金黄色葡萄球菌三联融合毒素的表达及免疫特性研究

发布时间：2018-01-05 18:01

本文关键词：金黄色葡萄球菌三联融合毒素的表达及免疫特性研究　出处：《吉林大学》2007年硕士论文　论文类型：学位论文

【摘要】： 以金黄色葡萄球菌肠毒素为研究对象,利用PCR方法分别扩增出金黄色葡萄球菌肠毒素SEA、SEB、SEE全基因片段。将克隆得到的三个毒素基因片段采用柔性的Linker序列(Gly4Ser)进行基因串联(SEA-SEB-SEE),通过PCR延伸法,扩增出了融合毒素基因T,目的基因全长序列2190bp,包括2个Linker序列,并定向克隆到表达质粒pET-28a(+)的EcoR I和XhoI位点之间,构建了重组表达质粒pET-28a(+)-T,且在表达宿主菌E. coli BL21 (DE3)中得到有效表达。表达蛋白主要以包涵体形式存在(表达量为26.4%)。将表达的包涵体蛋白经过纯化,免疫吉戎獭兔制备血清抗体,经ELISA和琼脂扩散试验检测表明,表达的融合毒素T具有较好的抗原性,其抗体能够与三种天然毒素发生免疫反应,与多种非目标菌和毒素不反应,具有较好的特异性。表明多个基因串联可达2190bp仍能在原核载体表达,表达产物可诱导机体产生具有广谱反应特性的抗体。本研究利用融合毒素广谱抗体建立食物中毒菌快速检测方法奠定基础。
[Abstract]:Staphylococcus aureus enterotoxin was amplified by PCR. SEE complete gene fragment. The three toxin gene fragments were cloned using the flexible Linker sequence Gly4Ser. to carry out the gene series SEA-SEB-SEE). The fusion toxin gene T was amplified by PCR extension. The target gene was 2190bp, including two Linker sequences. The recombinant expression plasmid pET-28a (pET-28a) was constructed by cloning into the EcoR I and XhoI sites of the expression plasmid pET-28a (pET-28a ()). The expressed protein was mainly in the form of inclusion body (26.4%). The expressed inclusion body protein was purified. Serum antibodies were prepared by immunizing Jjong Rex rabbits. ELISA and Agar diffusion tests showed that the expressed fusion toxin T had good antigenicity and its antibody could react with three natural toxins. No reaction with a variety of non-target bacteria and toxins showed that multiple genes could be expressed in prokaryotic vector in tandem with 2190bp. The expression product can induce the production of antibodies with broad-spectrum reaction. This study established a rapid detection method for food poisoning bacteria by using the fusion toxin broad-spectrum antibody.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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