本文選題：腸產(chǎn)毒性大腸埃希菌 + 菌毛亞單位FaeG　； 參考：《重慶醫(yī)科大學》2011年碩士論文

【摘要】：目的 克隆并表達腸產(chǎn)毒性大腸埃希菌(ETEC)F4ac菌毛蛋白亞單位FaeG,鑒定其抗原性和免疫原性,為制備預防幼畜ETEC感染的疫苗奠定基礎。 方法 以ETEC(C83902)基因組DNA為模板,PCR擴增faeG基因,插入原核表達質粒pGEX-6P-1,構建重組質粒pGEX-faeG。將pGEX-faeG轉化大腸埃希菌BL-21,IPTG誘導表達。SDS-PAGE分析表達蛋白的相對分子質量和表達形式,Western blot鑒定其抗原性。將表達菌超聲破碎后離心提取包涵體制備抗原,經(jīng)口灌喂免疫小鼠,檢測小鼠血清中抗FaeG的IgG、IgA,糞便IgA和腸粘液IgA,鑒定其免疫原性。重組蛋白FaeG口服免疫小鼠,進行動物免疫保護實驗。 結果 擴增的faeG基因全長786bp,與基因文庫中的faeG基因同源性達96%。重組質粒pGEX-faeG經(jīng)PCR及雙酶切鑒定確有插入片段,且序列完整。表達產(chǎn)物FaeG相對分子質量約53KD,主要存在于碎菌后的沉淀中,以包涵體形式表達。Western blot顯示該蛋白可與ETECF4ac陽性血清特異性結合,免疫后小鼠體內(nèi)抗FaeG IgG、IgA明顯高于PBS對照組和GST對照組。免疫小鼠至少能抵抗1MLD的大腸埃希菌強毒株C83902的攻擊。 結論 成功構建了ETEC F4ac菌毛蛋白亞單位FaeG的重組質粒pGEX-faeG,表達了重組蛋白FaeG,該蛋白具有良好的抗原性和免疫原性,可用于研制預防幼畜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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392.1

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