Expression And Biological Assay Of HrpN In Pichia Pastoris

Primers were designed according to the features of expression vector pPIC6αA of Pichia pastoris and ORF sequence of hrpN encoding harpin protein as templete. Hrp gene was obtained from pET-hrpN plasmid by PCR, then cloned into vector pUCm-T. pUCm-T-hrpN plasmid was sequenced and cut by Xba I and EcoR I. The hrp DNA fragement obtained by cutting was cloned into expression vector pPIC6aA of Pichia pastoris and located on the downstream of AOX1 promoter and α-factor secretion signal. The recombinant expression plasmid pPIC6αA-hrpN was constructed. It was linearized by Sac I and then transformed into X-33 Pichia strain by electroporation. After seleted by Blasticidin and identified by PCR, the steady engineered Pichia pastoris capable of secreting harpin_Ea protein into the medium was obtained. Concentration, initially purifying and SDS-PAGE analysis of the proteins in the culture supernatant of the methanol induced recombinant P.pastoris harboring gene fragement of hrpN indicated that the protein as long as harpin was secreted obviously by methanol induced X-33(pPIC6αA-hrpN) for 96 hours. The results of bioassay showed that the proteinhad the ability of eliciting hypersensitive response in tobacco.