The Expression Of Anti-aFGF ScFv Gene Fragment In Tobacco

Acidic fibroblast growth factor is a member of FGF family,which plays substantial roles in development, angiogenesis, hematopoiesis and tumorigenesis. It is over expressed in many cancer tissues according to recent research reports. Obviously, it is proved that there is a relationship between aFGF and the development and metastatic of cancer. The variable regions of light chain (VL) and heavy chain (VH) were cloned from an aFGF specific hybridoma and assembled into single chain variable fragment (scFv) with a flexible linker, which aims to produce the targeting therapy antibody medicine specific for aFGF. Due to the protein refolding in the downstream of recombinant protein expressed in E.coli system, here we report that the scFv was expressed in Nicotiana Benthmiana with plant virus-based vector (p35S-30B), which was low cost, convenient and efficient.p35S-30B is a tobacco virus vector which can efficiently transfer a portion of the plasmid (T-DNA) to plant cells near a wound area, and then the whole plant was infected. By inserting the scFv gene into the cloning site of p35S-30B, the recombinant plasmid p35S-30B-scFv was constructed. The recombinant plasmid was then introduced into Agrobacterium tumefaciens strains EHA105 by the freeze-thaw method. A. tumefaciens EHA105 harboring p35S-30B-scFv was used to infect the tobacco plants afterwards. In order to test the Agroinoculation method conveniently, GFP was used as a reporter gene. GFP expression in tobacco plants was observed by UV light (model B 100AP 365nm) each day in 25 days. In this study, the foreign proteins were accumulated at the highest level on the 8th day of post-inoculation. The scFv was expressed under the same condition. The plant total soluble proteins were collected from the leaves on the 10th day of post-inoculation using phosphate buffer (pH 7.4). The yield of scFv was reached about 0.5% of the plant total soluble proteins by ELISA assays.The anti-aFGF scFv was expressed by plant virus vector, which provides a helpful information for the scFv production in plant-bioreactor for the first time.