Expression Analysis And Subcellular Localization Of BmSTMN Gene From Silkworm (Bombyx Mori)

Stathmin is related to the proliferation, differentiation of cells. It has been proposed to be a relay integrating diverse intracellular signaling pathways. To date, most researches of stathmin were focused on rather vertebrate than invertebrate. The stathmin cDNA from the silkworm pupa cDNA library was named BmSTMN, whose ORF was 876 bp and encoded a protein of 291 amino acids. Its molecular weight calculated by the program DNAstar was 33.48 kD, and its pI was 8.005. The result of Multi BLAST was that it had the highest homology with Tribolium castaneum, and the homology was 50%. The entire ORF was obtained by PCR, and EcoR I- Hindâ…¢restrict sites were added by PCR. The ORF was digested by the two enzymes and linked into pET-28a(+) to construct recombinant plasmid pET-28a(+)-BmSTMN, which was identified by PCR, double digestion and sequencing. The recombinant plasmid was transformed into E.coli Rosetta(DE3). After inducing with IPTG, the analysis of SDS-PAGE showed that the fusion protein was expressed successfully in about 37 kD location. The BmSTMN was fused with His-tag and purified on chelating columns. The molecular weight of this fusion protein was 37191.0 Da (His?Tag was weight of 3560.0 Da, and BmSTMN was weight of 33483.99 Da), characterized by mass-spectrum after being further purified on 10 R1 FPLC. This result further proved that His?Tag BmSTMN was expressed in the right form. By immunization of New Zealand rabbit with purified fusion protein high titer poly-clonalantibodies were prepared (1:25600), and the specific reaction responsed very well detected in Western blotting analysis. Semi-quantitative and western blotting analysis revealed that the protein was the highest expressed in silkworm's pupa and head, followed by ovary and tesis, and was the lowest in silk gland. The result of real-time PCR revealed that BmSTMN mRNA was widespread in different tissues and growth stages of silkworm too, consistent with the results of semi-quantitative and western blotting. Finally, we carried out the subcellular localization about BmSTMN in BmN cell, positive signal to BmSTMN was mostly found around the membrane. It laid a good foundation for further researching the biological function of BmSTMN gene, which was in the growth and development process of silkworm.