Development Of A ERIC-PCR Genotyping Assay For Detection Of E.coli From Calf Origin And Evaluation Of Immunological Efficacy Of K99-987P-F41 Fusion Protein

We aimed to develop an enterobacterial repetitive intergenic consensus polymerase chain reaction(ERIC-PCR)DNA fingerprinting technique for genotyping ETEC in calves from Heilongjiang Province that can be applied to epidemiological and etiological investigations,and then epidemic strains were screened which were preparated for inactivated whole cell vaccine.Meanwhile,the K99-987P-F41co-expressive vector is constructed,K99,F41 and 987 P are as the target gene.Prepared K99-987P-F41 recombinant protein immunogen immunized mice and their immune effects were evaluated.ERIC primer was designed based on the literature,and then reaction conditions were combined to ERIC-PCR after being optimized.The ERIC-PCR method for genotyping clinical isolates of ETEC strains.Our results indicate that The ETEC ERIC-PCR could be amplified out 2~10 straps strains and grouped into 17 types,with type D predominant.The three pairs of primers were designed basing on the published sequence of ETEC adhesion K99,987 P and F41 in Gen Bank by PCR amplification.The three target genes of K99,987 P and F41 were subcloned into the prokaryotic expression vector pET30a(+).It was identified as a positive plasmid K99-987P-F41,then transformed into E.coli BL21(DE3).The recombinant bacteria induced by IPTG.It's results indicate that the fusion protein at about 60 Ku,it is consistent with the theoretical value.Detection of protein by Western Blot showed that it has good activity.This test using a conventional method preparing fusion protein K99-987P-F41,natural pili K99,987 P and F41 hybrid protein,inactivated whole cell,recombinant pili K99,987 P and F41 hybrid protein,then immuning in mice.Blood,feces and in the feces and lavage fluids were collected after immunization.IgG,IL-4 and IFN? in serum,SIgA in feces and lavage fluids were detected by indirect ELISA.MTT assay was applyed in mice spleen lymphocyte proliferation.In addition,evaluated the protection effect of several groups of vaccine with the challenge.The results showed that the test groups could induce the production of antibodies in mice,immune effect followed by inactivated whole cell> Natural pili K99,987 P and F41 hybrid protein> Recombinant pili K99,987 P and F41 hybrid protein> fusion protein K99-987P-F41,but the difference among the groups was not significant.MTT test results show that the test groups could produce significant humoral immunity.IL-4,IFN? were increased.The SIgA effect was not significant.It is provides a theoretical basis for the development of subunit vaccines effective in preventing disease caused by ETEC.