五种抗菌肽的设计、原核表达和抗菌作用机理研究

发布时间：2018-04-11 23:24

本文选题：五种 + 抗菌　；参考：《吉林大学》2014年博士论文

【摘要】：使用原核表达系统表达抗菌肽可以显著降低抗菌肽的生产成本。我们使用Ub-tag和人源SUMO1/2/3/4-tag融合表达抗菌肽A20L并比较了不同标签促可溶表达和表达总量的比较。园二色光谱实验表明其为α-螺旋抗菌肽。通过测定MIC和MHC，表明抗菌肽A20L具有良好的抗菌作用和较低的溶血活性；通过不同磷脂组分进行脂质体囊泡制备后，研究了抗菌肽与脂质体的作用。通过抗菌肽A20L与脂质体的相互作用，我们证明了“膜区分机理”可以较好的解释α-螺旋抗菌肽的作用机制，为抗菌肽与细胞膜相互作用的机理研究奠定了基础。研究表明XPF-St7蛙源肽序列中含有α-螺旋结构域XPF2。首先通过固相合成方式制备得到了XPF2,证实其具有抗菌活性，然后我们通过氨基酸替换方法提高α-螺旋结构域所带正电荷数量以其提高抗菌活性。将氨基酸替换后的α-抗菌肽通过与Ub-tag蛋白标签融合表达方式成功表达抗菌肽XPF4和XPF6。测定抗菌肽XPF4和XPF6的MIC和MHC,表明两条肽具有良好的杀菌活性和较低的溶血活性。细菌细胞膜渗透性实验和DNA凝胶阻滞分析实验证明两条抗菌肽作用靶点为细菌细胞膜。我们将AN5-1与泛素蛋白标签Ub-tag融合表达获得抗菌肽AN5-1。通过测定MIC，，确认了表达抗菌肽AN5-1的活性。我们通过细菌细胞外、内膜渗透性实验，研究了抗菌肽AN5-1与细胞膜的相互作用。通过紫外光谱，园二色光谱和荧光光谱研究了AN5-1与基因组DNA的相互作用，证明了AN5-1与大肠杆菌和金黄色葡萄球菌的基因组DNA可以结合，从而抑制细菌的生长。综合分析实验结果表明抗菌肽AN5-1对细菌细胞膜和DNA均有破坏作用，这说明抗菌肽AN5-1与细菌的相互作用时为多靶点作用。
[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 伪-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 伪 -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 伪 -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 伪 -helix domain by amino acid substitution method to improve its antibacterial activity.The 伪 -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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96;Q78

【共引文献】