The construction of Pichia pastoris expression system with E gene of Japanese encephalitis virus

Objective:To construct the Pichia pastoris expression system including E gene segment of Japanese encephalitis virus(JEV).Methods:Amplifying the E gene of the SA-14 strain by RT-PCR,which encodes for E protein.Then the RT-PCR products were cloned into the pPICZαA vector after being cutted by two enzymes.After linerized by Pme Ⅰ enzyme,the recombinant plasmid was transformed into the competent cell of Pichia pastoris GS115 strain,and the transforments were screened with zeocin.Results:The RT-PCR product was about 1 350 bp.We compared the sequence of the E gene which inserted into the pPICZαA vector with the ones reported in Genbank,their similarity was 100%.After the recombinant plasmid pPICZαA-E was transformed into Pichia pastoris,the recombinant strain GS115-E was obtained,and their genome could be proved to be correct by PCR.Conclusion:We construct the Pichia pastoris expression system including JEV E gene successfully and lay a foundation for further reaserches.