棉蚜His-CYP6J1融合蛋白的分离纯化及多克隆抗体的制备

Isolation and Purification of His-CYP6J1 Fusion Protein from Aphis gossypii and Preparation of Its Polyclonal Antibody

通过亲和层析法纯化棉蚜His-CYP6J1融合蛋白,制备及鉴定其多克隆抗体。采用镍离子金属螯合亲和层析柱分离纯化棉蚜His-CYP6J1融合蛋白,SDS-PAGE检测纯化产物。用纯化得到的His-CYP6J1融合蛋白免疫小鼠,制备抗棉蚜P450CYP6J1多克隆抗体;采用ELISA法检测抗体效价;免疫组化法检测抗血清特异性。结果表明,纯化的His-CYP6J1融合蛋白免疫小鼠后得到多抗血清,ELISA法检测抗血清效价达1∶200 000。免疫组化结果显示,此多克隆抗体能够与棉蚜P450 CYP6J1天然蛋白特异性结合,却在棉铃虫没有发现相应的结合反应。上述结果对研究棉蚜单一P450蛋白结构、功能及其在棉蚜抗药性形成过程的作用奠定基础。

This work aims to purify His-CYP6J1 fusion protein through affinity chromatography, and prepare and identify its polyclonal antibody. The fusion protein was purified by Ni-NTA chelating column, the purified product was detected by SDS-PAGE. Then the acquired fusion protein His-CYPrJ1 was used to immune mice for preparing its polyclonal antibody against Aphis gossypii. Finally, ELISA was used to test antibody＇ s titer, and immunohistochemistry to identify its specificity. As results, antiserum titer was 1 ： 200 000, the polyclonal antibody specifically bound to natural P450 CYP6J1 protein from A. gossypii, but not to the one from cotton bollworm. These results provide a foundation for studying the structure and function of a single P450 protein and its role in the insecticide- resistance ofA. gossypii.