Construction And Identification Of The Protoplast Fusion Of Salmonella And Escherichia Coli From Ducks

Salmonellosis (also known as paratyphoid) and colibacillosis of duck were two widespread bacterial diseases in duck farm and caused great economic loss to duck industry. Two kinds of bacteria vaccine were used respectively in the immune prevention, not only time-consuming but also high cost. It had an important significance to construct the protoplast fusion of Salmonella and Escherichia coli from ducks for providing an excellent strain to prevent the two kinds of bacterial disease.In this study, I selected S7 and D2 as parent bacterial strains for protoplast fusion by serological test and 17 kinds of antibiotics sensitive test from 15 strains of Salmonella and 10 strains of Escherichia coli which separated and saved by our laboratory from different duck farms in Sichuan and Chongqing. Gentamycin and Tetracycline as drug resistance tag induced salmonella S7 (6,7:l,v:e,n,x, GenR, TcyS) and Escherichia coli D2 (O78,GenS, TcyR).The best time of protoplast formation was obtained from bacterial different growth curve of OD600 value:14-16 h for S7 and 10～12 h for D2. And the result of the minimal inhibitory concentration of antibiotics in hyperosmotic regeneration selected medium was 16 ug/mL for Gen and 32 ug/mL for Tcy. On this basis of the practical concentration of Gen and Tcy added in hyperosmotic regeneration selected medium show that S7 can grow well with 16 ug/mL of Gen but completely unable to grow with 32 ug/mL of Tcy, and D2 can grow well with 32 ug/mL of Tcy but completely unable to grow with 16 ug/mL of Gen, both S7 and D2 completely unable to grow with 16 ug/mL of Gen and 32 ug/mL of Tcy in hyperosmotic regeneration selected medium at the same time.The parental bacterial S7 and D2 were digested by lysozyme (also called Muramidase) for protoplast with adding EDTA to a final concentration of 0.01 mol/L. And the result of the lysozyme concentration test was 3.0 ug/mL for S7 and 2.5 ug/mL for D2, the rate of protoplast preparation and regeneration of S7 were 62.88% and 33.55% respectively, and the rate of D2 were 51.69% and 27.79% respectively.Finally,7 fusions could stable genetic in hyperosmotic regeneration selected medium by subcultured continuously. The fusions were gram-negative bacilli with medium size and round on both ends of gram staining microscopic examination. The fusions could agglutinate both positive serum of the parental strains. Only hydrogen sulfide was positive, and others were negative from the result of biochemical test.A duplex PCR method was used to detect fusions part of genes by designing a pair of primers respectively from the outer membrane protein A gene conservative area of the parental strains. The results showed that 3 fusions ware detected the gene specific fragments of salmonella and E.coli simultaneously,3 fusions were could only detected a parent gene specific fragment, and 1 fusions was not detected any specific fragment. Sequencing results showed that the homology of the target gene of fusions were all 100% compared to the parental strains. This experiment extracted the OmpA from the fusions and parental strains, analysised the change by SDS-PAGE electrophoresis, three fusions could simultaneously express of salmonella and E.coli outer membrane protein.