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  本文選題：五種 + 抗菌�。� 參考：《吉林大學(xué)》2014年博士論文

【摘要】：使用原核表達(dá)系統(tǒng)表達(dá)抗菌肽可以顯著降低抗菌肽的生產(chǎn)成本。我們使用Ub-tag和人源SUMO1/2/3/4-tag融合表達(dá)抗菌肽A20L并比較了不同標(biāo)簽促可溶表達(dá)和表達(dá)總量的比較。園二色光譜實(shí)驗(yàn)表明其為α-螺旋抗菌肽。通過測定MIC和MHC，表明抗菌肽A20L具有良好的抗菌作用和較低的溶血活性；通過不同磷脂組分進(jìn)行脂質(zhì)體囊泡制備后，研究了抗菌肽與脂質(zhì)體的作用。通過抗菌肽A20L與脂質(zhì)體的相互作用，我們證明了“膜區(qū)分機(jī)理”可以較好的解釋α-螺旋抗菌肽的作用機(jī)制，為抗菌肽與細(xì)胞膜相互作用的機(jī)理研究奠定了基礎(chǔ)。 研究表明XPF-St7蛙源肽序列中含有α-螺旋結(jié)構(gòu)域XPF2。首先通過固相合成方式制備得到了XPF2,證實(shí)其具有抗菌活性，然后我們通過氨基酸替換方法提高α-螺旋結(jié)構(gòu)域所帶正電荷數(shù)量以其提高抗菌活性。將氨基酸替換后的α-抗菌肽通過與Ub-tag蛋白標(biāo)簽融合表達(dá)方式成功表達(dá)抗菌肽XPF4和XPF6。測定抗菌肽XPF4和XPF6的MIC和MHC,表明兩條肽具有良好的殺菌活性和較低的溶血活性。細(xì)菌細(xì)胞膜滲透性實(shí)驗(yàn)和DNA凝膠阻滯分析實(shí)驗(yàn)證明兩條抗菌肽作用靶點(diǎn)為細(xì)菌細(xì)胞膜。 我們將AN5-1與泛素蛋白標(biāo)簽Ub-tag融合表達(dá)獲得抗菌肽AN5-1。通過測定MIC，，確認(rèn)了表達(dá)抗菌肽AN5-1的活性。我們通過細(xì)菌細(xì)胞外、內(nèi)膜滲透性實(shí)驗(yàn)，研究了抗菌肽AN5-1與細(xì)胞膜的相互作用。通過紫外光譜，園二色光譜和熒光光譜研究了AN5-1與基因組DNA的相互作用，證明了AN5-1與大腸桿菌和金黃色葡萄球菌的基因組DNA可以結(jié)合，從而抑制細(xì)菌的生長。綜合分析實(shí)驗(yàn)結(jié)果表明抗菌肽AN5-1對細(xì)菌細(xì)胞膜和DNA均有破壞作用，這說明抗菌肽AN5-1與細(xì)菌的相互作用時為多靶點(diǎn)作用。
[Abstract]:Using prokaryotic expression system to express antimicrobial peptides can significantly reduce the production cost of antimicrobial peptides.We used Ub-tag and human SUMO1/2/3/4-tag to express antimicrobial peptide A20L and compared the total expression and expression of A20L with different tags.The circular dichroism spectrum showed that it was 偽-helical antibacterial peptide.The antimicrobial peptide A20L had good antibacterial activity and low hemolytic activity by the determination of MIC and MHC.The action of antimicrobial peptide and liposome was studied after the preparation of liposome vesicles with different phospholipid components.Through the interaction of antibacterial peptide A20L with liposome, we have proved that "membrane differentiation mechanism" can better explain the mechanism of 偽 -helix antibacterial peptide, which lays a foundation for the study of the mechanism of interaction between antibacterial peptide and cell membrane.The results showed that the XPF-St7 frog peptide sequence contained 偽 -helix domain XPF2.First, XPF2 was prepared by solid state synthesis, which proved its antibacterial activity. Then, we increased the number of positive charge in 偽 -helix domain by amino acid substitution method to improve its antibacterial activity.The 偽 -antimicrobial peptide after amino acid replacement was successfully expressed by fusion with Ub-tag protein label to express the antibacterial peptides XPF4 and XPF6.The determination of MIC and MHC of XPF4 and XPF6 showed that the two peptides had good bactericidal activity and low hemolytic activity.The bacterial membrane permeability test and DNA gel block analysis showed that the two antimicrobial peptides were the target of bacterial cell membrane.We fused AN5-1 with ubiquitin label Ub-tag to obtain the antibacterial peptide AN5-1.The activity of expressed antimicrobial peptide AN5-1 was confirmed by mics.The interaction between antimicrobial peptide AN5-1 and cell membrane was studied by endomembrane permeability experiment.The interaction between AN5-1 and genomic DNA was studied by UV spectra, circular dichroism and fluorescence spectra. It was proved that AN5-1 could bind to the genomic DNA of Escherichia coli and Staphylococcus aureus, thus inhibiting the growth of bacteria.The results of comprehensive analysis showed that the antibacterial peptide AN5-1 could destroy the cell membrane and DNA of bacteria, which indicated that the interaction between AN5-1 and bacteria was a multi-target action.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R96;Q78

【共引文獻(xiàn)】

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