Prokaryotic expression of pathogenesis related protein 1 gene from Nicotiana benthamiana: antifungal activity and preparation of its polyclonal antibody.

The nucleotide sequence of the pathogenesis-related protein 1(PR-1) gene was obtained from Nicotiana benthamiana using RT-PCR. Restriction enzyme cutting sites of EcoRI and NotI were introduced to the ORF fragments of PR-1, they were then linked together with pET-30a (+) and transformed into E. coli BL21 (DE3). The target protein was induced by 1.5 mM IPTG, at 37°C for 4 h. The expressed protein was purified by Ni-NTA and an anti-NbPR-1 polyclonal antibody was prepared using rabbits. The antibody could detect the expression of PR-1 in N. benthamiana and other Nicotiana plants. NbPR-1 protein has four α-helices and two β-sheets by homology modeling. Furthermore, the purified NbPR-1 protein exhibited a broad-spectrum antifungal activity.