禽源致病性大肠埃希菌安徽分离株I型菌毛pilA基因的序列测定与分析

Sequencing and analysis of typeⅠfimbrin pilA gene from avian pathogenic Escherichia coli isolated in Anhui province

目的探明大肠埃希菌I型菌毛pilA基因在不同宿主来源菌株间的同源性,为利用Ⅰ型菌毛基因诊断和防治大肠埃希菌病提供理论基础。方法以禽源致病性大肠埃希菌安徽分离株基因组DNA为模板,采用PCR方法扩增Ⅰ型菌毛pilA基因,并进行序列测定与分析。结果 13株不同血清型禽源致病性大肠埃希菌安徽分离株均携带pilA基因,彼此间该基因的核苷酸序列和氨基酸序列同源性分别介于84.9%～99.7%和86.2%～99.2%。pilA基因核苷酸序列在安徽分离株与鸡源参考株、猪源参考株以及人源参考株之间的同源性分别为85.9%～99.7%、85.9%～93.9%和87.5%～100%,氨基酸序列同源性分别为83.1%～99.2%、88.5%～93.8%和90%～100%。系统发育进化树分析显示JD16、JD34、JD11、JD24、YD2、JD8和YD1禽源安徽分离株与人源参考株sPH2的亲缘关系较近,在进化树的同一分支上;GD3、YD5、GD2、YD3、YD5和YD7禽源安徽分离株与鸡源参考株PDI-386和猪源参考株107/86的亲缘关系较近,在进化树中属于同一分支;GD1禽源安徽分离株与鸡源参考株HY1-2和MS2-1的亲缘关系较近,在进化树中属于另一分支。结论大肠埃希菌I型菌毛pilA基因在不同宿主来源菌株及不同血清型菌株之间高度保守,可作为大肠埃希菌病的诊断基因和疫苗候选基因。

Objective To investigate the homology of the typeⅠfimbrin pilA gene from Escherichia coli（E.coli） strains isolated from different hosts for the future diagnosis,prevention and treatment of colibacillosis with the typeⅠfimbrin pilA gene.Method The genomic DNA samples of avian Escherichia coli Anhui isolates were used as the templates for polymerase chain reaction（PCR） to amplify the pilA gene encoding the major subunit protein of type I fimbrin,and then pilA genes were sequenced and analyzed respectively.Result All 13 isolates carried the pilA gene.The homology of nucleotide sequence and amino acid sequence of pilA gene was from 84.9% to 99.7% and from 86.2% to 99.2% respectively among the 13 isolates.The homology of nucleotide sequence of pilA gene was 85.9% ～ 99.7%,85.9% ～ 93.9% and 87.5% ～ 100% respectively between the isolates and reference strains of chicken source,swine source and human source,and the homology of amino acid sequence of pilA gene ranged from 83.1% to 99.2%,from 88.5% to 93.8% and from 90% to 100% respectively.Analysis of the phylogenetic tree indicated that JD16,JD34,JD11,JD24,YD2,JD8 and YD1 isolates were more close to the reference strain sPH2,and they lied in the same branch of phylogenetic tree.GD3,YD5,GD2,YD3,YD5 and YD7 isolates were more close to the chicken reference strains PDI-386,as well as the swine reference strain 107 /86,and they lied in the same branch of phylogenetic tree.GD1 isolates was more close to the chicken reference strains HY1-2 and MS2-1,and they lied in the same branch of phylogenetic tree.Conclusion The pilA gene is highly conserved among different host isolates and different serotypes,which could serve as a target gene in rapid diagnosis of colibacillosis and a candidate gene in the vaccine development.