F18ac菌毛A亚单位基因的克隆与序列分析

Cloning and identification of the minor subunit fed A gene of the fimbrian F18ac

根据已经发表的F18ab菌毛A亚单位 (FedA/ab)的基因 (fedA/ab) [1] ,设计一对引物 ,利用PCR技术从表达F18ac菌毛的大肠杆菌 2 134P株[2 ] 、8199株[3 ] 、8813株[3 ] 中分别扩增到一段序列 ,并克隆至 pGEM_T载体 ,获得重组质粒T2 134PA、T8199A、T8813A。琼脂糖凝胶电泳、序列测定及分析表明 ,该 3个序列大小均为 5 16bp ,与fedA/ab(5 13bp)具有较高的同源性 ,分别为 96 3%、96 5 %、95 9% ,推导的Fed/ac氨基酸序列与FedA/ab同源性分别为 93 0 %、93 6 %、92 4 %。数据表明该实验所克隆的序列均为F18ac菌毛A亚单位 (FedA/ac)的基因 (fedA/ac)。

A pair of primers were designed and synthesized according to fed A of fimbriae F18ab(fedA/ab),and the coordinate subunit genes of F18ac(fedA/ac)were amplificated from three different Escherichia coli strains,2134P、8199 and 8813,which are all display fimbriae F18ac.The PCR products of fedA/ac were cloned into pGEM_T vector.Color screening,PCR and restriction endonucleases analysis were used to identify the recombinant plasmids,and three recombinant plasmids,T2134PA,T8199A and T8813A were obtained.The three recombinant plasmids sequences were analyzed,and compared with the published sequences of fedA/ab.The data of sequences for the three fedA/ac genes were shown that their size were 516 bp,and shared 96.3 %,96.5 % and 95.9 % identity at nucleotide level with fedA/ab respectively.The data of deduced amino acid were shown that they are shared 93.0 %,93.6 % and 92.4 % identity with FedA/ab respectively.All the data in this study conformed the three genes that derived from the three different Escherichia coli strains,2134P,8199 and 8813,are all fedA/ac genes of F18ac.