【作者】 黄颖桢；

【导师】 饶平凡；

【作者基本信息】 福州大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 从许多种类的两栖动物特别是蛙属(rana)动物中已经分离到一类线型,两性,带正电荷的抗菌肽。由于该类抗菌肽能够渗入并破坏细菌细胞膜,而且不易诱发细菌产生耐药性,所以其抗菌作用能力越来越引起人们的注意。沼水蛙(Hylarana guentheri)广泛分布于中国南部,在两栖动物种类和数量不断减少的同时,它通常仍是当地的优势物种。本文介绍了运用高效液相色谱和液质联用技术对沼水蛙皮肤分泌物中抗菌肽进行分离纯化,应用Edman降解法和串联质谱对多肽进行结构鉴定,经过互联网数据库的搜索和序列比对,结果证明分离到了5种新型抗菌肽AMP1 ( GFSSLFKAGAKYLLKSVGKAGAQQLACKAANNCA )、AMP2 (GVITDALKGAAKTVAAELLRKAHCKLTNSC)、AMP3 (VIDDLKKVAKKVRRELLCKKHHKKLN)、AMP4 (SIWEGIKNAGKGFLVSILDKVRCKVAGGCNP)和AMP5(FLPLLFGAISHLL-NH2),并且对其作了抗菌活性测定,发现它们对革兰氏阳性和阴性细菌具有一定的抗菌作用,对白色假丝酵母均无抗菌作用。AMP1对金黄色葡萄球菌FDA209P的MIC为14.9μg/mL;AMP2对金黄色葡萄球菌FDA209P的MIC为16.5μg/mL,对大肠杆菌O111的MIC为8.2μg/mL,对大肠杆菌ATCC25922的MIC为8.2μg/mL;AMP3对金黄色葡萄球菌FDA209P的MIC为35.5μg/mL;AMP4对金黄色葡萄球菌FDA209P的MIC为9.8μg/mL,对大肠杆菌O111的MIC为19.6μg/mL,对大肠杆菌ATCC25922的MIC为9.8μg/mL;AMP5对金黄色葡萄球菌FDA209P的MIC为44.3μg/mL。应用3’- and 5’- RACE策略对抗菌肽brevinin-2家族的新成员AMP2和AMP4进行了cDNA克隆。沼水蛙皮肤分泌物中抗菌肽的分离鉴定为首次报道。更多还原

【Abstract】 Linear, amphipathic and cationic antimicrobial peptides have been previously reported from a wide range of amphibian species especially frogs of the genus Rana. Such antimicrobial peptides are attracting increasing attention in pharmacological applications because they mainly act by permeabilizing and disrupting the target cell membrane with low degree of bacterial resistance. The guenther’s frog, Hylarana guentheri, is widely distributed in Southern China. It is commonly the dominating amphibian species even where the amphibian population is declining.The present study describes the purification and the structural and biological characterization of five novel peptides(AMP1, AMP2, AMP3, AMP4 and AMP5) with antimicrobial activity from electrically stimulated skin secretions of H. guentheri. Respectively, the primary structure of AMP1 ,AMP2, AMP3, AMP4 and AMP5 are GFSSLFKAGAKYLLKSVGKAGAQQLACKAANNCA, GVITDALK GAAKTVAAELLRKAHCKLTNSC, VIDDLKKVAKKVRRELLCKKHHKKLN, SIWEGIKNAGKGFLVSILDKVRCKVAGGCNP and FLPLLFGAISHLL-NH2. The minimum inhibitory concentrations (MICs) of the five antimicrobial peptides (AMPs) against strains of Staphylococcus aureus FDA209P range from 9.8μg/mL to 44.3μg/mL. The MIC value of AMP2 and AMP4 against Escherichia coli O111, Escherichia coli ATCC 25922 are 8.2μg/mL, 8.2μg/mL and 19.6μg/mL, 9.8μg/mL, respectively. The activity of all AMPs towards Bacillus subtilis ATCC 6633 are weak, the MIC values above 64μg/mL. Furthermore, the cDNAs of two novel members of brevinin-2 family, were subsequently cloned by using a 3’- and 5’- RACE strategy. This is the first report of antimicrobial peptides from Hylarana guentheri.更多还原