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目的:为了寻找高活性和长半衰期生的GHRH类似肽。方法:通过使用独特的酸敏感水解位点Asp-Pro的原核表达系统,构建了新的Pro-hGHRH(1-44)-Gly-Gly-Cys类似肽。通过重组细菌裂解、包含体洗涤、乙醇分级沉淀、酸水解、SP-SephadexC-25和SephadexG-10柱层析等技术,纯化了高纯度的Pro-hGHRH(1-44)-Gly-Gly-Cys肽。通过使用SDS-PAGE、离子化质谱、雌大鼠垂体和人流产胎儿垂体,测定了多肽的纯度、分子量、生长激素释放活性。结果:Pro-hGHRH(1-44)-Gly-Gly-Cys肽分子量5373Da与实际值吻合,0.1～10μg/ml的肽剂量不论是对人垂体还是大鼠垂体都增加了垂体生长激素的释放,大鼠垂体生长激素的释放具有剂量依赖性。与标准的hGHRH(1-40)肽比较,新的类似肽有较高的GH释放活性。结果也显示了,Pro-GHRH(1-44)Gly-Gly-Cys与Pro-hGHRH(1-44)肽的GH释放活性无统计学差异。结论:新的类似肽有较好的生长激素释放活性、功能选择性和种属特异性。
OBJECTIVE: To find GHRH-like peptides with high activity and long half-life. Methods: A novel Pro-hGHRH (1-44) -Gly-Gly-Cys analog peptide was constructed by using the prokaryotic expression system of Asp-Pro, a unique acid-sensitive hydrolysis site. High-purity Pro-hGHRH (1-44) -Gly-Gly-Cys was purified by recombinant bacterial lysis, inclusion body wash, ethanol fractional precipitation, acid hydrolysis, SP-Sephadex C-25 and Sephadex G- Peptide. The purity, molecular weight, and growth hormone releasing activity of the polypeptide were determined by SDS-PAGE, ionization mass spectrometry, pituitary of female rats and human pituitary of human abortion fetus. RESULTS: The molecular weight of Pro-hGHRH (1-44) -Gly-Gly-Cys peptide was 5373Da, which was consistent with the actual value. The peptide dosage of 0.1-10μg / ml increased the release of pituitary ghrelin both in pituitary and in rat pituitary, Rat pituitary growth hormone release in a dose-dependent manner. The new analogous peptides have higher GH release activity compared to the standard hGHRH (1-40) peptide. The results also show that there is no statistical difference in the GH release activity of Pro-GHRH (1-44) Gly-Gly-Cys and Pro-hGHRH (1-44) peptide. Conclusion: The new peptides have better growth hormone release activity, functional selectivity and species specificity.