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雷帕霉素高产菌株的选育及培养条件的优化.pdf77页
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雷帕霉素高产菌株的选育及培养条件的优化.pdf
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浙江大学 硕士学位论文
雷帕霉素高产菌株的选育及培养条件的优化 姓名：沈文和 申请学位级别：硕士 专业：生物化工 指导教师：徐志南 座机电话号码 浙江大学烦士学位论文 摘
要 雷帕霉素 Rapamycin 是从吸水链霉菌Streptomyces
出来的三烯大环内酯类抗生索，它是一种新型高效免疫抑制剂，同时又是一种有
效的抗真菌剂并具有抗肿瘤的作用。本论文对雷帕霉素产生菌的选育及发酵生产
进行了系统的研究，并取得了一定的效果。 建立了雷帕霉素的液相色谱检测方法与生物活性检测方法，并研究了两者之
间的联系，证实了两者具有一定的正相关性，基于这一前提，开发了一套高效的
新型琼脂块高通量筛选技术，为大规模筛选工作打下了基础。 从原始培养基出发，我们分别对刳面培养基，种子培养基和发酵培养基进行
考察，研究了菌种的生长特性，并对培养基进行了优化，经过培养基优化，雷帕
霉紊的产量比初始培养基中雷 吣霉素的产量提高了97％。 根据雷帕霉素在不同时期的生理状态及合成途径，设计出单一诱变剂连续诱
变、物理和化学诱变剂交替诱变、物理和化学诱变剂连续诱变等诱变路径，对雷
帕霉素产生菌进行多轮次的循环诱变，筛选到最高产量为279mg?L“的诱变菌株
一株，比出发菌株的产量提高了156％。 对雷帕霉素产生菌的原生质体制备和再生条件进#gT优化，研究了雷帕
霉素产生菌的热灭活和紫外灭活效应，对雷帕霉素产生菌的单亲灭活和双亲融合
进行了考察，根据雷帕霉素和红霉素在合成途径上的相似性，考察了两种菌株原
生质体的种间融合，对融合子生产能力进行测定，最高产量达到345mg。L～。 基于全基因组重排理论，经过三轮融合和再
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利用种子萌发法快速检测抗草铵膦转基因大豆-论文
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利用种子萌发法快速检测抗草铵膦转基因大豆-论文
官方公共微信Construction of cDNA library and antigen expression of Streptomyces Thermohydroscopicus in vitro--《Chinese Preventive Medicine》2013年06期
Construction of cDNA library and antigen expression of Streptomyces Thermohydroscopicus in vitro
LING Yuan,WANG Ling-ling,LIU Shuo,MA Lie,WANG Qun,ZHANG Li-jiao, HE Xiao-yu,ZHAO Ming-jing,WANG Xiao-ge *Department of Respiratory Medicine,the Fourth Affiliated Hospital of China Medical University,Shenyang,liaoning 110032,China
Objective To construct the cDNA library of Streptomyces Thermohydroscopicus and to express some of the specific antigens in vitro.Methods The cDNA library of Streptomyces Thermohydroscopicus was constructed by switching mechanism at 5′end of RNA transcript approach.The recombinant positive clones were selected randomly for EST sequencing and two candidate genes were sub-cloned into expression vector pET-28a.The recombinants were then transformed into BL2(DE3)and proteins were expressed by the induction of IPTG.Results A high-quality cDNA library of Streptomyces Thermohydroscopicus was constructed with the recombination rate of 96.3%.A set of 978 valid sequences were obtained out of total 1020 positive clones with the average length of 495 bp which contained 347 unique genes.The homology analysis revealed that 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein(51%)and transferring-binding protein B(42%)from Actinobacillus pleuropneumoniae serotype(APP).The molecular weights of the two expression products were 63kDa and 30kDa.Conclusions The cDNA library of Streptomyces Thermohydroscopicus harbors all EST sequences and most likely contains the majority of the virulence genes.It not only provides the basis for elucidating the etiology of farm′s lung disease,but also facilitates the development of serological diagnosis and vaccine for farm′s lung disease.
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