来航鸡FOXL2蛋白单克隆抗体的制备

Preparation of a monoclonal antibody specific to FOXL2 protein of Leghorn chicken

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 of Leghorn chicken FOXL2 protein.Methods： First,bioinformatics methods were used to predict its most possible epitopes,then polypeptides were synthesised,purity were analysised by liquid chromatography,coupled with KLH.Later,the mice were immunized by the polypeptides which coupled with KLH.After fused between the splenic cell of immunized mice and NS0 myeloma cell,the indirect ELISA and Western blot were used to detect whether the hybridoma cell can secrete antibody against polypeptide and FOXL2 protein.Last,the general nature,binding curve and IC50 of monoclonal antibody were measured.Results： It was found that the purity of artificial polypeptides were all 95%.After cell fusion,its fusion rate was 75%,and one cell lines were screened by indirect ELISA and Westen blot;cell line supernatant titer was 1/128,the ascites titer was 1/256,IgG1 type,had 102 cell chromosome,ascitic fluid protein content 1.576 g/L;its affinity constant was 2.12×106 L/mol,IC50 value was 0.082 47 μg/L.Conclusion： We successfully prepared the FOXL2 protein monoclonal antibody,this work should helped for a deep research to FOXL2.