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分别以卡介苗菌株(BCG)及结核分枝杆菌国际标准无毒株H37Ra菌株(H37Ra)为受体菌,制备上述两种菌株的原生质体,同时通过优化细菌菌龄、酶解浓度、酶解温度以及酶解时间等影响因素,探索出制备原生质体形成及再生的最优条件。结果显示:摸索出制备BCG和H37Ra菌株的原生质体条件:在对数生长期的两亲本菌株,经0.01 mol/L EDTA,0.01%β-巯基乙醇溶液预处理;酶解浓度为12 mg/mL,酶解温度为37℃,酶解时间为5 h,可制备出活性较高的两种菌株的原生质体。由此可知,成功制备了BCG与H37Ra菌株的原生质体,并能在高渗固体培养基上再生。本实验为进一步研究该两种菌株原生质体融合试验奠定了基础。
Protoplasts of Bacillus Calmette - Guerin (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra) were prepared respectively. The protoplasts of the two strains were prepared, and the optimal conditions of bacterium age, enzymolysis concentration, And enzymolysis time and other influencing factors to explore the protoplast protoplast formation and regeneration of the optimal conditions. The results showed that the protoplast conditions of BCG and H37Ra strains were found out: pre-treatment with 0.01 mol / L EDTA and 0.01% β-mercaptoethanol at the logarithmic growth phase; the enzymolysis concentration was 12 mg / mL , The enzymolysis temperature was 37 ℃, the enzymolysis time was 5 h, protoplasts of the two strains with higher activity could be prepared. Thus, protoplasts of BCG and H37Ra strains were successfully prepared and regenerated on hypertonic solid medium. This experiment laid the foundation for the further study on protoplast fusion experiment of the two strains.