STUDIES ON CALCIUM BINDING PROTEIN,OSTEOPONTIN,ITS DIAGNOSTIC AND THERAPEUTIC APPLICATION FOR BONE DISORDERS.

钙结合蛋白、骨桥蛋白的研究及其在骨疾病诊断和治疗中的应用。

• 批准号: 07557061
• 负责人: KOBAYASHI Kunihiko
• 金额: $ 7.62万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557061/
• 关键词: OSTEOPONTIN; CALCIUM BINDING PROTEIN; HUMAN MILK; OSTEOMALACIA; PHOSPHOPROTEIN; Ca結合蛋白; Ca-結合蛋白; Eta-1; ELISA; SSP; リン酸蛋白

Osteopontin (OPN hereafter) , a Ca binding phosphoprotein present in human milk as well as in serum is thought to be involved in the remodeling of the bony tissues by interacting with osteoblasts and osteoclasts, as well as with Ca ions. This forced us to investigate whether the serum OPN can be a marker for some bony disorders, such as osteomalacia and whether the milk OPN is involved in the osteogenesis of the infants.We isolated OPN with mol.wt of 78kD from human milk, of which N-terminal seven amino acids were identical to those deduced from consensus DNA sequence of OPN,and prepared polyclonal anti-OPN antibodies in rabbits. Using this antibody, we developed a sandwich type ELISA for measuring OPN in serum. However while the ELISA worked in determining levels of milk OPN,serum OPN could not be detected in this system. It was disclosed that while the antibody did not show precipitation reaction in agarose gel with human serum, it reacted with serum components with mol.wt.of 55 and 32kD by Western blot procedure. Furthermore, the serum components showed an inhibitory activity against reaction between the antibody and milk OPN in agarose gel as well as in the ELISA system. Thus, the serum components with mol.wt.of 55 and 32kD were defined both to be OPN homologue in serum, possibly the 32kD being fragment of the 55kD.During the antigenic analysis of OPN from serum and milk, it was notified that OPN especially that from serum was quite fragile and was easily degraded into small mol.wt.components. This would be a reason why serum OPN did not show precipitin reaction in agarose gel as well as binding reaction in ELISA system. It should be necessary to isolate OPN in serum and analyze its protein nature in comparison with that in milk.

骨桥蛋白（以后的OPN）是一种存在于人类牛奶中以及血清中的Ca结合磷酸蛋白，被认为通过与成骨细胞和成骨细胞和成骨细胞相互作用以及与CA离子相互作用。 This forced us to investigate whether the serum OPN can be a marker for some bony disorders, such as osteomalacia and whether the milk OPN is involved in the osteogenesis of the infants.We isolated OPN with mol.wt of 78kD from human milk, of which N-terminal seven amino acids were identical to those deduced from consensus DNA sequence of OPN,and prepared polyclonal anti-OPN antibodies in兔子。使用这种抗体，我们开发了一种三明治型ELISA来测量血清中的OPN。但是，尽管ELISA致力于确定牛奶OPN水平，但在该系统中无法检测到血清OPN。据披露，尽管抗体未显示琼脂糖凝胶与人血清中的降水反应，但它通过蛋白质印迹程序与55和32KD的血清成分反应。此外，血清成分在琼脂糖凝胶以及ELISA系统中表现出对抗体和牛奶OPN之间反应的抑制作用。因此，将55和32KD的摩尔wt.的血清成分定义为血清中的OPN同源物，可能是55kD的碎片。在血清和牛奶中对OPN进行抗原分析的片段，尤其是从血清和牛​​奶中的OPN抗原分析，特别是从血清和牛​​奶中，尤其是epn很脆弱，并且很容易脱脂地脱脂。这就是为什么血清OPN在琼脂糖凝胶中没有显示沉淀蛋白反应以及ELISA系统中的结合反应的原因。与牛奶中的蛋白质相比，必须分离血清中的OPN并分析其蛋白质的性质。