SCT-OGP的融合表达与活性的初步研究

根据天然鲑鱼降钙素(Salmoncalcitonin,SCT)和骨生长肽(OsteogenicGrowthPeptide,OGP)的序列,人工合成了6个DNA片段,进行退火、连接和转化后,获得含有SCT与OGP融合基因的pPIC9-SCT-OGP表达载体,经测序后将此重组质粒电击转化毕赤酵母GS115,通过培养、筛选以及SDS-PAGE鉴定,在毕赤酵母中获得了SCT-OGP融合蛋白的可溶性表达,经分子筛纯化后在5kD左右可见单一条带。将此条带进行了N端氨基酸组分分析,证实此目的蛋白为SCT-OGP融合蛋白。MTT法显示SCT-OGP融合蛋白在体外可以促进成骨细胞和成纤维细胞增殖,以小鼠为模型的试验显示SCT-OGP融合蛋白在体内可以提高碱性磷酸酶活性和降低血钙,表明该融合蛋白具有抑制破骨细胞的活性和刺激成骨细胞增殖的活性,从而提示该融合蛋白具有治疗骨质疏松症的药用潜力。

SCT-OGP Fusion Protein Expression and Its Potential Role as Osteoporosis Therapy

Osteoporosis is a disease of bone metabolic disorder, the incidence of which increases sharply in old people. Calcitonin(CT) is a peptide hormone containing 32 amino acid that can inhibit osteoclasts activity.Osteogenic Growth Peptide (OGP) is a peptide hormone with 14 amino acid. It is an autocrine mitogen for osteoblastic and firbroblastic cells which has anabolic activiy.Six SCT and OGP DNA segments were chemically synthesized and ligated into a yeast expression vector pPIC9.The recombinant plasmid was transformed into pichia pastoris GS115.Finally, we got two stable SCT-OGP high expression clones after screening. Purifed protein can stimulate the proliferation of osteoblastic and fibroblastic cells, and also stimulate serum ALP activity and decrease serum calcium level using mice as animal models, demonstrating its potential role in oteoporosis therapy.