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Virologica Sinica, 17 (3) : 198-201, 2002
Research Article
Secreted Expression of D2V Envelope Glycoprotein in Pichia pastoris
1.Guangdong Provincial People S Hospital,Guangzhou 510080,China
2.Department of Microbiology, Sun Yat—Sen University of Medical Sciences,Cmangzhou 510089,China
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.
  Published online: 5 Jun 2002
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