Prokaryotic expression and localization analysis of calcyphosphine gene from silkworm(Bombyx mori)

• 1、Biochemistry Institute of Zhejiang Sci-Tech University

Abstract：Bombyx mori calcyphosphine(BmCAPS) is a calcium binding protein containing two EF-hand conserved domains. The cDNA sequence of BmCAPS consists of 1038bp(GenBank accession No. DQ443221). It contains an open reading frame (ORF) of 720 bp encoding a protein of 239 amino acids with a predicted molecular mass of 26.874 kD. The ORF of BmCAPS gene was amplified by PCR and inserted into the prokaryotic expression vector pET-28a(+).The recombinant protein His-BmCAPS was expressed successfully in E.coli BL21 Star(DE3) after induced by IPTG. It was fused with His-tag and purified by affinity chromatography. the purified fusion protein was used to immunize a male New Zealand rabbit to obtain polyclonal antibody. The titer of the polyclonal antibody reaches over 1:6400 measured by indirect ELISA, and the recombinant protein was specifically recognized by antiserum in Western blotting analysis. We performed subcellular localization of BmCAPS protein in BmN cell and it was identified only in cytoplasm. The transcription level of mRNA and expression level of protein in different stages of Bombyx mori and different tissues of fifth instar larva was also analyzed by the real-time fluorescent quantificational PCR and Western blotting. We found that the transcription level of BmCAPS mRNA gradually increased from egg to moth in accord with the results of Western blotting. And the transcription level and protein expression level of spermary was highest compared to other tissues, but there was no message detected in the tissues of head, epidermis and silk glands by Western blotting. All the results above will provide us some evidences for further studies on the function of BmCAPS.

Keywords： Bombyx mori BmCAPS subcellular localization fluorescent quantificational PCR Western blotting