Expression And Identification Of CSFV E0 Protein In Pichia Pastoris And Mammalian Cell

Classical swine fever is a highly contagious infectious and mortality disease, it caused by Classical swine fever vius(CSFV), CSFV is a single strand RNA virus which belongs to Flaviviridae pestivirus, the CSFV E2 and EO protein is a protective protein inducing neutralizing antibodies, EO is the only glycoprotein can secreted into the culture supernatant classical swine fever virus-infected cells. In addition, the amino acid sequence coded EO is more conservative. Therefore, EO is one of the main target protein in the prevention and control of CSF. This paper mainly carried on the following several aspects of research:1:Expression and identification of classical swine fever virus EO protein in Pichia pastoris expression system. With the genetic engineering, we successfully constructed a recombinant plasmid pPIC9K-E0 which could express EO protein, and then successfully transformed it into Pichia pastoris GS115 strain by electroporation. Through optimizing the conditions such as the induction time, induction temperature and methanol induction concentration to improve the yield in Pichia pastoris, we ultimately found it made EO protein of yield reached the highest at the 28℃ under the 0.5% of methanol final concentration after the 120 hours. Identification of EO protein by Western blot and ELISA which the classical swine fever virus positive serum was used. The result showed that the recombinant EO protein had good immunogenicity, and it can be used as coating antigen to detect Classical swine fever virus.2:Expression and identification of Classical swine fever virus EO protein in PK-15 cells. We successfully constructed a recombinant plasmid PEGFP-EO which can express EO protein, and then it successfully transformed into PK-15 cells by chemical transfection method. A cell line expressing EO protein was obtained through culture. All of this lay the foundation for further expression and purification of EO glycoprotein, and for studying its biological function and activity.