Abstract Overlapping PCR technology was employed to splice IFN β and HSA genes in vitro. The spliced gene was inserted into Pichia pastoris secretory vector pPIC9K. The IFNβ-HSA gene was designed to be secretory expression under the control of promoter AOX1 and Mat α signal peptide in pPIC9K. The recombinant plasmid pPIC9K/IFNβ-HSA was linearized by restriction enzyme SalI and transformed into Pichia pastoris KM71 by electroporation. The recombinant strains identified by G418 selection and conformed by PCR analysis were induced by methanol to express fusion protein IFNβ-HSA. SDS-PAGE and Western blot analysis of the fusion protein showed that the expressed fusion protein IFNβ-HSA with an apparent 90kDa molecular weight had the antigenicity of HSA. The specific activity of culture supernatant was about 640IU/ml assayed by the standard antiviral activity test on WISH cells challenged with VSV virus.
Received: 21 February 2006
Published: 25 July 2006
