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目的纯化叉头盒蛋白O3(FOXO3)基因保守区蛋白,并制备其多克隆抗体。方法利用生物信息学分析预测FOXO3蛋白的保守区,通过PCR扩增编码FOXO3蛋白第290~472位氨基酸DNA片段并克隆至pET28a原核表达载体中,转化到大肠杆菌BL21,诱导表达并获得可溶性蛋白,经镍离子柱亲和纯化,免疫小鼠制备抗血清,随后采用ELISA、Western blot法和免疫沉淀实验检测抗体的效价、特异性以及应用范围。结果成功构建表达载体pET28a-FOXO3(aa290-472),实现可溶性FOXO3蛋白的表达和抗体制备。SDS-PAGE和Western blot实验结果证实,FOXO3蛋白与预期结果一致,抗血清能够特异性地识别原核、真核FOXO3蛋白以及其天然抗原表位。结论成功制备了小鼠抗人FOXO3的多克隆抗体,且抗体对内源性FOXO3具有较好的反应性和特异性。
Objective To purify the protein of conserved region of forkhead box protein O3 (FOXO3) gene and prepare its polyclonal antibody. Methods The conserved region of FOXO3 protein was predicted by bioinformatics analysis. The amino acid DNA fragment encoding 290-272 of FOXO3 protein was amplified by PCR and cloned into prokaryotic expression vector pET28a. The recombinant protein was transformed into E. coli BL21 and induced to express soluble protein. The antiserum was prepared by affinity purification of Ni (superscript +) ion column. The titer, specificity and application of the antibody were detected by ELISA, Western blot and immunoprecipitation. Results The expression vector pET28a-FOXO3 (aa290-472) was successfully constructed and the expression of soluble FOXO3 protein and antibody preparation were achieved. The results of SDS-PAGE and Western blot confirmed that the FOXO3 protein was consistent with the expected results. Antiserum could specifically recognize the prokaryotic and eukaryotic FOXO3 proteins and their natural epitopes. Conclusion The mouse anti-human FOXO3 polyclonal antibody was successfully prepared and the antibody had good reactivity and specificity to endogenous FOXO3.