High Level Expression And Biological Assay Of HrpZ In Escherichia Coli

HrpZ is a proteinaceous bacterial elitor from Pseudomonas syringae pv. Syringae, inducing a hypersensitive response (HR) in non-host plants, Characterized by the rapid, localized death of plant cells at the site of pathogen invasion. The HR can prevent further multiplication and restrict the spread of the pathogen. It was found that activation of other defencerelated responses often accompanies HR, for example, the oxidative burst, the production of antimicrobial compounds, and enzymes involved in the general phenylpropanoid pathway. The non-localized and long term induced protection, is similar to systematic acquired resistance(SAR).Primers were designed according to the features of expression vector pET32b(+)and ORF sequence of hrpZ encoding harpin protein. The hrpZ gene was amplified by PCR and inserted into pGEM-T vector. And then the recombinant plasmid was subconed into JM109 to amplified. The hrpZ fragment was digested with EcoR I /Xho I and cloned into the expression vector pET32b(+) of E.coli locating downstream of Trx.Tag. Then expression vector pET-hrpZ was constructed and transformed into host cell,BL21(DE3). The fusion hrpZ protein was expressed in recombinant BL21(DE3) with IPTG inducing. SDS-PAGE indicated that the hrpZ fusion protein was obviously highly expressed in BL21(DE3) and was soluble protein. The MR was 55.8KD, which was consistent with theoretical calculation. The result of bioassay showed that the fusion protein had the ability of inducing hypersensitiveresponse(HR) on tobacco by using punctures in laminae respectively .