关键词: 双委夜蛾, 气味结合蛋白, 精巢, 原核表达, 抗体, 表达谱

Abstract:

【Aim】 This study aims to carry out prokaryotic expression, antibody preparation and expression profiling of the odorant binding protein OBP6 in Athetis dissimilis, so as to facilitate the future study on the function of AdisOBP6. 【Methods】Bioinformatics software was used to analyze the structure characteristics of AdisOBP6 protein. AdisOBP6 antibody was prepared by immunizing New Zealand white rabbits four times using the recombinant protein obtained by prokaryotic expression. Fluorescence quantitative PCR and Western blot were used to detect the expression profiles of AdisOBP6 in the antennae of female and male adults, testes at different developmental stages and unfertilized and fertilized eggs of A. dissimilis. 【Results】 Homologous modeling prediction showed that AdisOBP6 has six conserved cysteine residues and seven α-helical structures, which are folded into a binding pocket. The prokaryotic expression results showed that the recombinant protein was expressed most highly and stably under induction with 0.5 mmol/L IPTG at 20℃. Antibody titers exceeded 1∶512 000, 1∶512 000, 1∶512 000, and 1∶64 000, respectively, in four immune replicates. The specific binding of the obtained antibody to AdisOBP6 protein was detected by Western blot. Fluorescence quantitative PCR results showed that the expression level of AdisOBP6 in the testis was much higher than that in the antennae. AdisOBP6 began to express in the testis of A. dissimilis at the larval stage, with a higher expression level in the larval testis than in the pupal testis, and its expression level gradually reached the peak in the testis of adults after eclosion, but it was scarcely expressed or expressed at a very low level in unfertilized eggs and fertilized eggs. Western blot analysis showed that AdisOBP6 protein was also mainly expressed in the testis, and its expression level was very low in the antennae of female and male adults, fertilized eggs and unfertilized eggs. 【Conclusion】 In this study, the prokaryotic expression of AdisOBP6 was realized, and its antibody was successfully prepared. This study also confirmed that AdisOBP6 is highly expressed in the testis, and the expression level of AdisOBP6 reaches the peak after adult eclosion, suggesting that AdisOBP6 may be involved in insemination process. These results lay a foundation for further studying the function of AdisOBP6.

Key words: Athetis dissimilis, odorant binding protein, testis, prokaryotic expression; antibody, expression profile