为探索大肠杆菌Nissle 1917株(EcN)鞭毛展示的应用,本研究以重组质粒p UC18-fli C为模板,反向PCR产物经DpnⅠ、Exnase^(TM)Ⅱ重组环化,将6His标签基因分别插入无质粒大肠杆菌Nissle 1917(EcNcured of its two cryptic plasmids p MUT1和...为探索大肠杆菌Nissle 1917株(EcN)鞭毛展示的应用,本研究以重组质粒p UC18-fli C为模板,反向PCR产物经DpnⅠ、Exnase^(TM)Ⅱ重组环化,将6His标签基因分别插入无质粒大肠杆菌Nissle 1917(EcNcured of its two cryptic plasmids p MUT1和p MUT2;EcNc)fli C高变区774 bp^775 bp和792 bp^811 bp之间,构建嵌合鞭毛基因片段fliC01和fliC02。分别将其克隆于自杀性载体pRE112中,构建重组质粒pRE112-fliC01和pRE112-fliC02,并分别转化至EcNc中。通过一次重组和二次重组将嵌合鞭毛基因整合于EcNc基因组中,构建无抗性筛选的重组菌株EcNc fliC01和EcNc fliC02。对重组菌株进行生长状况、鞭毛形成以及体外细胞黏附试验和小鼠体内定植试验检测,结果表明EcNc fliC01和EcNc fliC02的生长未受影响,表达的嵌合鞭毛能够被H1多抗或His-Tag单克隆抗体识别并组装形成鞭毛丝;重组菌对IPEC-J2细胞的黏附能力及小鼠肠道定植能力与亲本株EcNc相比无显著性差异。本实验结果为进一步研究EcNc鞭毛展示技术应用于活疫苗研制奠定了基础。展开更多
质粒消除旨在获得无质粒菌株,是对益生菌进行遗传改造所需的一项重要技术。本试验建立在质粒不相容性的基础上,引入自杀性载体pRE112作为辅助工具,使用分子生物学方法构建重组自杀质粒pMUT1-pRE112和pMUT2-pRE112,将其分别导入携带2个...质粒消除旨在获得无质粒菌株,是对益生菌进行遗传改造所需的一项重要技术。本试验建立在质粒不相容性的基础上,引入自杀性载体pRE112作为辅助工具,使用分子生物学方法构建重组自杀质粒pMUT1-pRE112和pMUT2-pRE112,将其分别导入携带2个大隐秘质粒pMUT1和pMUT2的益生菌大肠杆菌Nissle 1917(Escherichia coli Nissle 1917,EcN),利用重组自杀质粒去除EcN内原有隐秘质粒并在含有10%蔗糖的LB培养基上实施自杀质粒自身消除。结果试验成功获得EcN无质粒克隆菌株(Escherichia coli Nissle 1917cured of its two cryptic plasmids pMUT1and pMUT2,EcNc),为优质益生菌EcN更好的潜在应用研究奠定基础。展开更多
初步研究大肠杆菌Nissle 1917(E.coli Nissle 1917,EcN)鞭毛蛋白(Flagellin,FliC)高变区的功能,为探讨其作为表面展示系统的可行性打下基础。以EcN无质粒克隆(EcN was cured of its 2 cryptic plasmids pMUT1 and pMUT2 resulting in Ec...初步研究大肠杆菌Nissle 1917(E.coli Nissle 1917,EcN)鞭毛蛋白(Flagellin,FliC)高变区的功能,为探讨其作为表面展示系统的可行性打下基础。以EcN无质粒克隆(EcN was cured of its 2 cryptic plasmids pMUT1 and pMUT2 resulting in EcNc)为实验菌株,分别构建2个鞭毛蛋白基因(fl iC)高变区859-888位、829-858位的缺失突变株和回补菌株。分析突变区域对EcNc的生长性能、生化特性、运动性及抗原性等方面的影响。结果显示:EcNc、两个缺失株和回补株的生长特性无明显差异,且三者生化试验结果一致。缺失突变株在半固体培养基上不能运动,而回补株均恢复了原运动性;缺失突变株均不与H1单因子血清发生凝集,而互补菌株均能产生与EcNc一致的凝集反应。表明高变区的这两个区域尽管不影响菌株的生长性能和生化特性,但与EcN的运动性及抗原性密切相关。这为进一步探索EcN鞭毛展示技术的研究提供了一定的数据。展开更多
Ulcerative colitis (UC) is a chronic inflammatory disease, whose etiology is still unclear. Its pathogenesis involves an interaction between genetic factors, immune response and the “forgotten organ&#x...Ulcerative colitis (UC) is a chronic inflammatory disease, whose etiology is still unclear. Its pathogenesis involves an interaction between genetic factors, immune response and the “forgotten organ”, Gut Microbiota. Several studies have been conducted to assess the role of antibiotics and probiotics as additional or alternative therapies for Ulcerative Colitis. Escherichia coli Nissle (EcN) is a nonpathogenic Gram-negative strain isolated in 1917 by Alfred Nissle and it is the active component of microbial drug Mutaflor<sup>®</sup> (Ardeypharm GmbH, Herdecke, Germany and EcN, Cadigroup, In Italy) used in many gastrointestinal disorder including diarrhea, uncomplicated diverticular disease and UC. It is the only probiotic recommended in ECCO guidelines as effective alternative to mesalazine in maintenance of remission in UC patients. In this review we propose an update on the role of EcN 1917 in maintenance of remission in UC patients, including data about efficacy and safety. Further studies may be helpful for this subject to further the full use of potential of EcN.展开更多
AIM: To evaluate the effect of oral Escherichia coli (E. coli) Nissle application on the outcome of intestinal-borne dermatoses.METHODS: In a randomized, controlled, non-blinded prospective clinical trial 82 patients ...AIM: To evaluate the effect of oral Escherichia coli (E. coli) Nissle application on the outcome of intestinal-borne dermatoses.METHODS: In a randomized, controlled, non-blinded prospective clinical trial 82 patients with intestinal-borne facial dermatoses characterized by an erythematous papular-pustular rash were screened. At the initiation visit 37 patients entered the experimental arm and 20 patients constituted the control arm. All 57 patients were treated with a vegetarian diet and conventional topical therapy of the dermatoses with ointments containing tetracycline, steroids and retinoids. In the experimental arm patients received a one month therapy with oral E. coli Nissle at a maintenance dose of 2 capsules daily. The experimental group was compared to a non-treatment group only receiving the diet and topical therapy. The primary outcome parameter was improvement of the dermatoses, secondary parameters included life quality and adverse events. In addition the immunological reaction profile (IgA, interleucin-8 and interferon-α) was determined. Furthermore the changes of stool consistency and the microbiota composition over the time of intervention were recorded.RESULTS: Eighty-nine percent of the patients with acne, papular-pustular rosacea and seborrhoic dermatitis responded to E. coli Nissle therapy with significant amelioration or complete recovery in contrast to 56% in the control arm (P < 0.01). Accordingly, in the E. coli Nissle treated patients life quality improved significantly (P < 0.01), and adverse events were not recorded. The clinical improvement was associated with a significant increase of IgA levels to normal values in serum as well as suppression of the proinflammatory cytokine IL-8 (P < 0.01 for both parameters). In the E. coli Nissle treated group a shift towards a protective microbiota with predominance of bifidobacteria and lactobacteria (> 10<sup>7</sup> CFU/g stool) was observed in 79% and 63% of the patients, respectively (P < 0.01), compared to no change in the control group without E. coli Nissle. Moreover, the detection rate of a pathogenic flora dropped from 73% to 14 % of the patients in the experimental arm (P < 0.01) with no significant change in the control arm (accounting 80% before and 70% after the observation period, P > 0.05). Accordingly, stool consistency, color and smell normalized in the E. coli Nissle treated patients.CONCLUSION: E. coli Nissle protects the mucus barrier by overgrowth of a favorable gut microbiota with less immunoreactive potential which finally leads to clinical improvement of intestinal borne dermatoses.展开更多
文摘质粒消除旨在获得无质粒菌株,是对益生菌进行遗传改造所需的一项重要技术。本试验建立在质粒不相容性的基础上,引入自杀性载体pRE112作为辅助工具,使用分子生物学方法构建重组自杀质粒pMUT1-pRE112和pMUT2-pRE112,将其分别导入携带2个大隐秘质粒pMUT1和pMUT2的益生菌大肠杆菌Nissle 1917(Escherichia coli Nissle 1917,EcN),利用重组自杀质粒去除EcN内原有隐秘质粒并在含有10%蔗糖的LB培养基上实施自杀质粒自身消除。结果试验成功获得EcN无质粒克隆菌株(Escherichia coli Nissle 1917cured of its two cryptic plasmids pMUT1and pMUT2,EcNc),为优质益生菌EcN更好的潜在应用研究奠定基础。
文摘初步研究大肠杆菌Nissle 1917(E.coli Nissle 1917,EcN)鞭毛蛋白(Flagellin,FliC)高变区的功能,为探讨其作为表面展示系统的可行性打下基础。以EcN无质粒克隆(EcN was cured of its 2 cryptic plasmids pMUT1 and pMUT2 resulting in EcNc)为实验菌株,分别构建2个鞭毛蛋白基因(fl iC)高变区859-888位、829-858位的缺失突变株和回补菌株。分析突变区域对EcNc的生长性能、生化特性、运动性及抗原性等方面的影响。结果显示:EcNc、两个缺失株和回补株的生长特性无明显差异,且三者生化试验结果一致。缺失突变株在半固体培养基上不能运动,而回补株均恢复了原运动性;缺失突变株均不与H1单因子血清发生凝集,而互补菌株均能产生与EcNc一致的凝集反应。表明高变区的这两个区域尽管不影响菌株的生长性能和生化特性,但与EcN的运动性及抗原性密切相关。这为进一步探索EcN鞭毛展示技术的研究提供了一定的数据。
文摘Ulcerative colitis (UC) is a chronic inflammatory disease, whose etiology is still unclear. Its pathogenesis involves an interaction between genetic factors, immune response and the “forgotten organ”, Gut Microbiota. Several studies have been conducted to assess the role of antibiotics and probiotics as additional or alternative therapies for Ulcerative Colitis. Escherichia coli Nissle (EcN) is a nonpathogenic Gram-negative strain isolated in 1917 by Alfred Nissle and it is the active component of microbial drug Mutaflor<sup>®</sup> (Ardeypharm GmbH, Herdecke, Germany and EcN, Cadigroup, In Italy) used in many gastrointestinal disorder including diarrhea, uncomplicated diverticular disease and UC. It is the only probiotic recommended in ECCO guidelines as effective alternative to mesalazine in maintenance of remission in UC patients. In this review we propose an update on the role of EcN 1917 in maintenance of remission in UC patients, including data about efficacy and safety. Further studies may be helpful for this subject to further the full use of potential of EcN.
文摘AIM: To evaluate the effect of oral Escherichia coli (E. coli) Nissle application on the outcome of intestinal-borne dermatoses.METHODS: In a randomized, controlled, non-blinded prospective clinical trial 82 patients with intestinal-borne facial dermatoses characterized by an erythematous papular-pustular rash were screened. At the initiation visit 37 patients entered the experimental arm and 20 patients constituted the control arm. All 57 patients were treated with a vegetarian diet and conventional topical therapy of the dermatoses with ointments containing tetracycline, steroids and retinoids. In the experimental arm patients received a one month therapy with oral E. coli Nissle at a maintenance dose of 2 capsules daily. The experimental group was compared to a non-treatment group only receiving the diet and topical therapy. The primary outcome parameter was improvement of the dermatoses, secondary parameters included life quality and adverse events. In addition the immunological reaction profile (IgA, interleucin-8 and interferon-α) was determined. Furthermore the changes of stool consistency and the microbiota composition over the time of intervention were recorded.RESULTS: Eighty-nine percent of the patients with acne, papular-pustular rosacea and seborrhoic dermatitis responded to E. coli Nissle therapy with significant amelioration or complete recovery in contrast to 56% in the control arm (P < 0.01). Accordingly, in the E. coli Nissle treated patients life quality improved significantly (P < 0.01), and adverse events were not recorded. The clinical improvement was associated with a significant increase of IgA levels to normal values in serum as well as suppression of the proinflammatory cytokine IL-8 (P < 0.01 for both parameters). In the E. coli Nissle treated group a shift towards a protective microbiota with predominance of bifidobacteria and lactobacteria (> 10<sup>7</sup> CFU/g stool) was observed in 79% and 63% of the patients, respectively (P < 0.01), compared to no change in the control group without E. coli Nissle. Moreover, the detection rate of a pathogenic flora dropped from 73% to 14 % of the patients in the experimental arm (P < 0.01) with no significant change in the control arm (accounting 80% before and 70% after the observation period, P > 0.05). Accordingly, stool consistency, color and smell normalized in the E. coli Nissle treated patients.CONCLUSION: E. coli Nissle protects the mucus barrier by overgrowth of a favorable gut microbiota with less immunoreactive potential which finally leads to clinical improvement of intestinal borne dermatoses.
