Expression And Analysis Of Membrane Protein Gene BmP(echA)-like In Bombyx Mori Fused With Polynedron

The mass expression of membrane proteins would bring toxic effect to cells and it only shows low expression in eukaryotic expression systems. Cells can be avoided toxic infection and improved expression level by fused with polyhedrin. We found an 840 bp ORF fragment from the Bombyx mori membrane cDNA which was compared with M.tuberculosis BmP(echA)-like genes through BLAST in NCBI, then we named it BmP(echA)-like(Bombyx mori Protein of Enoyl-CoA hydratase like). This gene encoded 279 amino acid residues with the predicted molecular weight of 30.273 kDa and isoelectric point of 8.79 (GenBank accession number: DQ311163.1).Using the PCR method to amplificate the BmP(echA)-like gene. The ORF of the gene was cloned into the transfer vector pBacPAK 8-Polh as a recombinant transfer plasmid pBacPAK8-Polh-BmP(echA)-like, Then Polh-BmP(echA)-like was subcloned into the expression vector pET-32a and the transfer vector pFastBac HTb respectively, we also cloned the BmP(echA)-like gene along to the prokaryotic expression vector pET-32a and the transfer vector pFastBac HTb as a contrast of expression level. The recombinant prokaryotic expression plasmid was transformed into E.coli BL21(DE3), and detected by the SDS-PAGE after induce of the 1 mmol/L IPTG .The result showed that by contrast with the negative control, the fusion protein was corrected expreessed. Additionally,a recombinant transfer plasmid pFastBac HTb-Polh-BmP(echA)-like was transferred into DHl0Bac to get the recombinant shuttle plasmid Bacmid-Polh-BmP(echA)-like.Finally, the restructuring Bacmid-Polh-BmP(echA)-like was transfected into BmN cell to get the recombinant virus Bm-Polh-BmP(echA)-like. SDS-PAGE result showed that the level of expression of fusion form was higher than the other. The fusion protein can be easily purified by adjust the pH and by Ni-NTA affinity chromatography.Using TEV to cut the purified fusion protein, we can get the purified BmP(echA)-like by adjust the pH. Real-time RT-PCR was performed in different issues of the fifth instar larva of Bombyx mori.The analysis suggested that the protein was more expressed in every organizations, but the detection shows the expression in ovaries is the highest the pupal period is lowest.The distinction between two organizations could be 59 times. The fusion protein (His-Polh-BmP(echA)-like) was used to investigate the binding proteins of BmP(echA)-like by His-Pull down. Electrophoresis result showes that there were at least two BmP(echA)-like binding proteins which moleculer weight are approximately 35 kDa and 60 kDa.