关键词: 家蚕, 原核表达, 非典型嗅觉受体, Orco, 抗原蛋白

Abstract: 【Aim】 To establish an effective method to prepare the antigen protein of the atypical insect olfactory receptor Orco, and to provide the foundation for study on the immunolocalization and function of Orco. 【Methods】 The target fragment between the 4th-5th transmembrane regions of Orco gene was amplified from the silkworm, Bombyx mori, by RT-PCR using primers containing restriction sites of BamH I and Hind III. This target fragment was digested with BamH I and Hind III directly and ligated to the pET-28a (+) expression vector digested with the same two restrictive enzymes. The ligated product was transformed into the competent Escherichia coli cell DH5α, and the positive clones were testified by PCR and sequenced. The right recombinant expression plasmids were transformed into BL21(DE3) strains, which then were induced with IPTG. SDS-PAGE was used to identify the expressed recombinant protein. A large amount of the induced protein was purified by HisTrap HP affinity chromatography. 【Results】 The target protein whose size was consistent with the predicted protein was induced in E. coli prokaryotic expression system. The target protein was expressed in the form of inclusion body detected by SDS-PAGE. A large amount of purified protein which can be used for antibody preparation was obtained by HisTrap HP affinity chromatography under denaturing conditions. 【Conclusion】 The antigen protein used for antibody preparation of silkworm olfactory receptor Orco can be obtained by prokaryotic expression system.

Key words: Bombyx mori, prokaryotic expression, atypical olfactory receptor, Orco, antigen protein