PCR法对单核细胞增生李斯特菌食品分离株的鉴定

〔目的〕采用PCR法对常规法分离的八株单核细胞增生李斯特菌 (LM )再鉴定 ,以验证PCR法作为检测LM的有效性及可靠性。〔方法〕八株从食品中分离的LM在BHI培养基纯培养 2 4h后 ,提取基因组DNA ,用PCR扩增hly基因特异性 85 6bp的片段。〔结果〕八株从食品中分离并经传统方法鉴定的LMPCR扩增均出现特异性的条带 ,而其他属的李斯特菌和非李斯特菌未出现特异性的条带。〔结论〕采用hly基因作为PCR扩增的目标基因检测LM具有较好的特异性 ,可用于食品中LM的检测与分离。     

8类食品单核细胞增生李斯特菌流行特征及耐药性状研究

目的：了解扬州市8类食品中Lm污染状况、流行特征及耐药状况，为制定HACCP提供依据，以有效控制Lm的生物性危害。方法：从市场及宾馆饭店采集957份熟食、生肉制品、水产品等8类食品检测Lm，检出菌株以生化及PCR方法鉴定并做药物敏感试验。结果：共检出加43株，检出率为4．49％。其中熟肉制品检出率为9．62％，生肉制品为3．83％，蔬菜、牛奶、鲜鸡蛋及冰淇淋中未检出。17种抗生素中有9种抗生素出现了不同程度的耐药。结论：本市8种食品中存在一定程度Lm污染，以熟食污染率最高。生肉制品为食品链中Lm的主要来源，熟食是实施HACCP控制Lm的关键环节。Lm耐药现象在一定程度上存在。加强食品卫生管理尤其是熟肉制品的管理，控制动物饲料亚治疗抗生素的使用并严格遵守休药期对防止耐药菌株、控制食源性疾病很有必要。     

肠炎沙门菌随机扩增多态性DNA分子分型

目的对不同来源的12株肠炎沙门菌分离株进行分子分型。方法分别提取不同菌株基因组DNA,建立和优化随机扩增多态性DNA（RAPD）反应体系,试用22条随机引物对不同菌株进行RAPD扩增,筛选清晰稳定的扩增条带做统计分析。结果筛选到OPG-03、OPG-06、OPG-07和OPG-13共4条随机引物具有鉴别作用,每一菌株均可扩增出4~10条DNA片段,大多数片段在100~2000bp之间,共扩增出42条RAPD条带,其中共有条带7条,多态性条带35条,多态性为83.3%。不同来源的肠炎沙门菌分离株之间的遗传相似系数在42.6%~100%之间,在0.850的相似性水平上可将12株肠炎沙门菌分离株分为5个不同的亚型。结论应用RAPD技术在分子水平上对肠炎沙门菌进行鉴别和分型具有简便、快速和辨别能力高的优势,对肠炎沙门菌的分子流行病学研究具有较好的应用前景。     

柠檬酸和温度联合处理对海水鱼中副溶血性弧菌的杀灭作用

采用响应曲面分析法对副溶血性弧菌的抑制条件进行优化。对副溶血性弧菌死亡率影响因素进行单因素分析后,以Box-Benhnken 设计(BBD)评价温度、时间、pH3个因素显著性和交互作用,得出副溶血性弧菌最佳净化条件为:温度55.5 ℃、时间25 s、pH4.96。在该条件下, 副溶血性弧菌死亡率的预测值为97.36%,在三文鱼上验证该值达97.07％,并且该条件处理前后鱼肌原纤维蛋白的Ca²⁺-ATPase 活性没有显著差异。     

我国地方品种姜曲海猪TLR5基因的克隆、表达及鉴定

目的:克隆我国地方品种姜曲海猪TLR5基因,构建该基因的原核表达质粒,获得融合表达蛋白并鉴定其免疫特性,为进一步研制TLR5单克隆抗体(mAb)提供免疫原。方法:从全血中提取姜曲海猪的基因组,设计特异性引物,利用PCR方法扩增得到TLR5基因片段,PCR产物克隆到原核表达载体pET30a(+)中,构建重组表达质粒pET-TLR5,将重组表达质粒转化E.coli BL21,0.5 mmol/L IPTG诱导表达目的蛋白,应用SDS-PAGE和Western blot分析鉴定蛋白活性。结果:成功扩增出猪TLR5基因片段,大小为2 571 bp。酶切鉴定结果表明,猪的TLR5基因成功克隆入原核表达载体pET30a(+)中,重组质粒pET-TLR5在大肠杆菌中获得表达,SDS-PAGE结果显示出相对分子质量(Mr)大小约95 200;Western blot结果表明,表达产物与兔抗小鼠TLR5 mAb具有良好的反应性。结论:成功克隆并表达具有较好免疫原性的猪TLR5分子,为其mAb的制备提供生物材料。     

重组沙门菌表达结核分枝杆菌ESAT-6及其特异性免疫应答分析

Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.     

重组沙门菌表达结核分枝杆菌ESAT-6及其特异性免疫应答分析

Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.     

流式细胞术检测重组LLO溶血的效果

目的：分析rLLO在溶血过程中对CD45^＋细胞的影响。方法：利用流式细胞术，通过与商品化裂解液和自制NH4C1裂解液的比较，观察rLLO与以上2种裂解液裂解效果的差异。结果：rLLO具有较强的裂解红细胞的能力，裂解效果与商品化裂解液和自制NH4C1裂解液基本一致。另外，三种裂解液对细胞膜的渗透性均有较大影响。结论：rLLO对细胞膜CD45表面抗原的损伤程度低于商品化裂解液。