家蚕PMP22基因的表达研究

Membrane proteins play a very important role in the cell activities. The laboratory fused the polyhedrin protein with the Bombyx mori membrane protein PMP22 to explore high level expression methods of this membrane proteins. The open reading frame (ORF) of gene PMP22 was screened form the cDNA library of bombyx mori and was recombined with the polyhedrin gene Ph to obtain the recombinant gene Ph-PMP22. Then we successfully constructed the recombinant expression plasmid pET-28a-Ph-PMP22. The recombinant plasmids was respectively transformed into 3 kinds of E. coli. The engineering bacterias which was screened was induced by IPTG with a final concentration of 0.5 mmol / L respectively to express fusion proteins. Western blot detected some expression of fusion proteins in E. coli Rosetta, and the percents of fusion protein in total expression is about 2%. Through Bac-to-Bac system, we successfully constructed recombinant Bacmid with Ph-PMP22. Bombyx mori BmN cells was infected with the recombinant Bacmid, then was extracted and disrupted for western blotn analysis. The result suggested that there is no fusion proteins in upper fluid after disrupting and a small amount of fusion proteins in the precipitation which is lower than prokaryotic expression system.