沙门菌三种血清型鞭毛基因重组及免疫学检测方法研究

发布时间：2018-06-27 03:17

  本文选题：沙门菌鞭毛 + 基因重组　； 参考：《吉林大学》2008年硕士论文

【摘要】： 强致病性的甲型副伤寒沙门菌、鼠伤寒沙门菌和猪霍乱沙门菌Ⅰ相鞭毛抗原的血清型分别为a、i、c。针对这三种沙门菌Ⅰ相鞭毛抗原H1-a、H1-i、H1-c的编码基因设计三对扩增引物。首先,利用常规PCR技术扩增出三段Ⅰ相鞭毛抗原基因,再利用引物上的Linker和重叠延伸PCR扩增技术将此三段抗原决定区基因序列串联,获得鞭毛重组基因。构建鞭毛重组基因的原核表达载体pET-28a-F,并将重组表达质粒转化大肠杆菌BL21株。表达菌在终浓度为1mmol/L的IPTG,37℃下诱导6h后,SDS-PAGE电泳分析得到鞭毛融合蛋白大小约为95Ku,表达量约为11.2%。含有目的蛋白的包涵体经初步纯化后,免疫豚鼠获得血清抗体。琼脂扩散试验和间接ELISA测定抗体效价可达到1:512000,此时抗体的敏感性为8μg/mL;血清抗体稀释10000×时对甲型副伤寒沙门菌、鼠伤寒沙门菌、猪霍乱沙门菌检出值分别为:1.35×10~5个/mL、1.63×10~5个/mL、2.31×105个/mL;用10种沙门菌和15种非沙门菌属的其他食物中毒菌做免疫交叉反应,结果显示特异性较好,同时抗体显示很好的广谱性,即能和相同鞭毛血清型的沙门菌菌群反应。纯化血清抗体并进行胶体金免疫层析实验,对试纸条检测方法做了初步研究,为食品中沙门菌的快速检测技术奠定基础。
[Abstract]:The serotypes of strongly pathogenic Salmonella paratyphoid A, Salmonella typhimurium in mice and Salmonella cholerae in swine in the first phase of flagellum antigen were as follows. Three pairs of amplified primers were designed for the coding genes of the flagellum antigen H1-aH1-iH1-c of these three Salmonella species. Firstly, the three-segment I flagellum antigen gene was amplified by conventional PCR technique, and then the gene sequence of the three antigen-determining region was sequenced by Linker and overlapping extension PCR on the primer to obtain the flagellum recombinant gene. The prokaryotic expression vector pET-28a-Fof flagellum recombinant gene was constructed and transformed into Escherichia coli BL21 strain. The expression strain was induced at 37 鈩，

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