家蚕Ras oncoprotein（Bras1）基因的原核表达与功能分析

Bras1 (Bombyx mori ras oncoprotein) is one of the Ras protein super family in insects. Ras is one of small G proteins, which were believed to be involved in a growth promoting signal transduction process, to regulate the growth and proliferation of the cells. A unique new Bombyx mori cDNA was identified from the pupa cDNA library constructed in our laboratory. Sequence analysis revealed that the full length of the cDNA was 1459 bp. This gene codes a protein with 192 amino acids, which was named Bras1 (GenBank accession number NM_001043508). The protein has such secondary structures as α-helices, extended strand and random coli. The ORF of the gene was cloned into pET-28a (+) vector and transformed into E.coli BL21 (DE3). Recombinant protein was expressed successfully in E.coli BL21 (DE3) induced by IPTG with the final concentration of 1 mM. The recombinant protein was insoluble and was purified with metal-chelating affinity chromatography; its activity of GTPase was determined by (NH4)2MoO4-FeSO4 assay and the results showed that it had intrinsic activity of GTPase. New Zealand rabbit with recombinant protein generated high titer polyclonal antibodies (1:6400). The gene expression features in different organizations of the fifth instar larva and different stages were analyzed using Western blot method. The results showed that in all tissues Bras1 were expressed as following: pheromone gland, Malpighian tubule, brain, silk gland; in the four development stages, Bras1 was expressed highly in egg, pupa and adult; but in larval, low level of expression was detected. BmN cells were treated by mevastatin. The results showed mevastatin could inhibit isoprenylation of Bras1 to inhibit cell proliferation of BmN cells; and suggested Bras1 was essential to BmN cells proliferation. These results laid a good foundation for further study on function of this gene in silk worm.