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| A Secreted Expression Vector Construction of Pokeweed Antiviral Protein Gene and Its Expression in Pachia pastoris |
| Chen Dinghu Wang Xifeng Li Li Zhou Guanghe** |
| (State Key Laboratory of Biology of Plant Disease and Pest, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, China) |
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Abstract Abstract：The total RNA was isolated from pokeweed (Phytolacca americana ) leaves using the method of guanidine isothiocyante and used as template for amplification of the deleted mutant pokeweed antiviral protein (PAP ) gene by RT-PCR and then the amplified fragment was cloned into secreted expression pPIC9K vector to form pPIC9K-p, then the vector was transferred into Pachia pastoris GS115 strain. The specific expression protein was secreted into the medium after inducing with methanol and the protein amount reached about 50~60μg /mL measured by UV-absorbed methods in the supernatant of the medium via high density fermentation. SDS-PAGE results showed that there was one main protein band with molecular weight about 34 kD in the fermentation supernatant and it could specifically react with France PAP antiserum in Western blotting. Activity tests revealed that this protein could inhibit plant virus infection with high efficiency.
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Received: 01 January 1900
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