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【目的】细胞色素P450单加氧酶在昆虫生长发育和适应环境过程中发挥着重要功能。【方法】本研究克隆了褐飞虱Nilaparvata lugens细胞色素P450基因CYP4C62的开放阅读框(不含信号肽编码序列部分),在大肠杆菌Escherichia coli中实现了高效表达,经Ni-NTA琼脂糖凝胶亲和层析柱纯化得到了重组的CYP4C62蛋白。将该蛋白免疫日本大耳白兔Oryctolagus cuniculus雄兔,制备了兔抗CYP4C62血清抗体。采用间接ELISA方法检测了血清抗体的效价;并通过Western印迹杂交检测了该抗体的免疫学特异性。【结果】结果表明,通过大肠杆菌表达出的CYP4C62蛋白相对分子量为56 kD。间接ELISA法检测表明,制备的兔抗CYP4C62抗体的效价达到1∶100 000。Western印迹杂交证实,该抗体既可与异源表达的CYP4C62蛋白特异性结合,也可以与褐飞虱总蛋白中内源的CYP4C62特异性结合,表明具有较好的免疫反应特异性。【结论】CYP4C62多克隆抗体的成功制备,为后续分析CYP4C62在褐飞虱各组织中的时空表达水平,并通过免疫组织化学法定位分析该蛋白的组织、细胞及亚细胞分布规律,及最终解析CYP4C62的生物学功能奠定了基础。
【Objective】 Cytochrome P450 monooxygenase plays an important role in insect growth and adaptation to the environment. 【Method】 This study cloned the open reading frame of CYP4C62 of Nilaparvata lugens cytochrome P450 gene of BPN (excluding the part of signal peptide coding sequence), which was highly expressed in Escherichia coli. After Ni-NTA agarose gel affinity Purification by chromatography gave the recombinant CYP4C62 protein. Rabbit anti-CYP4C62 serum antibody was prepared by immunizing Japanese white rabbits with Oryctolagus cuniculus male rabbits. The titer of serum antibody was detected by indirect ELISA. The immunological specificity of the antibody was tested by Western blotting. 【Result】 The results showed that the relative molecular weight of CYP4C62 protein expressed by Escherichia coli was 56 kD. Indirect ELISA assay showed that the titer of the rabbit anti-CYP4C62 antibody reached 1: 100,000. Western blot hybridization confirmed that the antibody specifically binds to heterologous expressed CYP4C62 protein and specifically to endogenous CYP4C62 in the total RNA of N. lugens, indicating that the antibody has good immunoreactivity specificity. 【Conclusion】 The successful preparation of polyclonal antibody against CYP4C62 is to analyze the spatial and temporal expression of CYP4C62 in the brown planthopper, and to determine the distribution of CYP4C62 in tissues, cells and subcellular distribution by immunohistochemistry. Finally, Biological function has laid a foundation.