Citation: WEI Hui—yong, JIANG Li—fang, XUE Yao—hua, GUO Hui—yu. Secreted Expression of D2V Envelope Glycoprotein in Pichia pastoris .VIROLOGICA SINICA, 2002, 17(3) : 198-201.

Secreted Expression of D2V Envelope Glycoprotein in Pichia pastoris

  • Available online: 05 June 2002
  • 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.

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    Secreted Expression of D2V Envelope Glycoprotein in Pichia pastoris

    • 1. 1.Guangdong Provincial People S Hospital,Guangzhou 510080,China
    • 2. Department of Microbiology, Sun Yat—Sen University of Medical Sciences,Cmangzhou 510089,China

    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.

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