Abstract: 【Objective】The objective of this study is to explore the expression pattern of BmCPAP3-G and the binding mode of BmCPAP3-G with chitin, which will lay a foundation for the research of the cuticle proteins of silkworm (Bombyx mori).【Method】The sequence features of CPAP motif cuticular proteins and the conserved domains of BmCPAP3-G were analyzed by bioinformatics methods. The recombinant proteins were expressed by prokaryotic expression and purified by Ni affinity chromatography. The protein was identified by 5800 MALDI-TOF/TOF mass spectrometry, and then was used to prepare the polyclonal antibodies. The chitin-binding activity of BmCPAP3-G was verified by chitin affinity chromatography. The spatial and temporal expression patterns of BmCPAP3-G were analyzed by semi-quantitative RT-PCR and western blot. The binding mode of the domains of BmCPAP3-G with chitin was detected by using chitin affinity chromatography. 【Result】The BmCPAP3-G protein has a signal peptide consisting of 18 amino acids, the molecular weight of 27 kD and the isoelectric point of 4.82, the encoding gene located on the chromosome No.15. BmCPAP3-G protein has three ChtBD2 domains, in which cysteine and aromatic amino acid showed very high homology. the BmCPAP3-G was cloned, expressed and the active recombinant protein was purified. After being identified by mass spectrometer, the polyclonal antibody against BmCPAP3-G was prepared. The BmCPAP3-G was found to bind chitin by using chitin affinity chromatography. The spatial and temporal expression patterns of BmCPAP3-G were analyzed by semi-quantitative RT-PCR and western blot, revealing similar results at the transcriptional and protein levels. BmCPAP3-G was expressed highly in the head and cuticle, and minimally in the midgut, gonad, and silk gland. In the silk gland, BmCPAP3-G had a high expression level in the fourth molting and its expression decreased in the cuticle and silk gland as the fifth instar goes on. active recombinant proteins domain_3, domain_1-2, domain_2-3 were successfully expressed and it was found that all the domain_3, domain_1-2, and domain_2-3 could bind to chitin in vitro, but their binding abilities were different. Individual domains could bind with chitin, and two domains showed stronger chitin binding capacity than the single domain. 【Conclusion】BmCPAP3-G is a typical cuticular protein of CPAP family and may be involved in the degradation and formation process of chitin layer in the silk gland during molting stage. BmCPAP3-G could bind with chitin by a single ChtBD2 domain, and multiple domains in which makes it have stronger chitin binding capacity.

Key words: silkworm (Bombyx mori), cuticular proteins, CPAP, expression pattern, chitin-binding