Establishment And Study On The Protoplast Fusion Of Esherichia Coli And Pasteurella Multocida

The aim of the study was to construct a fusion stain with antigenicities of both parental strains derived from E.coli and P. multocida of duck to explore a new way of bacterial multiple vaccine. Drug sensitive test was used to find out an appropriate tag to select the fusion we wanted.ZYD2was sensitive on Neomycin as the bacteriostatic cycle was17mm,and YB2was seriously resistant on Neomycin as the bacteriostatic cycle was0.The result of the minimal inhibitory concentration of Neomycin was20μg/ml.On this basis, the practical concentration of Neomycin added in MACC show that ZYD2can’t be cultured, and YB2can be cultured rich in the borth medium contained5%laked rabbit blood with20μg/ml neomycin.The parental ZYD2(neos) and YB2(neor) were digested by lysozyme and EDTA to remove the cell wall, and the fusions were successfully obtained. The best concentration of lysozyme of ZYD2was2.5g/L acted for45min. The best concentration of lysozyme of YB2was3g/L acted for45min.0.1M EDTA was add to the second time to a final concentration of0.01M. The rate of protoplast preperation of ZYD2and YB2were95.32%and95.25%respectively,and the rate of regeneration were32.19%and33.52%.5of the11potential fusants were stable inheritance on Macconkey agar medium contained antibiotic. The fusant strains were still to be gram-negative and medium-sized bacilli like the parental strain ZYD2. The clones of the fusion strains in the common cultural medium were round, semitransparent, lacte and medium-sized.The clones were also obtained in MACC, which were round and faint yellow.Duplex PCR method was used to detect the part of the OmpA gene and OmpH gene of the fusions.2of them can be observed two target genes, and others were obtained the OmpA gene or none. Sequence alignment show the identity of the target genes of fusions were all100%compared to the parental strains.The antibodies of ZYD2and YB2can detected via microagglutination test after fusion inactived vaccine were vaccinated.The antibody level of both reach peak after35days in the secondary immune group,and the highest antibody titer of4.2for ZYD2,3.5for YB2.The antibody level fall quickly after about20days as immuned once,and the antibody titer were2.9for ZYD2,2.2for YB2.