›› 2014, Vol. 57 ›› Issue (6): 656-662.doi:

• 研究论文 • 上一篇    下一篇

褐飞虱细胞色素P450基因CYP4C62的原核表达及多克隆抗体的制备

孙海霞1, 陈   俊2, 杨之帆1,*   

  1. (1. 湖北大学生命科学学院, 生物资源绿色转化湖北省协同创新中心, 武汉 430062; 
    2. 武汉科技大学化学工程与技术学院, 武汉430081)
  • 出版日期:2014-06-20 发布日期:2014-06-20

Prokaryotic expression and polyclonal antibody preparation of cytochrome P450 gene CYP4C62 from Nilaparvata lugens (Hemiptera: Delphacidae)

SUN Hai-Xia1, CHEN Jun2, YANG Zhi-Fan1,*   

  1.  (1. College of Life Sciences, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Wuhan 430062, China; 2. College of Chemical Engineering and Technology, Wuhan University of Science and Technology, Wuhan 430081, China)
  • Online:2014-06-20 Published:2014-06-20

摘要: 【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。

关键词: 褐飞虱, 细胞色素P450基因, 原核表达, 蛋白纯化, 多克隆抗体, 免疫特异性

Abstract: 【Aim】 The cytochrome P450s are important metabolic enzymes in insects because of their involvement in the growth and development of insects and their adaptation to environments. 【Methods】 In this study, we cloned the open reading frame (ORF, excluding the coding sequence for the N-terminal signal peptide) of Nilaparvata lugens P450 gene CYP4C62 and expressed it in Escherichia coli. The recombinant protein was purified by Ni-NTA agarose gel affinity system. Polyclonal antibody of CYP4C62 was then generated by immunization of the Japanese white male rabbits (Oryctolagus cuniculus) with the purified protein. The antibody titer was monitored by indirect ELISA. The immune specificity of the antibody was determined by Western blot hybridization. 【Results】 The results showed that the relative molecular weight of the recombinant CYP4C62 protein is about 56 kD. The antibody titer was estimated as high as 1∶100 000 dilution ratio detected by indirect ELISA. Western blotting analysis showed that the antibody could bind specifically both the heterogeneously expressed CYP4C62 protein and the endogenous CYP4C62 from N. lugens. 【Conclusion】 This study lays a foundation for further investigation of CYP4C62 expression levels in various tissues of N. lugens, localization of the protein at tissue, cellular and subcellular levels by immunohistochemisty, and ultimately revealing its biological function.

Key words: Nilaparvata lugens, cytochrome P450 gene, prokaryotic expression, protein purification, polyclonal antibody, immune specificity