Expression of VP2 of Enterovirus 71 in Pichia.pastoris

The genes encoding the capsid protein VP2 were amplified by RT-PCR.After confirmed by DNA sequence analysis,the VP2 was inserted into the Pichia.pastoris expression vector pPIC9K.The sequence of ORF of α-factor and VP2 were confirmed by DNA sequencing with primer 5′-AOX1 and 3′-AOX1.Then the pPIC9K/VP2 were transformed into yeast GS115 by the of electro-transformation.After the PCR analysis and the screening for Mut+ phenotype of the transformants,multiple inserted transformants were fermented in flasks.By induction with methanol(96 h),recombinant protein could be effectively secreted into media.SDS-PAGE analysis showed that the gene VP2 could express as 30 kDa protein as the same as the natural protein.Western Blot analysis of EV71 VP2 protein expression in Pichia.pastoris demonstrated that they had the desired immunogenicity of EV71.