Expression Analysis And Subcellular Localization Of BmIBP From Silkworm Bombyx Mori

Insulin and related peptides are key hormones for the regulation of growth and metabolism. Insulin-related peptides have been identified in both vertebrates and invertebrates including mammals, protochordates, nematodes, mollusks, and insects. Many of these hormones have been shown to act through receptors structurally similar to the insulin receptor,being heterotetrameric,membrane-spanning tyrosine kinases. To date, most researches of insulin-related binding protein were focused on rather vertebrate than invertebrate.A pupae cDNA library of silkworm has been constructed in our Lab. We identified a gene with an open reading frame(ORF) of 768bp which encodes 255 amino acids containing two conserved IG domains. We nominated this gene as BmIBP (Bombyx mori insulin-related binding protein), and its accession number in Genbank is DQ443372.The ORF contains six exons and five introns The predicted protein has a molecular weight of 27.98kD and the theoretical pI lies at 7.36.The recombinant fusion protein His-tag BmIBP was successfully espressed, purified and then immuned a male New Zealand rabbit to get the antiserum(polyclonal antibodies).The titer of the polyclonal antibodies reaches 1:12800 measured by ELISA.Western blotting revealed the protein was expressed in some fully differentiated tissues of the fifth instar larvae,such as testicle,ovary,fat body, malpighian tubule and head,also in some differentiated growth stages of the silkworm,such as egg,pupa and adult.The result of real-time PCR revealed that BmIBP mRNA is widespread in different tisssues and growh stages of silkworm,which highest expression in the ovary,midgut, malpighian tubule and pupa. The subcellular localization experiment with polyclonal antibodies indicated that the BmIBP protein was distributed mainly in cytoplasm of the Bm5 cells.