Constructing Mutants Of HPI In Chromosome Of APEC And Evaluation Of Pathogenicity

Avian pathogenic Escherichia coli (APEC) is an serious bacterial diseases which cause colibacillosis in poultry, and easy mixed infections with other pathogens and viruses, it is a serious bacterial diseases in poultry industry.The define of high pathogenicity island (HPI) is pathogenicity island which can uptake,regulate and synthesze of Yersinia bacillin (Ybt). Yersinia HPI has a core function area about30.5kb, it carries with the gene cluster of Ybt synthesis, regulation and uptake, the gene cluster have11genes which is irp1~irp9, fyuA, ybtA, the HPI integration site is aspartate tRNA (asn-tRNA). In recent years, studies have shown that a variety of Enterobacteriaceae carrying the virulence gene cluster generally,for example E. coli, Klebsiella et.al, mainly with iron uptake and regulation,it is an important chromosome fragments that give the virulence and pathogenicity of bacteria.For research the function of high-pathogenicity islands in the development of colibacillosis. The wild-type E.coli (X0904EC02) as the strain,base on the known sequence of irp1~irp9virulence genes which in HPI. Using λ phage Red recombination system knockout the irp1~irp9gene of avian pathogenic Escherichia coli high pathogenicity island, constructed avian pathogenic Escherichia coli HPI-deficient mutant. The main construction steps are:(1) According to the sequence of HPI gene cluster, designed PCR primers, PCR products were obtained by using primers with50-bp extension which were homologous to E. coli HPI pathogenicity island gene.(2) pKD3as a template, with the help of the high-fidelity enzyme, accordance with the conditions for long PCR amplification, PCR primers containing FRT sites on both sides of the chloramphenicol resistance gene, PCR products were identified by agarose gel electrophoresis, purified by the DNA purification kit, and determined DNA concentration.(3)The PCR products were introduced into E.coli (X0904EC02) competent cells by electroporation.with the help of Red recombinant enzymes, the PCR product carrying chloramphenicol resistance gene flanked by FRT sites and homologous sequence, through it with the avian Escherichia coli chromosome by homologous recombination, screened positive clones by chloramphenicol resistant plate,(4)Plasmid pCP20which encoding Flp recombinase and trasformed into the positive clones, removal the resistance gene by second homologous recombination, Identification with PCR amplification and sequencing. the avian pathogenic mutant of HPI correct construction. Most of the mutant genes in chromosome HPI has been missing, which are less likely to cause reverse mutation.Based on the basis of comparative analysis of the mutant and the wild strains of different virulence. Results are as follows:(1)Through the LD50test showed that avian pathogenic E. coli standard strain E.coli (CVCC1565) which carrying HPI have pathogenic, a non-pathogenic strains does not have the pathogenicity of chicken However, after contamination, E.coli (X0904EC02-QS) mutant occurred with a small number of deaths also.(2) Mutant E.coli (X0904EC02-QS) after contamination, have been some corresponding pathological changes, but not obvious.(3)When the iron chelator2,2’-bipyridine’s concentration are0.3mmol/L,0.32mmol/L,SDS-PAGE protein electrophoresis found that the original strain occurring the protein band in240KD area,not seen any bands of Mutant strain.This study shows that, APEC virulence are closely associated with the HPI,In the contamination,the APEC which carrying HPI genes have different degrees of death,non-pathogenic E. coli not carrying HPI genes does not appear death. However, mutant also have different degrees of death, which prompted the virulence of pathogenic E. coli not only depends on the HPI, but many factors and overall performance.