抗菌肽Buforin Ⅱ衍生肽对细菌核酸的特异性作用机制研究

发布时间：2018-04-12 10:19

本文选题：抗菌肽 + 核酸作用机制　；参考：《江南大学》2012年硕士论文

【摘要】：研究已证明,很多抗菌肽通过作用于细胞膜来起到抑菌作用,除此之外,也有研究表明很多抗菌肽能穿过细胞膜进入胞内,通过作用于胞内物质,扰乱细菌生理状态而起到快速致命性的杀灭作用,但是关于抗菌肽对胞内核酸的具体作用机制及作用于核酸后对细胞周期的影响研究很少。据此本文探究了抗菌肽BuforinⅡ的衍生物BuforinⅡ-A (BF2-A)和BuforinⅡ-B (BF2-B)对细胞膜,尤其是穿过细胞膜后对胞内核酸的作用机制。首先用凝胶阻滞实验观察BF2-A和BF2-B与细菌DNA的结合。结果表明抗菌肽与金黄色葡萄球菌和大肠杆菌基因组DNA发生了结合。然后紫外光谱、圆二色谱和荧光光谱分别分析了这两种抗菌肽与金黄色葡萄球菌/大肠杆菌基因组DNA结构的影响及其结合方式。实验结果表明,加入肽后各谱图中特征峰的强度发生了变化,但峰位几乎没有变化。这证明了BF2-A和BF2-B在体外分别与金黄色葡萄球菌和大肠杆菌基因组DNA发生了结合,使DNA结构松散,但没有破坏其双螺旋结构; BF2-A和BF2-B均能与金黄色葡萄球菌和大肠杆菌基因组DNA结合并嵌入DNA碱基对中,且相同浓度的情况下这两种肽与金黄色葡萄球菌基因组DNA的结合及嵌入程度均强于大肠杆菌基因组DNA,初步推测与金黄色葡萄球菌基因组DNA的AT含量大于大肠杆菌基因组DNA有关。此外,BF2-B对基因组DNA的作用效果强于BF2-A。采用流式细胞术观察了抗菌肽作用于菌后对其细胞膜结构的影响。实验结果表明,在BF2-A和BF2-B的最低抑菌浓度时,细菌细胞膜几乎保持完整,尤其是BF2-A作用过的细菌。随着BF2-A和BF2-B浓度的分别升高,细菌的细胞膜受到一定影响,但并没有完全遭受破环。然后用凝胶阻滞电泳观察抗菌肽作用于菌后对基因组DNA的影响,结果表明BF2-A和BF2-B进入菌体内都没有引起细菌DNA的断裂。然后用流式细胞术观察抗菌肽作用于菌后对其细胞周期的影响,发现BF2-A和BF2-B将金黄色葡萄球菌和大肠杆菌抑制在细胞周期的DNA合成阶段,此为BF2-A和BF2-B产生抑菌作用的一个重要原因。同时,BF2-A和BF2-B对金黄色葡萄球菌DNA合成的抑制效果均好于大肠杆菌,再次说明这可能与金黄色葡萄球菌相关基因的AT含量大于大肠杆菌基因组DNA有关。同样,上述各实验中,BF2-B对细菌细胞膜及胞内的作用效果强于BF2-A。用PCR技术获得细菌DNA合成相关基因并通过凝胶阻滞实验验证抗菌肽与金黄色葡萄球菌和大肠杆菌DNA合成相关基因的结合。实验结果表明,BF2-A和BF2-B均能与DNA合成相关基因(DNA复制起始子基因、DNA聚合酶Ⅲ基因、DNA促旋酶基因等)结合,而且AT含量越高的DNA合成相关基因与BF2-A/BF2-B的结合强度越大。再一次证明BF2-A/BF2-B更容易与AT含量高的基因结合。此为金黄色葡萄球菌和大肠杆菌被抑制在DNA合成阶段的一个重要原因。同时,BF2-B对DNA合成相关基因的作用效果强于BF2-A。实验结果说明,BF2-A/BF2-B能在对细菌细胞膜只产生十分微小的破环作用的情况下穿过细胞膜,在胞内不断裂DNA,却能与DNA合成相关基因结合,而且DNA合成相关基因的AT含量越高,BF2-A/BF2-B与之结合的强度越大,最终将金黄色葡萄球菌和大肠杆菌的细胞周期抑制在DNA合成阶段。此外,从所有的实验中都可以看出,BF2-B的作用效果好于BF2-A,这是BF2-B的抑菌效果好于BF2-A的重要原因。
[Abstract]:Studies have shown that many antimicrobial peptides by acting on the cell membrane plays an inhibitory effect, in addition, studies have also shown that many antimicrobial peptides can cross the cell membrane into the intracellular acting through intracellular substances, bacteria and disrupt the physiological state to kill rapidly fatal, but the specific mechanism of antibacterial peptide on intracellular nucleic acid and nucleic acid in after effects on cell cycle. This paper explores the little research of antibacterial peptide Buforin II derivatives of Buforin II -A (BF2-A) and Buforin -B (BF2-B) on the cell membrane, especially the mechanism of intracellular nucleic acids across the cell membrane.
First, combined with gel retardation experiments to observe the BF2-A and BF2-B and DNA of bacteria. The results showed that the antibacterial peptide with Staphylococcus aureus and Escherichia coli DNA genome occurred. Then combined with UV spectroscopy, chromatography and fluorescence spectra of round two respectively and analyzes the two kinds of antibacterial peptide and Staphylococcus aureus Escherichia coli genome structure of DNA / influence and their combination. The experimental results show that the change of spectrum characteristic peak after the addition of peptide in strength, but almost no change in the peak position. It proved that BF2-A and BF2-B in vitro respectively with Staphylococcus aureus and Escherichia coli DNA genome occurred with DNA, the structure is loose, but no damage to the double spiral structure; BF2-A and BF2-B and Staphylococcus aureus and Escherichia coli genomic DNA binding and insertion of DNA base pairs, and the case of the same concentration of the two peptides with Staphylococcus aureus The binding and embedding degree of genomic DNA is stronger than that of Escherichia coli genome DNA. It is preliminarily speculated that the AT content of genomic DNA of Staphylococcus aureus is larger than that of Escherichia coli genome DNA. In addition, the effect of BF2-B on genomic DNA is stronger than BF2-A..
The observation of antibacterial peptides in bacteria on the cell membrane structure of flow cytometry. The experimental results show that the minimum inhibitory concentration of BF2-A and BF2-B, the bacterial cell membrane remained almost intact, especially BF2-A treated bacteria. With BF2-A and BF2-B concentration were increased, the bacterial cell membrane have a certain influence, but not completely damage. Then by gel electrophoresis were used to observe the effect of antimicrobial peptides to block bacteria on genomic DNA, the results showed that BF2-A and BF2-B in bacteria in vivo did not cause rupture of bacterial DNA. Then by flow cytometry on antimicrobial peptides on the cell cycle of bacteria effect of DNA, BF2-A and BF2-B found that the synthesis stage to Staphylococcus aureus and Escherichia coli in the inhibition of the cell cycle, an important reason for this is BF2-A and BF2-B have inhibitory effect. At the same time, BF2-A and BF2-B of golden color The inhibitory effect of Staphylococcus aureus DNA synthesis is better than that of Escherichia coli, which again indicates that this may be related to the AT content of Staphylococcus aureus related genes larger than that of Escherichia coli genomic DNA. Similarly, in these experiments, BF2-B has stronger effect on bacterial cell membrane and intracellular than BF2-A..
A combination of bacterial DNA synthesis related genes and antimicrobial peptides with Staphylococcus aureus and Escherichia coli DNA synthesis related genes by gel retardation experiments using PCR technology. The experimental results show that BF2-A and BF2-B can DNA synthesis related genes (DNA gene replication, DNA polymerase III gene, DNA gyrase gene etc.) with the content of AT is higher and the bonding strength of the DNA synthesis related genes and BF2-A/BF2-B more. Once again proved BF2-A/BF2-B more easily with high AT content. This is the combination of genes of Staphylococcus aureus and Escherichia coli were inhibited in one of the important reasons of DNA synthesis phase. At the same time, the effect of BF2-B on DNA synthesis the gene has a better effect than BF2-A.
The experimental results show that BF2-A/BF2-B can produce only on bacterial cell membrane of very small broken ring under conditions across the cell membrane, the intracellular DNA does not break, but can be combined with DNA synthesis related genes, the content of AT and DNA synthesis related genes with higher BF2-A/BF2-B with greater strength, will eventually the cell cycle of Staphylococcus aureus and Escherichia coli in inhibition of DNA synthesis stage. In addition, from all experiments we can see that the effect of BF2-B is better than BF2-A, which is an important reason for the inhibitory effect of BF2-B is better than BF2-A.

【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R151

【参考文献】