方格星虫低聚肽的分离、鉴定及其促骨发育作用评价

目的:从方格星虫中分离和筛选可促进骨形成的生物活性低聚肽。方法:通过酶解法和膜分离法制备寡肽，并通过HPLC-MS/MS鉴定肽段序列。通过在线数据库预测药物形成，并通过斑马鱼模型评估促进骨形成的作用。结果:共鉴定出10种低分子量肽，通过斑马鱼骨发育模型获得了两种潜在的候选肽，其氨基酸序列分别为GFAGDDAPR和GLGGLSPEK；分别命名为GSP1和GSP2；其分子量分别为905.04和857.06 Da；与空白对照组相比，1~50 μg/mL GSP1溶液浓度处理的AB斑马鱼幼鱼颅骨积分光密度和绿色荧光的面积均有所增加，GSP1浓度在5 μg/mL时促进骨发育的效果最好（P<0.05）。1~50 μg/mL GSP2溶液浓度处理的AB型斑马鱼幼鱼颅骨积分光密度和绿色荧光的面积均有所增加，且随着浓度的增加而增大，GSP2浓度在50 μg/mL时促进骨发育的效果最好（P<0.05）。结论:小于1000 Da的寡肽有望成为促进骨形成的候选药物。

Isolation and Identification of Oligopeptides from Squares Siphon Worm and Its Effect on Promoting Bone Growth

Objective: To isolate and screen the bioactive oligopeptides which can promote bone formation from square siphon worm. Methods: Oligopeptides were prepared by enzymatic hydrolysis and membrane separation, and peptide sequences were identified by HPLC-MS/MS. The online database was used to predict drug formation, and the zebrafish model was used to evaluate the effect of promoting bone formation. Results: A total of 10 low molecular weight peptides were given. Two potential candidate peptides were obtained by zebrafish bone development models. Their amino acid sequences were GFAGDDAPR and GLGGLSPEK, which named GSP1 and GSP2. Molecular weights of two potential candidate peptides were 905.04 and 857.06 Da. Two potential candidate peptides could significantly promote the osteogenesis of juvenile zebrafish. Compared with the blank group, when Juvenile of AB zebrafish were treated with GSP1 solution (1~50 μg/mL), the area of integral optical density and green fluorescence increased in the skull of juvenile fish. When the concentration of GSP1 was 5 μg/mL, it had the best effect of promoting bone development. Compared with the blank group, when the juvenile of AB zebrafish were treated with GSP2 solution (1~50 μg/mL) , the area of integral optical density and green fluorescence increased in the skull of juvenile fish. When the concentration of GSP2 was 50 μg/mL, it had the best effect of promoting bone development. Conclusion: The oligopeptides less than 1000 Da were expected to be candidate drugs for promoting bone formation.