High Level Expression of Major Antigenic Segment of Japanese Encephalitis Virus Strain SA 14-14-2 E Protein
Abstract: Based on a constructed prokaryotic expression plasmid which carries a ma-jor antigenic segment of E gene of JEV strain SA14-14-2 in our lab, we expressed the gene in methylotrophic yeast Pichia pastoris. The segment was choosn based on an- alysis provided by a computer software because of its higher antigenic index. The seg- ment was 1113bp, encoding 371 aa. The gene was subcloned into the vector pPICZα-A. The constructed plasmid was transformed into yeast X-33 by electrophoration. The recombinant transformants were selected by Zeocin. Induced by the addition of metha- nol every 24h, the product analyzed by SDS-PAGE was about 44kDa and 50kDa at a yield of 290 mg per litter of culture. The result of Western blotting indica- ted that the two bands both have specific antigenicity mainly owing to heterogeneous glycosylation. Coating with the supernatant, we can easily discriminate the positive serum from the negative, which should be useful for the production of diagnostic agents and genetically engineered vaccine vaccine of J EV.