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