肠炎沙门氏菌鸡源株ompR 基因缺失株的构建及生物学特性与亲本株的比较

【目的】为了探讨ompR基因在肠炎沙门氏菌生物被膜形成及毒力中的作用。【方法】以肠炎沙门氏菌作为母本,运用自杀性载体pGMB151构建了ompR基因缺失株,结晶紫染色法和扫描电镜观察测定缺失株的生物被膜形成能力,细胞的吸附和侵入及小鼠攻毒试验测定缺失株的毒力。【结果】RT-PCR和蛋白表达证明了ompR基因缺失株构建成功;该缺失株不表达纤维素和菌毛,不形成生物被膜;上皮细胞吸附和侵入试验表明缺失株与野生株具有相同的吸附和侵入率;BALB/c鼠腹腔感染性试验表明,缺失株的半数致死量为106.67CFU,而野生株的半数致死量小于2 CFU。【结论】ompR基因既是肠炎沙门氏菌生物膜形成的调控基因,又是重要的毒力基因。     

表达幽门螺杆菌黏附素保守区的减毒鼠伤寒沙门氏菌株的构建

为构建表达幽门螺杆菌（Hp）黏附素保守区（AB）的无抗性减毒鼠伤寒沙门氏菌疫苗,采用缺失腺苷酸环化酶基因（Δcya）、环腺苷酸受体蛋白基因（Δcrp）以及天冬氨酸β-半醛脱氢酶基因（Δasd）的鼠伤寒沙门菌（X4072）作为宿主,将编码AB的基因插入Asd⁺的组成型表达载体pYA248,通过两次转化引入宿主菌,构建了表达AB基因平衡致死的减毒鼠伤寒沙门重组菌X4072（pYA248-AB）,采用桥联法ELISA测定X4072（pYA248-AB）培养上清液和裂解上清液中AB的抗原性,参照Meacock叙述的方法及重组菌生长曲线的测定来确定重组菌株的稳定性,通过C57BL/6小鼠口服测定半致死量来确定重组菌的安全性。成功构建了表达AB的减毒鼠伤寒沙门菌重组菌株S.typhimurium X4072(pYA248-AB),桥联法ELISA测定表明重组菌X4072（pYA248-AB）培养上清中AB的含量高于菌体裂解液,重组菌pYA248-AB在没有选择压力的情况下培养100代,随机挑选的重组菌全部都能生长,且在ELISA测定AB抗原时均显阳性。重组菌的生长曲线测定表明,X4072（pYA248）和X4072（pYA248-AB）的生长状态基本一致;口服重组菌株X4072(pYA248-AB)1.0×10¹⁰cfu.30d后,C57BL/6存活率仍为100%。成功构建了表达AB的无抗性的减毒鼠伤寒沙门菌疫苗X4072（pYA248-AB）,体外实验表明重组质粒是稳定的,动物实验证明重组菌株是安全的;为防治幽门螺杆菌感染提供了口服活菌疫苗候选株。     

鹌鹑源鼠伤寒沙门氏菌的分离鉴定与耐药性分析

【背景】在鹌鹑养殖过程中,抗菌药物和消毒剂的不规范使用加剧了耐药菌株在动物、场所和食品之间的相互传播,因此,掌握致病菌株在养殖动物中的耐药状况至关重要。【目的】检测北京周边地区鹌鹑蛋源致病菌株的耐药特征和耐药基因的流行情况。【方法】在天津市武清区部分鹌鹑养殖场采集鹌鹑泄殖腔粪便、鹌鹑蛋表、养殖环境和鹌鹑饮水的样品,通过细菌分离培养、菌落形态观察、染色镜检、生化鉴定、血清分型、沙门氏菌inv A基因序列测定等方法对分离菌株进行鉴定。同时进行小鼠攻毒试验,测定小鼠半数致死量(median lethal dose, LD50)。再通过药敏试验和PCR方法对分离菌的耐药表型、耐药基因及毒力基因进行检测。【结果】分离菌株菌落颜色、镜检形态和生化试验结果符合沙门氏菌特性,沙门氏菌inv A基因序列测定与鼠伤寒沙门氏菌参考株相似度为99.44%,鉴定为鼠伤寒沙门氏菌,血清型为1,4,[5],[12]:i:l,2。该菌株对小鼠有致病作用,小鼠LD50为2.10×107 CFU/mL;药敏试验结果显示该菌株对氨苄西林、阿莫西林/克拉维酸、头孢噻呋、链霉素、磺胺甲啞唑、磺胺异啞唑、诺氟沙星、环丙沙星表现耐...     

短双歧杆菌对鼠伤寒沙门氏菌的抑制

【背景】鼠伤寒沙门氏菌是主要的肠道病原菌之一,利用益生菌治疗肠道病原菌感染已成为一种新型、绿色的微生态疗法。【目的】研究筛选出的短双歧杆菌无细胞发酵上清液(Cell-free supernatant,CFS)对鼠伤寒沙门氏菌的体外抑制作用及机制。【方法】采用微量稀释法测定短双歧杆菌YH68 CFS对鼠伤寒沙门氏菌的最小抑菌浓度(Minimum inhibitory concentration,MIC)和亚抑制浓度(Sub-inhibitory concentrations,SIC),并从鼠伤寒沙门氏菌的细胞形态、细胞膜通透性、膜完整性以及毒力基因表达的变化探讨YH68 CFS对鼠伤寒沙门氏菌的抑菌机理,同时检测YH68 CFS对鼠伤寒沙门氏菌粘附和侵袭肠上皮细胞HT29的影响。【结果】YH68 CFS (3×109 CFU/mL)对鼠伤寒沙门氏菌具有较好的抑制效果,抑菌圈直径为22.27±0.44 mm,最小抑菌浓度为250μL/mL,对鼠伤寒沙门氏菌的抑制机制是通过增加其细胞膜通透性破坏其完整性,形成难以修复的孔洞,最终达到抑菌的目的;亚抑制浓度为62.5μL/mL时YH68 CFS并不能影响鼠伤寒沙门氏菌的生长,但仍然能通过下调毒力基因表达的方式抑制其对肠上皮细胞的粘附和入侵。【结论】短双歧杆菌YH68对鼠伤寒沙门氏菌具有良好的抑菌作用,可作为治疗沙门氏菌感染的潜在益生菌。     

用体内激活的启动子调控HCV核心抗原表达可增强重组鼠伤寒沙门氏菌的免疫原性

为提高抗原表达质粒在重组伤寒沙门氏菌中的稳定性以增强重组伤寒沙门氏菌诱导的免疫应答 ,克隆鼠伤寒沙门氏菌pagC基因启动子 ,以其为转录调控元件构建HCV核心抗原表达质粒 ,转化到减毒鼠伤寒沙门氏菌中。体外培养时 ,Mg2 能够剂量依赖性抑制该重组菌表达HCV核心抗原。将该重组菌和组成性表达的重组菌分别口服接种BALB/c小鼠 ,观察质粒的稳定性和小鼠的免疫应答。结果表明 ,体内激活的pagC基因启动子能明显提高质粒在重组鼠伤寒沙门氏菌中的稳定性和增强重组菌诱导的体液和细胞免疫应答 ,这为发展高效免疫、成本低廉的口服丙肝疫苗提供了一个新思路     

表达大肠杆菌LT—B抗原的减毒鼠伤寒沙门氏菌株的构建

采用无毒的鼠伤寒沙门氏菌sR一1l△Cya,△Crp,△asd菌株作为宿主菌,选择相应的asd+表达载体构建了含大肠杆菌肠毒素B亚基基因(toxB)的重组质粒,并通过两次转化引入宿主菌,构成了平衡致死的重组体。特异性的测定表明,这种无抗药性的杂合菌株能较高水平地表达LT—B抗原,可望成为预防ETEC腹泻和相应的沙门氏菌病双价口服活疫苗的研究基础。     

表达大肠杆菌LT-B/ST融合抗原的减毒鼠伤寒沙门氏菌株的构建

编码LT-B/ST融合抗原的基因插入pYA248载体中,构建了重组质粒pXZL66。该重组质粒转入无毒鼠伤寒沙门氏菌SR-11,ΔCya,Δcrp,Δasd菌株X4072。此无抗药性的杂合菌株X4072(pXZL66)表达的LT-B/ST融合抗原具有LT和ST抗原性而没有生物毒性,可望成为预防ETEC腹泻和相应的沙门氏菌病双价口服活疫苗候选株。     

猪霍乱沙门氏菌DasdC500株的生物学特性及作为活疫苗表达载体的应用

为了评价猪霍乱沙门氏菌DasdC500株作为沙门氏菌Asd+平衡表达系统的可行性, 对DasdC500株和其亲本菌株C500的生物表型、生长特性、毒力、生物安全性、表达特性等进行比较研究。结果表明: DasdC500缺失株的生化特性和血清型与亲本菌株C500一致, 符合猪霍乱沙门氏菌的表型特征; 携带平衡表达质粒pYA3493的重组菌株DasdC500(pYA3493)与C500的生长速度没有明显差别; 根据Reed-Muench法, 测定DasdC500(pYA3493)腹腔感染BALB/c小鼠的LD50为1.1×107 CFU, 毒力稍低于C500; 口服接种ΔasdC500(pYA3493)和C500的所有仔猪未见任何发病症状, 两者没有显著差别; 携带重组质粒pYA-F1P2 (含有支气管败血波氏杆菌抗原基因fhaB的Type I区域和prn的R2区域)的重组菌株DasdC500(pYA-F1P2)能够稳定遗传重组质粒及其外源基因片段, 并能稳定、高效、分泌性表达外源保护性抗原。由此表明, DasdC500保留了亲本菌株C500的一系列生物学特征, 并可高效表达外源抗原, 可作为沙门氏菌Asd+平衡表达系统开发基因工程重组疫苗。     

鼠伤寒沙门氏菌ybi H基因缺失株的构建及其生物学特性

【背景】鼠伤寒沙门氏菌(Salmonella typhimurium)是一种重要的人兽共患病原菌,能够引起多种食源性疾病。ybiH基因在鼠伤寒沙门氏菌中的生物学功能尚未确定。【目的】构建鼠伤寒沙门氏菌ybiH基因的缺失株和回补株,研究ybiH基因在鼠伤寒沙门氏菌中的生物学功能。【方法】采用λ-Red同源重组系统构建鼠伤寒沙门氏菌CVCC541 ybiH基因缺失株STΔybiH,同时构建该基因缺失株的回补株STΔybi H/pybiH,并对缺失株STΔybiH的生长特性、运动性、生化特性和毒力情况等生物学特性进行比较分析。【结果】与标准株和回补株相比,缺失株STΔybiH生长速率略快,而其运动性、生化特性、耐药性均无明显差别。但是,ybiH基因的缺失明显提高了鼠伤寒沙门菌对IEC-6细胞和RAW264.7细胞的黏附力和侵袭力,qRT-PCR实验结果显示,缺失株中Inv H基因的表达量明显提高,表明ybiH基因的缺失使鼠伤寒沙门氏菌的侵袭力也有所提高。此外,在胞内存活实验中,标准株与缺失株在胞内的增长率变化不明显,表明ybiH基因的缺失对沙门氏菌在RAW 264.7细胞中存活的影响不大。【...     

鼠伤寒沙门氏菌群体感应复苏oxyR基因缺失引起的VBNC状态

探讨鼠伤寒沙门氏菌oxyR基因缺失株引起的VBNC状态及其与群体感应的关系。运用同源重组的方法构建oxyR基因无痕缺失的鼠伤寒沙门氏菌并检测该菌株对H_2O_2的敏感性;将oxyR基因缺失株和亲本株(WT)涂布或滴于LB固体培养基,观察其是否生长及其浓度依赖性生长情况;用swimming和swarming平板检测oxyR基因缺失株和WT的运动能力;检测固体培养基和液体培养基中沙门氏菌分解H_2O_2的能力。成功构建了oxyR无痕缺失菌株;oxyR基因缺失株在0.1 mmol/L H_2O_2的LB平板上形成的菌苔发生了变形,在1 mmol/L H_2O_2的LB平板上不能生长,而WT均能生长;6×10~6和6×10~5 cfu/mL的WT涂布于LB平板上能长满菌苔,而等量的oxyR缺失株不能生长菌落;不同浓度的WT滴于LB平板均能形成菌苔,而oxyR缺失株仅在OD_(600)≥10~(-1)浓度时才能形成菌苔;oxyR缺失株泳动距离无显著性变化,而群集运动距离显著性大于WT;固体培养的沙门氏菌比液体培养的沙门氏菌有更强的分解H_2O_2的能力。鼠伤寒沙门氏菌的群体感应系统通过调控其群集运动和H_2O_2分解能力来复苏由oxyR基因缺失引起的VBNC状态。     

The PhoQ histidine kinases of Salmonella and Pseudomonas spp. are structurally and functionally different: evidence that pH and antimicrobial peptide sensing contribute to mammalian pathogenesis

The PhoQ sensor kinase is essential for Salmonella typhimurium virulence for animals, and a homologue exists in the environmental organism and opportunistic pathogen Pseudomonas aeruginosa. S. typhimurium PhoQ (ST-PhoQ) is repressed by millimolar concentrations of divalent cations and activated by antimicrobial peptides and at acidic pH. ST-PhoQ has a periplasmic Per-ARNT-Sim domain, a fold commonly employed for ligand binding. However, substrate binding is instead accomplished by an acidic patch in the periplasmic domain that interacts with the inner membrane through divalent cation bridges. The DNA sequence encoding this acidic patch is absent from Pseudomonas phoQ (PA-PhoQ). Here, we demonstrate that PA-PhoQ binds and is repressed by divalent cations, and can functionally complement a S. typhimurium phoQ-null mutant. Mutational analysis and NMR spectroscopy of the periplasmic domains of ST-PhoQ and PA-PhoQ indicate distinct mechanisms of binding divalent cation. The data are consistent with PA-PhoQ binding metal in a specific ligand-binding pocket. PA-PhoQ was partially activated by acidic pH but not by antimicrobial peptides. S. typhimurium expressing PA-PhoQ protein were attenuated for virulence in a mouse model, suggesting that the ability of Salmonella to sense host environments via antimicrobial peptides and acidic pH is an important contribution to pathogenesis.     

Molecular analysis of the Escherichia coli phoP-phoQ operon.

The phoP-phoQ operon of Salmonella typhimurium is a member of the family of two-component regulatory systems and controls expression of the phoN gene that codes for nonspecific acid phosphatase and the genes involved in the pathogenicity of the bacterium. The phoP-phoQ operon of Escherichia coli was cloned on a plasmid vector by complementation of a phoP mutant, and the 4.1-kb nucleotide sequence, which includes the phoP-phoQ operon and its flanking regions, was determined. The phoP-phoQ operon was mapped at 25 min on the standard E. coli linkage map by hybridization with the Kohara mini set library of the E. coli chromosome (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The predicted phoP and phoQ gene products consist of 223 and 486 amino acids with estimated molecular masses of 25,534 and 55,297 Da, respectively, which correspond well with the sizes of the PhoP and PhoQ proteins identified by the maxicell method. The amino acid sequences of PhoP and PhoQ of E. coli were 93 and 86% identical, respectively, to those of S. typhimurium.     

鼠伤寒沙门菌spvBC基因编辑株的构建

利用λRed重组系统和pBAD原核表达载体构建鼠伤寒沙门菌spvBC质粒毒力基因修饰菌株,为深入探究沙门菌毒力基因spv的功能和致病机制及宿主抗感染免疫提供工具菌。以pKD4为模板,PCR扩增含spvBC同源臂的卡那霉素抗性基因以构建同源打靶片段,再将其电转入含有质粒pKD46的鼠伤寒沙门菌中进行同源重组,随后将质粒pCP20电转导入阳性转化子,消除卡那霉素抗性基因,PCR鉴定敲除株的构建。PCR扩增含酶切位点的spvBC基因片段,扩增产物与原核表达载体pBAD/gⅢ分别双酶切后连接构建pBAD-spvBC重组质粒,PCR筛选阳性菌落并测序鉴定。将构建成功的pBAD-spvBC重组质粒电转导入spvBC敲除株中,Western blot测定不同浓度L-阿拉伯糖诱导SpvB和SpvC蛋白表达情况。PCR结果表明鼠伤寒沙门菌spvBC基因敲除成功;PCR及测序结果表明pBAD-spvBC重组质粒构建成功,Western blot结果表明13 mmol/L L-阿拉伯糖可诱导SpvB和SpvC蛋白正常表达。λRed重组系统可用于沙门菌质粒上大片段基因的敲除,pBAD原核表达载体可用于沙门菌质粒上大片段基因的回补,丰富了细菌质粒的基因修饰和编辑策略。     

减毒鼠伤寒沙门氏菌全长hpaA基因工程菌的构建

为构建表达HpaA蛋白的重组减毒鼠伤寒沙门氏菌 ,并探讨以减毒鼠伤寒沙门氏菌为载体构建H .pylori疫苗株的意义 ,应用PCR法从H .pylori基因组DNA中扩增 783bp的hpaA基因 ,经酶切 连接反应将其克隆入原核表达质粒pTrc99A的NcoⅠ SalⅠ位点 ,并进行了核苷酸序列测定。重组质粒转化减毒鼠伤寒沙门氏菌SL3 2 6 1 ,提取重组菌质粒 ,PCR和酶切鉴定 ,筛选阳性克隆。用SDS PAGE电泳和Westernblot进行HpaA表达分析和鉴定 ,用薄层扫描分析HpaA含量。重组菌C5 7BL 6小鼠喂灌 ,分批两d和 1 0d后处死小鼠 ,取脾和末段回肠进行细菌培养 ,挑菌落提质粒鉴定。结果表明 ,经PCR和酶切证实 ,构建了含 783bphpaA基因的重组原核表达质粒 ,并将后者成功转化了减毒鼠伤寒沙门氏菌。重组菌能表达约3 0kDHpaA蛋白 ,重组HpaA量约占全菌体蛋白量的 3 8 9% ,Westernblot证实其有免疫反应性。小鼠重组菌喂灌两d或 1 0d后 ,脾和末段回肠均发现携目的基因的菌落。这些结果提示 ,构建了表达H .pyloriHpaA的重组减毒…     

鼠伤寒沙门氏菌asd基因的克隆及序列分析

以鼠伤寒沙门氏茵标准株基因组DNA作为模板,用PCR的方法扩增鼠伤寒沙门氏菌的asd基因并克隆入质粒pUCl9,并对其进行测序,序列与献报道一致。同时将质粒pYA248上的链球菌asd基因进行了置换,观察了分别含有链球菌asd基因与鼠伤寒沙门氏菌asd基因的质粒在减毒鼠伤寒沙门氏菌X4072中的生长情况,结果表明含有鼠伤寒沙门氏菌的asd基因的高拷贝质粒pUCl9的菌株生长情况更好。为完善染色体／质粒平衡致死系统,构建减毒鼠伤寒沙门氏活菌疫苗奠定了基础。     

Molecular analysis of the Salmonella typhimurium phoN gene, which encodes nonspecific acid phosphatase.

The phoN gene of Salmonella typhimurium encodes nonspecific acid phosphatase (EC 3.1.3.2), which is regulated by a two-component regulatory system consisting of the phoP and phoQ genes. We cloned the phoN region into a plasmid vector by complementation of a phoN mutant strain and determined the nucleotide sequence of the phoN gene and its flanking regions. The phoN gene could encode a 26-kDa protein, which was identified by the maxicell method as the product of phoN. Results of the enzyme assay and Southern hybridization with chromosomal DNA of Escherichia coli K-12 suggests that there is no phoN gene in E. coli. The regulatory pattern of phoN in E. coli and Southern hybridization analysis of the E. coli chromosome with the S. typhimurium phoP gene suggest that E. coli K-12 also harbors the phoP and phoQ genes.     

Biological properties of plasmid-free strains of Salmonella typhimurium and S. dublin

The study of Salmonella virulent strains has revealed that the characteristic feature of such strains is the presence of plasmids with a molecular weight of 90.2-91.5 kb for S. typhimurium and 77.2-78.5 kb for S. dublin. From Salmonella strains harboring only a single plasmid, variants with no plasmid at all have been obtained. These variants possess lower virulence for mice infected through enteral and intraperitoneal routes; besides, they lose their capacity for penetration into epithelial cells of HeLa line. S. typhimurium and S. dublin have shown decreased multiplication rate in vivo in comparison with the parent strains, while the multiplication rates in vitro were similar. These results suggest that the products of plasmid genes are either responsible for the virulent properties of salmonellae, or they have regulatory functions, thus controlling the work of chromosomal genes.