Establishment And Study On The Protoplast Fusion Of Salmonella And Pasteurella Multocida

The aim of the study was to construct a fusion stain with antigenicities of both parental strains derived from Salmonella 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. S6 was sensitive on Spectinomycin as the bacteriostatic cycle was 26mm,and CBa was seriously resistant on Spectinomycin as the bacteriostatic cycle was Omm.The result of the minimal inhibitory concentration of Spectinomycin was 32μg/ml.On this basis, the practical concentration of Spectinomycin added in MACC show that S6 can’t be cultured, and CBa can be cultured rich in the borth medium contained 5% laked rabbit blood with 32μg/ml spectinomycin.The parental S6 and CBa were digested by lysozyme and EDTA to remove the cell wall, and the fusions were successfully obtained. The best concentration of Spectinomycin of S6 was 2.5g/L. The best concentration of Spectinomycin of CBa was 3g/L.0.1M EDTA was add to the second time to a final concentration of 0.01M. The rate of protoplast preperation of S6 and CBa were 95.32% and 95.25%respectively,and the rate of regeneration were 32.19% and 33.52%.5 potential 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 S6.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. Only oxidase was positive, and others were negative from the result of biochemical test.Duplex PCR method was used to detect the part of the OmpA gene and OmpH gene of the fusions.3 of them can be observed two target genes, and others were obtained the OmpA or OmpH. Sequence alignment show the identity of the target genes of fusions were all 100% compared to the parental strains.