自噬基因融合蛋白重组多克隆抗体的纯化与检测

为了提纯并鉴定自噬基因融合蛋白重组多克隆抗体，探讨其在部分组织中的免疫定位．首先合成带谷胱甘肽转移酶标签的自噬基因融合蛋白，免疫新西兰大白兔，制备多克隆抗体，然后用聚丙烯酰胺凝胶电泳检测抗体浓度，最后用免疫亲和纯化方法提纯抗血清中的多克隆抗体，酶联免疫吸附法检测抗体效价，蛋白印迹检测抗体特异性及在组织中的表达情况．结果表明：制备得到自噬基因融合蛋白重组多克隆抗体经聚丙烯酰胺凝胶电泳检测其质量浓度为每10μL含5μg，用酶联免疫吸附法显示此抗体效价及特异性较高，蛋白印迹显示蛋白大小为55kD左右．

Purification and detection of autophagy-related gene fusion protein restructured polyclonal antibody

The aim of the study is to purify and identify the autophagy-related gene fusion protein restructured polyclonal antibody, and to detect the immune localization in tissues. Firstly, the autophagy-related gene fusion protein restructured polyclonal antibody with glutathione-S-transferase tag was synthesized, the New Zealand white rabbits was immuned and the polyclonal antibody was prepared. Secondly, the antibody concentration was detected by polyacrylamide gel electrophoresis. Finally, the antibody was purified in the anti-serum by using immunoaffinity purification, antibody titer was detected by enzyme linked immunosorbent assay methods, the antibody specificity and the organization expression were tested by Western blotting. The result shows that autophagy-related gene fusion protein restructured polyclonal antibody concentration is 10 μL per 5 μg, which is detected by polyacrylamide gel electrophoresis; the antibody is proved to have higher titer and specificity by enzyme linked immunosorbent assay~ the molecular weight of the protein is about 55 kD from Western blotting results.