大肠杆菌野生株K99菌毛蛋白结构基因的克隆与序列分析

根据产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)K99菌毛蛋白的全基因序列设计产肠毒素大肠杆菌主要菌毛K99的一对引物。PCR扩增K99菌毛蛋白的全基因序列大小为546bp。将PCR扩增的目的片断克隆于pMD18-T载体、pET-32a载体中,分别转化大肠杆菌,经酶切鉴定、PCR鉴定及DNA序列分析,筛选出阳性克隆。经过序列比对,所克隆的外源基因与报道的K99菌毛蛋白结构基因序列同源性达99.3%。成功克隆分离到的产肠毒素大肠杆菌菌毛的K99基因,为当地产肠毒素性大肠杆菌病疫苗的选择及基因疫苗的研制提供了坚实的理论基础,为产肠毒素性大肠杆菌病检测、预防及基因工程疫苗的研制开辟新的途径。

Structure Gene Cloning and Sequence Analysis of K99Pili Protein of ETEC from Wild Strains

According to the reported sequence of K99 pili protein gene of Enterotoxigenic Escherichia coli(ETEC),a pair of primers of K99 were designed and synthesized. The K99 sequence of 546bp from isolated strain was amplified by polymerase chain reaction (PCR). The fragment was cloned to pMD 18 -Tvector and pET-32a vector respectively,then the recombinant plasmid was transferred into E. coli. The recombinant plasmid was identified by the methods of restriction endonuclease digestion,PCR and sequence analysis. The homologies between the reference strain C83912 and isolated strain were 99.3%. This work provided a new method for selecting of local vaccine,detecting and preventing ETEC that cause neonatal diarrhea in calves.