【作者】 刘晓；

【导师】 崔言顺；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2016， 硕士

【摘要】 抗生素滥用导致细菌耐药性增加已是全球公共卫生问题。因此,寻求广谱、高效的新一代抗菌剂已成为全世界亟待解决的问题。近年来,抗菌肽作为一种新型的抗菌剂引起了国内外相关研究者的广泛关注。抗菌肽(Antimicrobial peptides AMPs),又称肽类抗生素(peptide antibiotics),是一类具有广谱抗菌活性的小分子短肽,是宿主对抗外源性病原体的天然免疫防御系统的重要分子屏障。目前,在家禽体内已发现两大类抗菌肽,即defensins和cathelicidins。相关研究表明,cathelicidins类抗菌肽在体外具有较强的广谱抗革兰氏阴性菌(G-)和革兰氏阳性菌(G+)活性,cathelicidins类抗菌肽具有极大的应用潜力,值得进行更深入的研究。本试验选取乌骨鸡cathelicidins类抗菌肽为目的基因,构建真核重组表达质粒,应用巴斯德毕赤酵母真核表达系统表达目的蛋白并与多种抗生素联用检测其与抗生素联合应用,检测其抑菌作用,以寻求从实质上降低抗生素使用的新方法。根据GenBank中收录的乌骨鸡Cathelicidin-1、-2、-3的基因序列(GenBank序列号:FJ938357、FJ938358、FJ938359),设计三对特异性引物,在其基因两端分别加入酶切位点EcoR I和Xba I。无菌采集乌骨鸡腿骨新鲜骨髓提取其RNA,并以此为模板进行RT-PCR扩增,并克隆至pMD18-T载体上,构建重组质粒pMD18-T-Cath L-1、-2、-3,并测序鉴定。用限制性内切酶EcoR I和Xba I双酶切重组质粒pMD18-T-CathL-1、-2、-3获得目的基因Cathelicidin-1、-2、-3,将目的基因连接到经EcoR I和Xba I双酶切的真核表达载体pPICZα-A上,构建重组表达载体pPICZα-A-CathL1、L2、L3,并再次测序鉴定序列的准确性。然后,利用内切酶Sac I对重组表达载体pPICZα-A-Cath L1、L2、L3、质粒pPICZα-A分别进行单酶切,获得线性化质粒。将线性化质粒pPICZα-A-CathL1、L2、L3和pPICZα-A分别电击转入毕赤酵母X-33株感受态细胞中。分别对得到的阳性转化子,利用PCR鉴定阳性转化子。阳性转化子再进行Mut+表型和高拷贝筛选,得到能够正常利用甲醇的高拷贝重组酵母菌株。将筛选到的阳性重组酵母菌株进行甲醇诱导表达,每24 h追加1次甲醇(终浓度分别为1.0%、2.0%),并取1 mL菌液,离心收集上清,直至培养96 h。收集表达上清利用DOC-TCA浓缩蛋白方法处理上清,获得表达的蛋白并进行Tricine-SDS-PAGE鉴定。Tricine-SDS-PAGE电泳结果显示,出现三条特异性条带,大小分别为7.6 KD、8.1 KD、7.6 KD,与预期结果相符。同时送华大基因公司做质谱鉴定,结果显示表达蛋白准确。此外,结果还显示阳性转化子在1.0%、2.0%甲醇浓度下72 h时蛋白表达量最高且2.0%甲醇浓度下蛋白表达量高于1.0%甲醇浓度下的蛋白表达量。利用96孔板微量稀释法来测定表达产物的抑菌效果。试验结果显示,3种Cathelicidin都对大肠杆菌、绿脓杆菌、奇异变形杆菌表现出不同程度的抑菌效果,其MIC值在2-64μg/mL之间,并且抗菌肽Cathelicidin-1对大肠杆菌1505具有明显的抑菌效果,其MIC值为2μg/mL,优于环丙沙星(MIC=4μg/mL)、氨苄西林(MIC=32μg/m L)、氨苄青霉素(MIC=32μg/m L)、头孢他啶(MIC=32μg/m L)、庆大霉素(MIC=16μg/m L)、左氧氟沙星(MIC=8μg/mL)。将三种cathelicidin抗菌肽与阿莫西林、阿米卡星、阿齐霉素等11种抗生素联用,研究其对大肠杆菌、沙门氏菌、多杀性巴氏杆菌、藤黄微球菌、绿脓杆菌、金黄色葡萄球菌等菌株的联合抑菌效果,以抗生素的抑菌效果为对照。联合抑菌结果显示,针对不同的菌株,抗生素和抗菌肽联合有协同作用效果,也有拮抗作用效果。在联合抑菌效果中其中,对于绿脓杆菌,阿米卡星单独使用时没有抑菌效果,但是和重组抗菌肽cathelicidin-1、-2、-3联用时,表现出极显著的抑菌效果(P<0.01);对于大肠杆菌1507、0914株,头孢西丁单独使用时没有抑菌效果,但是和重组抗菌肽cathelicidins-1、-2、-3联用时,表现出抑菌效果,并且协同效果极为显著(P<0.01)。但是,对于大肠杆菌1504株,氧氟沙星单独使用时具有抑菌效果,但是和重组抗菌肽cathelicidins-1、-2、-3联用时,表现为无抑菌效果,拮抗效果极显著(P<0.01)。结果表明,禽源cathelicidin抗菌肽与不同抗生素之间存在相互作用,某些特定的组合表现出明显的协同抑菌潜力。针对不同的细菌,探索具有明显抑菌效果的Cathelicidin抗菌肽与抗生素组方是一个降低抗生素使用量的可行方法。更多还原

【Abstract】 The increased bacterial resistance caused by the abuse of antibiotics had become a global public health problem. Therefore, seeking for a broad-spectrum, highly effective new generation of antimicrobial agents has become a serious problem over the world. In recent years, antimicrobial peptides as a novel antibacterial agent caused widespread concern in domestic and foreign researchers. Antimicrobial peptides(Antimicrobial peptides, AMPs), also known as peptide antibiotics, are a class of small molecule peptides with broad-spectrum antimicrobial activity against exogenous pathogens, and play an important role in natural immune defense system of the host. Currently, two kinds of antimicrobial peptidesit had been found in poultry, defensins and Cathelicidins. Research indicated that, the Cathelicidins antimicrobial peptides showed a strong and broad-spectrum activities on Gram-negative(G-) and gram-positive(G+) bacteria in vitro, and they also showed great potential. In the current study, we selected Silky Cathelicidins genes as the purpose genes to constructe eukaryotic expression plasmids to express the purpose proteins by Pichia pastoris(P. Pastoris) eukaryotic expression system. The expressed purpose proteins were combined with a variety of antibiotics to analyze their antibacterial effects, hoping to provide a substantial help to solve the problem of antibiotic resistance, and lay the foundation for further studies in combination with antibiotics.To amplify the Cathelicidin-1,-2,-3 genes, three pairs of primers were designed according to the Cathelicidin-1,-2,-3 genes sequences(GeneBank accession number: FJ938357、FJ938358、FJ938359). EcoR I and Xba I restriction enzyme sites were introduced at the respective 5′-termini. The RNA of Silky were collected from its leg bine marrow and then amplify the Cathelicidin-1,-2,-3 genes through RT-PCR. The RT-PCR products were cloned into the pMD18-T vector, and the resultant plasmids were confirmed by sequencing. The resultant plasmids pMD18-T-Cathelicidin-1,-2,-3 were digested with EcoR I and Xba I, and cloned into the pPICZα-A vector, which was digested with the same restriction enzymes. The recombinant plasmids were confirmed by restriction digestion and sequencing. The recombinant plasmids pPICZα-A-Cath L1,L2,L3 and plasmid pPICZα-A were digested with Sac I to obtain the linearized plasmids. Then the linearized plasmids were transformed into competent P. Pastoris cells by Electroporation, respectively. Correct positive transformants were by PCR. The Mut+ phenotype and high copies selecting were going with the correct positive transformants to obtain the final strain, which can use the methanol.The positive recombinant yeast strains were induced by methanol to express the protein, with adding methanol(final concentration of 1.0%, 2.0%) per 24 h and collecting 1 m L cell culture supernatants, until to 96 h. Then the expressed proteins in the cell culture supernatants was purified with DOC-TCA, and verified by Tricine-SDS-PAGE. The results showed that three single protein bands with apparent molecular weight of 7.6 KD, 8.1 KD, 7.6 KD were obtained after the purification procedure, which was consistent with the expected results. In addition, the results of HuaDa company showed that the expressed proteins were correct. Notably, the results showed that the positive transformants in 1.0%, 2.0% methanol had highest protein expression at 72 h, and the protein expression in 2.0% methanol was higher than in 1.0% methanol.Through the orifice 96 micro-dilution method is used to determine the bacteriostatic effect of expression products.Test results show that the three kinds of Cathelicidins are Escherichia coli, Pseudomonas aeruginosa, Singular proteus showed different degrees of bacteriostatic effect, the MIC value between 2-64 μg/mL, and antibacterial peptide Cathelicidin-1 for E.coli 1505 has obvious antibacterial effect, the MIC value of 2 μg/m L, is better than that of ciprofloxacin(MIC=4 μg/mL), ampicillin(MIC=32 μg/m L), ampicillin(MIC=32μg/mL), cephalosporins he organism(MIC=32 μg/m L) and gentamycin(MIC=16 μg/m L), levofloxacin(MIC=8 μg/mL).Three kinds of antibacterial peptide cathelicidin and amoxicillin, amikacin, archie, and other 11 kinds of antibiotics and research on E.coli、P.aeruginosa、S.pullorum、P.multocida、Micrococcus luteus 、S.aureus strains of antibacterial effect, the bacteriostatic effect of antibiotics as control.United bacteriostasis results show that for different strains of antibiotic and antimicrobial peptides have synergy effect, also have antagonism effect.Among them, in the joint antibacterial effect for pseudomonas aeruginosa, amikacin has no antibacterial effect when used alone, but and restructuring of antimicrobial peptides Cathelicidin-1,-2,-3, when used, showed a very significant bacteriostatic effect(P < 0.01); For 1507, 0914 strains of E.coli, cefoxitin no antibacterial effect when used alone, but and restructuring of antimicrobial peptides cathelicidin-1,-2,-3, when used, show that the bacteriostatic effect, and synergy effect is extremely significant(P < 0.01). But for 1504 strains of E.coli, ofloxacin bacteriostatic effect when it is used alone, but and restructuring of antimicrobial peptides cathelicidin-1,-2,-3, when used, expression is no antimicrobial effect, antagonism effect is very significant(P < 0.01). The results showed that the poultry cathelicidin antimicrobial peptides and the interaction between different antibiotics, the combination of certain showed obvious bacteriostatic potential synergy. According to different bacterias, exploration has obvious antibacterial effect of cathelicidin antimicrobial peptide antibiotics and prescription is a feasible way to reduce the antibiotic usage.更多还原