Expression Antigen VP3 of Enterovirus 71 in Pichia. pastoris

The genes encoding the capsid protein VP3 were amplified by RT-PCR.After con-firmed by DNA sequence analysis,the VP3 was inserted into the Pichia.Pastoris expression vector pPIC9K.The sequence of ORF of-factor and VP3 were confirmed by DNAsequencing with primer 5'-AOX1 and 3'-AOX1.Then the pPIC9K/VP3 weretransformed into yeast GS115 by the of electro-transformation.After the PCR analysis and the screening for Mut+ phenotype of the transformants,Then multiple inserted transformants were fermented in flasks.After induction by methanol(96 hours),recombinant protein could be effectively secreted into media.SDS-PAGE showed that the gene VP3 could expressas 31kD protein as same as thenatural protein,ELISA analysisof the culture supernatants confirmed that they had the desired immunogenicity of EV71.