Knockout Of Irp1 Gene On HPI Virulence Island Ofbovine Escherichia Coli And Its Effect On Virulenceand Biological Characteristics

Dairy cow endometritis is one of the most common and most costly diseases in dairy farming and production worldwide,and 65%to 80%of cow infertility is caused by endometritis in China.It is generally believed that the main causes of cow endometritis are pathogenic microorganisms,including bacteria,mycoplasma,leptospirosis virus,fungi,and trichomoniasis,and E.coli,Proteus,Streptococcus,and Staphylococcus aureus are the most common among the bacteria.In order to determine the relationship between the pathogenicity of E.coli and the irp1gene of pathogenicity island HPI,the suicide plasmid p RE112 was used in this experiment to construct the deleted strain and the complementary strain of the irp1 gene of E.coli.And the functional verifications of irp1 gene were carried out.The results obtained are as follows:1. The chloramphenicol resistance and irp1 gene of 14 E.coli strains?E1-E14?were detected.Strains E1,E4,E6,E8,E9 and E12 were sensitive to chloramphenicol and positive for irp1 gene.And the strain E4 was selected for irp1 gene knockout and repair.2. Using overlap PCR method and E4 genome as template to fuse the up and down homologous arms of irp1.The fusion fragment was cloned into suicide plasmid p RE112 and successfully constructed the recombinant suicide plasmid p RE?irp1.E.coli X7213containing recombinant suicide plasmid p RE?irp1 was used as donor bacteria and E.coli E4was used as recipient bacteria for co-culture,and the deletion strain E4?irp1 was screened by two-step homologous recombination.Finally,the complementary strain E4?irp1-C was also constructed by suicide plasmid p RE112.3. The biological characteristics of parent strain,deletion strain and complementary strain were studied.The results showed that the deletion of irp1 gene in E.coli would cause:?1?the LD₅₀of E.coli to mice was reduced by 10 times;?2?in addition to genes hly A and csg B,the expression levels of genes ecp A,fim A,kps D,fae G,clb D,stf G,sfm H,ipf A and fim H decreased to a certain extent;?3?when determining biofilm formation ability by crystal violet staining,its OD₆₀₀decreased by 29.8%.The function is restored after repaired.In addition,the biochemical characteristics,drug sensitivity,growth regularity and siderophore forming ability of the parent strain,deletion strain and complementary strain have not changed.These indicated that the irp1 gene is closely related to the pathogenicity of E.coli.The deletion or expression inhibition of irp1 gene can reduce the virulence of E.coli,but has no effect on its biochemical characteristics,drug sensitivity,growth regularity and siderophore formation ability.