Expression And Identification Of Membrane Protein Bax Inhibitor-1 In BEVS,Tagged With Polyhedrin

Baculoviral polyhedrin (Polh), derived from the Bombyx mori nuclear polyhedrosis virus, is known to protect virus particles from physical and biochemical degradation, thus allowing virions to remain infectious outside the host previously. The BmN insect cells were infected with the wide Bombyx mori nuclear polyhedrosis virus, infected cells were collected and disrupted by cell lysis buffer. After being centrifuged, the precipitation was collected and dissolved by buffer solution(pH 10.8), then collected supernatant after centrifuged. Purified Polh was obtained and the purified protein was used to immunize BALB/c mouse, it had been showed that Polh could be successfully used as a fusion partner for the formation of recombinant proteins as insoluble inclusion bodies in E. coli expression system. In addition, the recombinant Polh protein showed almost the same characteristics (rapid solubilization under alkaline conditions) as native baculoviral Polh. We characterized the expression of recombinant polyhedron protein fused to Enhance Green Fluorescent Protein (EGFP) and insert TEV protease between them. The polyhedrin fusion protein (60 kDa) was successfully expressed as an insoluble inclusion body comprising approximately 35% of the total cellular protein. To purify the fusion protein, only differential centrifugation and differential pH buffer were used. EGFP purified By Ni-NTA sepharose chromatography after digested purified fusion protein by TEV protease. We contributed a new method to purify polyhedrin fusion protein quickly and efficiently.The Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that is located in endoplasmic reticulum (ER) membranes. It’s a neotype regulating factor concerned with cell death which controlled by Bcl2 and Bax. When screening Silkworm cDNA library, we found a gene which has a higher homology with Bax inhibator-1, named BmBI-1(Bombyx mori Bax inhibator-1) after bioinformatics analysis. Baculoviral polyhedrin was used as a novel fusion partner for facilitating expression of BmBI-1 in Bac-to-Bac baculovirus expression system. We constructed the baculovirus transfer vector pFastBac HTb-Polh-BmBI-1 and pFastBac HTb-BmBI-1. The recombinant transfer plasmid was transformed into the BmDH10Bac competent cells, and inserted into the Bacmid DNA by transposition recombination. Then we constructed two recombinant virus vBmPolh-BmBI-1 and vBmBmBI-1. The BmN insect cells were infected with the recombinant virus discernly. The result of Western blotting showed that the BmBI-1 fused with Polh could be expressed in infected BmN cell, but only BmBI-1 can not express. The silkworm larvae and pupa were infected with the recombinant virus vBmPolh-BmBI-1. The result of Western blotting showed that the BmBI-1 fused with Polh could be expressed in infected silkworm larvae and pupa. The result of real-time quantitative PCR indicated that the mRNA of Polh-BmBI-1 was highest after 72 h infected by recombinant virus.