金黄色葡萄球菌多联融合毒素的表达及免疫特性研究

发布时间：2018-09-19 14:41

【摘要】： 以食物中毒菌金黄色葡萄球菌毒素为研究对象,利用PCR方法分别扩增出金黄色葡萄球菌肠毒素SEB1、SEB2、SED1、SED2、SEE基因片段。将克隆得到的五个毒素基因片段采用柔性的Linker序列(Gly4Ser)进行基因串联(SEB1-SEB2-SED1-SED2-SEE),通过PCR重叠延伸法扩增出了融合毒素T,目的基因全长序列1835bp,包括2个Linker序列,并定向克隆到表达质粒pET-28a(+)的NdeI和HindIII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29.6%)。将表达蛋白的包涵体经过纯化,分别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以不同的抗体效价证明保留了五种天然毒素的各自抗原性,与多种非目标菌和毒素不反应,具有较好的特异性。说明多个基因串联至少可达2000 bp仍能在原核载体表达,至少包含2个Linker而不影响串联基因的抗原结构特性,表达产物可诱导机体产生广谱反应特性的抗体。为融合毒素T的进一步应用研究以及利用融合毒素的方法检测食物中毒菌,进而建立食物中毒菌广谱、快速的检测方法奠定了基础。
[Abstract]:Staphylococcus aureus enterotoxin SEB1,SEB2,SED1,SED2,SEE gene fragment was amplified by PCR method from Staphylococcus aureus toxin of food poisoning bacteria. The five toxin gene fragments were amplified by using flexible Linker sequence (Gly4Ser) for gene tandem (SEB1-SEB2-SED1-SED2-SEE). The fusion toxin T was amplified by PCR overlap extension. The full-length sequence of the target gene was 1835 BP, including two Linker sequences. The recombinant expression plasmid pET-28a () -Twas constructed between the NdeI and HindIII sites of the expression plasmid pET-28a (), and was effectively expressed in E. coli BL21 (DE3). The expressed protein was mainly in the form of inclusion body (29.6%). The inclusion bodies of the expressed protein were purified, and the serum antibodies were prepared by immunizing Jjong Rex rabbits. The results of ELISA and Agar diffusion test showed that the expressed fusion toxin T retained the antigenicity of the five natural toxins with different antibody titers. It does not react with a variety of non-target bacteria and toxins and has good specificity. The results showed that at least 2000 bp could be expressed in prokaryotic vector, and at least 2 Linker could be expressed in the prokaryotic vector without affecting the antigenic structure of the tandem gene. The expressed product could induce the body to produce antibodies of broad-spectrum reaction. It lays a foundation for the further application of fusion toxin T and the detection of food poisoning bacteria by the method of fusion toxin, and the establishment of a broad spectrum of food poisoning bacteria and a rapid detection method.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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