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目的制备兔抗人Snapin的多克隆抗体,检测其特异性以及Snapin蛋白在心脏组织中的分布。方法构建pGEX-4T-1-Snapin N/C质粒,通过原核表达和纯化分别获得带有snapin基因N端(1-54Aa)和C端(60-136Aa)表达产物(融合蛋白GST-Snapin N/C),通过多点皮下注射免疫新西兰家兔,多次免疫后获得血清,用Protein G Agrose亲和纯化血清,获得IgG型高效价抗Snapin多克隆抗体。结果经过ELISA检测发现二种抗体效价高,滴度在1:256000时仍有较强信号。用Western blotting以及免疫组织化学检测发现二种抗体的特异性良好,能特异性识别小鼠心脏组织Snapin蛋白,其中尤以抗Snapin C抗体为佳。结论成功制备出高效价、强特异性的抗Snapin N/C抗体,为进一步研究Snapin蛋白在心脏中的作用提供了条件。
Objective To prepare polyclonal anti-human Snapin antibody and test its specificity and the distribution of Snapin protein in cardiac tissues. Methods Plasmid pGEX-4T-1-Snapin N / C was constructed and expressed in E.coli BL21 (DE3). The expression of the fusion protein GST-Snapin N / C). New Zealand rabbits were immunized by multiple subcutaneous injections. Serum was obtained after multiple immunizations. The serum was purified by affinity chromatography with Protein G Agrose to obtain IgG high titer anti-Snapin polyclonal antibody. Results After ELISA test, the titer of the two antibodies was high, and the titer was still strong at 1: 256000. Western blotting and immunohistochemistry showed that the two antibodies showed good specificity and could specifically recognize Snapin protein in mouse heart tissue, especially anti-Snapin C antibody. Conclusion The successful preparation of anti-Snapin N / C antibody with high titer and specificity provided the conditions for the further study of the role of Snapin in the heart.