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Microcin C7七肽及其类似物对大肠杆菌生长的影响
冉仁森
学位类型硕士
导师徐霞
2017-07
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化工
关键词Microcin C7 七肽 细菌生长 细胞膜完整性
摘要

抗生素耐药性已成为全世界广泛关注的一个问题,严重地威胁着人类以及动物健康,因此迫切地需要新的解决策略和新的抗生素。Microcin C7(McC)是广泛分布于肠杆菌属细菌中一种很有希望解决抗生素耐药性问题的特洛伊木马式抗菌肽。已有研究报道McC七肽(MR)具有抑制蛋白质合成的能力,但是对于大肠杆菌的生长没有影响,只特异性识别靶细菌细胞膜上的转运子YejABEF,从而主动转运McC进入靶细菌细胞内,保护McC在靶细菌细胞外不受酶解作用。然而本文就MR具有抑制蛋白质合成但不影响大肠杆菌生长为问题出发点,设计了一系列的活性实验,深入研究了MR及其类似物f-MR、a-MR、MK、f-MK、a-MK对于大肠杆菌生长的影响。首先利用多肽固相合成技术合成了MR以及MR的类似物f-MR、a-MR、MK、f-MK、a-MK,利用半制备反相高效液相色谱分离纯化获得目标产物后,经过质谱鉴定,确定正确合成目标产物。继而进行了一系列对于大肠杆菌生长影响的实验,实验结果表明:MR、f-MR、a-MR、a-MK分别在5.34mM、8mM、6.47mM、6.72mM时,能够杀死大肠杆菌,并且MR和a-MR在10min之内杀死大肠杆菌;MR和f-MR的浓度分别为2.67mM、4mM以及更低时,可以显著减少约36.77%的大肠杆菌数量,表明低浓度的MR、f-MR可以抑制大肠杆菌的生长;然而富营养环境下,MR、f-MR、a-MR、MK、f-MK、a-MK对于大肠杆菌的生长没有影响,添加0.1%的BSA也不会影响MR、f-MR、a-MR、MK、f-MK、a-MK的抑制活性。进一步测定了MR、f-MR、a-MR处理后的大肠杆菌的β-半乳糖苷酶、6-磷酸葡糖脱氢酶、呼吸链脱氢酶的酶活性,实验结果表明,MR、f-MR、a-MR分别在5.34mM、8mM、6.47mM时完全抑制β-半乳糖苷酶、6-磷酸葡糖脱氢酶、呼吸链脱氢酶的酶活性,且所有酶的酶活性都随着多肽浓度的降低而升高至正常水平,所以MR、f-MR、a-MR可以抑制大肠杆菌酶的合成。最后,利用β-半乳糖苷酶活性实验以及扫描隧道电子显微镜实验进一步研究MR、f-MR、a-MR与大肠杆菌细胞膜的作用方式,实验结果表明:虽然MR、f-MR、a-MR不改变大肠杆菌细胞膜的渗透性,但是可以抑制β-半乳糖苷酶的合成;扫描隧道电子显微镜观察用5.34mM以及更低浓的MR处理过的大肠杆菌,可以直接确定MR对大肠杆菌细胞膜的完整性没有影响。从而可以得到结论,MR以及MR的部分类似物可通过大肠杆菌细胞膜上转运子的转运进入大肠杆菌细胞内,抑制蛋白质的合成,最终影响大肠杆菌的生长,或是导致大肠杆菌死亡。

其他摘要

There is an urgent need for new solutions and new antibiotics to overcome antibiotic resistance which has been becoming a widespread and serious menace to human and animal health. Microcin C7 (McC), widely distributed in enterobacteria, is a promising and Trojan horse antibiotic against antibiotic resistance. Previous studies have demonstrated that the heptapeptide of McC (MR) has the capacity of inhibiting protein synthesis, but lacks the capacity for inhibiting microbial cell growth. Additionally, MR can protect Microcin C7 from degradation. However, in this study, we fully investigated the novel proverty of MR and its analogues on microbial cell growth in terms of cell viability, enzyme activity and cell membrane integrity.First, MR and its analogues of MR, f-MR, a-MR, MK, f-MK and a-MK were synthesized using a solid-phase peptide synthesis method. The synthesized peptides were purified by a semi-preparative high performance liquid chromatography and then identified by a mass spectrometry. The effects of MR, f-MR, a-MR and a-MK on the growth of Escherichia coli were investigated. The experimental results show that MR, f-MR, a-MR and a-MK can kill Escherichia coli at 5.34 mM, 8 mM, 6.47 mM and 6.72 mM, respectively. MR and a-MR kill Escherichia coli completely within 10 min at 2.67 mM and 4 mM, respectively. Even at the low concentration, the number of Escherichia coli is significantly reduced to about 36.77%. The presence of 0.1% BSA in the culture medium has not effect on the inhibitory activity of MR, f-MR, MR, MK, f-MK, a-MK. However, in the rich media MR, f-MR, a-MR, MK, f-MK and a-MK have no effect on the growth of Escherichia coli.Furthermore, the β-galactosidase, 6-phosphogluconate dehydrogenase, and respiratory chain dehydrogenase of Escherichia coli treated by MR, f-MR and a-MR at different concentrations were detected by determining the intracellular enzyme and constituent enzyme activity. The activitives of β-galactosidase, 6-phosphate dehydrogenase and respiratory chain dehydrogenase are completely inhibited by MR, f-MR and a-MR at 5.34mM, 8mM and 6.47mM, respectively. In contrast, the activities of all enzymes are increased with decreasing concentration of the peptides.Finally, the effects of MR, f-MR and a-MR on cell membrane integrity of Escherichia coli were further evaluated by β-galactosidase activity assay and scanning tunneling electron microscopy. Both β-galactosidase activity assay and electron microscopy assay indicate that the cell membrane of Escherichia coli remains integrity even in the presence of MR, f-MR and a-MR at the lethal concentration.

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/24220
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冉仁森. Microcin C7七肽及其类似物对大肠杆菌生长的影响[D]. 北京. 中国科学院研究生院,2017.
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