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

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-faeC，表达了重组蛋白FaeG，该蛋白具有良好的抗原表达了重组蛋白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。

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

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 for...