 (21.7%) isolates that did not have any of the above virulence genes. These results suggest a broad array of virulence genes associated with PWD in pigs.     

Hormonal profiles of late gestation ewes following intra-uterine inoculation with and without lux-modified Escherichia coli

The objectives of these investigations were to develop an ovine model for Escherichia coli (E. coli)-induced preterm delivery, and monitor ewe hormonal response. EXP 1: Ewes (105 +/- 13 days of gestation) were allotted to the following intra-uterine inoculations: Saline-(CON; n=5); 1 x 10(6) CFU/ml (Low Treatment, LT; n=6); or 1 x 10(7) CFU/ml (High Treatment, HT; n=6) E. coli. Twenty-four h after inoculation, the HT ewes had increased (P<0.05) cortisol compared to LT and CON ewes, and HT and LT ewes had increased (P<0.05) progesterone compared to CON ewes. Preterm delivery was 33% for LT ewes and 0% for HT and CON ewes. EXP 2: Ewes (124 +/- 18 days of gestation) were allotted to the following intra-uterine inoculations using lux-modified E. coli: Trial-1: Luria Broth (LB; CT1; n=5); 4.0 x 10(6) CFU (n=5), 20.0 x 10(6) CFU (n=5); and Trial-2: LB (CT2; n=5), 1.2 x 10(6) CFU (n=5), and 5.6 x 10(6) CFU (n=5) E. coli-lux. Preterm delivery occurred between 48 and 120 h post-inoculation in 60, 25, 60 and 75% of ewes infected with 1.2, 4.0, 5.6, and 20 x 10(6) CFU, respectively. Serum cortisol and progesterone did not differ (P>0.05) between CT1 or CT2 and inoculated ewes. In summary, 25 to 75% of ewes inoculated preterm delivered. However, variable results in cortisol and progesterone profiles between Control and inoculated ewes were observed between the two studies.     

重组大肠杆菌不耐热肠毒素的表达及对小鼠胚胎稳定性的影响

为研究大肠杆菌不耐热肠毒素（LT）对小鼠胚胎稳定性的影响,运用大肠杆菌表达系统制备了具有生物活性的重组大肠杆菌LT,用重组LT通过腹腔注射处理妊娠小鼠,分析了LT对小鼠胚胎稳定性的影响。首先采用PCR方法从产毒大肠杆菌菌株44815基因组中扩增LT的A、B亚基基因,将其插入到pET-20b（＋）原核表达载体pelB信号肽的下游,分别构建成LTA和LTB分泌型表达载体;将载体分别转化大肠杆菌BL21（DE3）pLysS,在IPTG的诱导下进行表达,利用Ni-NTA琼脂糖凝胶从细菌周质释放液中提取和纯化重组LTA和LTB蛋白;利用细胞毒性试验检测重组蛋白的生物活性。然后用制备的重组LT注射妊娠6d的小鼠,连续注射3d后,统计小鼠的胚胎存活率。同时用ELISA方法检测小鼠血清中与胚胎稳定性密切相关的Th1型细胞因子（IFN-γ、IL-2）和Th2型细胞因子（IL-4、IL-10）及IL-β的表达水平。结果表明,采用分泌性表达策略实现了重组LT在大肠杆菌中的高效分泌表达,LTA和LTB的表达量分别达68、62mg/L。表达的重组LT具有明显的细胞毒性作用;用LT处理妊娠小鼠,胚胎的存活率为32%,明显低于对照组;小鼠血清中Th1型细胞因子IFN-γ和IL-2的含量明显高于对照组（P〈0.01）,分别为对照组的2、3倍。同时细胞因子IL-1β为对照组的2倍（P〈0.01）,而稳定胚胎发育的Th2型细胞因子IL-4、IL-10没有明显变化（P〉0.05）。据此推测,LT可能与大肠杆菌性肠炎引起妊娠家畜流产有关,LT对胚胎稳定性的影响除与LT的直接毒性有关外,可能还与LT介导的免疫调节异常有关。     

Evaluation of procholeragenoid against experimental colibacillosis in piglets of vaccinated dams

A toxoid prepared from the toxin of Vibrio cholera was adjuvanted with aluminium hydroxide and used for immunisation of pregnant gilts. Litters of these and of non-vaccinates were experimentally challenged with Escherichia coli producing either heat labile and heat stable (LT and ST) enterotoxins or ST enterotoxin only. Both the challenge strains of E coli produced high rates of mortality (64 and 68 per cent) and morbidity (80 and 100 per cent) in litters of non-vaccinated dams. Statistically highly significant protection against the LT/ST enterotoxin producing strain of E coli was obtained accompanied by the absence of colonisation of the small intestine by the pathogen. No protection against the ST enterotoxin producing strain was found. It is suggested that this vaccine would not confer passive protection to piglets against K99 and 987-positive E coli which usually produce ST enterotoxin only.     

Prevalence of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene in isolates in weaned pigs with diarrhea and/or edema disease

A total of 476 Escherichia coli isolated from weaned pigs with diarrhea and/or edema disease were screened for the presence of the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) gene by polymerase chain reaction (PCR). E. coli strains that carried EAST1 genes were also tested by PCR for the presence of genes for five fimbriae (F4, F5, F6, F18 and F41), two heat-stable (STa and STb) and one heat-labile (LT) enterotoxin, and Shiga toxin 2e (Stx2e). One hundred and forty nine (31.3%) of the 476 E. coli isolates carried the gene for EAST1. Of these 149 isolates, 66 (44.3%) carried the east1 gene only and 83 (55.7%) carried genes for the fimbrial adhesins or enterotoxins. E. coli which carried east1 gene also possessed genes for STa or F4 frequently. EAST1 may represent an additional determinant in the pathogenesis of E. coli diarrhea in weaned pigs.     

大肠埃希菌不耐热肠毒素作为黏膜免疫佐剂的研究进展

免疫佐剂是一类通过刺激机体免疫系统而非特异性地增强机体对抗原免疫应答的活性物质,也是近年来研究的热点.免疫佐剂种类众多,利用细菌及其代谢产物作为黏膜免疫佐剂具有很好的发展前景,研究较多的是霍乱毒素(CT)和大肠埃希菌不耐热肠毒素(LT),从研究效果看,LT比CT更为理想.但是LT和CT一样本身具有毒性,这是其作为黏膜佐剂的主要缺点.目前,国内外研究工作者对其突变体做了大量研究,以期找到具有黏膜佐剂活性但低毒或无毒的大肠埃希菌不耐热肠毒素突变体.文章对大肠埃希菌不耐热肠毒素分子及其突变体的研究做一综述.     

Prevalence of genotypes for fimbriae and enterotoxins and of O serogroups in Escherichia coli isolated from diarrheic piglets in Korea.

Polymerase chain reaction for 4 fimbriae (F4, F5, F6, F41), 2 heat-stable enterotoxins (STa, STb), and 1 heat-labile enterotoxin (LT) were performed on 400 Escherichia coli isolates to determine their genotype prevalence among enterotoxigenic E. coli isolates from preweaned pigs with diarrhea in the Republic of Korea. A total of 200 of the 400 E. coli isolates were also selected for characterization of the O serogroup. Of these 200 isolates, serogroup could be determined in 139 (69.5%) but not in 61 isolates (30.5%). Isolates of serogroup O101 were the most common, followed in descending order by 08, 020, 0162, 0141, and 0149. Ninety-seven (24.3%) of the 400 E. coli isolates carried genes for at least 1 of the entertoxins or fimbrial adhesins. Of these 97 isolates, 27 carried genes for at least 1 of the fimbrial adhesins and entertoxins. Sixty-six percent of the isolates that carried fimbrial adhesin genes carried genes for at least 1 of the enterotoxins, and 71% of the isolates that carried enterotoxin genes carried genes for at least 1 of the fimbrial adhesins. Genes for the F6 fimbriae were detected in 6% of the E. coli isolates, and F4+, F41+, and F5+ genes were detected in 4.3%, 3.3%, and 2% of the isolates, respectively. Genes for STa, STb, and LT were detected in 10%, 8.5%, and 4.3% of the isolates, respectively. The 6 major genotypes observed in this study (in decreasing order) were F6+, STb+, F41+, STa+STb+, F6+STa+, and STa+.     

Detection with nonradioactively labeled probes of the toxin genes of different E. coli pathotypes from swine]

We tested hemolytic E. coli from 86 pigs with edema disease or colidiarrhea. They were tested serologically and with nonradioactive digoxigenin-dUTP labelled probes for the presence of enterotoxin or Shiga-like-toxin genes. By slide-agglutination we detected 38 cases with E. coli O149:K88, 28 with E. coli O139:82B and 20 with E. coli O141. E. coli of serogroup O149:K88 isolated from diarrheic pigs, reacted with the probes for LT and STb genes. Edema disease E. coli O139:82B reacted with the SLTII probe. E. coli O141, isolated from colidiarrhea or edema disease showed a diversity of toxin gene patterns. All the E. coli O141 from diarrheic pigs reacted with the probes for LT and STap in addition to SLTII. No strains isolated from pigs with edema disease possessed any of these enterotoxin genes. Gene probe technique confirmed the serological method as useful tool for diagnosing E. coli O149:K88 and O139:82B as ETEC or VTEC, respectively. On the other hand only the demonstration of toxin genes with probes could explain the pathological findings in the pigs shedding E. coli of serogroup O141.     

Genotypic prevalence of the adhesin involved in diffuse adherence in Escherichia coli isolates in pre-weaned pigs with diarrhoea in Korea

A total of 1002 Escherichia coli strains isolated from pre-weaned pigs with diarrhoea on 1114 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of five fimbriae (F4, F5, F6, F18 and F41), heat-stable (STa, STb) and heat-labile (LT) enterotoxin, enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1), and Shiga toxin 2 oedema disease (Stx2e) genes. Twenty-three (2.3%) of the 1002 E. coli isolates carried the gene for AIDA. Among 23 isolates shown to carry genes for AIDA, three carried the AIDA gene as the only shown virulence factor. Other isolates carried other virulence factor genes in addition to AIDA. Four isolates carried genes for at least one of the fimbrial adhesins and enterotoxins. Sixteen isolates carried genes for enterotoxins only. The AIDA may represent an additional virulence determinant in pre-weaned pigs with diarrhoea.