Expression of Noro Virus Capsid Protein in Tn5 Cells

Abstract:

The ORF2 gene sequence of NoVs VA387, at a length of 1 623 bp, was synthesized and inserted into vector pFastBac1. Transformed DH10Bac competent cells by pFastBac1-NoV-ORF which was identified by sequencing to obtain Bacmid-NoV-ORF2. The constructed recombinant plasmid Bacmid-NoV-ORF2 was transfected to sf9 cells by co-precipitation with calcium phosphate. Harvested the supernatant of cells and frozen storage recombinant baculovirus at -20 ℃. Tn5 cells were infected by Ac-NoV-ORF2 virus and the cells was harvested 5 d ~6 d after infection. Break the cells, and the supernatant was harvested and purified by MS purification. Various components were collected, observed by electron microscopy，and determined for protein content by 10% SDS-PAGE. The Tn5 cells transfected with Ac-NoV-ORF2 virus showed specific protein by 10%SDS-PAGE profile. The capsid protein expression was significantly and gradually increase since 3 d after infected, and the production reached stable in 4 d. The 10% SDS-PAGE showed that the capsid protein was mainly located in two bands with relative molecular masses of 59 KD and 56 KD. Electron microscopy showed that the expressed capid protein was assembled to VLPs at sizes of about 40-50 nm. So the capsid protein of NoVs was successfully expressed in Tn5 cells and assembled into VLPs.