基于密码子优化策略的斑马鱼FOXP3A分子的原核表达及多克隆抗血清制备

Prokaryotic Expression and Polyclonal Antiserum Production of Codon Optimized FOXP3A from the Zebrafish(Danio rerio)

目的:构建斑马鱼FOXP3A融合蛋白的原核表达系统,诱导表达并制备多克隆抗血清。方法:选取斑马鱼foxp3a基因cDNA的894 bp特异区段(s-foxp3a),对该片段进行密码子优化后构建原核表达载体pET-32a-s-foxp3a,转化大肠杆菌BL21(DE3),诱导表达斑马鱼S-FOXP3A-His融合蛋白并纯化,纯化后的蛋白免疫家兔,制备斑马鱼FOXP3A蛋白的多克隆抗血清,4次免疫后取血清,采用间接ELISA法检测抗血清效价。结果:在16℃经0.5 mmol/LIPTG诱导10 h的条件下,SDS-PAGE分析表明斑马鱼S-FOXP3A-His融合蛋白以包涵体形式产生;间接ELISA检测结果显示,FOXP3A蛋白多克隆抗血清的效价在1.6×10~5以上。结论:制备了高效价的斑马鱼FOXP3A多克隆抗血清,为进一步解析斑马鱼FOXP3A蛋白的功能提供了基础工具。

Objective:To obtain polyclonal antibodies,a prokaryotic expression system for the zebrafish FOXP3A protein was constructed.Methods:The 894 bp specific region of zebrafish foxp3a(s-foxp3a)cDNA was selected,and its codons were optimized for E.coli and cloned into a pET-32a vector.The recombinant expression vector was transformed into E.coli BL21(DE3)competent cells and IPTG was applied to induce recombinant zebrafish S-FOXP3A-His fusion protein.The purified fusion protein was used as antigen to immunize healthy rabbits(Oryctolagus cuniculus f.domesticus).The antiserum was isolated after the fourth immunization and its titers were detected by ELISA.Results:SDS-PAGE analysis showed that the zebrafish S-FOXP3A-His recombinant fusion protein was efficiently expressed as inclusion bodies in the presence of 0.5 mmol/L IPTG at 16℃ for 10 h,and successfully purified.The results of indirect ELISA showed that the titer of the antiserum against zebrafish FOXP3A were all above 1.6×10^5.Conclusion:The zebrafish FOXP3A antiserum was successfully obtained,which provided a powerful tool for the further analysis of the function of zebrafish FOXP3A protein.