【作者】 于文媛；

【导师】 周永灿； 王世锋；

【作者基本信息】 海南大学 ， 水产养殖学， 2011， 硕士

【摘要】 马氏珠母贝是我国生产海水珍珠的主要养殖贝类,开展马氏珠母贝抗菌肽的研究有助于揭示马氏珠母贝的免疫机制,发现具有良好活性的新型抗菌药物,提高贝体的健康水平,改善海水珍珠品质,为珍珠养殖业的持续发展提供保障。本文利用酸性缓冲液制备马氏珠母贝外套膜和鳃组织的抗菌肽粗提液,利用491制备型电泳槽从粗提液中分离出含有抗菌活性成分的蛋白类物质,并对分离的抗菌蛋白进行了性质测定、溶血活性检测和抑菌活性分析。1、马氏珠母贝组织抗菌粗提液的制备使用无乳链球菌(Streptococcus agalactiae)对马氏珠母贝进行浸泡刺激。在冰浴条件下,分别利用玻璃匀浆器、研钵和IKAT-18组织匀浆机制备马氏珠母贝外套膜和鳃组织抗菌肽粗提液,采用径向扩散法(Radial Diffusion Assay, RDA)测定粗提液的抑菌活性。通过加热和冻融的方法,研究确定提高组织粗提液抗菌效果的处理条件。结果表明：以终浓度为1.0×106fu·mL-1的无乳链球菌对马氏珠母贝刺激14h,马氏珠母贝组织粗提物抗菌活性无显著变化；马氏珠母贝外套膜或鳃组织抗菌肽粗提物制备时,IKAT-18匀浆机的匀浆效果要优于玻璃匀浆器和研钵；100℃水浴处理组织粗提物10 min可显著提高其对溶藻弧菌的抗菌活性；反复冻融则会显著降低外套膜组织粗提物的抗菌效果,因此,在研究过程中应尽量减少样品的冻融次数。2、马氏珠母贝抗菌肽的分离与鉴定根据抗菌物质分子量大小的不同,使用491制备型电泳槽对马氏珠母贝组织中含有的肽类抗菌物质进行分离。检测分离得到的抗菌物质(PM-AMP-Ⅱ)后,采用聚丙烯酰胺凝胶电泳法确定其分子量,利用紫外分光光度计测定抗菌物质中的蛋白浓度。研究结果表明,利用491制备型电泳槽分离得到了抗菌物质,其中单峰值样品经SDS-PAGE分析,胶上显示单一条带,分子量为1124Da,该单峰值样品能被蛋白酶K水解,属于小分子的抗菌蛋白,蛋白含量为2.10mg/mL3、马氏珠母贝抗菌成分的抑菌活性研究为了确定所分离得到的单峰值样品的溶血活性和抑菌效果,使用血琼脂板孔穴扩散法和液体培养基倍比稀释法分别测定了样品的溶血活性、抑菌谱,得出了该样品的最低杀菌浓度和最低抑菌浓度。研究结果表明,分离得到的马氏珠母贝抗菌肽具有广谱抗菌活性,对藤黄微球菌(Micrococcus luteus)、哈氏弧菌(Vibrio harveyi)和绿脓杆菌(Pseudomonas aeruginosa)的最低抑菌浓度为0.63mg/mL,最低杀菌浓度为0.75mg/mL;对金黄色葡萄球菌(Staphyloccocus aureus)、无乳链球菌(Streptococcus agalactiae)、大肠杆菌D31(Escherichia coli D31)的最低抑菌浓度为0.75mg/mL,最低杀菌浓度为0.87mg/mL;对枯草芽孢杆菌(Bacillus subtilis)和溶藻弧菌(Vibrio alginolyticus)的最低抑菌浓度为0.52mg/mL,最低杀菌浓度为0.63mg/mL。抗菌肽的溶血效果表明,溶血圈随样品浓度的降低而减小,当样品中蛋白浓度低于0.87mg/mL时,溶血圈完全消失。更多还原

【Abstract】 In China, Pinctada martensii is the major economic shellfish in aquaculture. The study about the antimicrobial peptides from Pinctada martensii can reveal the immune mechanism of Pinctada martensii, can help in finding a kind of curative new drugs, and can improve the health of shellfish and the quality of pearls. The study also makes the cultured pearl industry develop in a virtuous circle. In this paper, the crude extracts with antimicrobial peptide was abstracted from the mantle and gill tissues of Pinctada martensii by 1% acetic acid. The antimicrobial peptides were separated from the crude extracts by 491 Prep Cell. The character, the antibacterial activity and the hemolytic activity of antimicrobial protein were studied. 1. The preparation of the tissue extraction from Pinctada martensiiThe Pinctada martensii were stimulated by putting them in the water which included Streptococcus agalactiae. The crude extractions from the mantle and gill tissues of Pinctada martensii were prepared by the glass tissue grinder, the mortar and the IKAT-18 homogenate machine at 0℃. The antimicrobial activities of the crude extracts were detected by Radial Diffusion Assay. The conditions, which can improve the antimicrobial activity of crude extracts, were confirmed by heating method, freezing and thawing method. The results showed that the antimicrobial activity of crude extracts were not changed significantly when the P. martensii was stimulated by S. agalactiae with 1.0×106 cfu·mL-1 within 14 h. The antibacterial activity of crude extracts homogenated by IKAT-18 homogenate machine was higher than that by the tissue grinder and the mortar. The activities of crude extracts against Vibrio alginolyticus can be improved significantly when the crude extractions were heated at 100℃for 10 min; The activity against V. alginolyticus was reduced when the crude extractions of the mantle tissues from P. martensii were freezed and thawed repeatedly. Therefore, the times of freezing and thawing the samples should be reduced in the process of the antimicrobial peptide purification. 2. Isolation and identification of antimicrobial peptides from Pinctada martensiiAccording to the different molecular weight of antimicrobial substances, the antimicrobial substances of tissue extraction were separated by 491 Prep Cell. The nature of the antimicrobial substances was detected, then the molecular weight of antimicrobial peptide was determined by SDS-PAGE. The protein concentration of antimicrobial substances was identified by UV spectrophotometer. The results showed that some antimicrobial substances were isolated by 491 Prep Cell. A kind of the antimicrobial substances formed a single band on the SDS-PAGE gel and its molecular weight is 1124Da. Its antibacterial activity disappeared after it was handled with proteinase K for 10 min. The protein concentration of the sample is 2.10mg/mL. 3. Some biological activities of antimicrobial peptide from Pinctada martensiiThe hemolytic activity of the antimicrobial peptide sample was determined by the blood agar diffusion assay. The antibacterial activity of the antimicrobial peptide sample was identified by broth gradient dilution assay. Then the MBC (Minimal Bacteriocidal Concentration) and MIC (Minimal Inhibitory Concentrations) were confirmed. The results showed that the antimicrobial peptide from Pinctada martensii had broad-spectrum antimicrobial activities. Its MIC and MBC against Micrococcus luteus, Vibrio harveyi and Pseudomonas aeruginosa were 0.63mg/mL and 0.75mg/mL respectively; its MIC and MBC against Staphyloccocus aureus, Streptococcus agalactiae and Escherichia coli D31 were 0.75mg/mL and 0.87mg/mL respectively; and its MIC and MBC against Bacillus subtilis and Vibrio alginolyticus were 0.52mg/mL and 0.63mg/mL respectively. The hemolysis activity of the antimicrobial peptide reduced when the protein concentration in the sample decreased, and the hemolytic ring disappeared while the protein concentration in the sample was less than 0.87mg/mL.更多还原