棉蚜His-CYP6J1融合蛋白的分离纯化及多克隆抗体的制备

doi:10.13560/j.cnki.biotech.bull.1985.2016-0920

摘要/Abstract

摘要： 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P450 CYP6J1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。

关键词: P450 CYP6J1融合蛋白, 蛋白纯化, 多克隆抗体

Abstract: 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-CYP6J1 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 of A. gossypii.

Key words: P450 CYP6J1 fusion protein, protein purification, polyclonal antibody

王亚美,魏原杰,艾新宇,刘小宁. 棉蚜His-CYP6J1融合蛋白的分离纯化及多克隆抗体的制备[J]. 生物技术通报, 2017, 33(5): 164-169.

WANG Ya-mei WEI Yuan-jie AI Xin-yu LIU Xiao-ning. Isolation and Purification of His-CYP6J1 Fusion Protein from Aphis gossypii and Preparation of Its Polyclonal Antibody[J]. Biotechnology Bulletin, 2017, 33(5): 164-169.

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