本文選題：抗菌肽 + 光滑爪蟾�。� 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：抗菌肽作為抗生素最佳候選藥物,廣泛存在于兩棲動(dòng)物體內(nèi)及表面腺體中,在其適應(yīng)陰暗潮濕環(huán)境中,發(fā)揮著重要作用。同時(shí),抗菌肽是兩棲動(dòng)物非特異性免疫系統(tǒng)的重要組成部分,在受到外界刺激時(shí),即可大量分泌,并具有多樣性。至今為止,仍有眾多種類抗菌肽未被發(fā)現(xiàn)。本實(shí)驗(yàn)通過(guò)電刺激誘導(dǎo)光滑爪蟾皮腺抗菌肽的分泌,并進(jìn)行純化。首先利用超速離心除去雜質(zhì)及膠原蛋白,再通過(guò)制備型高效液相色譜配合半制備型C18柱進(jìn)行粗分離,最后將具有抗菌活性的組分通過(guò)分析型高效液相色譜結(jié)合分析型C18柱進(jìn)一步純化。最終獲得3個(gè)單一組分抗菌肽樣品,并將其進(jìn)行MALDL-TOF/TOF檢測(cè)及氨基酸測(cè)序。最終共有2條抗菌肽是未被報(bào)道過(guò)的新抗菌肽,命名為P1,P2。為了對(duì)抗菌肽進(jìn)行活性研究,本實(shí)驗(yàn)通過(guò)固相合成法合成并純化抗菌肽P1及P2,分別探究其抑菌活性、溶血活性、對(duì)乳腺癌細(xì)胞MCF-7的抑制活性。結(jié)果表明,P1及P2不僅能抑制標(biāo)準(zhǔn)株生長(zhǎng),同時(shí)對(duì)一些典型的多重耐藥菌株同樣具有顯著的抑制活性。而溶血活性結(jié)果表明,當(dāng)達(dá)到8倍針對(duì)于金黃色葡萄球菌MIC值時(shí),P1及P2才會(huì)表現(xiàn)6.2%及9.3%的溶血率。而在腫瘤細(xì)胞抑制實(shí)驗(yàn)中,抗菌肽濃度越高,其表現(xiàn)出細(xì)胞增殖的抑制作用越強(qiáng),在50μg/m L,抑制率高達(dá)90%,對(duì)MCF-7細(xì)胞系抑制作用顯著。盡管在濃度低至5μg/m L時(shí),其抑制率仍能維持在42%及45%。透射電鏡結(jié)果表明,處理后大腸桿菌細(xì)菌及金黃色葡萄球菌的細(xì)胞膜結(jié)構(gòu)發(fā)生變化,失去典型菌體形態(tài)。其中相比于大腸桿菌,P1、P2對(duì)于金黃色葡萄球菌的破壞作用更加顯著,與MIC值相吻合,同時(shí)也符合本實(shí)驗(yàn)以金黃色葡萄球菌作為篩選測(cè)試菌的實(shí)驗(yàn)?zāi)康摹１緦?shí)驗(yàn)利用高壓均質(zhì)機(jī)將抗菌肽與一定比例的角鯊烯分子及表面活性劑混合乳化,形成均一穩(wěn)定的納米乳。對(duì)金黃色葡萄球菌的MIC值證明乳化后抗菌肽乳劑具有緩釋作用。在最初的1h內(nèi),未被包裹的P1抑菌活性迅速降低,而在4 h后幾乎失去抑菌活性(MIC值大于320μg/m L)。相比之下,納米乳包裹P1在孵育8 h后MIC值仍能保持在40μg/m L,證明其能夠在8 h內(nèi)平穩(wěn)而高效地發(fā)揮作用。而孵育48 h后,其MIC值為320μg/m L,證明其至少在2天的時(shí)間內(nèi)維持著一個(gè)相對(duì)有效的抑菌環(huán)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q51

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