Construction of Pichia pastoris expression system of G1 glycoprotein of hantavirus

Objective To construct the Pichia pastoris expression system of G1 glycoprotein of hantaan virus and Seoul virus.Methods The gene of the Z10 strain and L99 strain were amplified by RT-PCR,which encoded for Glycoproterin G1.Then the RT-PCR products were cloned into the pMD18-T simple vector.After identifing by sequence analysis,the heterologous gene in the recombinant plasmid was cutted by two enzymes and inserted into pPICZαA respectively.After linerized by BstX I enzyme,the recombinant plasmid was transformed into the competent cell of Pichia pastoris-X33,and the multi-copy transformants was screened with Zeocin.Results The Z10 strain RT-PCR product was about 1 937 bp,and the L99 strain RT-PCR product was about 1 991 bp.The sequence of the gene which inserted into the pMD18-T simple vector were compared with the ones reported in Genbank,their similarity was 99.5% and 99.7% respectively.Their target protein's second structure was not changed.After the recombinant plasmid pPICZαA-Z10G1 and pPICZαA-L99G1 were transformed into Pichia pastoris,the recombinant strain Ppx33-Z10G1 and Ppx33-L99G1 were obtained,and their genome could be proved to be correct by PCR.Conclusion The Pichia pastoris expression system of hantavirus G1 glycoprotein has been constructed successfully in the present study.