The Cloning, Expression And Subcellular Localization Of The Bmeif3s5 Gene From Silkworm (bombyx Mori)

Eukaryoctic initiation factors (eIF3s) are the key elements in proten synthesis.Among identified 13 initiation factors, eIF3,composed cf 8 or more subunits, is the largest one and plays a central role in the initiation of translation.eIF3s5 is one subunits of eIF3 complex and mediates most functions of eIF3. The full open reading frame (ORF) of Bombyx mori eIF3s5 was obtained from silkworm pupa by RT-PCR and named BmeIF3s5, of which the accession number was DQ443287. This ORF contained 834 bp, encoding a polypeptide of 278 amino acids with a predicted molecular weight of 29.60 kD. Moreover, the sequence of the amino acids encoded by this gene was more conservative in invertebrates through the homology comparision. Using pET-28a(+) as the expression vector, and. constructed the procaryotic expression plasmid pET-28a(+)-BmeIF3s5 which was identified by PCR, double digestion and sequencing.The recombinant plasmid was transformed into E. coli Rosetta. After inducing with IPTG, the analysis of SDS-PAGE showed that the recombinant protein was expressed in about 33.28 kD location, which was compared with the negative control. The recombinant protein was insoluble, so it was washed,dissolved and centrifuged.The supernatant was purified with metal-chelating affinity chromatography. The result showed that the molecular weight of this fusion protein was 34.03 kD, characterized by mass-spectrum after being further purified on 10 RI FPLC. It was consistent with thecoretical value 33.28 kD (His-tag 3.81 kD, BmeIF3s5 29.60 kD). By immunization of New Zealand rabbit with purified recombinant protein high titer poly-clonalantibodies were prepared (1:25600). Western blotting validated the antibody was specific to His-BmeIF3s5. The total proteins from eight developmental stages and ten different tissues of silkworm were extracted for Western blotting, in order to analysis the expression level of BmeIF3s5. Our results showed that BmeIF3s5 was existed in all development stages and organizations test at different levels of expression, especially higher in silk gland, egg and adult.The experiment with ploy-antibody indicated that BmeIF3s5 was mainly distributed in cytoplasm through the method of subcellular localization. It laid a good foundation for further researching the biological function of BmeIF3s5 gene, which was in the growth and development process of silkworm.