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目的 雷帕霉素 (RPM)是吸水链霉菌产生的一种强效三烯含氮大环内酯类免疫抑制剂和基因治疗药 ,它的免疫抑制结合蛋白 (FK5 0 6 - binding proteins,FKBPs)是种肽酰 -脯氨酰顺 -反异构酶 (peptidyl- prolyl- cis- trans- isomerase,PPIase)。雷帕霉素对 FKBPs有很高的亲和力 ,结合后 FKBPs的酶活性明显受抑制 ,而另一类免疫抑制剂环孢素 (Cs A)不与 FKBPs结合 ,对FKBPs的酶活性无明显影响。目前研究表明雷帕霉素需与其中的 FKBP12结合后才能发挥其免疫抑制效应。本研究从雷帕霉素产生菌吸水链霉菌中分离具有 PPIase活性的结合蛋白 FKBPs,并探讨分析雷帕霉素产量与 FKBPs含量的可能关系。 方法 采用硫酸铵分段沉淀 (终饱和度分为 5 0 % ,80 % )、制备性不连续聚丙烯酰胺凝胶电泳分离蛋白质 ,利用紫外 /可见光分光光度计及 Reaction kinetics酶动力软件测蛋白质的 PPIase酶活性 ,高效液相色谱仪测不同菌株的 RPM产量 ,对比高产突变菌株和野生菌株中蛋白质电泳图谱及 FKBPs含量的差异。 结果 吸水链霉菌菌体蛋白质制备、分离 ,所得蛋白质样品 ,应用SDS- PAGE以标准分子量蛋白质确定样品蛋白质的分子量 ,以底物寡肽 N- Succinyl- Ala- Ala- Pro- Phe 4- nitro- anilide测样品的酶活性 ,获得具有 PPIase?
OBJECTIVE: Rapamycin (RPM) is a potent triene nitrogen macrolide immunosuppressant and gene therapy drug produced by Streptomyces hygroscopicus. Its immunosuppressive binding protein (FKBPs) ) Is a peptidyl-prolyl-cis-trans-isomerase (PPIase). Rapamycin had high affinity to FKBPs, and the enzyme activity of FKBPs was obviously inhibited. However, CsA, another immunosuppressant, did not bind to FKBPs and had no significant effect on the enzyme activity of FKBPs. Current research shows that rapamycin needs to bind with FKBP12 to exert its immunosuppressive effect. In this study, FKBPs with PPIase activity were isolated from Streptomyces hygroscopicus, a rapamycin-producing strain, and the possible relationship between rapamycin production and FKBPs content was explored. Methods The protein was separated by preparative discontinuous polyacrylamide gel electrophoresis using ammonium sulfate fractionation (final saturation was 50%, 80%). The protein was detected by UV / Vis spectrophotometer and Reaction kinetics enzyme kinetic software PPIase activity and HPLC were used to measure the RPM production of different strains. The differences of protein electrophoretogram and FKBPs in high-yield mutant strains and wild-type strains were compared. Results The protein of Streptomyces hygroscopicus was prepared and separated. The resulting protein sample was analyzed by SDS-PAGE using standard molecular weight proteins to determine the molecular weight of the sample protein. The substrate oligopeptide N-Succinyl-Ala-Ala-Pro-Phe 4-nitroanilide The enzyme activity of the sample was measured to obtain a sample with PPIase?