Molecular Cloning, Expression And Function Analyses Of EIF4E From Silkworm Pupa (Bombyx Mori)

The full length open reading frame (ORF) of Bombyx mori eukaryotic translation initiation factor 4E cDNA was obtained from silkworm pupa by RT-PCR and was named BmeIF4E (Bombyx mori eIF4E), the accession number was DN443192. The ORF of this gene was blasted with the GenBank database on NCBI. Bioinformatics analysis results showed that the gene encode 210 amino acids with a isoelectric point of 5.97 and a predicted molecular weight of 24.44 kD, which has a conserved domains namely IF4E superfamily. Homogeneous analysis showed that BmeIF4E shares highly homology with the amino acid sequences from Spodoptera frugiperda (AF281654) (about 90%).In order to study the function of BmeIF4E, the ORF of this gene was cloned into the pET-28a(+) vector, and constructed the procaryotic expression plasmid pET-28a(+)-BmeIF4E. The recombinant plasmid was transformed into E.coli BL21 (DE3). After inducing with IPTG, the result of SDS-PAGE showed that the recombinant protein rBmeIF4E was expressed successfully. Because rBmeIF4E in E.coli was soluble, the protein was purified with metal-chelating affinity chromatography directly. The result of purification showed that the molecular weight of this fusion protein was 27.87 kD, characterized by mass-spectrum after being further purified on 10RI FPLC. It was consistent with theoretical value 28 kD (His?tag 3.56 kD, BmeIF4E 24.44 kD). The purified fusion protein was used to immunize a male New Zealand rabbit. Then, antisera (polyclonal antibody) was harvested and IgG was prepared by affinity chromatography using immobilized protein A. The titer of the polyclonal antibody reaches 1:12800 measured by ELISA.At the same time, the gene expression features in different stages and different organizations of silkworm were analyzed using Real-time RT-PCR and Western blot analyses. BmeIF4E expression begins during the egg stage, increases during the larvae stage and pupa stage, and reaches a peak during the moth stage. Additionally, BmeIF4E are expressed in all tissues and the expression levels are highest in Malpighian tubes, followed by epidermis, fat body, spiracle, ovary, gut and head, and are lowest in the silk glad. Therefore, we propose that BmeIF4E may play an important role in the whole life cycle of Bombyx mori as a valuable house-keeping gene. These results may lay an important foundation of further function studies of BmeIF4E protein. With immunofluorescence method, subcellular localization by anti-BmeIF4E polyclonal antibody in Bm5 suggested that BmeIF4E protein could be found in both the cytoplasm and nucleus.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, Western blot result revealed that the expression level of BmeIF4E was decreased previously. The MTT assay also indicated that when BmeIF4E was knocked down in Bm5 cells, the cell viability was significantly decreased.