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Gene cloning, prokaryotic expression and subcelluar localization of a class C scavenger receptor BmSR-C in the silkworm, Bombyx mori.

Abstract

Aim: This study aims to clone and identify a class C scavenger receptor gene BmSR-C in the silkworm, Bombyx mori, so as to lay a foundation for the further exploration of the function of scavenger receptor class C in the silkworm immunity. Methods: The full-length cDNA of BmSR-C was cloned by RACE and analyzed by bioinformatics. The spatial and temporal expression profiles of BmSR-C were detected by RT-PCR and qPCR. The BmSR-C recombinant protein was obtained by prokaryotic expression and Ni+ affinity chromatography. The antibody was acquired by using the recombinant protein to immunize mice, and its titer and specificity were detected by ELISA and Western blot, respectively. Simultaneously, the subcellular localization of the protein was analyzed after BmSR-C eukaryotic vector was constructed and then transfected into BmE cells of the silkworm. Results: We cloned the full-length cDNA of BmSR-C (GenBank accession no.: BGIBMGA004577) from B. mori, which contains an ORF of 1 821 bp encoding a putative protein of 606 amino acid residues. BmSR-C has the typical structural features of scavenger receptor class C family, including CCP, MAM and SO domains, as well as a single transmembrane region in the near end of the C-terminus. Evolutionary analysis showed that SR-C proteins from lepidopteran insects clustered alone, and BmSR-C was most closely related to its homologues from Spodoptera frugiperda and Danaus plexippus. The spatial and temporal expression profiles showed that BmSR-C was highly expressed in the Malpighian tubules and hemocytes, but showed no obvious expression in other detected tissues. In addition, BmSR-C was expressed in hemocytes during different developmental stages, with the expression peak at the 4th instar larval molting. The titer of the antibody was estimated as high as 1:128 000 dilution ratio through ELISA, and the result of Western blot showed that the antibody could specifically recognize the recombinant protein. The subcellular localization result in BmE cells showed that BmSR-C is mainly located on the cell membrane. Conclusion: BmSR-C was cloned and its expression patterns were investigated. The anti-mouse polyclonal antibody was obtained. The subcellular localization of BmSR-C was investigated in BmE cells of the silkworm. It is inferred that BmSR-C may be involved in the growth and development and immune response to the invasion of pathogenic microorganisms in the silkworm. This study provides a foundation for further research of the biological function of BmSR-C in the silkworm.