Poster: Plant Pathogen/Symbiont Interactions

Abs # 761: Production of human GM-CSF protein using PVX vector on N. benthamiana plants

Presenter:	Zhou, Fengyong , fengyong123@yahoo.com
Authors	Zhou, Fengyong  (A)   Wang, Ming-Li  (A)   Albert, Henirik  (B)   Moore, Paul  (B)   Zhu, Judy  (A)
Affiliations:	(A): Hawaii Agriculture Research Center, Aiea, HI 96701 (B): USDA-ARS, Pacific Basin Agricultural Research Center, Aiea, HI 96701

Plant virus-based vectors can be used to introduce and express genes for protein production in plants. We are evaluating the potato virus X (PVX) as a viral vector for plant production of the granulocyte macrophage colony-stimulating factor (GM-CSF) protein, a clinically import human cytokine. Two constructs were made for GM-CSF gene based on pPVX201 vector. One construct contained PCR-amplified GM-CSF gene with a 6Xhis tag at the 3' end and the other contained the original GM-CSF cDNA. Leaves #5 & #6 of 4-week-old N. benthamiana plants were inoculated with plasmid DNA. Leaf samples were collected for analyses at 7 and 11 days after inoculation (DAI). Protein was extracted and analyzed with the DuoSet ELISA Development SystemŽ kit. Large differences in the level of GM-CSF were observed as functions of leaf position and DAI. On 7-DAI, Removal of leaves #7 & #8, immediately above inoculated leaves #5 & #6, decreased the amount of GM-CSF protein in leaves #9 - #11 by 47.9%. Alternatively, maintaining leaves #7 & #8 until 11-DAI, increased the amount of GM-CSF protein in leaves #9 - #11 by 159.5%. This result is compatible with the possibility that the leaves (#7 & #8) between those inoculated (#5 & #6) and the top (#9 - #11) leaves may be a source of viral RNA. The amount of GM-CSF protein in leaves #7 & #8 on 11-DAI declined 29.0% compared to 7-DAI. This effect of aging was confirmed with data showing that top young leaves accumulate more recombinant protein than lower leaves on the same plant.