F18菌毛粘附素F基因(fedF)在大肠杆菌中的表达及对小鼠的免疫保护效果  

Expression of F18 Fimbrial Adhesion F Antigen Gene(fedF) in Escherichia coli and Its Immunoprotection in BALB/c Mice(Mus musculus)

作　　者：李卫芬[1] 朱海燕[1] 余东游[1] 傅罗琴[1] 李梅[1] 高研[1] 姜中其[1] 

机构地区：[1]浙江大学动物科学学院教育部动物分子营养重点实验室,杭州310029

出　　处：《农业生物技术学报》2012年第9期1065-1071,共7页Journal of Agricultural Biotechnology

基　　金：浙江省重大科技专项(No.2006C12086)

摘　　要：引起早期仔猪断奶腹泻(PWED)的主要致病菌是产肠毒素型大肠杆菌(ETEC),免疫预防是防治腹泻最主要的手段。本研究采用通过大肠杆菌(Escherichia coli)表达的重组F18菌毛粘附素F(FedF)作为抗原,在小鼠(Mus musculus)中探索以此为靶标免疫防治仔猪腹泻的可能性。根据相应F18菌毛粘附素基因(fedF)设计了一对添加EcoRⅠ和SalⅠ酶切位点的引物,以E.coli F18为模板扩增出全长908bp含20个信号肽序列的fedF片段,并与原核表达载体pET-30a(+)连接,导入大肠杆菌BL21,得到基因工程菌E.coli[pET-fedF],后经IPTG诱导及菌液蛋白电泳分析,结果表明,大小约为32.9kD的重组蛋白FedF可成功诱导表达;将重组工程菌口服免疫BALB/c小鼠,可有效地诱导小鼠血清中FedF特异性IgG的产生(P/N值>2.0)及小鼠肠粘膜sIgA显著增加了2.07倍(与空载体组pET-30比较)(P<0.05);攻毒试验表明,口服基因工程菌E.coli[pET-fedF]可显著地有效保护小鼠免予攻毒死亡,免疫保护率可达62.5%。研究结果提示,FedF黏附蛋白基因工程菌可通过口服诱导小鼠特异的体液和粘膜免疫反应,以此保护小鼠免受产肠毒素型大肠杆菌的进攻,因而有望作为一种口服疫苗防治产肠毒素型大肠杆菌引起的仔猪腹泻。Enterotoxigenic Escherichia coli （ETEC） is the main cause of Piglet early weaning diarrhea （PWED） and immunoprophylaxis is one of the most efficient measure to solve this problem. This paper aims to use E. coli F18 fimbrial adhesion F（FedF） recombinantly expressed as antigen to explore in mice （Mus musculus） the possibility of using FedF as the target to prevent PWED. Primers added with EcoR I and Sol I sites were devised and synthesized according to the sequence F18 fimbriae antigen genefedF on GenBank. Full lengthfedF gene fragment of 908 bp with 20 signal peptide sequence was amplified by using E. coli F18 genome as template. Then the target fragment was subcloned into the prokaryotic expression vector pET-30a （＋） and the recombinant plasmid pET-fedF was transformed into E.coli BL21. After induction with IPTG, recombinant FedF about 32.9 kD was detectable. Then BALB/c mice were orally immunized by the recombinant strain E. coli [pET-fedF]. The results showed that the recombinant strain could induce high level of sera FedF specific IgG （P/N 〉2.0） and significant increase in intestinal sIgA by 2.07 fold （P〈0.05）; finally oral immunization of the recombinant strain could greatly protect mice against lethal dose ofenterotoxin E. coli administration, the immune protective rate reached up to 62.5%. All the above results implied that the recombinant strain E. coli [pET-fedF] can protect mice from ETEC attach by induction of sera FedF specific IgG and intestinal sIgA, and it may be a promising weapon against PWED.

关 键 词：F18菌毛 粘附素F基因(fedF) 克隆表达 免疫 

分 类 号：S852.612[农业科学—基础兽医学]