【摘要】 为准确鉴别海绵造骨细胞,分别提取了繁茂膜海绵、多皱软海绵和澳大利亚厚皮海绵的硅聚合酶,以繁茂膜海绵硅聚合酶为抗原制备抗体,效价为1∶9600。SDSPAGE显示三种海绵硅聚合酶的亚基分布在28kD左右;建立竞争抑制性检测方法并结合WesternBlotting检测,显示该抗体可与繁茂膜海绵硅聚合酶特异性结合,且与另外两种海绵硅聚合酶几乎无交叉反应。利用该抗体对繁茂膜海绵组织和体外培养细胞进行免疫组织化学染色,均可显示造骨细胞的分布。结果提示硅聚合酶抗体可以特异性与繁茂膜海绵造骨细胞内的硅聚合酶结合,因此,该抗体可以用于造骨细胞的鉴别。更多还原

【Abstract】 Silicatein is a key enzyme which catalyzes the formation of silicic spicules in marine sponges, and a specific protein of the spicule-producing sponge cells, the sclerocytes. To establish a new and accurate method to identify sclerocytes among a mixed sponge cell population, the silicateins of three marine sponges, Hymenidcidon perleve, Halichondria hugosa and Craniella australiensis were purified. SDS-PAGE showed that the silicatein subunits were about 28 kD. The prepared mice anti-silicatein polyclonal antibody of H.perleve, with a high titer of 1∶9 600, could specifically recognize the silicatein from this marine sponge. The low cross-reaction between the antibody and the other two types of silicateins, detected by competitive inhibition ELISA and Western Blotting, suggested that silicatein is highly species-specific. The antibody could clearly reveal sclerocytes in marine sponge tissue and cell suspension of H.perleve by immunohistochemical assay, demonstrating the feasibility of using this antibody to identify the sclerocytes of marine sponges. Moreover, the antibody and competitive inhibition ELISA method can be further used for the quantitative detection of the silicatein in marine sponge under different conditions .更多还原