Abstract: 【Aim】 Odorant binding proteins (OBPs) are involved in the identification of odor molecules, and play an important role in bees’ olfaction. This study was designed to clone and express an OBP3 gene from the Chinese honeybee Apis cerana cerana, in order to prepare the polyclonal antibody.【Methods】 The OBP3 gene was amplified by RT-PCR from total RNA from head of A. cerana cerana, then sub-cloned into prokaryotic expression vector pET-28a and expressed in Escherichia coli Rosetta (DE3) host cells. The fusion protein was purified and immunized into New Zealand white rabbits so as to prepare polyclonal antibody. The sensitivity and specificity of the polyclonal antibody were detected through indirect ELISA and Western blot, respectively. The expression profiles of AccOBP3 in different tissues were detected by real-time quantitative PCR. 【Results】 AccOBP3 (GenBank accession no. KJ026357) was cloned and a 444 bp fragment was obtained. The recombinant vector pET-OBP3 was successfully constructed. SDS-PAGE analysis showed that the fusion protein was well expressed. The anti-OBP3 polyclonal antibody showed high titer (higher than 1∶40 000) and specificity. Real-time quantitative PCR results showed that the expression level of AccOBP3 gene was significantly higher in legs and antennae (P<0.01), moderate in thorax, and significantly lower in heads and abdomen (P<0.01). The expression level of AccOBP3 in heads and abdomen had no significant difference (P>0.05). 【Conclusion】 The OBP3 gene has high transcript expression in the antennae of A. cerana cerana. In this study, AccOBP3 gene has been cloned and expressed, and the rabbit anti-ApisCeranaOBP3 polyclonal antibody has been prepared by immunization with the purified recombinant OBP3 protein, which lays the foundation for further studies on functions of OBP3 gene.

Key words: Apis cerana cerana, OBP3 gene, gene cloning, prokaryotic expression, tissue expression profiles, antibody preparation