Construction Of Double Drug-resistance Of Fusion Cell Strains Of Riemerella Anatipestifer

Riemerella anatipestifer (RA) infection is a contagious disease caused by RA which infringes on farm ducks, it also occurrs in other species of domestic poultry and wild species. The pathogeny of RA distributes over worldwide. RA can be divided to 21 serotypes by slide agglutination and tube agglutination.The cross-protection is deficient between each serotype of RA, which can cause failure in immunization and hazard the development of duck industry severely. The serotype 1 and 2 of RA can be found in China commonly. We selected serotype 1 (RA-JX strain) and serotype 2 (RA-2 strain) strain of RA as test strains and adopted protoplast fusion technology to construct bivalent strains of RA contained double antigens, which laid foundation to the development of new generation vaccine for RA disease, the main results as followings:(1) Selecting chloramphenicol and erythromycin respectively to make the drug resistant marker for RA-JX strain and RA-2 by antibacterial sensitivity test. Stable heredity of RAC (Chl~r, Ery~s) strain and RAE (Ery~r, Chl~s) strain were obtained by the induction in TSB liquid medium contained different concentration of antibiotics. The resistant concentration of chloramphenicol to RA-JX strain was increased to 80μg/mL and erythromycin to RA-2 strain was increased to 160μg/mL.(2) Using RAC strain and RAE strain as parental strains and adopting lysozyme-EDTA method to prepare their protoplasts. To ascertain the proper concentration of lysozytme and EDTA was 5μg/mL and 0.01 mol/L respectively by the optimization between working concentration of lysozyme-EDTA during 25 minutes. In this optimized condition, the preparation rate of RAC strain and RAE strain protoplasts was 85% and 81%, the regeneration rate of RAC strain and RAE strain protoplasts was 43% and 40%.(3) Protoplast fusion was accomplished between the protoplasts of RAC strain and RAE strain with 40% PEG 4000 as assistant solvent, 8 fusion cell strains were obtained through indirect selection method in plate medium, they were F1,F2,F3,F4,F5,F6,F7,F8. All the fusion cell strains could be resistant to chloramphenicol and erythromycin, they owned double properties of drug-resistance of parental strains . In addition, their hereditary properties were identical with the two parental strains, such as morphology, staining, biochemical, etc. It was approved that all the fusion cell strains owned stable drug-resistance by the test of stability.In this research, we had obtained fusion cell strains which owned stable drug-resistance of parental strains by protoplast fusion. It provided theoretical foundation to develop new generation vaccine of RA and prevent the disease of RA through fusion cell strains with double properties of drug-resistance of parental strains were constructed successfully.