本文選題：MicrocinC7 + 七肽　； 參考：《中國科學(xué)院大學(xué)(中國科學(xué)院過程工程研究所)》2017年碩士論文

【摘要】：抗生素耐藥性已成為全世界廣泛關(guān)注的一個問題,嚴(yán)重地威脅著人類以及動物健康,因此迫切地需要新的解決策略和新的抗生素。MicrocinC7(McC)是廣泛分布于腸桿菌屬細(xì)菌中一種很有希望解決抗生素耐藥性問題的特洛伊木馬式抗菌肽。已有研究報道McC七肽(MR)具有抑制蛋白質(zhì)合成的能力,但是對于大腸桿菌的生長沒有影響,只特異性識別靶細(xì)菌細(xì)胞膜上的轉(zhuǎn)運子YejABEF,從而主動轉(zhuǎn)運McC進入靶細(xì)菌細(xì)胞內(nèi),保護McC在靶細(xì)菌細(xì)胞外不受酶解作用。然而本文就MR具有抑制蛋白質(zhì)合成但不影響大腸桿菌生長為問題出發(fā)點,設(shè)計了一系列的活性實驗,深入研究了 MR及其類似物f-MR、a-MR、MK、f-MK、a-MK對于大腸桿菌生長的影響。首先利用多肽固相合成技術(shù)合成了 MR以及MR的類似物f-MR、a-MR、MK、f-MK、a-MK,利用半制備反相高效液相色譜分離純化獲得目標(biāo)產(chǎn)物后,經(jīng)過質(zhì)譜鑒定,確定正確合成目標(biāo)產(chǎn)物。繼而進行了一系列對于大腸桿菌生長影響的實驗,實驗結(jié)果表明:MR、f-MR、a-MR、a-MK 分別在 5.34mM、8mM、6.47mM、6.72mM 時,能夠殺死大腸桿菌,并且MR和a-MR在10min之內(nèi)殺死大腸桿菌;MR和f-MR的濃度分別為2.67mM、4mM以及更低時,可以顯著減少約36.77%的大腸桿菌數(shù)量,表明低濃度的MR、f-MR可以抑制大腸桿菌的生長;然而富營養(yǎng)環(huán)境下,MR、f-MR、a-MR、MK、f-MK、a-MK對于大腸桿菌的生長沒有影響,添加0.1%的BSA也不會影響 MR、f-MR、a-MR、MK、f-MK、a-MK 的抑制活性。進一步測定了 MR、f-MR、a-MR處理后的大腸桿菌的β-半乳糖苷酶、6-磷酸葡糖脫氫酶、呼吸鏈脫氫酶的酶活性,實驗結(jié)果表明,MR、f-MR、a-MR分別在5.34mM、8mM、6.47mM時完全抑制β-半乳糖苷酶、6-磷酸葡糖脫氫酶、呼吸鏈脫氫酶的酶活性,且所有酶的酶活性都隨著多肽濃度的降低而升高至正常水平,所以MR、f-MR、a-MR可以抑制大腸桿菌酶的合成。最后,利用β-半乳糖苷酶活性實驗以及掃描隧道電子顯微鏡實驗進一步研究MR、f-MR、a-MR與大腸桿菌細(xì)胞膜的作用方式,實驗結(jié)果表明:雖然MR、f-MR、a-MR不改變大腸桿菌細(xì)胞膜的滲透性,但是可以抑制β-半乳糖苷酶的合成;掃描隧道電子顯微鏡觀察用5.34mM以及更低濃的MR處理過的大腸桿菌,可以直接確定MR對大腸桿菌細(xì)胞膜的完整性沒有影響。從而可以得到結(jié)論,MR以及MR的部分類似物可通過大腸桿菌細(xì)胞膜上轉(zhuǎn)運子的轉(zhuǎn)運進入大腸桿菌細(xì)胞內(nèi),抑制蛋白質(zhì)的合成,最終影響大腸桿菌的生長,或是導(dǎo)致大腸桿菌死亡。
[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.
【學(xué)位授予單位】：中國科學(xué)院大學(xué)(中國科學(xué)院過程工程研究所)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TQ465
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本文編號：1912734