光滑爪蟾皮腺抗菌肽的分离纯化及其活性研究

发布时间：2018-05-27 22:12

本文选题：抗菌肽 + 光滑爪蟾　；参考：《吉林大学》2017年硕士论文

【摘要】：抗菌肽作为抗生素最佳候选药物,广泛存在于两栖动物体内及表面腺体中,在其适应阴暗潮湿环境中,发挥着重要作用。同时,抗菌肽是两栖动物非特异性免疫系统的重要组成部分,在受到外界刺激时,即可大量分泌,并具有多样性。至今为止,仍有众多种类抗菌肽未被发现。本实验通过电刺激诱导光滑爪蟾皮腺抗菌肽的分泌,并进行纯化。首先利用超速离心除去杂质及胶原蛋白,再通过制备型高效液相色谱配合半制备型C18柱进行粗分离,最后将具有抗菌活性的组分通过分析型高效液相色谱结合分析型C18柱进一步纯化。最终获得3个单一组分抗菌肽样品,并将其进行MALDL-TOF/TOF检测及氨基酸测序。最终共有2条抗菌肽是未被报道过的新抗菌肽,命名为P1,P2。为了对抗菌肽进行活性研究,本实验通过固相合成法合成并纯化抗菌肽P1及P2,分别探究其抑菌活性、溶血活性、对乳腺癌细胞MCF-7的抑制活性。结果表明,P1及P2不仅能抑制标准株生长,同时对一些典型的多重耐药菌株同样具有显著的抑制活性。而溶血活性结果表明,当达到8倍针对于金黄色葡萄球菌MIC值时,P1及P2才会表现6.2%及9.3%的溶血率。而在肿瘤细胞抑制实验中,抗菌肽浓度越高,其表现出细胞增殖的抑制作用越强,在50μg/m L,抑制率高达90%,对MCF-7细胞系抑制作用显著。尽管在浓度低至5μg/m L时,其抑制率仍能维持在42%及45%。透射电镜结果表明,处理后大肠杆菌细菌及金黄色葡萄球菌的细胞膜结构发生变化,失去典型菌体形态。其中相比于大肠杆菌,P1、P2对于金黄色葡萄球菌的破坏作用更加显著,与MIC值相吻合,同时也符合本实验以金黄色葡萄球菌作为筛选测试菌的实验目的。本实验利用高压均质机将抗菌肽与一定比例的角鲨烯分子及表面活性剂混合乳化,形成均一稳定的纳米乳。对金黄色葡萄球菌的MIC值证明乳化后抗菌肽乳剂具有缓释作用。在最初的1h内,未被包裹的P1抑菌活性迅速降低,而在4 h后几乎失去抑菌活性(MIC值大于320μg/m L)。相比之下,纳米乳包裹P1在孵育8 h后MIC值仍能保持在40μg/m L,证明其能够在8 h内平稳而高效地发挥作用。而孵育48 h后,其MIC值为320μg/m L,证明其至少在2天的时间内维持着一个相对有效的抑菌环境。
[Abstract]:As the best candidate for antibiotics, antimicrobial peptides are widely found in amphibians and their surface glands, and play an important role in their adaptation to dark and humid environments. At the same time, antimicrobial peptide is an important part of amphibian nonspecific immune system. Up to now, many kinds of antimicrobial peptides have not been found. In this study, the secretion and purification of antimicrobial peptides from Xenopus smooth skin gland were induced and purified by electrical stimulation. Firstly, the impurities and collagen were removed by ultracentrifugation, and then the crude separation was carried out by high performance liquid chromatography (HPLC) and semi-preparation C18 column. Finally, the antibacterial components were further purified by analytical high performance liquid chromatography (HPLC) combined with analytical C 18 column. Finally, three single component antimicrobial peptides were obtained and detected by MALDL-TOF/TOF and amino acid sequencing. A total of 2 antimicrobial peptides were unreported new antimicrobial peptides, named P 1 P 2 2. In order to study the activity of antimicrobial peptides, antimicrobial peptides P1 and P2were synthesized and purified by solid phase synthesis method. The antibacterial activity, hemolytic activity and inhibitory activity on MCF-7 of breast cancer cells were investigated respectively. The results showed that P _ 1 and P _ 2 could not only inhibit the growth of the standard strain, but also had significant inhibitory activity against some typical multidrug resistant strains. The results of hemolytic activity showed that when the MIC value of Staphylococcus aureus reached 8 times, the hemolysis rates of P1 and P2 were 6.2% and 9.3% respectively. In the tumor cell inhibition test, the higher the concentration of antimicrobial peptide, the stronger the inhibition of cell proliferation, at 50 渭 g / mL, the inhibition rate was as high as 90%, and the inhibitory effect on MCF-7 cell line was significant. Even when the concentration was as low as 5 渭 g / mL, the inhibition rate remained at 42% and 45%. The results of transmission electron microscope showed that the cell membrane structure of Escherichia coli and Staphylococcus aureus changed after treatment and the typical cell morphology was lost. The destruction of Staphylococcus aureus was more significant than that of Escherichia coli P1P _ 2, which was consistent with the MIC value and the purpose of screening Staphylococcus aureus as test bacteria. In this experiment, the antibacterial peptides were mixed with squalene molecules and surfactants in a high pressure homogenizer to form homogeneous and stable nano-emulsion. The MIC value of Staphylococcus aureus showed that the emulsified antibacterial peptide emulsion had slow release effect. In the first 1h, the unencapsulated P1 bacteriostatic activity decreased rapidly, but after 4 h, the MIC value was more than 320 渭 g / m L ~ (-1). In contrast, the MIC value of P1-coated nanoemulsion remained at 40 渭 g / mL after incubation for 8 h, which proved that it could function smoothly and efficiently within 8 h. After incubation for 48 h, its MIC value was 320 渭 g / mL, which proved that it maintained a relatively effective bacteriostasis environment for at least 2 days.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q51

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