减毒沙门氏菌为载体在Vero细胞中表达新城疫病毒融合蛋白

RT PCR扩增了新城疫病毒 (NDV)F4 8E9株的融合蛋白 (F)基因并插入到 pcDNA3的CMV启动子下游 ,构建成真核表达质粒pcDNA3 F ,高压电转化dam和 phoP基因双突变株减毒鼠伤寒沙门氏菌 (ZJ111株 ) ,并直接感染Vero细胞 ,分别提取细胞总DNA和总RNA ,DIG标记探针均可检测到阳性杂交信号。FITC标记的羊抗鸡IgG进行间接免疫荧光试验 ,可检测到特异性的黄绿色荧光。ELISA检测F蛋白结果表明 ,转染后 4 8h开始表达 ,随后逐渐增多。SDS PAGE和Western印迹可检测到 5 5kD的蛋白质条带。上述试验结果证实减毒沙门氏菌不仅可将目的基因呈递给Vero细胞 ,而且还得到了转录和表达 ,表达的F蛋白具有免疫反应性 ,为研制减毒沙门氏菌为载体的口服NDVDNA疫苗创造了条件。     

2018年辽宁省沙门菌血清型、分子分型及耐药性

目的 分析辽宁省2018年沙门菌血清型、脉冲场凝胶电泳(PFGE)及耐药特征.方法 对2018年辽宁省分离到的35株沙门菌菌株进行鉴定后,采用脉冲场凝胶电泳及药物敏感试验对沙门菌进行分子分型及耐药性分析.结果 经鉴定35株沙门菌分布于13种血清型,数量居前3位的血清型为肠炎沙门菌(37.14％)、鼠伤寒沙门菌(14.2...     

Determinants of immunity to murine salmonellosis: studies involving immunization with lipopolysaccharide-lipid A-associated protein complexes in C3H/HeJ mice

Abstract We have earlier demonstrated that the C3H/HeJ Salmonella hypersusceptible mouse can be protected against infection with this organism by prior immunization with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes, but not with LPS alone. In the current studies, protection has been shown to correlate with the induction of LPS-specific antibody in immunized mice. LPS was demonstrated to be a relevant target antigen for Salmonella immunity since C3H/HeJ mice were afforded higher survival rates when they were challenged with Salmonella that shared the same LPS O-antigen as the vaccine. Although low levels of LPS-specific antibody can be detected 14 days after immunization with LAP-LPS, significant antibody is present only after 21–28 days. In addition, anti-LAP specific antibodies can be detected after 14 days of immunization with LAP-LPS. Adoptive transfer of either day 28 anti-LAP-LPS immune serum or day 28 LAP-LPS immune splenocytes alone to naive recipients affords mice minimal, if any, survival against lethal S. typhimurium LT2 challenge. In contrast, transfer of day 28 anti-LAP-LPS immune serum and day 28 LAP-LPS immune splenocytes together is able to transfer Salmonella immunity to naive C3H/HeJ mice. Further, equivalent transfer of only day 28 anti-LAP-LPS immune serum to C3H/HeJ mice immunized 7 days previously with LAP-LPS provides protection similar to that found in mice adoptively transferred with immune cells and serum. These results suggest that a host cellular factor or factors responsive to LAP-LPS, in addition to day 28 anti-LAP-LPS immune serum, may contribute to the protection afforded C3H/HeJ mice following immunization with LAP-LPS.     

In vitro adherence study using formalin-treated human small intestine: Fimbria-meidated adherence of enterotoxigenic Escherichia coli and Salmonella typhi oral-route vaccine strain possessing E. coli colonization factor antigen to villi and lymphoid follicle epithelium

Abstract Enterotoxigenic Escherichia coli possessing colonization factor antigen (CFA)/I or CFA/II adhered to formalin-fixed human ileal villi with their CFA/I- or CFA/II-fimbriae. This adherence was host-specific. Adherence to formalin-fixed human ileal lymphoid follicle epithelium or mucus was also observed, although to a lesser extent. Salmonella typhi oral-route live vaccine strain possessing E. coli CFA/I manifested increased adherence to the human ileal mucosa.     

沙门菌外膜蛋白D的原核表达及多克隆抗体制备

目的:在大肠杆菌中表达沙门菌外膜蛋白(OMP)D,纯化后制备兔抗OMPD抗体。方法:用PCR方法从鼠伤寒沙门菌中扩增出ompD基因,并插入融合表达载体pET-28a(+)的多克隆位点,构建重组表达质粒pET28a(+)-ompD;以重组质粒转化大肠杆菌BL21(DE3),筛选阳性重组菌株,经IPTG诱导目的蛋白表达,在变性条件下对目的蛋白进行亲和层析纯化;以表达的OMPD蛋白免疫家兔,制备抗OMPD的多克隆抗体并进行鉴定。结果:扩增了ompD基因,测序证实正确后亚克隆于表达载体pET-28a(+)中,经PCR筛选和酶切鉴定获得阳性克隆,经诱导在大肠杆菌中表达出相对分子质量为40×103的目的蛋白并进行纯化;纯化的OMPD免疫家兔后,能有效地刺激特异性抗体的产生,抗血清的效价达到1∶10000以上,且具有良好的特异性。结论:构建ompD基因的原核表达载体,并在大肠杆菌中获得高效表达;制备出兔抗OMPD抗体,效价及特异性均良好,为进一步制备肠黏膜高亲和力疫苗奠定了基础。     

猪霍乱沙门氏菌ΔasdC500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ΔasdC500株作为沙门氏菌Asd+平衡表达系统的可行性,对ΔasdC500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:AasdC500缺失株的生化特性和血清型与亲本菌株C500一致,符合猪霍乱沙门氏茵的表型特征;携带平衡表达质粒pYA3493的重组菌株ΔasdC500(pYA3493)与C500的生长速度没有明显差别;根据Reed-Muench法,测定ΔasdC500(pYA3493)腹腔感染BALB/c小鼠的LD50为1.1 x 107CFU,毒力稍低于C500;口服接种AasdC500(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ΔasdC500(pYA-FIP2)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,ΔasdC500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,可作为沙门氏茵Asd+平衡表达系统开发基因工程重组疫苗.     

2018-2022年佛山地区小儿沙门氏菌感染的临床特征和耐药性分析

摘要 目的：分析2018-2022年佛山地区小儿沙门氏菌感染的临床特征和耐药性,为小儿沙门氏菌感染的临床防治提供依据。方法：对2018年1月1日至2022年12月31日期间佛山市妇幼保健院收集的小儿沙门氏菌感染病例186例进行回顾性分析,分析患儿一般资料、小儿沙门氏菌感染的时间分布特征、临床特征、血清型分布及耐药性。结果：186例小儿沙门氏菌感染患儿年龄分布,1~3岁构成比最高,占37.63%,>6岁构成比最低,占1.61%。2018年发病33例,2019年发病19例,2020年发病39例,2021年发病44例,2022年发病51例,沙门氏菌感染病例数总体呈现上升趋势;一年四季均有沙门氏菌感染病例,夏秋两季发病率最高,分别占43.55%、30.11%。发热、腹泻、解粘液便为主要症状,分别占90.32%、74.73%和63.98%。血清型B群最高,为118例,占63.44%。通过对分离出的191株沙门氏菌药物敏感性试验分析,只有3株（1.57%）对所检测抗菌药物均不耐药,耐3类或3类以上的多重耐药沙门氏菌有157株（82.20%）,庆大霉素、头孢唑啉、头孢替坦耐药率均为100%。不同年份沙门氏菌对左氧氟沙星、环丙沙星、四环素的耐药率差异有统计学意义（P<0.05）。结论：近年来佛山地区小儿沙门氏菌感染呈上升趋势,1~3岁儿童是主要群体,夏季和秋季是发病的高峰季节,主要血清型是B群,儿童感染后以发热、腹泻、解粘液便为主要症状,沙门氏菌的耐药性较为严重,应引起相关部门重视。     

稳定携带H5亚型禽流感病毒候选DNA疫苗减毒沙门氏菌的构建及其免疫原性

采用PCR技术从重组质粒pVAX1-HA扩增出禽流感病毒JSGO(H5N1)株的血凝素(HA)基因,将其克隆入真核表达质粒pmcDNA3.1 中,获得重组表达质粒pmcDNA3.1-HA。通过电穿孔转化法将重组质粒转入减毒鼠伤寒沙门氏菌SL7207*,构建成功携带DNA疫苗的重组沙门氏菌SL7207*(pmcDNA3.1-HA)。经体内体外试验证实,重组质粒pmcDNA3.1-HA在沙门氏菌中的稳定性显著高于pcDNA3.1-HA。将重组菌SL7207*(pmcDNA3.1-HA)和SL7207*(pcDNA3.1-HA)分别以2×109CFU剂量两次口服免疫BALB/c小鼠,免疫小鼠可产生针对禽流感病毒HA蛋白的黏膜抗体。重组菌以5×109CFU剂量两次口服免疫试验鸡,免疫鸡的小肠样品中可测到针对禽流感病毒HA蛋白的黏膜抗体,且SL7207*(pmcDNA3.1-HA)免疫组的抗体效价高于SL7207*(pcDNA3.1-HA)免疫组。免疫保护试验结果显示,SL7207*(pmcDNA3.1-HA)和SL7207*(pcDNA3.1-HA)免疫组的免疫保护率均与空载体组之间存在显著性差异(P<0.05),且SL7207*(pmcDNA3.1-HA)免疫组的保护率较SL7207*(pcDNA3.1-HA)免疫组提高了22.6%,说明稳定携带H5亚型禽流感病毒DNA疫苗的减毒沙门氏菌具有良好的免疫原性和免疫保护性。