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通过葡聚糖Sephadex G-50凝胶过滤和反相高效液相析(RP-HPLC)等手段,对云南小狭口蛙(Callueslla yunnanensisBoulenger)皮肤分泌液进行分离纯化,得到了有促胰岛素释放活性的多肽分离峰。运用质谱仪测定该促胰岛素释放肽的相对分子质量为1 768.08,一级结构鉴定测得其氨基酸基本序列为:FLPIVGRSYA GLSFKL-NH2,并将其命名为Callin,是一个C末端酰胺化的十六肽。分别采用体外培养的大鼠胰岛和INS-1胰岛β细胞系检测的促胰岛素释放活性,结果表明,Callin既可以增加大鼠胰岛的胰岛素释放量,又可以促进INS-1细胞的胰岛素分泌,经统计学分析具有显著性差异(P<0.05),且存在一定的剂量依赖性。该研究分离得到的促胰岛素释放肽Callin将为研制能治疗2型糖尿病患者的新型多肽药物提供基础数据和理论支持。
Sephadex G-50 gel filtration and reversed-phase high performance liquid chromatography (RP-HPLC) were used to separate and purify the skin exudates of Callueslla yunnanensis Boulenger. Polypeptide separation peak. The relative molecular mass of the insulinotropic peptide was determined by mass spectrometry to be 1 768.08. The primary amino acid sequence of FLPIVGRSYA GLSFKL-NH2 was identified as FLPIVGRSYA GLSFKL-NH2 and named as Callin, a C-terminal amidated XVI Peptide. The insulin-releasing activity of rat pancreatic islets and INS-1 pancreatic β-cell lines were detected respectively in vitro. The results showed that Callin can increase the insulin release of insulin islets and the insulin secretion of INS-1 cells. Statistical analysis showed significant differences (P <0.05), and there is a certain dose-dependent. Callin, an insulinotropic release peptide isolated from this study, will provide basic data and theoretical support for the development of novel peptide drugs that can treat patients with type 2 diabetes.