Study On The Construction And Breeding Of New Tuberculosis Vaccine Strains

Objective: To explore the preparation, protoplast fusion and identification of fusion strains cultured of twoparental strains, Bacillus Calmette-Guérin strains (BCG) and international standard Mycobacteriumtuberculosis strains H37Ra (H37Ra)Methods:1. In order to explore the optimization of BCG strains protoplast and H37Ra strains protoplast preparationconditions and the factor of influence in preparation conditions of fungus age, enzyme concentration,enzymatic hydrolysis time by using the Protoplast technology to prepare BCG strains protoplast andprotoplast H37Ra strains protoplast.2. Use three different kinds of medium, namely the regeneration Roche solid medium, the regenerativeSauton solid medium and the regeneration Sauton liquid medium, to explore the BCG strains protoplast andH37Ra strains protoplast regeneration research and optimizing its regeneration condition.3. Label the two parental protoplasts strains with different fluorescent dye using fluorescence stainingtechnique.4. Use the electroporation technology on two labeled parental strains protoplast electrofusion, preparationof the fusion strains and optimizing the suitable conditions of preparation.5. Use confocal laser scanning microscope to conduct observation, identification and explore the fusionstrains protoplast regeneration research.Results:1. Successfully obtained BCG strains protoplast, for18d in fungus age, enzyme solution concentration of12mg/mL, enzymolysis time for5h, were the optimal condition of preparation of BCG protoplast. The bestcondition of preparation of H37Ra strains protoplast: fungus age for21d, enzyme solution concentration of12mg/mL, enzymolysis time was6h.2. Use the regenerative Roche solid medium, the regenerative Sauton solid medium, regeneration Sautonliquid medium to cultivate BCG strains plasmid and H37Ra strains protoplast respectively, found thatRoche solid medium and regenerative Sauton solid medium, the BCG strains protoplast and H37Ra strainsprotoplast grew well, and in the regeneration Sauton almost no growth in liquid medium.3. Use fluorescence staining techniques respectively to label BCG strains protoplast and H37Ra strainsprotoplast: FDA dye labeled active BCG strains protoplast appeared yellow-green fluorescence; rhodamineB dye labeled active H37Ra strains protoplast appeared red fluorescent.4. Set fusion voltage U=2.2KV, electric shock time t=0.8ms observed the fusion efficiency of two parentalstrains. In this condition, we maintained high activity fusion strains. Conduct observation the fusion strainswith confocal laser scanning microscope.5. Cultured the fusion strains of BCG strains protoplast and H37Ra strains protoplast on the regeneration ofRoche solid culture medium. We got the fusion strains which grew well.Conclusions:1. Using protoplast technology successfully prepared the BCG strains protoplast and H37Ra strainsprotoplast and optimized the preparation conditions. Optimize the regeneration conditions of BCG strainsprotoplast and H37Ra strains protoplast.2. Successfully obtained the fusion strains of BCG strains protoplast and H37Ra strains protoplast usingelectroporation technology, cultured the regeneration strains which grew well.