鸡致病性大肠杆菌肠毒素(LT)的鉴定及其基因的克隆

鸡致病性大肠杆菌分离株O78经家兔肠袢结扎试验(RILT)证实,该菌株产生热敏性肠毒素(LT)。用PCR技术从该菌株中扩增出1.2kb的LT基因,然后将纯化的PCR产物克隆到pGEM-T载体中,转化至受体菌JM109中。用Amp/IPTG/X-gal琼脂平板蓝白菌落筛选得到阳性重组菌株,提取质粒用SphI和SalI双酶切鉴定,结果证实,构建的克隆质粒pXCLT1含有LT基因。     

新生仔猪产肠毒素性大肠埃希氏菌致病因子的分子生物学研究进展

阐述了新生仔猪产肠毒素性大肠埃希氏菌（ETEC）菌毛的结构、分子生物学特性、基因检测技术、菌毛抗原受体的生化特性以及菌毛载体系统的优点；另外，从分子结构与功能、作用机理、基因检测三方面叙述了新生仔猪ETEC不耐热肠毒素和耐热肠毒素的研究进展。     

大肠杆菌不耐热肠毒素(LT)突变体LTR72的表达与鉴定

将重组质粒PLTR72和PLT分别转化大肠杆菌JM101，DH5α和C600，并接种于LB和CAYE-2中培养，用GM1-ELISA法检测菌体裂解液中2者的表达情况，将表达产物纯化后用SDS-PAGE等方法鉴定。结果表明，3种大肠杆菌在2种培养基中都可表达出LT突变体和LT，并可将产物分泌到大肠杆菌胞周质中，成为具有GM1结合活性的突变体蛋白，鉴定纯化后的突变体的组成形式与野生型LT相同。均是1A：5B。     

大肠埃希菌不耐热肠毒素研究进展

大肠埃希菌不耐热肠毒素(LT)是产毒性大肠埃希菌产生的一种能导致人畜腹泻的毒素,由六聚体蛋白形成AB5型结构.A亚基具有ADP核糖基转移酶活性,而B亚基为五聚体,主要与表达于真核细胞表面的神经节苷脂GM1结合.LT具有很强的免疫原性和黏膜免疫佐剂活性.为了将其毒性与佐剂活性分开,研究者构建了一系列无毒或减毒的突变体.证明突变体具有佐剂活性,并发现没有毒性的AB复合物以及酶活性在佐剂活性中发挥的作用是分开的.野生型LT、LT突变体以及B亚基与外源抗原共同免疫机体后,对机体免疫系统具有调节作用.     

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。     

表达大肠杆菌K88ac-ST1-LTB融合蛋白工程菌株的免疫原性研究

已构建的能表达大肠杆菌K88ac-ST1-LTB融合蛋白的工程菌株BL21（DE3）（pXKST3LT5)及其表达产物经动物试验证实没有毒性反应。用从IPTG诱导的工程菌中提取的包涵体或经甲醛灭活的工程菌制成抗原，免疫小鼠，结果免疫小鼠至少能抵抗2MLD的大肠杆菌强毒株C83902（K88ac,ST^ ,L^ )的攻击，用提取的包涵体免疫家兔后，采集的血清能够中和天然ST1的毒性，这表明构建的工程菌株BL21（DE3）（pXKST3LT5)可以作为预防幼畜大肠杆菌性腹泻基因工程菌苗的候选株。     

电针对大肠杆菌热敏肠毒素致肠水、钠分泌的拮抗作用

新生仔猪下痢是严重危害养猪业的常见病、多发病,而肠致病性大肠杆菌产生的肠毒素特别是热敏肠毒素(Heat-Labileenterotoxin,LT)则是导致幼畜腹泻的直接致病因子。在兽医临床上,由于广泛使用抗菌药物治疗仔猪下痢,大肠杆菌对这些药物普遍地产生了耐药性,致使临床疗效越来越差。针灸疗法是我国传统医学的宝贵遗产,已有不少文献报道了针灸对哺乳仔猪下痢良好的防治效果,但有关针灸防治仔猪下痢机理的研究则相对滞后。为此,本试验应用兔回肠结扎实验动物模型观察了电针刺激对大肠杆菌热敏肠毒素致肠水、钠分泌的拮抗作用,以便为针灸防治…     

Relationship between production of enterotoxin and various drug resistance patterns in strains of Escherichia coli isolated from piglets suffering from colienterotoxaemia.

The relationship between production of enterotoxin and drug resistance patterns in 92 E. coli strains isolated from the jejunal content from piglets suffering from colienterotoxaemia was investigated. The drugs included were Oxytetracycline, streptomycin, sulphaisodimidin, neomycin, ampicillin and chloramphenicol. The frequency with which transmission of drug resistance occurred as well as the O-groups of these strains were also recorded.Fifty-one E. coli strains produced enterotoxin. Of these, 42 (approx. 82 %) produced the LT. The same number of strains also produced the ST. Strains simultaneously producing both enterotoxins thus constituted approx. 64 % of the enterotoxin-producing strains. The E. coli strains were divided in 7 groups, each representing different drug resistance patterns. Statistical analysis showed that the various categories of enterotoxin production mentioned above were more frequently found in strains simultaneously resistant to Oxytetracycline, streptomycin and sulphaisodimidin than in strains belonging to the other drug resistance groups in general.Transmission of drug resistance was demonstrated in all resistance pattern groups. Approx. 50–70 % of the enterotoxin-producing strains showed drug transmission. The O-groups 138 and 149 constituted 8 and 54 out of the 62 typable strains, respectively.     

生物素标记LT基因探针检测产肠毒素性大肠杆菌

用碱变性法提取重组质粒pEWD299,经Hind Ⅲ酶切后,进行琼脂糖凝胶电泳,用电洗脱法回收LT基因850bp的核酸片段,再用二步法缺口翻译反应制备生物素标记的LT基因探针.通过菌落杂交试验表明,在严格去蛋白和RNA的条件下,该探针与试验中所有产LT和ST,或仅产LT的ETEC发生杂交反应;而与所有仅产ST的ETEC、侵袭性大肠杆菌(EIEC)、普通大肠杆菌、沙门氏菌、志贺氏菌、小肠结肠耶尔森氏菌、霍乱弧菌均无杂交反应.其检测提纯的LT—DNA的敏感性水平为25pg,检测食品模拟标本中产LT大肠杆菌的敏感性达到130个细菌/g的水平.     

产肠毒素大肠杆菌菌毛的DNA疫苗研究进展

肠毒素大肠杆菌是导致婴幼儿及旅游者急性腹泻,仔猪腹泻和水肿的主要病原菌之一。菌毛定居因子是该病原菌主要的致病因素。DNA疫苗既能激发机体的细胞免疫,也能诱导特异性的体液免疫。目前用于预防ETEC腹泻的DNA疫苗的研究已取得一定进展,作者从重组质粒的构建、免疫应答、疫苗的接种系统以及基因佐剂4个方面简单的概述了产肠毒素大肠杆菌菌毛的DNA疫苗的研究现状。     

基于猪小肠上皮细胞炎症模型分析产肠毒素大肠杆菌诱导的转录组表达变化

【目的】分析产肠毒素大肠杆菌（enterotoxigenic Escherichia coli，ETEC）诱导猪小肠上皮细胞（IPEC-J2）炎症反应的主要宿主调控基因及相关分子通路，为进一步揭示ETEC感染引发炎症反应的作用机制提供理论依据。【方法】用感染复数（multiplicity of infection，MOI）为100的ETEC菌液感染IPEC-J2细胞，18 h后收集细胞上清，通过ELISA方法检测炎性因子白细胞介素1β（interleukin 1 β，IL-1β）的分泌水平。由Illumina PE150测序平台进行高通量转录组测序，通过edgeR v 3.12.1软件对测序结果进行差异表达分析，利用GOseq和KOBAS 2.0软件对得到的差异表达mRNA进行GO功能和KEGG通路富集分析，随机挑选5条差异表达mRNA，利用实时荧光定量PCR对其验证。【结果】试验成功建立了ETEC感染诱导的IPEC-J2细胞炎症模型。与对照组相比，炎症模型组共发现529条差异表达mRNA，其中236条表达上调，293条表达下调，包括HSP90AA1、CGNL1、CADPS2、EHBP1等免疫相关基因。GO功能分析表明，ETEC感染后差异表达mRNA显著富集于细胞组分和生物过程，包括细胞内成分（intracellular part）、细胞质（cytoplasm）、核成分（nuclear part）、胞内膜结合细胞器（intracellular membrance-bounded organelle）、膜结合细胞器（membrance-bounded orgabelle）及细胞进程（cellular process）等。KEGG通路分析表明，炎症模型中差异表达基因大多数被注释到疾病相关通路及Toll样受体信号通路、mTOR信号通路、细胞周期、黏附连接等免疫相关信号通路。利用实时荧光定量PCR验证随机挑选的5条差异表达mRNA，结果与测序报告中差异表达mRNA变化趋势一致，证明测序结果可信。【结论】HSP90AA1、CGNL1、CADPS2和EHBP1等基因可能在ETEC黏附IPEC-J2细胞并诱导炎症反应中发挥关键作用，并通过Toll样受体、mTOR等信号通路进行免疫调控。研究结果为进一步揭示ETEC感染诱导炎症反应的分子机制及相关调控网络提供参考依据。     

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。     

不同地区猪源大肠埃希菌磺胺类药物耐药性研究

对不同地区猪源大肠埃希菌磺胺药物耐药性进行系统性调查研究.对从山东、内蒙古、山西分离到的161株猪源大肠埃希菌测定磺胺药物对其最低抑菌浓度(MIC值),结果表明大肠埃希菌对磺胺药物高度耐药,对磺胺异恶唑(SF)和新诺明(SMZ)的耐药率分别为91.30%和90.06%.同时根据GenBank上已有的二氢叶酸合成酶基因S...     

Effects of dietary supplementation of bacteriophages against enterotoxigenic Escherichia coli (ETEC) K88 on clinical symptoms of post‐weaning pigs challenged with the ETEC pathogen

The present study was performed to investigate the effects of dietary supplementation of bacteriophages (phages) against enterotoxigenic Escherichia coli (ETEC) K88 as a therapy against the ETEC infection in post‐weaning pigs. Two groups of post‐weaning pigs aged 35 days, eight animals per group, were challenged with 3.0 × 10¹⁰ colony forming units of ETEC K88, a third group given the vehicle. The unchallenged group and one challenged group were fed a basal nursery diet for 14 days while the remaining challenged group was fed the basal diet supplemented with 1.0 × 10⁷ plaque forming units of the phage per kg. Average daily gain (ADG), goblet cell density and villous height:crypt depth (VH:CD) ratio in the intestine were less in the challenged group than in the unchallenged group within the animals fed the basal diet (p < 0.05); the reverse was true for rectal temperature, faecal consistency score (FCS), E. coli adhesion score (EAS) in the intestine, serum interleukin‐8 (IL‐8) and tumour necrosis factor‐α (TNF‐α) concentrations and digesta pH in the stomach, caecum and colon. The ETEC infection symptom within the challenged animals was alleviated by the dietary phage supplementation (p < 0.05) in ADG, FCS, EAS in the jejunum, serum TNF‐α concentration, digesta pH in the colon, goblet cell density in the ileum and colon and VH:CD ratio in the ileum. Moreover, the infection symptom tended to be alleviated (p < 0.10) by the phage supplementation in rectal temperature, EAS in the ileum and caecum, and VH:CD ratio in the duodenum and jejunum. However, EAS in the colon, digesta pH in the stomach and caecum, and goblet cell density in the jejunum did not change due to the dietary phage. Overall, results indicate that the phage therapy is effective for alleviation of acute ETEC K88 infection in post‐weaning pigs.     

感染产肠毒素大肠杆菌的猪小肠上皮细胞microRNA表达谱分析

试验旨在探索产肠毒素大肠杆菌（enterotoxigenic Escherichia coli，ETEC）感染猪小肠上皮细胞（IPEC-J2）诱导的microRNA （miRNA）表达谱变化，为解析宿主miRNA在ETEC感染过程中的调控作用提供理论基础。利用Illumina 6000 Novoseq SE50测序平台分别对ETEC感染前后的IPEC-J2进行高通量测序，用Bowtie与参考基因组比对，用DESeq R Package进行miRNA差异性分析。通过miRanda和RNAhybid共同预测差异表达miRNA的靶基因，对差异表达miRNA靶基因进行GO功能和KEGG通路分析。随机选取5个miRNAs，对测序结果进行实时荧光定量PCR验证。结果显示，IPEC-J2在感染前后的sRNA文库经过滤分别得到12 889 260和11 203 056条clean reads。感染前后文库中，miRNA所占比例最高，分别为73.16%和54.10%；分别有97.98%和69.83%长度为18~40 nt的sRNA可比对到参考基因组，表明测序质控良好。长度在22~24 nt的序列大部分首位碱基偏向U，2~8位点出现频率最高的碱基分别为AGCUUAU。共发现311个已知miRNAs，128个新miRNAs。在2个文库中，长度为23 nt的miRNA序列占比最高，分别为41.42%和23.56%。感染后共筛选到140个差异表达miRNAs，其中74个表达上调，66个表达下调。GO分析表明，miRNA靶基因显著富集于代谢过程、正向调节代谢过程、细胞成分或生物合成、免疫系统、细胞内部分和细胞器等功能。KEGG分析表明，差异表达miRNA靶基因显著富集于赖氨酸降解、生产IgA的肠道免疫网络、NF-κB信号通路和T细胞受体信号通路等。实时荧光定量PCR验证结果表明，随机选取的5个miRNAs表达趋势与测序结果一致，表明测序准确可靠。综上所述，IPEC-J2的miRNAs参与了ETEC感染过程，为进一步揭示调控ETEC感染的关键miRNA及其作用机制提供科学依据。     

Effects of Recombinant E. coli Expressing Heat-stable Enterotoxin on Intestinal Morphology and Antioxidant Capacity in Seven Days old Piglets

This experiment was conducted to investigate the effects of recombinant E.coli expressing heat-stable enterotoxin(STa) on intestinal absorption and barrier function, intestinal morphology and antioxidant capacity of 7 days old piglets. Twenty-four 7 days old piglets were allotted to four treatments:control group (artificial milk), STa group (artificial milk +2×10⁹ CFU E.coli LMG194-pBAD-STa), LMG194 group (artificial milk +2×10⁹ CFU E.coli LMG194), and K88 group (artificial milk +2×10⁹ CFU E.coli K88).The pigs were treated with E.coli on the 5th day and slaughtered on the 7th day. The results showed that, compared with the control group, villus height in jejunum, ileum and duodenum, crypt depth and villus height/crypt depth in duodenum, and small intestine villi surface area were significantly decreased in STa group (P<0.05). The activities of catalase (CAT) in ileum and colon, total nitric oxide synthase (TNOS) in serum, ileum, jejunum and colon, inducible nitric oxide synthase (iNOS) in colon were significantly decreased in STa group (P<0.05). STa group also had a higher levels of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) in serum (P<0.05). These results suggested that recombinant E.coli expressing STa could lead to intestinal injury and oxidative stress of 7 days old piglets.     

表达耐热肠毒素的重组大肠杆菌对7日龄仔猪肠道形态结构及抗氧化功能的影响

试验旨在研究表达耐热肠毒素（STa）的重组大肠杆菌对7日龄仔猪肠道形态结构及抗氧化功能的影响。选取24头7日龄仔猪,随机分在4个日粮处理组,分别为对照组（人工乳）,STa组（人工乳+2×10⁹ CFU重组菌LMG194-pBAD-STa）,LMG194组（人工乳+2×10⁹ CFU大肠杆菌LMG194）,K88组（人工乳+2×10⁹ CFU大肠杆菌K88）。试验第5天进行攻毒,第7天屠宰取样,测量小肠黏膜的绒毛高度和隐窝深度,检测空肠、回肠、结肠及血清中的过氧化氢酶（CAT）、总一氧化氮合成酶（TNOS）与诱导型一氧化氮合酶（iNOS）的活力及血清中丙二醛（MDA）与过氧化氢（H₂O₂）的含量。试验结果表明,与对照组相比,STa组仔猪各肠段绒毛高度均显著降低（P<0.05）,十二指肠隐窝深度及绒毛高度与隐窝深度的比值显著降低（P<0.05）,十二指肠、空肠、回肠绒毛表面积显著降低（P<0.05）;同时,STa组回肠、结肠CAT活力显著降低,血清、空肠、回肠和结肠中的TNOS活力显著降低（P<0.05）,STa组结肠iNOS活力显著降低（P<0.05）,STa组血清中的MDA与H₂O₂含量显著提高（P<0.05）。结果显示,表达STa的重组大肠杆菌可导致7日龄仔猪肠道结构损伤和抗氧化能力下降。     

Effect of added dietary threonine on growth performance,health, immunity and gastrointestinal function of weaning pigs with differing genetic susceptibility to Escherichia coli infection and challenged with E. coli K88ac

Threonine (Thr) is important for mucin and immunoglobulin production. We studied the effect of added dietary Thr on growth performance, health, immunity and gastrointestinal function of weaning pigs with differing genetic susceptibility to E. coli K88ac (ETEC) infection and challenged with ETEC. Forty‐eight 24‐day‐old weaned pigs were divided into two groups by their ETEC susceptibility using mucin 4 (MUC4) gene as a marker (2 MUC4^?/?, not‐susceptible, and 2 MUC4^+/+, susceptible, pigs per litter). Within genotype, pigs were fed two different diets: 8.5 (LThr) or 9.0 (HThr) g Thr/kg. Pigs were orally challenged on day 7 after weaning and slaughtered on day 12 or 13 after weaning. Before ETEC challenge, HThr pigs ate more (p < 0.05). The diet did not affect post‐challenge growth, but HThr tended to increase post‐challenge feed efficiency (p = 0.087) and overall growth (p = 0.087) and feed efficiency (p = 0.055). Before challenge, HThr pigs excreted less E. coli (p < 0.05), while after challenge, diet did not affect the number of days with diarrhoea and ETEC excretion. MUC4^+/+ pigs responded to the challenge with more diarrhoea, ETEC excretion and anti‐K88 IgA in blood and jejunal secretion (p < 0.001). HThr pigs had a higher increase of anti‐K88 IgA values in jejunal secretion (p = 0.089) and in blood (p = 0.089, in MUC4^+/+ pigs only). Thr did not affect total IgA and IgM values, morphometry of jejunum, goblet cells count in colon, total mucin from jejunum and colon, but varied jejunal goblet cells counts (p < 0.05). In the first two post‐weaning weeks, 8.5 g Thr/kg diet may be not sufficient to optimize initial feed intake, overall feed efficiency and intestinal IgA secretion and to control the gut microbiota in the first post‐weaning week, irrespective of the pig genetic susceptibility to ETEC infection.     

载铜蒙脱石对大肠杆菌和仔猪腹泻的影响

对通过阳离子交换反应合成的载铜蒙脱石 (MMT- Cu) ,应用 XRD衍射分析显示 ,载铜反应前后 ,蒙脱石的(0 0 1)面网间距从原先的 1.5 4 4 nm增加到了 1.5 88nm ,表明铜是以水合阳离子或复合阳离子形式进入蒙脱石晶格层间位置。体外试验表明 ,MMT- Cu对大肠杆菌 K88具有较强的抗菌能力。MMT- Cu对仔猪的试验表明 :与对照组相比 ,日粮中添加 0 .2 % MMT- Cu使仔猪日增重提高了 13.89% (P<0 .0 1) ,料重比下降了 8.38% (P<0 .0 5 ) ,平均腹泻率降低了 71.80 % (P<0 .0 1) ,结肠内容物中大肠杆菌数显著下降 (P<0 .0 1) ,空肠绒毛高度及绒毛高度与腺窝深度的比率分别提高了 19.2 1% (P<0 .0 5 )和 37.10 % (P<0 .0 1)。     

1株感染多种致病性大肠杆菌噬菌体的生物学特性研究

旨在测定此前从断奶前仔猪粪样中分离得到的1株大肠杆菌噬菌体C6在致病性大肠杆菌上的生物学特性,并比较该噬菌体在不同致病菌上的感染特性.利用电镜形态观察和基因组测序确定其分类,用点滴法和双层平板法测定其在致病性大肠杆菌上的宿主谱,通过噬菌斑形态、最佳感染复数(MOI)、成斑率(EOP)、吸附率、一步生长曲线以及抑菌曲线等...