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目的 克隆、表达重组融合蛋白ActRⅡA的C末端肽 ,制备抗ActRⅡAC末端抗体 ,并检测其在小鼠巨噬细胞中的表达。方法 构建GST ActRⅡAC末端蛋白表达质粒 ,经SDS PAGE分析表达蛋白。以GST ActRⅡAC末端蛋白为抗原免疫家兔制备抗ActRⅡAC抗体 ,采用免疫细胞化学染色技术观察ActRⅡA在巨噬细胞的分布。结果 克隆得到的ActRⅡAC末端cDNA ,经DNA测序证实所克隆的cDNA与GenBank收录的ActRⅡAC末端基因序列完全相符。SDS PAGE分析表达的GST ActRⅡAC蛋白相对分子质量约为 3 70 0 0。采用亲和层析纯化的抗ActRⅡAC末端抗体进行免疫细胞化学染色显示 ,ActRⅡA在小鼠巨噬细胞中高水平表达。结论 已成功地构建了ActRⅡAC末端表达质粒 ,制备了抗ActRⅡAC末端抗体并证实小鼠巨噬细胞表面存在ActRⅡA。
Objective Cloning and expression of the recombinant fusion protein ActR Ⅱ A C-terminal peptide, preparation of anti-ActR Ⅱ AC terminal antibody and its expression in mouse macrophages. Methods The GST ActRⅡAC expression vector was constructed and analyzed by SDS PAGE. ActRⅡAC antibody was prepared by immunization of rabbits with GST ActR Ⅱ A terminal protein and the distribution of ActRⅡA in macrophages was observed by immunocytochemical staining. Results The cDNA of ActRⅡAC was cloned and confirmed by DNA sequencing. The cloned cDNA was completely consistent with the ActRⅡAC sequence contained in GenBank. The relative molecular mass of GST ActRⅡAC protein expressed by SDS PAGE was about 3 70 000. Immunocytochemistry using anti-ActRIIAC end-antibody purified by affinity chromatography showed that ActRIIA was highly expressed in mouse macrophages. Conclusion The ActRⅡAC expression plasmid was successfully constructed and the anti-ActRⅡAC terminal antibody was prepared and the ActRⅡA was confirmed on the surface of mouse macrophages.