3株致病性鸡大肠杆菌P型菌毛结构基因的克隆、序列测定及同源性分析  

Cloning and Sequence Analysis of Type P Pilin Structural Gene in Pathogenic Escherichia coli from Chichen

作　　者：苗玉和[1] 王文成[1] 张贵刚[1] 

机构地区：[1]辽宁省益康生物制品厂,辽宁辽阳111000

出　　处：《中国兽医学报》2005年第3期250-252,共3页Chinese Journal of Veterinary Science

摘　　要：用鸡致病性大肠杆菌分离株O1、O2和O78的基因组DNA做为模板,PCR分别扩增出了0.55kb的P型菌毛结构基因(papA)。将扩增得到的3个papA基因片段分别克隆进pGEM-T○R载体中,转化至受体菌JM109中,用Amp/IPTG/X-gal琼脂平板蓝白菌落筛选法,得到含阳性重组子的菌株,提取质粒后用BamH和Sal双酶切进行鉴定。所得阳性重组子进行DNA序列测定,测序结果经DNAStar核酸分析软件包分析比较,结果表明,所构建的克隆质粒中均含有相应完整papA基因,其开放式阅读框架大小为549bp,编码182个氨基酸。此3株菌的P型菌毛结构基因的同源性为98.9%～100%,其中O1株和O78株的P型菌毛基因的ORF序列100%相同,O2株有2个碱基与前两者不同。Three type P pilin structural genes(papA) were amplified by TD-PCR whose templates came from three pathogenic E.coli isolates-O_1,O_2,and O_~78 .The three amplified P genes were all 0.55 kb,which were inserted into pGEM-T vector,then transformed to JM109 bacteria and screened with Amp/IPTG/X-gal agar plate.Recombinant strains JM109 and Recombinant plasmids were obtained.These recombinant plasmids were identified by restriction endonuclease BamHⅠ and SalⅠ and sequenced.Sequence analysis showed that these recombinant plasmids carried completely type P pilin structural gene containing a ORF of 549 bp.These type P genes encoded 182 aa.Homologous were analyzed with the DNAStar programs.These P genes had very high homologous about 98.9%-100%.The O_1 P ORF nucleotide sequence was 100% homologous with that of the O_~78 .There were only 2 bases different between O_2 and O_1,O_~78 .

关 键 词：结构基因 P型菌毛 鸡大肠杆菌 同源性分析 克隆 鸡致病性大肠杆菌 DNA序列测定 基因组DNA 分析软件包 基因片段 琼脂平板 IPTG 分析比较 Star 菌毛基因 重组子 分离株 PCR 受体菌 筛选法 Amp Sal 双酶切 开放式 氨基酸 

分 类 号：S852.612[农业科学—基础兽医学] Q78[农业科学—兽医学]