【作者】 潘凌子；

【导师】 王关林；

【作者基本信息】 辽宁师范大学 ， 植物学， 2007， 硕士

【摘要】 本文通过绘制抑菌曲线和牛津杯法,研究了蜂毒肽对金黄色葡萄球菌、酵母菌GS115、番茄叶霉孢子等多种病原菌和基因工程菌的抑杀作用及抑菌效力的影响因素。利用SDS-聚丙烯酰胺凝胶电泳、细胞流式仪、透射电子显微镜和扫描电子显微镜等实验技术,从蛋白质合成和表达、DNA复制和细胞超微结构的角度系统分析了蜂毒肽的抑杀菌机理。将蜂毒肽前体蛋白基因导入原核表达载体pET-42a(+)中,经PCR、测序鉴定后转化至大肠杆菌BL21(DE3)中。结果表明:蜂毒肽对多种病原菌和基因工程菌都具有强烈、速效的抑杀作用,抑菌效果受pH值、温度等因素的影响。经流式细胞仪分析,蜂毒肽对菌体作用后,集中于Ⅰ(或G1)期的细菌数量增加,进入R(或S)期细菌数量减少,说明蜂毒肽的作用发生在I (或G1)期;而SDS-PAGE结果显示,药物作用后菌体蛋白谱带明显变浅,甚至消失,且这种作用是速效的;通过电子显微镜观察到经蜂毒肽作用后,菌体细胞穿孔、破裂或席卷式瓦解,细胞质外流,菌体裂解死亡。蜂毒肽能透过细胞壁,作用于细胞膜,在膜上形成孔洞,使膜破裂或席卷式瓦解,细胞内容物外泄死亡;进入细胞内,作用于蛋白质,抑制蛋白质的合成与表达或降解细胞内原有的蛋白,使细胞内蛋白质含量显著降低,细胞质固缩凝集;使DNA复制受阻而能不通过细胞周期的检查点,滞留在DNA复制期不能进入细胞分裂期,从而抑制细胞的分裂,这条途径可能是由于蜂毒肽对蛋白质的作用引起的。综上,蜂毒肽具有广谱、速效、高效的抑杀菌特性,可以克服当前生物农药存在的不足,对温度的不敏感和抑菌作用的稳定性都体现出蜂毒肽作为新型生物农药的开发潜力。为了解决蜂毒肽的来源这一瓶颈问题,我们将蜂毒肽前体蛋白基因和pET-42a(+)载体重组后,导入大肠杆菌BL21(DE3)中,在异丙基硫代-β-D-半乳糖苷(IPTG)的诱导下,实现了蜂毒肽前体蛋白与谷胱甘肽-S-转移酶(GST)的融合表达。更多还原

【Abstract】 The inhibiting effect and possible effective factors of melittin were investigated on several pathogens and engineered bacteria, such as Staphylococcus aureus, GS115 and Fulvia fulva, by means of cylinder- plate on series dilutions and growth curve monitoring. We explored the mechanisms by examining protein expression level, cell cycle and cell ultrastructural alterations, using SDS-PAGE, flow cytometry and electron microscopy. Promelittin geen was inserted into expression vector pET-42a(+) and transformed into E.coli BL21(DE3) after identified by PCR and sequencing.The experimental results show that the inhibitory activity of melittin was insensitive to temperature change within room range, which could be applied in each season. At 37℃, bacteriostatic activity of melittin began to decline after 5 d and dropped precipitously after 10 d. The inhibition curve suggests that pathogens went into decline phase directly instead of entering the normal logarithmic phase. From flow cytometry analysis, after several hours treatment with melittin, the percentage of cells in phase I (or in phase G1) increased, while those in phase R(or in phase S) decreased. This indicates that melittin disrupted phase I (or phase G1) rather than phase R (or phase S), causing most cells to remain in phase I (or in phase G1). Most protein expression in pathogens declined quickly with melettin treatment, some bands on SDS-PAGE gels almost vanished especially. These results suggest that melittin inhibited gene expression quickly, blocking cell division after 4 h. Electron microscopy revealed that melittin interacted with and disrupted the cytoplasmic membrane and caused the dissolution of proton motive force and leakage of essential molecules, resulting in cell death.In summary, it was confirmed that three pathways could be involved in inhibition: melittin integrated with the cell membrane causing cell bursting and cytoplasm release, inhibited the synthesis of proteins and caused the cytoplasm to condense, and delayed pathogens in phase I (or phase G1) so that they could not complete the cell cycle. These results suggest that melittin could serve as a broad-spectrum biological pesticide with fast-action and high-efficiency.In order to solve the problem of lacking natural resource, the fusion protein of promelittin and GST was expressed in E.coli under IPTG inducing.更多还原