Secreted Expression of D2V Envelope Glycoprotein in Pichia pastoris
Abstract: Abstract:The E gene of dengue一2 virus was amplified using RT—PCR mothod from the C6/36 cells in— fected by D2V NGC strain and inserted into pPICZ a B vector.The recom binant plasm id was integret— ed into Pichia pastoris X一33 by electroporation and the expressed products were analyzed w ith SDSPAGE and W estern blotting.High level secreted expression was performed by determining the M ut phenotype and screening m ulti—copy integrants in the recombinant Pichia strains.The molecular m ass of recombinant E glycoprotein was approx. 69kD and secreted into supernatants when induced with methano1.The expression product was able tO reacted with D2V polyclone antibody and D2V E specif— ic M cAb.M CAC purified E—hisidine—tagged protein from the expressed product w ith HisTrap Kit can bind im m unologically tO H is antibody and D2V E specific M cAb in W estern—blot as ay. This study suggests the entire E gene coding D2V envelope glycoprotein can be expressed efficiently in Pichia pastoris and the expressed products of E fusion protein could amount to 0.1 g/L.