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目的:制备来航鸡FOXL2蛋白的单克隆抗体。方法:利用生物信息学的方法分别预测来航鸡FOXL2蛋白最有可能的线性表位,并人工合成多肽,高效液相色谱分析其纯度,与KLH偶联后免疫小鼠制备其单克隆抗体,用间接ELISA方法和免疫印迹进行筛选,然后对筛选出来的细胞株进行一般性质测定、亲和常数测定、IC50测定。结果:合成多肽纯度>95%,细胞融合后,其平均融合率为75%,并用间接ELISA方法和Western blot筛选出1条可与FOXL2蛋白发生明显反应的细胞株C2;经测定,C2细胞株上清效价为1/128,腹水效价为1/256,其亚型属于IgG1,杂交瘤细胞染色体为102条,腹水单抗的蛋白含量为1.576 g/L,其亲和常数为2.12×106L/mol,IC50值为0.082 47μg/L。结论:成功制备了FOXL2蛋白单克隆抗体,为下一步深入研究FOXL2蛋白奠定基础。
Objective: To prepare monoclonal antibody to FOXL2 protein from Laihang chicken. METHODS: Bioinformatics methods were used to predict the most likely linear epitopes of Leghorn FOXL2 protein. Synthetic peptides were synthesized and purified by high performance liquid chromatography (HPLC). After conjugation with KLH, mice were immunized to prepare monoclonal antibodies. Indirect ELISA and Western blotting were used to screen the selected cell lines for general property determination, affinity constant determination and IC50 determination. Results: The purity of the synthesized peptide was> 95%. After fusion, the average fusion rate was 75%. One of the cell lines C2 that could react with FOXL2 protein was screened by indirect ELISA and Western blot. The titer of supernatant was 1/128, the titer of ascites was 1/256, the subtype was IgG1, the hybridoma cell chromosome was 102, the protein content of ascites McAb was 1.576 g / L, and the affinity constant was 2.12 × 106L / mol, IC50 value of 0.082 47μg / L. Conclusion: The FOXL2 monoclonal antibody was successfully prepared, which laid the foundation for further study of FOXL2 protein.