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| Cloning, Expression and Function Analyses of Eukaryotic Translation Initiation Factor 4E(BmeIF4E) Gene from Silkworm Pupa (Bombyx mori) |
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Abstract The full open reading frame (ORF) of eukaryotic translation initiation factor 4E (eIF4E) was obtained from silkworm(Bombyx mori) pupal cDNA library and was named BmeIF4E (GenBank accession No. DN443192). In order to study the biological function of BmeIF4E, the ORF of this gene was cloned and the procaryotic expression recombinant plasmid pET-28a(+)-BmeIF4E was constructed to express His-BmeIF4E fusion protein in Escherichia coli BL21(DE3). Because the rBmeIF4E in E.coli was soluble, the protein was purified with metal-chelating affinity chromatography directly. rBmeIF4E was used to generate anti-BmeIF4E polyclonal antibodies, to determine the subcellular localization of BmeIF4E. Immunostaining indicated that BmeIF4E protein could be found in both the cytoplasm and nucleus. The gene expression features in different organizations and different developmental stages were analyzed using Real-time RT-PCR and Western blotting analyses. The results showed that BmeIF4E were expressed in all tissues and the expression levels were highest in malpighian tubes, followed by epidermis, fat body, spiracle, ovary, gut and head, and were lowest in the silk glad. Additionally, BmeIF4E expression began during the egg stage, increased during the larvae stage and pupa stage, reached a peak during the moth stage. Furthermore, RNA interference technology was applied to silence the target mRNA and the cell viability was assessed by Western blot and MTT assay. After 72 h of RNAi, the MTT assay indicated that when BmeIF4E was knocked down in Bm5 cells, the cell viability was decreased significantly. BmeIF4E may play an important role in the whole life cycle of Bombyx mori as a valuable house-keeping gene.
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Received: 26 July 2010
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