| In this study, 13 Riemerella anatipestifer strains and 19 Esherichia coli strains were isolated from Zhejiang province, drug sensitivity test results showed that the drug resistance of Riemerella anatipestifer and Esherichia coli in Zhejiang province was very serious. Riemerella anatipestifer had serious resistance on polymyxin, kanamycin, amikacin, gentamicin, cotrimoxazole; Esherichia coli has more serious resistance to erythromycin, Midecamycin, norfloxacin, ampicillin, penicillin G, lincomycin, amoxicillin, Riemerella anatipestifer could tolerate 6 kinds of antibiotics, the proportion of types of antibiotics accounted for 31.6 %; Esherichia coli could tolerate 11 kinds of antibiotics which accounted for 57.9 % of all antibiotics, so serious multiple drug resistance is bound to cause great difficulties in prevention and treatment.For the development of bacterial protoplast fusion technology, to explore the development of new methods and new ways of bacterial multiple vaccine, this study selected Riemerella anatipestifer and Esherichia coli dominant serotype TXRA1 and ZBO78 for fusion experiments, and used chloramp- henicol (Chl) and erythromycin (Ery) to produce drug resistance tag strains induced Riemerella anatipestifer TXRA1 (Chlr, Erys) and duck Esherichia coli ZBO78 (Eryr, Chls) strains. The purpose of this study was to estabilish a bivalent strain of these two diseasease through optimization of conditions for the protoplast preparation and regeneration.The concentration and action time of lysozyme and EDTA were syste- matic optimized, Riemerella anatipestifer TXRA1(Chlr,Erys) added with lysozyme 0.1 g/L acted for 20 minutes, EDTA was then added slowly to a final concentration of 0. 01 mol/L for another 45 minutes as the best experimental condition, 92 % protoplast formation frequency was obtained; Esherichia. coli ZBO78(Chls,Eryr)added with lysozyme 0.2 g/L acted for 20 minutes, EDTA was then added slowly to a final concentration of 0. 01 mol/L for another 16 minutes as the best experimental condition, 91.94 % protoplast formation frequency was obtained. Based on further optimization of protoplast preparation methods, hypertonic buffers, sucrose concentration in the medium, agar concentration, pH, ion concentration and other factors, 38.77 % and 37.29 % regeneration frequency were obtained espectively.Based on the optimization of protoplast preparation and regeneration conditions, we successfully constructed Riemerella anatipestifer and Esherichia. coli fusion strains by PEG method, 31 fusions had two types of drug resistance. In view of 31 fusions had Self-agglutination phenomenon, we established a duplex PCR detection method which based on the outer membrane protein (ompA) of the two strains, 11 fusions could express two parental strain's ompA gene and another 20 strains could only express Esherichia. coli ompA gene by the douplex PCR amplification, Dnase I was added during the whole experiment to exclude the transformation of exogenous DNA ,which showed that recombination of fusion and chromosome recombination had taken place. The 670 bp and 408 bp fragment amplified from fusions F3, F13, F17, F31 were sequenced, the results showed that the homoeology of the gene were both 100 % by sequence analysis compared with their parental stains TXRA1and ZBO78, that proved the fragments DNA amplified by duplex PCR were aimed fragments. It's the first time to identify 11 positive fusions which could express their two parental strains's genome at home and abroad, that not only developed bacterial protoplast fusion technology and establish the foundation for further development of efficient bivalent or multiple bacterial vaccine. Another 20 fusions which could express Esherichia. coli ZBO78 gene might not be negative fusions, maybe due to their genomes reorganization through the other way, and the primers we designed could not be detected the other segments of the reconstruction. The duplex PCR method established in this study can be learned by the other fusion experiments.
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