肠炎沙门氏菌SEF14菌毛的表达及其对该菌的特异性检测研究

为获得肠炎沙门氏菌SEF14菌毛蛋白并检测其生物学功能,本研究以肠炎沙门氏菌标准株SE50336为模板,PCR扩增其含分泌信号肽的sef14操纵子基因片段,并克隆至表达载体pBR322中,构建重组质粒pBR322-sef14,将该重组质粒转化至经修饰且不含任何菌毛的大肠杆菌SE5000M株中,获得重组菌pBR322-s...     

肠炎沙门氏菌SEF14菌毛单克隆抗体的制备及初步应用

肠炎沙门氏菌现已成为全球性食品安全问题的主要病原之一。本试验基于肠炎沙门氏菌特异性SEF14菌毛,应用淋巴细胞杂交瘤技术成功制备了3株单克隆抗体杂交瘤细胞株,Westermhlotting检测结果显示3株单克隆抗体均具有良好的特异性。同时,用制备的单抗建立了双抗体夹心间接ELISA方法,利用方阵滴定确定捕获抗体和SEF14蛋白的最佳工作浓度分别为8μg／ml和2μg／ml。用该方法检测100份鸡血清样本,59份为阳性,与肠炎沙门氏菌特异性基因片段sdfI的PCR符合率达93％。本试验建立的方法特异性强、灵敏性高,可用于临床诊断,在肠炎沙门氏菌特异性检测方面具有潜在的应用前景。     

肠炎沙门氏菌SEF14菌毛及SefD蛋白的研究进展

SEF14菌毛是血清型肠炎沙门氏菌所特有的一种菌毛。文章不仅综述了SEF14菌毛的分布、功能及组装形成,还综述了目前针对SEF14菌毛上的顶端蛋白Sef D所进行的研究及成果,包括确定了Sef D蛋白的功能、解析了Sef D蛋白的结构及证实了含有Sef D蛋白的疫苗的有效性。最后在此研究基础上还提出了以后关于Sef D蛋白的研究前景及方向。     

肠炎沙门菌SEF14菌毛的抽提、纯化及生物学活性检测

试验从肠炎沙门菌国内标准株50336中抽提和纯化SEF14菌毛,并制备SEF14蛋白鼠源高免血清.结果发现,肠炎沙门菌在CFA培养液中,静止培养50 h左右,SEF14菌毛表达量相对较多,在机械高速匀浆后,经进一步的透析纯化,得到较纯的SEF14菌毛蛋白,蛋白大小和相关报道一致,约14 ku左右.Westem-blotting检测发现纯化的重组蛋白rSEFA(体外表达的SEF14菌毛主要亚单位蛋白SEFA)免疫小鼠得到的高免血清能识别标准株肠炎沙门菌SEF14菌毛蛋白以及纯化的重组蛋白rSEFA,说明体外抽提的SEF14菌毛蛋白和体外表达的rSEFA蛋白同样具有较好的免疫原性和反应原性.     

肠炎沙门氏菌SEFA基因表达和间接ELISA检测方法的初步建立

为了给临床检测肠炎沙门氏菌的感染和血清学调查提供一种切实可行的方法,本研究根据肠炎沙门氏菌SEF14菌毛操纵子亚单位sefA基因序列设计一对引物,利用PCR技术从国内标准株CMCC(B)50336中扩增sefA基因,并按预定的阅读框插入表达载体pET22b+中,获得重组质粒pET-sefA,限制性内切酶结合琼脂糖凝胶电泳分析和序列测定结果表明,该序列大小为498bp,与已发表的sefA结构编码序列完全一致。重组质粒pETsefA转化大肠杆菌E.coli BL21(DE3)并能获得高效诱导表达,通过对菌体裂解上清液SDS-PAGE和western blot分析鉴定,该重组菌可以表达大小为15.2ku的可溶性重组蛋白rSEFA。纯化的rSEFA免疫小鼠所得高免血清以及标准株CMCC(B)50336感染小鼠后所获阳性血清,均能识别标准株肠炎沙门氏菌SEF14菌毛蛋白以及纯化的重组蛋白rSEFA,结果表明明体外表达的rSEFA蛋白有较好的免疫原性和反应原性。而基于rSEFA介导的间接ELISA有较好的特异性,对肠炎沙门氏菌特异性抗体检测有潜在的应用前景。     

肠炎沙门菌SEF14菌毛与肠上皮细胞IPEC-J2和Caco-2的体外黏附作用研究

为研究肠炎沙门菌SEF14菌毛对肠上皮细胞的黏附作用,本试验利用肠炎沙门菌50336株、突变株50336△sefA、50336△sefD以及互补株50336△sefA (pBRA)、50336△sefD (pACYCD)与肠上皮细胞系细胞(IPEC-J2和Caco-2)进行了黏附作用.结果显示:上述菌株均能与IPEC-J2、Caco-2细胞进行有效黏附,并且随时间延长黏附数量有所增多,相同时间各菌株与IPEC-J2细胞的黏附数量明显多于Caco-2细胞;但细菌和细胞共感染1和4h后,肠炎沙门菌野生株、相应的突变株和互补株与IPEC-J2和Caco-2细胞黏附的数量差别很小,未到达显著差异水平(P＞0.05).结果表明:SEF14菌毛并不特异性介导肠炎沙门菌与肠上皮细胞系IPEC-J2和Caco-2的黏附作用,或者不是介导黏附作用的主要因子.     

肠炎沙门菌间批ELISA检测方法的建立

本试验建立和优化了重组蛋白rSEFA介导的间接ELISA方法以特异性检测肠炎沙门菌感染血清.确定其抗原最佳包被量为7.5 μg/mL,血清最适稀释度1:100,作用时间为60 min;酶标二抗最适稀释度为1:4 000,作用时间为60 min;判定标准为OD值≥0.346.基于rSEFA介导的间接ELISA有较好的特异性,能特异性识别肠炎沙门菌和都柏林沙门菌感染血清,不能识别相近的鸡白痢沙门菌感染血清,此方法与菌体凝集反应同时检测临床血清样品100份,二者阳性率分别为67%和54%,结果符合率为80.6%.这一方法的建立对肠炎沙门菌特异性抗体检测有潜在的应用前景.     

小鼠肠炎沙门氏菌1和3型菌毛的粘附和致病作用

以红细胞凝集试验检测肠炎沙门氏菌的两个同源株证明分别有１和１．３型菌毛。该两菌株均能粘附于人颊上皮细胞和小鼠小肠粘膜上皮细胞，Ａ株较Ｖ株更容易发生粘附且粘附的菌数更多；在Ｄ－甘露糖存在的条件下，两菌株极易粘附，而经鞣酸预处理的上皮细胞对粘附人颊粘膜和小鼠小肠上皮细胞的抗Ｄ－甘露糖型的肠炎沙门氏菌Ａ株的粘附力下降，通过半数致死量测定表明，口服接种小鼠。Ａ株呈明显的毒力，而腹腔接种对Ｖ株的毒力则明显加     

基于Red同源重组和高效自杀性载体系统构建肠炎沙门氏菌突变株方法的比较

Red同源重组系统和高效自杀性载体系统都能对革兰氏阴性茵染色体直接进行修饰,本试验利用上述2种系统敲除不同来源肠炎沙门氏茵菌株SEF14茵毛操纵子亚单位sefD或sefA基因,并对构建肠炎沙门氏茵突变株方法进行比较.本研究成功构建了国际、国内标准株,鸡源和人源国内分离株sefD和sefA缺失株各4株.Red同源重组系统中能将PCR产物一步法直接转化肠炎沙门氏茵,通过同源重组序列发生同源重组交换.但对PCR产物和感受态细胞的质量要求很高,一次重组效率很低,而二次重组过程则相对简单、高效.自杀性载体系统若将构建好的合同源DNA片段的重组质粒转化宿主茵,其一次重组效率高,二次重组也只需蔗糖筛选传代丢失抗性质粒.Red同源重组系统将在染色体中残留FRT位点;而自杀性栽体系统在染色体中可不留任何痕迹.所以2个系统都能对不同源肠炎沙门氏茵染色体进行直接修饰,而且易删除抗性选择标志,与传统的高效自杀性载体系统比较,Red同源重组系统更方便快捷,省时省力.     

肠炎沙门菌sef14基因缺失株生物学功能

构建了肠炎沙门菌参考株SE50336的SefA主要亚单位编码基因sefA的缺失株SE50336ΔsefA,进一步探究野生株及SEF14菌毛缺失株的生物学功能。以SE50336为模板,利用λ-red同源重组技术构建sefA缺失株,通过绘制生长曲线、生物被膜测定试验、刚果红-考马斯亮蓝无盐培养基菌落表型试验、RT-qPCR(real-time quantitative polymerase chain reaction)、人结肠腺癌上皮细胞(Caco-2)的体外黏附测定来检测和比较2种菌株的生物学特性,并利用野生株和缺失株分别攻毒小鼠,探究SEF14菌毛对肠炎沙门菌致病力的影响。结果显示,野生株及缺失株生长情况无明显差异。与野生株相比,ΔsefA缺失株的生物被膜形成能力有所下降;菌落表型试验中野生株表现出典型的rdar(red, dry and rough)表型,而ΔsefA缺失株表现出saw(smooth and white)表型;RT-qPCR试验中,野生株的生物被膜形成主要关联基因csgA、bcsA、pgaC、fimA的转录量均显著高于ΔsefA缺失株。但缺失株与野生株对于Caco-...     

肠炎沙门菌非编码小RNA isrC基因缺失株的致病性

细菌非编码小RNA(sRNA)是一类可在转录后水平调控基因表达的调节子。IsrC是存在于鼠伤寒沙门菌毒力岛上的一种与毒力相关的sRNA,可调节巨噬细胞存活蛋白MsgA的表达。本研究克隆了肠炎沙门菌50336菌株isrC基因,通过λ-Red同源重组系统构建了isrC缺失突变株50336△isrC,并利用表达载体pBR322构建了回补株50336△isrC/pisrC。将野生株,isrC突变株和回补株分别接种1日龄清远麻鸡,比较三者之间的致病性差异。结果显示,肠炎沙门菌isrC基因与鼠伤寒沙门菌同源性为100%;成功构建了isrC缺失突变株和回补株;野生株,突变株和回补株对雏鸡的半数致死量(LD50)分别为2.81×108 CFU,2.02×108 CFU和3.10×108 CFU,相比野生株50336,突变株50336△isrC的LD50明显下降,表明isrC基因的缺失增强了肠炎沙门菌的毒力。     

肠炎沙门菌非编码小RNA RyhB-1和IsrE对清远麻鸡的致病性

RyhB是一种大小为90bp左右的细菌非编码小RNA,已有研究表明存在于鼠伤寒沙门菌和霍乱弧菌中的2种RyhB同源物RyhB-1和IsrE可调控细菌生物膜形成、趋化性和耐酸性。为研究RyhB在肠炎沙门菌致病性上的作用,以及2种同源物能否对毒力调控发挥协同作用,本试验利用λ-Red同源重组系统构建了ryhB-1和isrE单、双缺失株,利用表达载体pBR322和pACYC184分别构建了回补质粒并导入缺失株50336△ryhB-1,50336△isrE,50336△ryhB-1/△isrE中,获得各突变株相应的回补菌株,检测了野生株和各突变株对1日龄雏鸡半数致死量(LD50)、致死率、体内分布等致病性差异。结果发现,ryhB-1和isrE基因缺失后导致肠炎沙门菌毒力减弱,双基因缺失株毒力下降更为明显,表明2种小RNA均对肠炎沙门菌致病性具有增强作用,且二者对毒力的调控具有协同作用。     

55 kb Plasmid and Virulence-Associated Genes are Positively Correlated with Salmonella enteritidis Pathogenicity in Mice and Chickens

Twenty-four strains of Salmonella enteritidis, isolated from several outbreaks of salmonellosis from different poultry farms in India, were checked for the plasmid profile and detection of virulence gene(s) by PCR. Most of the strains contained only a single plasmid of 55 kb. Additional plasmids of 23.2 kb and 8.7 kb were seen in one of the strains, and another strain carried only two plasmids of 23.2 kb and 8.7 kb. Four strains did not carry any plasmid. PCR amplification showed the presence of virulence-associated genes in all the isolates harbouring the 55 kb plasmid. Intraperitoneal inoculation of mice, with most of the strains carrying the 55 kb plasmid, caused 100% mortality. Most strains lacking the 55 kb plasmid were avirulent. In chickens, oral inoculation of the S. enteritidis strains carrying the 55 kb plasmid produced 40–100% mortality, with characteristic signs of salmonellosis. Oral inoculation of strains lacking the 55 kb plasmid did not cause any mortality. Hence, it appears that the large plasmid of S. enteritidis probably contributes towards virulence in mice and chickens.     

禽肠炎沙门菌外膜蛋白OMPX基因的克隆及其在乳酸乳球菌中的表达

以禽肠炎沙门菌基因组DNA为模板,采用PCR技术扩增得到外膜蛋白OMPX基因片段,并将其克隆到乳酸乳球菌表达载体pMG36e中,构建重组质粒pMG36e-OMPX。将重组质粒电转入乳酸乳球菌MG1614,得到重组基因工程乳酸乳球菌,在GM17培养基中培养12h后,经SDS-PAGE分析显示表达的蛋白约为16 000,与预期相符,经Western-blot检测表明,表达的蛋白具有良好的反应特异性。     

Pathogenicity of Salmonella enteritidis phage types 4, 8, and 23 in broiler chicks.

Four hundred fifty day-old Hubbard broiler chicks were subdivided into 15 groups of 30 chicks each. Six groups of chicks received 0.5 ml of broth culture containing 5 x 10(6) colony-forming units (CFU) of Salmonella enteritidis (SE) phage types (PTs) 4, 8, and 23 by crop gavage. Similarly, six other groups received 0.5 ml containing 5 x 10(8) CFU of SE. One group was inoculated with 0.5 ml containing 5 x 10(6) CFU of Salmonella pullorum, and another group received 0.5 ml containing 5 x 10(8) CFU of S. pullorum. A group of 30 chicks were kept as uninoculated controls. Chicks were observed daily for clinical signs and mortality. All birds were weighed at 7, 14, and 21 days postinoculation 21 (DPI). Four chicks were randomly selected from each treatment group, euthanatized, and necropsied at 7 and 14 DPI. Gross lesions were recorded and selected tissues were collected for histopathology. The higher rates of illness and mortality were observed in chicks inoculated with 5 x 10(6) and 5 x 10(8) CFU of S. pullorum, followed by SE PT4 of human origin and SE PT4 of chicken origin. Moderate to high mortality was observed in chicks inoculated with the higher dose of SE isolates that belonged to PT8 and one SE of PT23. Variable mortality was evident in groups inoculated with the lower dose of salmonella. The most consistent gross and histopathologic changes, including fibrinous pericarditis and perihepatitis, were seen in the dead birds from various treatment groups. The lower mean body weights were present in all treatment groups compared with uninoculated controls. No illness or mortality was observed in uninoculated control groups.     

The SEF14 fimbrial antigen of Salmonella enterica serovar Enteritidis is encoded within a pathogenicity islet

The DNA sequence of the chromosomal gene cluster encoding the SEF14 fimbriae of Salmonella enterica serovar Enteritidis was determined. Five contiguous open reading frames, sefABCDE, were identified. The sefE gene shared significant homology with araC-like positive regulators. Serovar-associated virulence plasmid (SAP) genes orf7,8,9 and pefI were identified immediately adjacent to the sef operon. The pefI gene encoded a putative regulator of the Plasmid-encoded fimbrial antigen (PEF) expression. The entire sef--pef region, flanked by two IS-like elements, was inserted adjacent to leuX that encoded a transfer RNA molecule. The organisation of this region was suggestive of a classic pathogenicity islet. Southern hybridisation confirmed two copies of the SAP derived orf7,8,9 and pefI region in S. Enteritidis, one in the chromosome and one on the SAP. Of other group D Salmonella, only S. Blegdam and S. Moscow harboured both chromosomal and plasmid copies of pefI--orf9 region although polymorphism was evident.