关键词: 家蚕, 肠道免疫, 双重氧化酶, 序列分析, 多克隆抗体, 表达模式

Abstract: 【Aim】 To explore the expression pattern and roles of dual oxidase (DUOX) in gut immunity in the silkworm, Bombyx mori. 【Methods】 The amino acid sequence of BmDUOX was analyzed by bioinformatic methods, and the outside region encoding gene, BmDUOX_OM, was cloned from B. mori and transferred into Eescherichia coli Transetta (DE3) to obtain the fusion protein. After purification by Ni affinity chromatography, the purified protein was treated as antigen to immunize mouse to prepare polyclonal antibodies, which were utilized for analyzing the expression and localization of BmDUOX in BmE cells. Semi-quantitative RT-PCR was employed to assay the expression patterns of BmDUOX in different developmental stages and different tissues and the pathogen-induced expression profiles. Additionally, the content of reactive oxygen species (ROS) in BmE cells after induction by Nosema bombycis was analyzed by using ROS detection kit. 【Results】 Bioinformatics analysis showed that BmDUOX has conserved peroxidase, ferric_reduct, EF-hand, FAD-binding and NAD-binding domains. There are also six transmembrane regions in BmDUOX, and their transmembrane form is consistent with that of hDUOX2 and DmDUOX. Multiple sequence alignment analysis showed that the peroxidase domain of BmDUOX has conserved activity sites. BmDUOX_OM gene was cloned, the recombinant protein was purified, and mouse-origin polyclonal antibody with high specificity was prepared. Indirect immunofluorescence assay (IFA) showed that BmDUOX was located on the plasma membrane of BmE cells. Expression pattern analysis showed that BmDUOX was highly expressed in the 5th instar day-3 larvae and adults of the silkworm, and in the tissues of integument, testis, ovary, head of larvae, and the ovary, testis, integument, fat body of its adults. Expression of BmDUOX in the larval midgut of the silkworm could be continuously induced by microsporidia Nosema bombycis, and the content of ROS in BmE cells was obviously increased after induction by N. bombycis, suggesting that DUOX-regulated intestinal epithelial ROS response may play important roles in the resistance against the infection of N. bombycis. 【Conclusion】 BmDUOX has the typical functional domains and active sites, showing its conservative biological function in B. mori. Expression profiles of BmDUOX in B. mori in different developmental stages, different tissues, and under induction by diverse pathogenic microorganisms suggest that BmDUOX may be involved in the immune response of host intestinal epithelium to the invasion of pathogenic microbes.

Key words: Bombyx mori; gut immunity, dual oxidase, sequence analysis, polyclonal antibody, expression pattern