14-3-3 zeta蛋白C-端多肽抗体的制备与鉴定

目的制备14-3-3 zeta蛋白C-端多肽多克隆抗体,并鉴定其特异性。方法用生物信息学分析14-3-3 zeta蛋白的同源性、亲水性和抗原性,设计并合成zeta蛋白C-端特异性的氨基酸序列,将其与牛血清白蛋白铰链;分别转化14-3-3的7个亚型质粒于大肠杆菌BL21(DE3)中,经IPTG诱导表达,并分离纯化His-zeta融合蛋白;分别以zeta C-端多肽及zeta融合蛋白为抗原,免疫新西兰大耳白兔,制备多克隆抗体;用Western blot分析zeta蛋白C-端多肽抗体亚型特异性,并用该特异性抗体检测zeta蛋白在COS-7细胞中的分布规律。结果在14-3-3的7个亚型蛋白存在的条件下,zeta蛋白C-端多肽抗体仅识别zeta亚型蛋白,而zeta融合蛋白抗体可以识别gamma、zeta、tau3种亚型;14-3-3zeta蛋白主要分布在COS-7细胞质一侧。结论已获得了亚型特异性的14-3-3zeta多克隆抗体,该抗体可用于14-3-3zeta免疫细胞化学等分析。

Preparation and Characterization of Polyclonal Antibody against Polypeptide at C-terminus of 14-3-3 zeta Protein

Objective To prepare and characterize the polyclonal antibody against polypeptide at C-terminus of 14-3-3 zeta protein.Methods Based on the bioinformatic analysis of homology,hydrophilicity and antigenicity of 14-3-3 zeta protein,the specific amino acid sequence at C-terminus of zeta protein was designed,synthesized and cross-linked with bovine serum albumin(BSA).Transform seven recombinant plasmids with genes encoding the 14-3-3 protein of seven isoforms respectively to E.coli BL21(DE3)for expression under induction of IPTG.Purify the expressed His-14-3-3 zeta fusion protein.Polyclonal antibodies were prepared by immunizing New Zealand rabbits with the purified fusion protein and polypeptide at C-terminus of 14-3-3 zeta protein respectively.The polyclonal antibody against polypeptide at C-terminus of 14-3-3 zeta protein was tested for specificity by Western blot and used for the analysis of distribution of zeta protein in COS-7 cells.ResultsWestem blot proved that the polyclonal antibody against polypeptide at C-terminus of 14-3-3 zeta protein recognized only 14-3-3 zeta isoform,while the antibody against His-14-3-3 zeta fusion protein recognized 14-3-3 gamma,zeta and tau isoforms.14-3-3 zeta protein was mainly distributed at one side of cytoplasm.Conclusion The polyclonal antibody against polypeptide at C-terminus of 14-3-3 zeta protein was successfully prepared and could be used for the immunocytochemical analysis of 14-3-3 zeta protein.