【作者】 陈利华；

【导师】 赖仞；

【作者基本信息】 南京农业大学 ， 动物学， 2008， 博士

【摘要】 两栖类动物属于一类可持续利用的自然资源,它们具有药用、食用、美学等多种科研及利用价值。此外,它们的组织中含有大量分子结构特殊、功能复杂多样的生物活性物质,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325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,因此我国两栖类动物资源的开发利用有很大的潜力。目前,已从两栖类动物中分离鉴定出许多具有多种生物活性的蛋白及多肽。通过生物化学,分子生物学的手段和研究方法,从两栖动物黑带蛙（Rananigrovittata）皮肤分泌液中,我们得到了两种皮肤生物活性多肽类物质,包括一个胆囊收缩素和一个缓激肽,并分别对这两种活性多肽类物质进行了研究。通过Sephadex G-50凝胶过滤、反相高压液相色谱等手段,从黑带蛙皮肤分泌液中分离到一个胆囊收缩素（cholecystokinin,CCK）。Edman降解测序结果显示:CCK的N-末端氨基酸残基序列为RVDGNSDQKAVIGAMLAKDLQTRKAX。BLAST分析结果表明,该氨基酸残基序列与从牛蛙（Rana catesbeiana）和非洲爪蟾（Xenopus laevis）中得到的胆囊收缩素具有显著的同源性。根据胆囊收缩素（CCK）的保守性,我们以编码牛蛙胆囊收缩素（CCK）信号肽起始密码子开始的21个碱基设计5’特异性引物,从黑带蛙皮肤cDNA文库中克隆到了编码CCK的cDNA全序列。我们首次从两栖动物皮肤中发现了胆囊收缩素,并也在皮肤中克隆得到了它的cDNA序列。通过基质辅助激光解吸附电离飞行时间质谱得知从黑带蛙皮肤分离的到胆囊收缩素（CCK）的分子量为6584.07 Da,该分子量与由CCK的cDNA序列推导得到的分子量6502.26 Da多80 Da。已知从牛蛙的大脑、腹腔中分离到的CCKs在氨基酸序列中有一个酪氨酸残基硫酸化（Tyr-SO₃）位点。当酪氨酸残基硫酸化之后,将导致约80 Da分子量的增加,因此这极有可能是黑带蛙CCKs在氨基酸残基序列中Tyr处也发生了硫酸化（Tyr-SO₃）的结果。通过分子筛、反相高压液相色谱等分离纯化方法,从黑带蛙皮肤分泌物中分离得到了一种缓激肽,命名为Ranakinin-N,具有促离体豚鼠回肠肌收缩的活性,并且它对离体豚鼠回肠肌收缩的活性的影响具有浓度依赖性。Edman降解和快原子轰击质谱法分析结果:氨基酸序列为RAEAVPPGFTPFR,理论分子量为1442.8 Da。Ranakinin-N与从其它两栖动物分离得到的缓激肽在结构上具有很高的相似性。像其它的缓激肽类似肽一样,Ranakinin-N也具有PPGF四氨基酸残基的保守区域。将Ranakinin-N的cDNA序列与另一种两栖动物花臭蛙（Odorrana schmackeri）中编码缓激肽cDNA的序列进行BLAST分析,发现Ranakinin-N的cDNA成熟肽编码区少了15个核苷酸（agaatgatcagacgc）,而这15个碱基存在于花臭蛙Odorranaschmackeri编码缓激肽的cDNA序列中,这种缺失导致了一个类胰蛋白酶位点的缺乏,并且在Ranakinin-N的N-末端有四个氨基酸AEAV的插入,AEAV的插入导致Ranakinin-N的N-末端呈中性净电荷的状态。Ranakinin-N是首次报道在N-末端呈中性净电荷状态存在的缓激肽。更多还原

【Abstract】 Amphibian is a biological treasure with great potentials. To be important natural resources, the ecological, academic, economic and cultural values of this kind of organism become more and more important. Varieties of bio-active substances in amphibian skins which play an important role in defense against invading factors have attracted the researchers all over the world. In this study, two bioactive proteins/ploypeptides were purified and identified by biochemical and molecular biological methods. By usuing a combination of gel filtration chromatography and reverse phase high performance liquid chromatography （RP-HPLC） steps, a new CCK ploypeptides were purified. Four completed precursors that encoding CCK cDNAs were cloned from Rana nigrovittata skin by molecular methods. Amino acid sequence for purified CCK was determined by using automated Edman degradation methods. The partial N-minal amino acid sequence of the purified CCK was RVDGNSDQKAVIGAMLAKDLQTRKAX. The amino acid fragment of the new purified CCK displayed significant similarity to that of CCKs from Rana catesbeiana and Xenopus laevis in the database by BLAST search. First discovery of CCK was purified and cloned cDNAs encoding CCKprecursors from R. nigrovittata skin.The predicted molecular weight （6502.26 Da） was about 80.8 Da lower than the observed molecular weight （6583.07） determinted by MALDI-TOF mass spectrometry analysis. The observed molecular weight of 6583.07 in this study may be resulted from the Tyr-SO₃ H modification of skin CCK of R. nigrovittata.A novel bradykinin-like peptide was also obtained from skin secretions of the Rana nigrovittata. The new purified bradykinin-like peptide was named as Ranakinin-N. Its primary structure （RAEAVPPGFTPFR） was also determined by Edman degradation and FAB-MS spectrometry. Sequence of Ranakinin-N is composed of 13 amino acid residues and it showed high similarity to the bradykinin purified from the skin secretions of Odorrana schmackeri, which is composed of 9 amino acid residues. Ranakinin-N was found to exert concentration -dependent contractile effects on isolated guinea pig ileum. The cDNA sequence encoding the precursor of ranakinin-N was cloned from a skin cDNA library of R. nigrovittata. The amino acid seqence deduced from the cDNA sequence match well with the result from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that a deficiency of an 15-nucleotide fragment （agaatgatcagacgc in the cDNA encoding bradykinin from O. schmackeri） in the peptide-coding region resulted in an absence of a di-basic site for trypsin-like proteinases and an unusual -AEAV-insertion in the N-terminal part of ranakinin-N. The-AEAV- insertion resulted in neutral net charge at the N-terminus of ranakinin-N. Ranakinin-N is the first report of bradykinin-like peptide with a neutral net charge at the N-terminus.更多还原