抗菌肽MET在毕赤酵母中的表达及其与抗生素的体外联合抑菌作用

发布时间：2018-11-13 10:36

【摘要】：抗生素滥用导致细菌对抗生素产生耐药性,对医疗和畜牧业产生较大影响,开发新模式的抗菌药物成为急需要解决的问题。抗菌肽能够作用于细菌的细胞膜使其发生裂解,导致细胞死亡,不容易产生耐药性,而且具有广谱抗菌活性,可以成为抗生素的理想替代品。但已知抗菌肽大多数是天然分离的,不仅含量小,成本极高,而且有些天然抗菌肽对细胞有一定毒性,不利于抗菌肽在临床和畜牧业的应用。很多学者致力于研究人工合成抗菌肽、提高其生物活性。本试验将人工设计合成的抗菌肽MET在毕赤酵母中进行表达,并分离纯化对其抗菌活性和与抗生素的联合使用做了研究。根据Genbank中抗菌肽MET的氨基酸序列,人工设计合成一段可编码表达MET的核酸序列。将MET基因序列克隆到真核表达载体pPICZa-A中,并对克隆结果进行酶切鉴定来验证,然后将重组载体电击转化入毕赤酵母GS-115中,提取阳性重组子的基因组做PCR,结果表明转化成功。筛选阳性酵母重组子,定期添加甲醇发酵培养,提取发酵上清通过SDS-PAGE电泳对条带进行分析。电泳结果表明,目的抗菌肽得到成功表达,而且蛋白分子量的大小与预期相符。用考马斯亮蓝法测定抗菌肽MET的表达量约为132μg/mL。测定抗菌肽MET和抗生素(Ampicillin、Cephalexin、ciprofloxacin、Doxycycline hyclate、Florfenicol、Polymyxin B、Roxithromycin、Streptomycin sulfate)的最小抑菌浓度(MIC)。分析抗菌肽MET与抗生素联合使用时的分级抑制浓度指数(FIC),研究抗两者之间的联合药效:(1)抗菌肽MET与盐酸强力霉素结合时对致病性大肠杆菌的抑菌效果表现为协同作用;(2)抗菌肽MET与多粘菌素B结合时对S.aureus的抑菌效果表现出协同作用,与罗红霉素结合时对S.aureus的抑菌效果表现出增强作用;(3)抗菌肽MET与罗红霉素结合时对猪沙门氏菌的抑菌效果表现出协同作用,与多粘菌素B结合时对猪沙门氏菌的抑菌效果表现出增强作用;(4)抗菌肽MET与氨苄青霉素、罗红霉素、硫酸链霉素联合使用对副溶血性弧菌的抑菌效果表现为协同关系,与头孢美唑、丙环沙星、盐酸强力霉素、氟苯尼考、多粘菌素B联合使用对副溶血性弧菌表现出增强作用;(5)抗菌肽MET与氨苄青霉素、多粘菌素B联合作用于痢疾志贺氏菌表现出增强作用;(6)抗菌肽MET与氟苯尼考、多粘菌素B联合作用于嗜水气单胞菌表现出协同作用,与氨苄青霉素、头孢美唑、硫酸链霉素联合作用于嗜水气单胞菌表现出增强作用;(7)抗菌肽MET与氟苯尼考、多粘菌素B联合作用于铜绿假单胞菌表现出增强作用;(8)抗菌肽MET与多粘菌素B联合作用于产II气荚膜梭菌表现出协同关系,与盐酸强力霉素、氟苯尼考、罗红霉素联合作用于产气荚膜梭菌表现出增强作用。结果表明,抗菌肽与不同抗生素联合使用对不同的菌株有不一样的效果。本研究为人工合成抗菌肽MET在毕赤酵母中的表达、为进一步研究与抗生素联合使用提供了参考,对抗菌肽用于治疗一些细菌疾病奠定了基础。
[Abstract]:The abuse of antibiotics has led to the drug resistance of bacteria to antibiotics, and has a great effect on the medical and animal husbandry, and the development of new mode of anti-bacterial drugs has become a problem to be solved urgently. The antibacterial peptide can act on the cell membrane of the bacteria to cause the cell to crack, cause cell death, is not easy to generate drug resistance, and has broad-spectrum antibacterial activity, and can be an ideal substitute for antibiotics. but most of the known antibacterial peptides are naturally separated, the content is small, the cost is high, and some natural antibacterial peptides have a certain toxicity to the cells, and the antibacterial peptide is not beneficial to the application of the antibacterial peptide in clinical and animal husbandry. Many scholars are committed to the research of synthetic antibacterial peptides and to improve their biological activity. In this study, the synthetic antibacterial peptide MET was expressed in Pichia pastoris, and its antibacterial activity and the combined use of antibiotics were studied. According to the amino acid sequence of the antimicrobial peptide MET in Genbank, a fragment of a nucleic acid sequence encoding the expression MET can be artificially designed. The MET gene sequence was cloned into the true nuclear expression vector pPICZa-A and the result of the cloning was identified by enzyme digestion. The recombinant vector was then electroshock transformed into Pichia pastoris GS-115, and the genome of the positive recombinant was extracted for PCR, and the results showed that the transformation was successful. the positive yeast recombinant is screened, the methanol fermentation culture is periodically added, the fermentation supernatant is extracted and the strip is analyzed by SDS-PAGE electrophoresis. The results showed that the target antibacterial peptide was successfully expressed, and the molecular weight of the protein was consistent with the expected. The expression of the antimicrobial peptide MET was determined to be about 132. m u.g/ mL with the Coomassius bright blue method. The minimum inhibitory concentration (MIC) of the antimicrobial peptide MET and the antibiotics (Amplicillin, Cephalan, ciprofloxacin, Doxycycline, Florfenicol, Polymyxin B, Roxidecin, Streptin sulfinate) was determined. The results showed that (1) the antibacterial effect of the antimicrobial peptide MET on the pathogenic E. coli was shown to be a synergistic effect when the combination of the antimicrobial peptide MET and the doxycycline hydrochloride was analyzed. (2) when the antibacterial peptide MET is combined with the polymyxin B, the antibacterial effect of the antibacterial peptide MET on the S. aureus shows a synergistic effect, and the antibacterial effect of the antibacterial peptide MET with the roxithromycin is enhanced; (3) the antibacterial effect of the antibacterial peptide MET and the roxithromycin is synergistic, when combined with the polymyxin B, the antibacterial effect of the salmonella in the pig is enhanced; (4) the antibacterial effect of the antibacterial peptide MET and the aminopicillin, the roxithromycin and the streptomycin sulfate on the vibrio parahaemolyticus is shown to be in a synergistic relationship, The combined use of the doxycycline hydrochloride, the florfenicol and the polymyxin B has an enhanced effect on the vibrio parahaemolyticus; (5) the antibacterial peptide MET is combined with the aminobilin and the polymyxin B to show the enhancement effect on the Shigella flexneri; (6) the antibacterial peptide MET and the florfenicol, The combined action of the polymyxin B on the Aeromonas hydrophila has a synergistic effect, and the combined action of the polymyxin B on the Aeromonas hydrophila shows an enhanced effect; (7) the antibacterial peptide MET and the florfenicol, The combination of the polymyxin B and the polymyxin B has an enhanced effect on the pseudomonas aeruginosa; (8) the antibacterial peptide MET and the polymyxin B act in a synergistic relationship with the production of the II gas-producing membrane shuttle bacteria, and the antibacterial peptide MET and the polymyxin B are combined with the doxycycline hydrochloride and the florfenicol, The combination of roxithromycin and roxithromycin in the production of air-producing membrane shuttle bacteria shows an enhanced effect. The results showed that the combination of antibacterial peptide and different antibiotics had different effects on different strains. In this study, the expression of synthetic antibacterial peptide MET in Pichia pastoris was studied. It provided a reference for further study on the combination of antimicrobial peptide and antibiotic, and laid a foundation for the treatment of some bacterial diseases.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5;S859.79

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