家蚕BmE(spl)-like基因的原核表达定位及功能分析

We cloned a novel gene with an open reading frame (ORF) of 606bp and encoding 201 amino acids with a predicted molecular weight of 22.3kD, and isoelectric point 9.70 from the silkworm pupae cDNA library constructed by our laboratory. After blasting the sequence homology in the NCBI database, we identified it was a novel gene in the Bombyx mori. The protein presented here exhibit a high degree of sequence similarity to proteins encoded by E(spl). It was found to encode highly conserved helix-loop-helix (HLH) proteins and the homology includes, besides the bHLH motif, orange domains, and a sequence of four amino acids, tryptophan-arginine-proline-tryptophan (W-R-P-W) which occurs at the carboxy terminus. We named it BmE(spl)-like (Bombyx mori Enhancer of Split like) gene. The ORF of the gene was cloned into the prokaryotic expression vector pET-28a and the recombinant plasmid was transformed into E.coli BL21(DE3). After being induced with IPTG, this gene was successfully expressed. The fusion protein was purified by affinity chromatograph and 10 RI FPLC. After the fusion protein characterized by mass-spectrum, the results indicated that the real molecular mass of the fusion protein was concordance with predicted. The purified protein was injected into the New Zealand white rabbit to produce the polyclonal antibodies. We extracted the total protein and RNA from different tissues and developmental stages of Bombyx mori, and found that the levels of transcription and expression were great difference in the different tissues and developmental stages of Bombyx mori by Western blotting and real-time PCR. Meanwhile, we located the protein in the Bm5 cells, the results indicated that it was distributed in the cytoplasm.