Microcin C7七肽及其类似物对大肠杆菌生长的影响

发布时间：2018-05-20 02:14

  本文选题：MicrocinC7 + 七肽　； 参考：《中国科学院大学(中国科学院过程工程研究所)》2017年硕士论文

【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。MicrocinC7(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。
[Abstract]:Antibiotic resistance has become a worldwide concern, seriously threatening human and animal health. Therefore, there is an urgent need for new strategies and a new antibiotic, MicrocinC7 McC1), which is a Trojan horse antimicrobial peptide widely distributed in Enterobacter bacteria, which is a promising solution to the problem of antibiotic resistance. It has been reported that McC has the ability to inhibit protein synthesis, but it has no effect on the growth of Escherichia coli. It only specifically recognizes the transporter Yej ABEFon on the cell membrane of the target bacteria, and thus transports McC into the target bacterial cells. To protect McC from enzymatic hydrolysis outside the target bacteria. However, in view of the problem that Mr can inhibit protein synthesis without affecting the growth of Escherichia coli, a series of active experiments have been designed, and the effect of Mr and its analogues, f-MRA, MK, on the growth of Escherichia coli has been studied in depth. At first, Mr and its analogues, f-MRA- MRA-MRMK, were synthesized by solid phase peptide synthesis. The target products were separated and purified by semi-preparative RP-HPLC. The target products were identified by mass spectrometry. Then a series of experiments on the growth of Escherichia coli were carried out. The results showed that the concentration of Mr and a-MR killing Escherichia coli was 2.67mM4mm in 10min and 2.67mM4mm in 10min, respectively. The amount of Escherichia coli was significantly reduced by 36.77%, which indicated that the low concentration of MRF MRF Mr could inhibit the growth of E. coli, but the growth of E. coli was not affected by the addition of 0.1% BSA, and the inhibitory activity of MRF MRF MRK f MK was not affected by the addition of 0.1% BSA. The activity of 尾 -galactosidase 6-glucophosphate dehydrogenase and respiratory chain dehydrogenase in Escherichia coli treated with MRF-MRF-Mr was further determined. The experimental results showed that MRF-MRN-MRa-MR completely inhibited 尾-galactosidase 6-glucophosphate dehydrogenase at 5. 34 mm ~ 8 mm ~ 6. 47 mm, respectively. The enzyme activity of respiratory chain dehydrogenase and the enzyme activity of all enzymes increased to the normal level with the decrease of the concentration of polypeptide, so MRF-MRN-Mr could inhibit the synthesis of Escherichia coli enzyme. Finally, 尾 -galactosidase activity test and scanning tunneling electron microscope (SEM) were used to further study the interaction between MRF and E. coli cell membrane. The experimental results showed that MRF MRF MRA Mr did not change the permeability of E. coli cell membrane. However, the synthesis of 尾 -galactosidase was inhibited, and scanning tunneling electron microscopy (SEM) was used to observe Escherichia coli treated with 5.34mM and lower concentration of Mr, which directly confirmed that Mr had no effect on the integrity of E. coli cell membrane. It can be concluded that Mr and some analogs of Mr can transport transporters on the membrane of Escherichia coli into Escherichia coli cells, inhibit protein synthesis, and ultimately affect the growth of E. coli, or lead to the death of Escherichia coli.
【学位授予单位】：中国科学院大学(中国科学院过程工程研究所)
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ465
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本文编号：1912734