双团棘胸蛙皮肤胸腺肽-β_4的基因克隆及活性检测

Gene cloning and bioactive analysis of thymosin-β_4 from skin of Nanorana yunnanensis

目的研究双团棘胸蛙(Nanorana yunnanensis)皮肤胸腺肽-β4的基因和促进血管生成作用。方法通过基因扩增的方法克隆出双团棘胸蛙的2个皮肤胸腺肽-β4(p Tβ4-1和p Tβ4-2)的c DNA;根据推导蛋白序列合成多肽在鸡胚尿囊膜和内皮细胞实验系统进行血管生成活性分析。结果 p Tβ4-1和p Tβ4-2的c DNA长度分别为662 bp和673 bp,开放阅读框都为135 bp,编码44个氨基酸残基。成熟蛋白拥有四足动物胸腺肽β4的典型特征。p Tβ4-1和p Tβ4-2的c DNA序列及成熟蛋白与来源于人类、家鼠、家鸡、爪蟾、斑马鱼、大比目鱼和棕点湍蛙的胸腺肽-β4高度相似;合成的p Tβ4-1和p Tβ4-2都能促进鸡胚尿囊膜血管生长和内皮细胞小管形成。结论双团棘胸蛙皮肤含有两个具有促血管生成的胸腺肽-β4。

Aim To investigate the gene sequences and pro-angiogenic activities of thymosin-β4from skin of Nanorana yunnanensis. Methods Two c DNA sequences of thymosin-β4from Nanorana yunnanensis designated as p Tβ4-1 and p Tβ4-2 were obtained by PCR. The tube formation and proliferation of human umbilical vein endothelial cells( HUVEC) and chicken chorioallantoic membrane( CAM) model were used to observe the pro-angiogenic effects of synthetic pep-tides according to the reduced amino acid sequences of p Tβ4-1 and p Tβ4-2 in vitro and in vivo,respectively.Results The c DNA sequences of Tβ4-1 and p Tβ4-2were composed of 662 bp and 673 bp,respectively,and their deduced amino acid sequences contained 44 residues which shared highly similarity with those from human,mouse,chicken,claw frog,zebra fish,turbot and Amolops loloensis. Furthermore,the mature amino acid sequences of p Tβ4-1 and p Tβ4-2 had highly con-served structural regions when compared with other thymosin-β4previously reported. Synthetic peptides could significantly promote proliferation and tube formation of HUVEC and angiogenesis of CAM. Conclusion There are two thymosin-β4peptides in skins of Nanorana yunnanensis which have pro-angiogenic activities.