肠产毒性大肠埃希菌F4ac菌毛蛋白FaeG亚单位的原核表达及免疫学鉴定

发布时间：2018-06-07 06:18

  本文选题：肠产毒性大肠埃希菌 + 菌毛亚单位FaeG　； 参考：《重庆医科大学》2011年硕士论文

【摘要】：目的 克隆并表达肠产毒性大肠埃希菌(ETEC)F4ac菌毛蛋白亚单位FaeG,鉴定其抗原性和免疫原性,为制备预防幼畜ETEC感染的疫苗奠定基础。 方法 以ETEC(C83902)基因组DNA为模板,PCR扩增faeG基因,插入原核表达质粒pGEX-6P-1,构建重组质粒pGEX-faeG。将pGEX-faeG转化大肠埃希菌BL-21,IPTG诱导表达。SDS-PAGE分析表达蛋白的相对分子质量和表达形式,Western blot鉴定其抗原性。将表达菌超声破碎后离心提取包涵体制备抗原,经口灌喂免疫小鼠,检测小鼠血清中抗FaeG的IgG、IgA,粪便IgA和肠粘液IgA,鉴定其免疫原性。重组蛋白FaeG口服免疫小鼠,进行动物免疫保护实验。 结果 扩增的faeG基因全长786bp,与基因文库中的faeG基因同源性达96%。重组质粒pGEX-faeG经PCR及双酶切鉴定确有插入片段,且序列完整。表达产物FaeG相对分子质量约53KD,主要存在于碎菌后的沉淀中,以包涵体形式表达。Western blot显示该蛋白可与ETECF4ac阳性血清特异性结合,免疫后小鼠体内抗FaeG IgG、IgA明显高于PBS对照组和GST对照组。免疫小鼠至少能抵抗1MLD的大肠埃希菌强毒株C83902的攻击。 结论 成功构建了ETEC F4ac菌毛蛋白亚单位FaeG的重组质粒pGEX-faeG,表达了重组蛋白FaeG,该蛋白具有良好的抗原性和免疫原性,可用于研制预防幼畜ETEC感染的疫苗。
[Abstract]:Purpose To clone and express Escherichia coli ETECF4ac fimbrial protein subunit FaeG to identify its antigenicity and immunogenicity, and to lay a foundation for the preparation of vaccine against ETEC infection in young animals. Method The faeG gene was amplified from the genomic DNA of ETEC-C83902 and inserted into the prokaryotic expression plasmid pGEX-6P-1 to construct the recombinant plasmid pGEX-faeG. The expression of pGEX-faeG transformed into Escherichia coli BL-21 was induced by IPTG. SDS-PAGE was used to analyze the relative molecular weight and expression form of the expressed protein. Western blot was used to identify its antigenicity. The antigen was extracted from the inclusion bodies by ultrasonic fragmentation, and the immunogenicity of the immunized mice was determined by oral administration of IgGG FaeG, fecal IgA and intestinal mucus IgA. Mice were immunized orally with recombinant protein FaeG, and the animal immune protection test was carried out. Result The total length of the amplified faeG gene was 786 BP, which was similar to that of the faeG gene in the gene library. The recombinant plasmid pGEX-faeG was confirmed by PCR and double enzyme digestion and its sequence was complete. The relative molecular weight of the expressed product FaeG was about 53kD, which mainly existed in the precipitate after breaking bacteria. The protein was expressed in the form of inclusion body. Western blot showed that the protein could specifically bind to ETECF4ac positive serum. The anti-IgA of FaeG in immunized mice was significantly higher than that in PBS and GST control groups. The immunized mice could resist at least the attack of Escherichia coli strain C 83902 of 1MLD. Conclusion The recombinant plasmid pGEX-faeG of ETEC F4ac pili protein subunit FaeG was successfully constructed, and the recombinant protein FaeG was expressed. The recombinant protein has good antigenicity and immunogenicity, and can be used to develop a vaccine to prevent ETEC infection in young animals.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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