The PCR, Clone, And Sequence Analyse Of PilA Gene Of Type 1 Pili Of Pathogenic Escherichia Coli Of Chick Origin

Type 1 pili is the important virulence factors on the E.coli in fection in chicken. Through the adhering of pili, E.coli adhered on the epidermic cell of aspiratory tract, which was the first step of invading in host. In this paper ,45 E.coli strains isolated from chicken farms in Sichuan province were determined to be the pathogenic E.coli by animal test.Type 1 pili of 45 strains isolated was detected by MSHA. The pilA gene of 45 avian pathogenic E.coli strains were amplified by the polymerase chain reaction(PCR) with primers designed according to the sequence of the pilA gene in GenBank. Results showed that PCR was more sensitive, faster and more characteristic than MSHA to detect type 1 pili. The positive rate of type 1 pili detection was 80% by MSHA,but was 95.5% by PCR.. Five amplified fragment of dominant serotype strains in sichuan province were purified, cloned and sequenced respectively. The results showed that the coding region is 459 bp in size.The pilA genes of 5 avian pathogenic E.coli strains and their amino acid sequence have been accessed by GenBank(Accession: MS2-1: AY082805; LS3-1: AY082806; LS2-1: AY082807; HY1-2: AY082808; HY1-1: AY082809).The pilA gene sequence ,amino acid sequence,secondry sructure and antigen characteristic were analyzed by DNASTAR, a kind of bio-software. The result showed that the homology rate of pilA gene among the 5 avian pathogenic E.coli strains tested and one human E.coli were from 89.8% to 91.1%,and the homology rate of amino acid were from 88.5% to 91.8%. The homology rate of pilA gene sequence among 5 avian pathogenic E. coli strains tested and avian pathogenic E.coli reported (serotype O1,O2,O78) were from 87.8% to 90.2%,and the homology rate of amino acid were from 84.6% to 91.2%. There had homology in avian pathogenic E.coli.There had some common antigen side in type 1 pili of avian pathogenic E.coli.In this study,the meaningful results have been achieved, which is the important basic work to research the pili subunit vaccine of avian E.coli,and detect pilA gene by nucleric acid probe.Moreover,it is significant to research molecular epidemiology,to diagnose ,to prevent and treat avian colibacillosis.