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目的:体外原核表达小鼠Foxp3蛋白,并以其免疫家兔,制备多克隆抗体。方法:从质粒Foxp3-pMD-18T扩增小鼠Foxp3全长基因,亚克隆至原核表达载体pET-32 a,转化E.coliBL-21;IPTG诱导表达目的蛋白,N i2+-NTA柱纯化及蛋白质印迹鉴定蛋白;纯化蛋白免疫家兔,制备多克隆抗体,ELISA法检测抗体效价,蛋白质印迹检测抗体特异性。结果:成功构建了Foxp3-pET-32 a原核表达载体,表达和纯化了目的蛋白;ELISA法检测抗体效价为1∶20 000,蛋白质印迹检测结果显示抗体特异性与小鼠Foxp3蛋白结合。结论:成功表达小鼠Foxp3蛋白并制备了多克隆抗体。
OBJECTIVE: To express mouse Foxp3 protein in vitro and to immunize rabbits to prepare polyclonal antibody. Methods: Foxp3 full-length gene was amplified from plasmid Foxp3-pMD-18T, subcloned into prokaryotic expression vector pET-32a and transformed into E.coli BL-21. IPTG was used to induce expression of target protein, N i2 + -NTA column purification and protein The purified protein was used to immunize rabbits to prepare polyclonal antibody. The antibody titer was detected by ELISA and the specificity of antibody was detected by Western blotting. Results: The prokaryotic expression vector of Foxp3-pET-32 a was successfully constructed and the target protein was expressed and purified. The titer of the fusion protein was 1:20 000 by ELISA. Western blotting showed that the antibody specifically binds to the mouse Foxp3 protein. Conclusion: Mouse Foxp3 protein was successfully expressed and polyclonal antibody was prepared.