新型蛙皮素多肽QUB2995的重组表达及其促胰岛素分泌活性鉴定

Identification of a novel bombesin bioactive peptide QUB2995 and its insulin secreting activity

目的从阔褶水蛙皮肤分泌物中发现新型蛙皮素多肽,并鉴定其对胰岛细胞的促胰岛素分泌作用。方法通过电刺激法提取阔褶水蛙皮肤分泌物,以分子克隆大量制备蛙皮素多肽单链并测序。通过固相合成法合成已知序列多肽QUB2995,并以反相高效液相色谱(HPLC)纯化合成多肽,通过基质辅助激光解析飞行时间质谱(MALDI-TOF)确认合成多肽是否为之前发现的新型多肽QUB2995。用qPCR以及ELISA检测QUB2995对小鼠胰岛细胞MIN6及大鼠胰岛细胞INS-1的促胰岛素分泌作用。结果通过分子克隆技术,在亚洲阔褶水蛙的皮肤分泌物中发现了一种新型蛙皮素多肽,命名为QUB2995(GAFGDFLKGAAKAGALKILSIAQCKLSGTC)。该蛙皮素多肽对小鼠胰岛细胞MIN6及大鼠胰岛细胞INS-1具有显著的促进胰岛细胞增殖以及胰岛素分泌作用,在10-5 mol/L浓度下效果最为显著。结论从阔褶水蛙中发现了新型蛙皮素多肽并命名为QUB2995,其对小鼠胰岛MIN6及大鼠胰岛INS-1细胞具有显著促胰岛素分泌作用,显示出作为治疗糖尿病药物的潜在价值。

Objective To identify a novel bombesin bioactive peptide from the skin secretion of Hylarana Latouchii,and to explore its effect on insulin secretion in islet cells.Methods The skin secretion from Hylarana Latouchii was extracted by electrical stimulation,and the single chain of bombesin peptide was cloned and sequenced.The peptide QUB2995 was synthesized via solid-phase synthesis,then purified using reversed-phase high performance liquid chromatography(HPLC).Matrix assisted laser desorption time-of-flight mass spectrometry(MALDI-TOF)was applied to validate.QPCR and ELISA were used to probe the effect of QUB2995 on insulin secretion in MIN6 and INS-1 cells.Results A novel bombesin peptide named QUB2995(GAFGDFLKGAAKA GALKILSIAQCKLSGTC)was found in the skin secretion of Hylarana Latouchii through molecular cloning.The bioactive peptide could significantly promote the proliferation and insulin secretion from mouse islet MIN6 cells and rat islet INS-1 cells.The effect reached a climax at the concentration of 10-5 mol/L.Conclusion A novel bombesin bioactive peptide named QUB2995 was found from Hylarana Latouchii.It could significantly promote insulin secretion in MIN6 cells of mouse islets and INS-1 cells of rat islets,indicating its potential in the treatment of diabetes.