卡介苗菌株和结核分枝杆菌H37Ra菌株原生质体的制备研究

Study on Formation of Protoplast of BCG Strains and the Mycobacterium Tuberculosis H37Ra Strains

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。

To explore the formation of protoplasts from BC G strains （hereinafter referred to as the BCG） and the Mycobacterium tuberculosis H37Ra strains （hereinafter referred to as the H37Ra strain）.Optimal conditions for the formation and regeneration of protoplasts were studied via optimizing of the fungus age,enzyme solution concentration, temperature and action time,the optimal conditions of the formation of BCG and H37Ra strains protoplasts were proved to be as follows：the logarithmic phase of two parent strain, pretreated with 0.01mol/L EDTA and 0.01% β-mercaptoethanol solution,under enzyme concentration 12mg/mL,enzymolysis temperature 37 ℃ and enzymatic hydrolysis time 5h,that can achieve high activity protoplasts.The two protoplasts,successfully obtained from BCG strain and Mycobacterium tuberculosis H37Ra strain,possess the ability of regeneration in hypertonic solid medium,which laid the foundation for further protoplasts fusion experiment.