Expression Of A RNA Silencing Virus Suppressor P25 Protein In Eschcrichia Coli And Preparation Of Polyclonal Antibody Against It

The 25 kDa movement protein (P25) of potato virus X (PVX),a suppressor ofposttranscriptional gene silencing, which can help PVX to succeed in infecting plant, wasessential for the cell-to-cell movement of PVX in its host. Protein P25 was expressed byprokaryotic expression system, purified in vitro, and be used as antigen for making itspolyclonal antibodies. The p25 gene from the plasmid p25, was inserted into the pET-28a (+)vector between the site of BamH Ⅰ and Sac Ⅰ , to create a recombinant plasmidpET-28a(+)-p25. Then the recombinant pET-28a (+)-p25 was confirmed by PCR, restrictionenzyme digestion and DNA sequencing,showing that the p25 gene was fused with 6×Histag and expressed as fusion protein. The recombinant plasmid was transferred into thecompetent BL21 (DE3) cells and approximately a 28.6 kDa fusion protein was induced by theaddition of isopropyl-β-D -thiogalactoside (IPTG) (1.0 mM) during cultivation. The solublefusion protein P25 was purified after expending cultivation by Ni-NTA affinitychromatography under the denaturing condition. The concentration of the fusion protein P25was about 180 μg /ml determined by Bradford assays. The purified fusion protein P25 wasinjected into three 6 to 8 weeks old BALB/c mice (15 μg/mouse, time) three times to makepolyclonal antibody. The antibody titration was up to 1:12000 determined by ELISA assays.Western hybridization of antibody and P25 showed a single band in the nitrocellulose (NC)membrane. These results showed that we have obtained the P25 protein by in vitro expressionsystem and the expressed P25 showed the expected antigenicity and immunogenicity. Thepolyclonal antibody against the RNA silencing suppressor (P25) has been obtained, and willbe used in the research of the mechanism of gene silencing.