重组小鼠脑14-3-3γ蛋白单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody of mouse brain 14-3-3γ isoform protein

目的制备重组小鼠脑14-3-3γ蛋白单克隆抗体(McAb),为神经系统退行性疾病的诊断提供标志物。方法采用重组小鼠脑14-3-3γ蛋白免疫小鼠,制备抗小鼠脑14-3-3γ蛋白的多克隆抗体,并应用多克隆血清检测14-3-3蛋白在大鼠脑中的分布。进一步应用杂交瘤技术制备其单克隆抗体,并鉴定其特异性以及与天然抗原的亲合力。结果制备的多克隆血清能有效识别大鼠脑组织的14-3-3蛋白,免疫组化显示在黑质,蓝斑,室周核,背外侧核染色阳性。制备的单克隆抗体可特异性识别重组14-3-3γ蛋白、大鼠脑及人脑中14-3-3蛋白。结论重组小鼠脑14-3-3γ蛋白多克隆血清和单克隆抗体成功制备;此可用于神经系统退行性疾病的诊断。

Objective To prepare monoclonal antibody （MeAb） against recombinant mouse brain 14-3-3γ protein, and to provide diagnosis marker for the nervous system degenerative diseases. Methods Mouse were immunized with recombinant mouse brain 14-3-3γ protein to prepare the anti-mouse brain 14-3-3γ protein polyclonal serum, and the distribution of 14-3-3γ protein in mouse brain were detected by using the polyelonal serum. Then, the monoclonal antibody were prepared by hybridoma technique, its specificity and affinity with natural antigen were identified. Results The polyclonal serum prepared can identify the mouse brain 14-3-3γ protein effectively, immunohistochemistry shows positive staining in the substantia nigra, locus coeruleus, periventricular nucleus, dorsal lateral nucleus. The monoclonal antibodies prepared can recognize the recombinant 14-3-3γ protein , 14-3-3γ protein in mouse and human brain specifically. Conclusion Successful preparation of recombinant mouse brain 14-3-3γ protein polyclonal serum and monoclonal antibodies can be used for the diagnosis of nervous system degenerative diseases.