【作者】 韩曜平；

【导师】 夏德全； 吴婷婷；

【作者基本信息】 南京农业大学 ， 水生生物学， 2006， 博士

【摘要】 两栖类动物属于一类可持续利用的自然资源，它们具有多种多样的科研及利用价值。此外，它们的组织中含有大量的分子结构特殊、功能复杂多样的生物活性物质，它们是有待于开发的资源宝库。中国拥有丰富的两栖类动物资源，共有284个种(亚种)，其中2／3是特有种类，它是我国生物资源的重要组成部分，也是人类的宝贵财富，对我国两栖类动物资源的开发利用有很大的潜力。目前，已分离鉴定出许多具有各种生物活性的蛋白及多肽。 我们收集分布于中国西南境内的2种常见的药用、食用两栖类：山溪鲵(Batrachuperus pachunii)及无指盘臭蛙(Rana grahami)，并对这2种两栖类皮肤分泌液及其卵进行了如下一系列的生物活性筛选研究：(1) 以发色底物对2种两栖类动物皮肤分泌液进行丝氨酸蛋白酶水解活性和丝氨酸蛋白酶抑制剂活性测定，山溪鲵皮肤分泌液无丝氨酸蛋白酶水解活性，无指盘臭蛙皮肤分泌液及卵匀浆液显示较强的丝氨酸蛋白酶抑制剂活性；(2) 2种两栖类皮肤分泌液具有较强的抗菌活性，而其卵匀浆液则无明显抗菌作用；(3) 2种两栖类的皮肤分泌液不具备出血活性，无指盘臭蛙卵匀浆液显示较弱的溶血活性，而山溪鲵卵匀浆液无溶血活性。(4) 2种两栖类皮肤分泌液及其卵匀浆液对正常人血浆的复钙时间没有明显的影响，即它们对正常人血液没有明显的促凝和抗凝作用。 进一步通过生物化学，分子生物学手段和研究方法，分别从2种两栖动物皮肤分泌液或卵匀浆液中，我们得到了3种生物活性多肽类或蛋白类物质，包括两个抗菌肽、两个丝氨酸蛋白酶抑制剂及β-microseminoprotein样蛋白。并分别对这几类活性多肽类或蛋白类物质进行了进一步研究。 通过Sephadex G-50凝胶过滤、反相高压液相等生化技术手段，从无指盘臭蛙皮肤分泌波中得到两个具有广谱抗菌活性的抗菌肽。该两个抗菌肽被命名grahamin 1 and grahamin 2。Edman降解法测序结果grahamin 1的氨基酸序列为GLLSGILGAGKNIVCG LSGLC；grahamin 2的氨基酸序列为GLLSGILGAGKHIVCGLSGLC；快原子轰击质谱分析表明，grahamin 1及grahamin 2的分子量分别为1942Da和1965Da，结果与理论分子量(1942.5Da和1965.6Da)基本相符。grahamin 1及grahamin 2氨基酸序列比较分更多还原

【Abstract】 Amphibian is a biological treasure with great potentials as well as important natural resources with ecological, research, economic and cultural value. A variety of bio-active substances from amphibian skins which play an important role in defense against invading factors have attracted significant attention of the researchers all over the world. In this study, three bioactive proteins/ploypeptides were identified by biochemical and molecular biological methods.Two antimicrobial peptides manifesting a broad spectrum of antimicrobial activity against various microorganisms have been isolated from skin secretions of Rana grahami. These antimicrobial peptides were named grahamin 1 and grahamin 2. Their primary structures are GLLSGILGAGKNIVCGLSGLC (1942Da) and GLLSGILGAGKHIVCGL SGLC (1965Da), respectively, determined by Edman degradation and mass spectrometry. They are structurally related to nigrocins identified from skin secretions of the darkspotted frog, Rana nigromaculata. The cDNA clones encoding the precursor of grahamins were screened and sequenced from the skin cDNA library of R. grahami. The amino sequences deduced from the cDNA sequences match well with the results from Edman degradation. As other antimicrobial peptides from Rana species, grahamins contain a C-terminal loop region delineated by an intra-disulfide bridge named Rana box. Based on structural comparison of grahamin with other known antimicrobial peptides, grahamins could be classified into the family of antimicrobial peptides containing a single intra-disulfide bridge.Two proteins with an apparent molecular weight of 40kDa and 14.4kDa were purified from the egg of Rana grahami by a combination of gel filtration chromatography, AKTA(?) Resource-Q ion chromatography and reverse phase high performance liquid chromatography (RP-HPLC) steps. The purified two proteins showed a trypsin inhibitor更多还原