鸡致病性大肠杆菌O_1、O_2和O_(78)Ⅰ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 1 pili structure gene of chicken pathogenic Escherichia coli serotype O_1,O_2 and O_(78)

提取鸡致病性大肠杆菌分离株O1、O2和O78的基因组DNA作模板,用TD-PCR技术从其中分别扩增出0.55KB的I型菌毛结构基因(piliA)。将扩增得到的piliA基因片段,用TA克隆的方法分别克隆进pGEM-T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,提取质粒用PstI单酶切及NcoI和PstI双酶切鉴定,结果证实,所构建的克隆质粒中均含有相应piliA基因。经DNA序列分析,其结构基因阅读框架大小为549bp,但其中O1菌株I型菌毛基因在第72位发生突变,有6个碱基插入。经DNAStar核酸分析软件分析,3个基因同源性为89.9%～92.0%。

Type 1 pili structre gene （piliA） from chicken pathogenic Escherichia coli O1,O2 and O78 by Polymerase Chain Reaction （PCR）, PCR products were isolated by 1% agarose gel eleetrophoresis and recovered by PCR Preps DNA Purification System, then They were inserted pGEM-T vector. By transformation of JM109 and screening with Amp/IPTG/X-gal agar plate, we got recombinant strains JMI09 （pTFimO1, pTFimO2 and pTFimOTs）. These recombinant plasmids pTFimO1, pTFimO2 and pTFimO78 were studied in detail by restriction endonuclease analysis. The results have shown that these recombinant plasmids carried type 1 pili structure gene. Homologous were compared and analyzed with the help of DNAStar programs. Similarity of piliA gene were 89.9%-92.0%.