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  

Prokaryotic expression and immunological identification of F4ac Fimbrial Subunit FaeG of ETEC

作　　者：于拽拽[1] 杨致邦[1] 周裕珍 熊玉霞[1] 胡洪铭 蒋任举[1] 

机构地区：[1]重庆医科大学神经科学研究中心,重庆400016 [2]海南裕昌龙实业有限公司大昌养猪场,海南海口570125

出　　处：《中国微生态学杂志》2011年第3期204-207,共4页Chinese Journal of Microecology

基　　金：海口市重点科技计划项目(海科立[2009]29号)

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,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-21I,PTG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IgGI、gA,鉴定其免疫原性。结果扩增的faeG基因全长786 bp,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53 kD,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ETEC F4ac阳性血清特异性结合,免疫后小鼠血清抗FaeG IgGI、gA明显高于PBS和GST对照组。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。Objective To clone and express F4ac fimbrial subunit gene faeG of Enterotoxigenic Escherichia coli（ETEC） in order to establish a foundation for preparing the vaccine to prevent ETEC infection of young animals. Method The faeG gene fragment of ETEC（C83902） genomic DNA was amplified by PCR and inserted into prokaryotic expression plasmid pGEX-6P-1.The constructed recombinant plasmid pGEX-faeG was transformed into E.coli BL-21 and expressed through induction of IPTG.The expressed product was determined for form and relative molecular mass by SDS-PAGE and identified for antigenicity by Western blot.The mice were immunized with the inclusion-bodies extracted from the sonicated bacterial cells.The IgG and IgA levels in sera were determined to identify the immunogenicity of the expressed product. Result The sequence of the amplified faeG gene was about 786 bp and shared 96% identity with the pulished sequence in GenBank.Both PCR and restriction enzyme analysis showed that the recombinant plasmid pGEX-faeG contained the insertion element with complete sequences.The expressed product with a relative molecular mass of 53 kD existed mainly in the form of inclusion body.Western blot showed specific reaction of the expressed recombinant FaeG protein with the F4ac-positive sera of pigs.The IgG and IgA levels of mice immunized with FaeG were significantly higher than those of control mice. Conclusion The recombinant plasmid pGEX-faeG contained F4ac fimbrial subunit gene faeG of ETEC was constructed and expressed successfully.The recombinant protein show good antigenicity and immunogenicity,and may be used for the development of ETEC vaccine to prevent ETEC infection of young animals.

关 键 词：肠产毒性大肠埃希菌 菌毛亚单位FaeG 表达 抗原性 免疫原性 

分 类 号：R378.21[医药卫生—病原生物学]