Expression, Purification And Characterization Of Ras Oncogene (bras2) From Silkworm (bombyx Mori)

The ras superfamily is an important kind of functional proteins. They play essential roles in a variety of cellular responses including cell growth, differentiation, survival and proliferation. We found a member of ras superfamily named ras oncogene (Bras2) from bombyx mori through analyzing the NCBI database and we called ras oncogene (Bras2) as BmBras2. The cDNA of BmBras2 from Bombyx mori consists of 1,412 bp. The open reading frame (ORF) contains 603 bp, encoding 200 amino acid residues with a predicted molecular weight of 22.9 kD and theoretical isoelectric point (pI) of 6.62. Its accession number in GenBank is AB206960. The ORF of BmBras2 was cloned by PCR and then inserted into the sites of EcoRⅠand HindⅢof pET-28a(+). A recombinant expression plasmid, pET-28a(+)-BmBras2, was constructed and transformed into E.coli Rosseta (DE3). Recombinant His-tag BmBras2 (rBmBras2) was expressed successfully in E.coli Rosetta (DE3) induced by IPTG. The purified rBmBras2 was obtained through Ni2+ affinity chromatography. Then this recombinant BmBras2 was used as an antigen for preparation of rabbit polyclonal antibody. After purified with the Protein A HP, the titer of the polyclonal antibodies reaches 1:12,800 measured by ELISA. The immunoblotting results indicated that the antibody was specific to rBmBras2. Western blot and Real time PCR analyses showed that BmBras2 was expressed during four developmental stages, and the expression level of BmBras2 was highest in pupae and lower in the fifth instar larvae, moth and nascent egg; BmBras2 was expressed in all eight tissues, and it was highly expressed in head, intestine and epidermis. The result of subcellular localization indicated that BmBras2 mainly localized at the nuclei of Bm5 cells, partly at cytoplasms, but not at plasma membranes. The related cell proliferation assay showed that rBmBras2 could stimulate the proliferation of hepatoma cells (HepG2 and PLC) but not the normal hepatic cells (L02). These results will lay a foundation for further study of the function of cellular BmBras2 protein.