抗菌肽MET在畢赤酵母中的表達(dá)及其與抗生素的體外聯(lián)合抑菌作用
發(fā)布時(shí)間：2018-11-13 10:36
【摘要】：抗生素濫用導(dǎo)致細(xì)菌對抗生素產(chǎn)生耐藥性,對醫(yī)療和畜牧業(yè)產(chǎn)生較大影響,開發(fā)新模式的抗菌藥物成為急需要解決的問題�？咕哪軌蜃饔糜诩�(xì)菌的細(xì)胞膜使其發(fā)生裂解,導(dǎo)致細(xì)胞死亡,不容易產(chǎn)生耐藥性,而且具有廣譜抗菌活性,可以成為抗生素的理想替代品。但已知抗菌肽大多數(shù)是天然分離的,不僅含量小,成本極高,而且有些天然抗菌肽對細(xì)胞有一定毒性,不利于抗菌肽在臨床和畜牧業(yè)的應(yīng)用。很多學(xué)者致力于研究人工合成抗菌肽、提高其生物活性。本試驗(yàn)將人工設(shè)計(jì)合成的抗菌肽MET在畢赤酵母中進(jìn)行表達(dá),并分離純化對其抗菌活性和與抗生素的聯(lián)合使用做了研究。根據(jù)Genbank中抗菌肽MET的氨基酸序列,人工設(shè)計(jì)合成一段可編碼表達(dá)MET的核酸序列。將MET基因序列克隆到真核表達(dá)載體pPICZa-A中,并對克隆結(jié)果進(jìn)行酶切鑒定來驗(yàn)證,然后將重組載體電擊轉(zhuǎn)化入畢赤酵母GS-115中,提取陽性重組子的基因組做PCR,結(jié)果表明轉(zhuǎn)化成功。篩選陽性酵母重組子,定期添加甲醇發(fā)酵培養(yǎng),提取發(fā)酵上清通過SDS-PAGE電泳對條帶進(jìn)行分析。電泳結(jié)果表明,目的抗菌肽得到成功表達(dá),而且蛋白分子量的大小與預(yù)期相符。用考馬斯亮藍(lán)法測定抗菌肽MET的表達(dá)量約為132μg/mL。測定抗菌肽MET和抗生素(Ampicillin、Cephalexin、ciprofloxacin、Doxycycline hyclate、Florfenicol、Polymyxin B、Roxithromycin、Streptomycin sulfate)的最小抑菌濃度(MIC)。分析抗菌肽MET與抗生素聯(lián)合使用時(shí)的分級抑制濃度指數(shù)(FIC),研究抗兩者之間的聯(lián)合藥效:(1)抗菌肽MET與鹽酸強(qiáng)力霉素結(jié)合時(shí)對致病性大腸桿菌的抑菌效果表現(xiàn)為協(xié)同作用;(2)抗菌肽MET與多粘菌素B結(jié)合時(shí)對S.aureus的抑菌效果表現(xiàn)出協(xié)同作用,與羅紅霉素結(jié)合時(shí)對S.aureus的抑菌效果表現(xiàn)出增強(qiáng)作用;(3)抗菌肽MET與羅紅霉素結(jié)合時(shí)對豬沙門氏菌的抑菌效果表現(xiàn)出協(xié)同作用,與多粘菌素B結(jié)合時(shí)對豬沙門氏菌的抑菌效果表現(xiàn)出增強(qiáng)作用;(4)抗菌肽MET與氨芐青霉素、羅紅霉素、硫酸鏈霉素聯(lián)合使用對副溶血性弧菌的抑菌效果表現(xiàn)為協(xié)同關(guān)系,與頭孢美唑、丙環(huán)沙星、鹽酸強(qiáng)力霉素、氟苯尼考、多粘菌素B聯(lián)合使用對副溶血性弧菌表現(xiàn)出增強(qiáng)作用;(5)抗菌肽MET與氨芐青霉素、多粘菌素B聯(lián)合作用于痢疾志賀氏菌表現(xiàn)出增強(qiáng)作用;(6)抗菌肽MET與氟苯尼考、多粘菌素B聯(lián)合作用于嗜水氣單胞菌表現(xiàn)出協(xié)同作用,與氨芐青霉素、頭孢美唑、硫酸鏈霉素聯(lián)合作用于嗜水氣單胞菌表現(xiàn)出增強(qiáng)作用;(7)抗菌肽MET與氟苯尼考、多粘菌素B聯(lián)合作用于銅綠假單胞菌表現(xiàn)出增強(qiáng)作用;(8)抗菌肽MET與多粘菌素B聯(lián)合作用于產(chǎn)II氣莢膜梭菌表現(xiàn)出協(xié)同關(guān)系,與鹽酸強(qiáng)力霉素、氟苯尼考、羅紅霉素聯(lián)合作用于產(chǎn)氣莢膜梭菌表現(xiàn)出增強(qiáng)作用。結(jié)果表明,抗菌肽與不同抗生素聯(lián)合使用對不同的菌株有不一樣的效果。本研究為人工合成抗菌肽MET在畢赤酵母中的表達(dá)、為進(jìn)一步研究與抗生素聯(lián)合使用提供了參考,對抗菌肽用于治療一些細(xì)菌疾病奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5;S859.79
本文編號：2328841
[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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5;S859.79
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