A New Bombyx Mori Immune-Related Protein Expression With Polyhedrin Fusion And It’s Primary Function Research

During the late phase infection of Baculovirus, there are two highly expressed proteins, which are polyhedrin (Polh) and P10 protein. Polyhedrin is the main component of the polyhedra, the size of gene sequences is 738 bp, encoding 246 amino acids, and the molecular weight is 29 kD. Polyhedrin promoter is highly efficient promoter, it promote the high expression of polyhedrin. The amount of polyhedrin accumulates to very high level, becoming 30% to 50% of the total protein in cell, and they forms crystals, this crystal structure called polyhedra. Such polyhedra crystals can be easily precipitated by low speed centrifugation, and it can be dissolved under alkaline conditions. These unique properties suggest that baculoviral polyhedrin might be an advantageous fusion partner for expression and purification of foreign proteins.Based on the above properties of polyhedrin, select a new gene which encoding immune-related protein from the cDNA library of Bombyx mori constructed by our lab. Named this gene BmIRP(GenBank:NM_ 0010 98349.1), the length of this gene is 775 bp and the ORF size is 516 bp, encoding 172 amino acids, predicted molecular weight is 18.49 kD. Construct a recombinant expression vector pET-28a-Polh-BmIRP using BmIRP fusion with Polh, and among them is a TEV cleavage site. Transformed recombinant plasmids into E.coli BL21 for prokaryotic expression, SDS-PAGE and Western blotting shows a specificity band. Compare with the recombinant pET-28a-BmIRP,the polyhedrin fusion expression is much more efficient. The expressed polyhedrin fusion protein formed polyhedra structure. It has the same feature that can dissolve under alkaline conditions with the natural polyhedron crystal. It can dissolve in pH 11.0 glycine-NaOH buffer, when adjust the pH value of dissolved supernatant to 7.4, the fusion protein will precipitate. Washing the fusion protein gradiently by different pH value glycine-NaOH buffer can remove the other protein easily from fusion protein, the final purity of the fusion protein is about 92%. The purified fusion protein Polh-BmIRP is successfully identification by MALDI-TOF-MS, and the fusion protein present polyhedron structure under Scanning Electron Microscope. Meanwhile, apply this method to Bac-to-Bac baculovirus expression system to attempt highly expression of polyhedrin fusion protein. The fusion protein has successfully expressed in Bombyx mori BmN cells. This fusion expression way can be used to highly expression of foreign gene and simply purification of target protein. The bioinformatics analysis, RNAi research and analysis of transcription levels in different tissue of Bombyx mori will pave the way for the future structure and fuction research of this protein.