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聚合酶链反应扩地大肠杆菌志贺样毒素基因 总被引：5，自引：0，他引：5
两对寡核苷酸经物通过聚合酶链反应扩增大肠杆菌志贺样毒素Ⅰ（ＳＬＴⅠ）和志贺样毒素Ⅱ（ＳＬＴ Ⅱ）基因,在单一反应中,两对引物分别扩增出１３０ｂｐ（ＳＬＴⅠ）和３４６ｂｐ（ＳＬＴⅡ）的ＤＮＡ片段,扩增片段经地高辛标记寡核苷酸探针Ｓｏｕｔｈｅｒｎ印迹杂交分析,证实了ＳＬＴⅠ和ＳＬＴⅡ基因产物。 相似文献
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应用复合引物扩增大肠杆菌肠毒素基因的研究 总被引：1，自引：0，他引：1
以两对合成的不同寡核苷酸引物通过聚合酶链反应（ＰＣＲ）、扩增肠毒素大肠杆菌（ＥＴＥＣ）不耐热肠毒素（ＬＴ）和耐热肠毒素（ＳＴ）基因；两对引物从ＥＴＥＣＬＴ和ＳＴ基因中分别扩增出３１４ｂｐ，２３７ｂｐ的ＤＮＡ片段，均能与相应的ＬＴ和ＳＴ基因探针杂交。ＬＴ扩增产物（３１４ｂｐ）和ＳＴ扩增产物（２３７ｂｐ）分别和ＳｍａⅠ和ＨｉｎｃⅡ酶切后，产生１９０ｂｐ和１２４ｂｐ，１４７ｂｐ和９０ｂｐ的ＤＮＡ片段。同一扩增反应中应用ＬＴ和ＳＴ复合引物进行扩增，３种基因型ＬＴ、ＳＴ和ＬＴＳＴＥＴＥＣ从样品中鉴定出。１０９份动物腹泻粪样分别用ＰＣＲ，核酸杂交和ＥＬＩＳＡ进行测定，结果表明，ＰＣＲ是最为灵敏和快速的测定ＥＴＥＣ的方法。 相似文献
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利用PCR法鉴定产SLT—Ⅱe大肠杆菌 总被引：18，自引：4，他引：14
民贺氏菌毒素Ⅱ型变异性，是仔猪水肿病的主要致病历子。根据ＳＬＴ－Ⅱｅ基因序列，合成一对针对ＳＬＴ－Ⅱｅ操纵子基因保守区的寡核苷酸引物，建立了聚合酶链反应扩增方法。通过对产ＳＬＴ－Ⅱｅ毒素大肠杆菌菌株ＴＢ１，产ＳＬＴ－Ⅱ毒素大肠杆菌菌株９３３Ｗ和产ＳＬＴ－Ⅰ毒素大肠杆菌菌株Ｈ３０的试验，结果表明用所设计的引物建立的ＰＣＲ方法可特异性鉴定产ＳＬＴ－Ⅱｅ大肠杆菌。 相似文献
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利用PCR法鉴定产SLT-IIe大肠杆菌 总被引：3，自引：0，他引：3
类志贺氏菌毒素Ⅱ型变异体（ Ｓｈｉｇａｌｉｋｅ Ｔｏｘｉｎ Ⅱ Ｖａｒｉａｎｔ, Ｓ Ｌ ＴⅡｖ ｏｒ Ｓ Ｌ ＴⅡｅ）,是仔猪水肿病的主要致病因子。根据 Ｓ Ｌ ＴⅡｅ 基因序列,合成一对针对 Ｓ Ｌ ＴⅡｅ 操纵子基因保守区的寡核苷酸引物,建立了聚合酶链式反应（ Ｐ Ｃ Ｒ）扩增方法。通过对产 Ｓ Ｌ ＴⅡｅ 毒素大肠杆菌菌株 Ｔ Ｂ１、产 Ｓ Ｌ ＴⅡ毒素大肠杆菌菌株 ９３３ Ｗ 和产 Ｓ Ｌ ＴⅠ毒素大肠杆菌菌株 Ｈ３０ 的试验,结果表明用所设计的引物建立的 Ｐ Ｃ Ｒ方法可特异性鉴定产 Ｓ Ｌ ＴⅡｅ 大肠杆菌。用此法对本实验室从患水肿病仔猪肠道分离的 １５ 株大肠杆菌进行了鉴定,结果可从 １５ 个菌株的 １２ 株中扩增到 Ｓ Ｌ ＴⅡｅ 的特异性保守序列基因片段,而其它菌株未见此保守序列的基因片段。 相似文献
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应用PCR检测产vero毒素大肠杆菌 总被引：5，自引：0，他引：5
根据产ｖｅｒｏ毒素大肠杆菌（ＶＴＥＣ）产生的ｖｅｒｏ毒素１（ＶＴ１）和ｖｅｒｏ毒素２（ＶＴ２）的基因序列，合成了２对寡核苷酸引物，建立了聚合酶链式反应（ＰＣＲ）扩增方法。共对４株产ｖｅｒｏ毒素大肠杆菌和其他７个属的４１株肠道致病菌中的ＶＴ基因进行了检测，结果仅从用传统组织培养方法确定为ＶＴ阳性的产ｖｅｒｏ毒素大肠杆菌和痢疾１型志贺氏菌提取的ＤＮＡ中观察到了ＰＣＲ扩增产物，而从其他肠道致病菌提取的模板ＤＮＡ中未扩增出任何ＤＮＡ片段。用这２对引物可以清楚地区分产ＶＴ１、ＶＴ２或ＶＴ１／ＶＴ２的大肠杆菌。ＰＣＲ扩增方法的检测敏感性为３２０ｐｇ全细菌ＤＮＡ，用ｖｔ１引物可以检测出低达３２ｐｇ的全细菌ＤＮＡ。 相似文献
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采用蛋白酶Ｋ法从母牛肝中提取染色体ＤＮＡ,将其纯化后作为ＰＣＲ扩增模板。以引物设计软件从牛β－酪蛋白基因上游６００ｂｐ至第１个外显子设计引物,引物长度１９ｎｔ,跨度６３７ｂｐ。扩增产物电泳结果显示,在分子量Ｍａｒｋｅｒ６５７ｂｐ带附近,出现一明显亮带,这一结果与设计产物大小基本一致。用低温冷冻法纯化ＰＣＲ产物,煮沸裂解法提取载体ｐＢｌｕｅｓｃｒｉｐｔⅡＫｓ＋质粒,ＥｃｏＲＶ酶切质粒ＤＮＡ,Ｔ４ＤＮＡ连接酶连接ＰＣＲ扩增片段和质粒ＤＮＡ。将重组质粒ＤＮＡ转化到ＪＭ１０１大肠杆菌中,经筛选、酶切、测序鉴定,结果表明所克隆的片段为牛β－酪蛋白基因上游调控序列。 相似文献
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大肠杆菌不耐热性肠毒素LT_B基因的克隆及其核苷酸序列分析 总被引：12，自引：0，他引：12
用内切酶ＳａｃⅠ和ＨｉｎｄⅢ双酶切大肠杆菌质粒ｐＥＷＤ２９９，回收５０５ｂｐ的ＬＴＢ基因片段，再将载体ｐＵＣ１８用ＳａｃⅠ和ＨｉｎｄⅢ双酶切，最后将ｐＵＣ１８ＤＮＡ与５０５ｂｐ的ＬＴＢＤＮＡ进行连接，转化至受体菌ＤＨ５α中，经ＳａｃⅠ／ＨｉｎｄⅢ、ＥｃｏＲⅠ／ＨｉｎｄⅢ酶切反应鉴定重组子，得到了理想重组子质粒ｐＸＬＴ１。再将ｐＸＬＴ１进行ＥｃｏＲⅠ酶切、大肠杆菌聚合酶ⅠＫｌｅｎｏｗ大片段补平、ＨｉｎｄⅢ酶切处理，然后与用ＨｉｃⅡ和ＨｉｎｄⅢ酶切的ｐＵＣ１８连接，转化至受体菌ＤＨ５α中，经ＸｂａⅠ／ＨｉｎｄⅢ酶切反应鉴定重组子，得到了理想重组子质粒ｐＸＬＴ１－１。将质粒ｐＸＬＴ１－１进行核苷酸序列分析，确定了插入的ＬＴＢ基因与载体的连接向位及其全部核苷酸序列 相似文献
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牛结核分枝杆菌特异性核酸探针的克隆与分析 总被引：7，自引：0，他引：7
牛结核分枝杆菌是引占结核病的致病源，其染色体ＤＮＡ中含有插入序列ＩＳ１０８１，属于结核杆菌类特异性基因片段，在牛结核杆菌染色体中含有６个拷贝。目前国外普遍有杉该片段作为探针对牛结核病进行鉴定和诊断。本试验采用牛结核分枝杆菌染色体Ｄ避的特民插入片段即ＩＳ１０８１为模闰于ＩＳ１０８１中４３８－４５９和６６４－６８５碱基 的两个片段为引物。利用ＰＣＲ技术扩增出一段２４８ｂｐ的特异性片段，将其克隆进ｐＵ 相似文献
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猪水肿病（ＥＤ）是断奶仔猪常见的一种传染病，与类志贺氏毒素Ⅱ变种（ＳＬＴ－Ⅱｖ）密切相关。本试验用多粘菌素Ｂ从基因工程菌大肠杆菌ＴＢ１中提取ＳＬＴ－Ⅱｖ，作中和试验检测来自江苏等地２６７份屠宰猪血清，发现９份血清有ＳＬＴ－Ⅱｖ的抗体，滴度由１∶４～１∶２５６不等，显示致猪水肿病毒素在猪群的实际存在 相似文献
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Escherichia coli K12 strains TB1(pCG5), with the genes for Shiga-like toxin IIv from an edema disease isolate of E. coli and TB1(pCG5-1), with the toxin genes inactivated by transposon mutagenesis, were used to test the hypothesis that Shiga-like toxin IIv was the same as edema disease principle. Ammonium sulfate precipitated culture supernatants from the pair of E. coli K12 strains and from a wild edema disease isolate of E. coli (E145) were tested for their ability to induce signs and lesions of edema disease in intravenously inoculated weaned pigs. Similar preparations from E. coli which produce Shiga-like toxins I and II were also tested. Preparations from E. coli TB1 (pCG5) and E145 contained high levels of Shiga-like toxin IIv and induced signs and lesions similar to those seen in edema disease, whereas preparations from E. coli TB1 (pCG5-1) failed to induce signs or lesions of edema disease. All Shiga-like toxin preparations produced delayed neurological signs, fibrinoid necrosis of arterioles and hemorrhages in the cerebellum of pigs. High doses of Shiga-like toxin IIv were associated with superficial necrosis of the colonic epithelium and vasculitis. Shiga-like toxins I and II resulted in kidney lesions but no enteric pathology. Shiga-like toxin II preparations had the lowest median lethal dose for pigs, Shiga-like toxin IIv was intermediate and Shiga-like toxin I was the least toxic. 相似文献
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Immunization of pigs with a purified Shiga-like toxin II variant toxoid. 总被引：14，自引：0，他引：14
Passive transfer of neutralizing antibodies and active immunization with a toxoid of purified Shiga-like toxin II variant (SLT-IIv, edema disease toxin) were used to protect pigs against challenge with SLT-IIv. Six 6-week-old pigs were passively immunized by intraperitoneal administration of an immunoglobulin preparation from porcine antiserum against purified SLT-IIv. Six 6-week-old pigs and twelve 2-week-old pigs were actively immunized by two intramuscular injections of 25 micrograms of SLT-IIv toxoid given 2 weeks apart. The 24 immunized pigs and an equal number of age-matched unimmunized control pigs were all challenged by intravenous injection of purified SLT-IIv (6 ng/kg body weight). The six passively immunized pigs acquired neutralizing SLT-IIv antibody titers of 1280 or 2560 and the 18 actively immunized pigs mounted a serum immune response with toxin neutralizing titers ranging from 128 to 10,240. All 24 immunized pigs survived the challenge but nine pigs with the lowest titers showed mild clinical signs of edema disease. All 24 control pigs died within 30 hours of challenge. 相似文献
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本研究旨在建立猪水肿毒素抗体的快速检测方法.以纯化的猪水肿毒素A亚基基因片段的原核表达产物作为抗原免疫BALB/c小鼠制备单抗,并利用表达蛋白和猪水肿毒素A亚基单抗酶结合物建立了竞争ELISA方法检测猪水肿毒索抗体.经过研究确定抗原包被浓度为0.32 btg·mL-1,待检血清最佳稀释度为1:2,酶标单抗工作浓度为1:3 200,血清抑制率大于40 %为阳性.应用单抗竞争ELISA和细胞毒性中和试验同时对60份血清进行猪水肿毒素抗体检测,竞争ELISA的检出率为33.8%,细胞毒性中和试验检出率为30.9%,两者符合率达88.2 %.试验结果表明,该ELISA方法特异性强,敏感性高,稳定性和重复性好,操作简便.本方法的建立在实验室诊断的标准化、猪水肿病疫苗免疫效果的评价及流行病学调查方面具有较高的应用价值. 相似文献
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Twenty-one weaned Yorkshire-Landrace pigs were injected intravenously with graded doses of purified Shiga-like toxin-II variant (edema disease toxin). In a preliminary study, three pigs (Nos. 1, 2, 3) were injected with 48, 24, and 12 ng, respectively, of SLT-IIv/kg of body weight. Subsequently, three groups (Nos. 4, 5, 6) of six pigs each were injected with 6, 3, and 1.5 ng, respectively, of SLT-IIv/kg of body weight. Severe clinical signs and histologic lesions characteristic of edema disease developed in pigs Nos. 1, 2, and 3, and all six pigs in group No. 4. Eight of these pigs were euthanatized in extremis (mean time to death was 34 hours) and one died of the disease (52 hours). Moderate signs and lesions of edema disease were observed in all pigs in group No. 5, and three pigs were euthanatized (mean time to euthanasia was 42 hours). Mild signs and lesions were observed in three pigs in group No. 6. The most common gross pathologic changes were edema of the eyelids, submucosa of the stomach, and mesentery of the spiral colon and hemorrhage of the colon and cerebellum. Microscopic lesions were associated with vascular injury and included vessel necrosis, perivascular edema and hemorrhage, and superficial colonic and cecal erosions. The vascular lesions were observed in the cerebellar folia, submucosa and mucosa of the stomach, cecum, colon, and sporadically in the retina. None of the clinical signs associated with endotoxin were observed. 相似文献
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Characteristics of the Shiga-like toxin produced by Escherichia coli associated with porcine edema disease 总被引：4，自引：0，他引：4
Shiga-like toxin (SLT-IIv) from Escherichia coli strains associated with edema disease of pigs was characterized and compared with SLT-I, SLT-II, and the SLT of E. coli strain HI8 (SLT-HI8). SLT-IIv from an E. coli K12 in which the genes for SLT-IIv had been cloned was indistinguishable from SLT-IIv of wild strains of E. coli from edema disease. There was cross-neutralization among all SLTs except SLT-I. The different SLTs could be distinguished by heat lability, with the descending order of heat lability being SLT-IIv, SLT-II, SLT-I, and SLT-HI8. SLT-IIv and SLT-HI8 had lower cytotoxic titers on HeLa cells compared with Vero cells and were more active on MDBK cells than were the other SLTs. All SLTs were enterotoxic in rabbit but not in pig intestine and SLT-IIv was less enterotoxic than SLT-I. SLT-IIv had a lower LD50 in mice than did the other SLTs. 相似文献
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Shiga-like toxin production and attaching effacing activity of Escherichia coli associated with calf diarrhea 总被引：11，自引：0，他引：11
J G Mainil C J Duchesnes S C Whipp L R Marques A D O'Brien T A Casey H W Moon 《American journal of veterinary research》1987,48(5):743-748
Four hundred twenty-nine isolates of Escherichia coli from calves were tested for the production of HeLa cell cytotoxin(s). Isolates that produced enough cytotoxin to be detected in culture supernatants of iron-depleted broth were considered to produce increased amounts of cytotoxins. Isolates also were tested for homology with a DNA probe for a gene that encodes localized adherence of human enteropathogenic E coli. Four isolates produced increased amounts of cytotoxin that was neutralized by Shiga antitoxin (toxin designated as Shiga-like toxin-I [SLT-I]). A 5th isolate produced increased amounts of cytotoxin (SLT+) that was not neutralized by the Shiga antitoxin, but was neutralized by antitoxin against a variant of SLT (toxin designated as SLT-II). None of the isolates hybridized with the probe for the localized adherence gene. Three of the SLT+ isolates belonged to human enteropathogenic E coli serogroups O26 and O111. All 5 of the SLT+ isolates were from calves with diarrhea, but none of the 5 SLT+ isolates contained genes for classic heat-labile or heat-stable enterotoxins, for K99 fimbriae, or for invasiveness; neither did any of them adhere to HeLa cells in culture. Three of the 5 SLT+ isolates had attaching and effacing activities when inoculated into ligated intestinal loops of rabbits. One of the isolates with attaching and effacing activity in rabbits was originally isolated from a calf with lesions characteristic of those produced by attaching effacing E coli (AEEC). Calves inoculated with this SLT+ AEEC isolate developed focal colonic lesions characteristic of those produced by AEEC, but did not develop diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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无抗性表达致PWD和ED大肠杆菌保护性抗原基因的重组菌构建 总被引：2，自引：0，他引：2
利用平衡致死系统成功地将构建表达致PWD和ED大肠杆菌保护性抗原基因的重组菌BL21(pFSFaeG)中所含氨苄抗性基因敲除,并转化沙门氏菌宿主菌X4550。经过质粒酶切分析和表达产物的SDSPAGE证实,氨苄抗性基因已经成功敲除,并且目的蛋白仍与谷胱甘肽转移酶相融合,得到有效表达,重组融合蛋白的分子质量约为80ku。经体外连续传代试验,重组菌X4550(pFSFaeG)均能在无抗生素压力下大量扩增并稳定传代,质粒稳定性和蛋白表达稳定性良好。无抗性重组菌的成功构建解决了基因工程疫苗抗性基因潜在危险问题,并有望通过沙门氏菌载体的递送作用,为研制口服疫苗奠定了基础。 相似文献