Expression Of A Pokeweed Antiviral Protein Gene (PAP) In Pichia Pastoris

Plant virus disease is one primary crop disease. It distributes wildly and does great harm to economy of agriculture yield. People search after ways to prevention and cure virus diseases for long time but not develop effective reagent yet. Many studies revealed that pokeweed antiviral protein (PAP) can control plant virus effectively. But PAP in nature is limited which could not apply cosmically. Using bioreacter to produce PAP is one efficacious way to get a mass of PAP. Someone tried this way but the yield is low. Francis expressed truncated mutant pokeweed antiviral protein gene in Pichia pastoris. The yield reached 10-12mg/L. Dr. Chen ding hu in plant virus group of Institute of plant protection of the CAAS (Chinese academy of agriculture sciences) selected PAP Muts recombinants and wanted to produce PAP through fermentation to prevention and cure plant virus disease. But as to Muts recombinants, the periods of fermentation is too long and the efficiency of using methanol is low. So using Muts recombinants to produce PAP in factory is not felicitousness.Linearized the expression vector pPIC9K-P including the truncated mutant pokeweed antiviral protein (PAP) gene by restriction endonuclease Sal I and transformed it electrically into Pichia pastoris GS115.Mut+recombinants were selected by PCR and high yield Mut+ recombinant was picked out by double film immunoblotting.To find out the difference between Mut+ and Muts recombinants we compared their expression of pokeweed antiviral protein in the same conditions. SDS-PAGE results showed that there was a clear target protein band in Mut+ recombinant supernatant after 48 hours of culturing, while a faint band only in Muts recombinant after 72 hours. Western-blotting result showed that there was no remarkable difference of yield between Mut+ and Muts recombinants after 6 days induced. Anti-virus activity tests revealed that culture supernatants of Mut+ and Muts recombinants could inhibit TMV infection with high efficiency in the same concentration and there was no significant difference between them.Different cultivation condition such as induced time, methanol concentration and pH of culture medium was studied at shake flask cultivation. SDS-PAGE results showed that as to Mut+ recombinant highest yield was obtained after 4 days inducing and with the culturetime prolonged it reduced. Pokeweed antiviral protein gene expressed well when methanol concentration reached 10g/L. Pokeweed antiviral protein obtained high yield in thin acidic culture medium (pH6.0-6.4) and its quantity in total mass of secrete protein exceeded 30%.