Production and characterization of antibodies against murine dentine phosphoprotein.
Production and characterization of antibodies against murine dentine phosphoprotein.
复制标题
抗鼠牙本质磷蛋白抗体的产生和表征。
作者：
M. Macdougall;M. Zeichner‐David;H. Slavkin
实验旨在产生并鉴定一种针对小鼠牙本质磷蛋白的多克隆抗体，牙本质磷蛋白是牙本质细胞外基质的主要非胶原蛋白。用0.5 M乙酸提取出生2天的瑞士 - 韦伯斯特小鼠牙器官的牙细胞外基质蛋白，随后用4 M氯化胍/0.5 M乙二胺四乙酸提取。通过用1 M氯化钙沉淀进一步提高小鼠牙本质磷蛋白的产量。通过从制备性十二烷基硫酸钠/尿素/聚丙烯酰胺凝胶中切下并洗脱牙本质磷蛋白多肽带来实现最终纯化。小鼠牙本质磷蛋白是一种约72 kDa的单一组分，其特征性氨基酸组成是33%的天冬氨酸和55%的丝氨酸/磷酸丝氨酸。在兔体内针对纯化的小鼠牙本质磷蛋白制备了一种多克隆抗体，并且通过酶联免疫吸附测定、斑点免疫结合测定和“蛋白质印迹转移”测定显示其具有单一特异性。该抗体用于检测出生1天的小鼠牙器官中牙本质磷蛋白的表达和定位。该抗原在成牙本质细胞单层内定位，成牙本质细胞排列在牙髓乳头间充质的周边，并且在穿过前期牙本质基质的成牙本质细胞突起内定位。新形成的矿化牙本质基质也与牙本质磷蛋白特异性抗体发生交叉反应。未矿化的前期牙本质基质不包含任何可检测到的交叉反应性抗原。
Experiments were designed to produce and characterize a polyclonal antibody directed against mouse dentine phosphoprotein, the major non-collagenous protein of the dentine extracellular matrix. Dental extracellular matrix proteins from 2-day-postnatal Swiss-Webster-mouse tooth organs were extracted with 0.5 M-acetic acid, followed by 4 M-guanidinium chloride/0.5 M-EDTA. Mouse dentine phosphoprotein yields were further increased by precipitation with 1 M-CaCl2. Final purification was achieved by excising and eluting dentine phosphoprotein polypeptide bands from preparative sodium dodecyl sulphate/urea/polyacrylamide gels. Mouse dentine phosphoprotein is a single component of approx. 72 kDa and has a characteristic amino acid composition of 33% aspartic acid and 55% serine/phosphoserine. A polyclonal antibody was raised in rabbits against purified mouse dentine phosphoprotein and was shown to be monospecific by enzyme-linked immunoabsorbent, dot-immunobinding and 'Western transfer' assays. This antibody was used to detect the expression and localization of dentine phosphoprotein in 1-day-postnatal mouse tooth organs. This antigen was localized intracellularly within the monolayer of odontoblasts, which line the perimeter of the dental papilla mesenchyme, and within the odontoblastic cell processes, which traverse the predentine matrix. Newly forming mineralized dentine matrix was also cross-reactive with the dentine phosphoprotein specific antibody. The non-mineralized predentine matrix did not contain any detectable cross-reactive antigens.