The Transient Expression Of Anti-aFGF ScFv Gene Fragment In Tobacco And Pea

Single chain variable fragment(sc Fv) is a kind of small molecular engineered antibody by synthesized using the technology of recombinant DNA technology and protein engineering. Recently sc Fv was applied widely in the diagnosis and treatment of tumors. The anti-a FGF sc Fv was expressed in Nicotiana Benthmiana and Pisum sativum L. with the plant virus-based vector, which was a safe, efficient and large-scale expression syetem.The sc Fv was expressed in Nicotiana Benthmiana with TMV-based vector(p35S-30B), vertfied the feasibility of expression of sc Fv in plants. Then we established the transient expression system in pea by p CAPE1 and p CAPE2-GFP, and expressed sc Fv through injection to search more suitable plants in transient expression. A large-scale expression system was established in pea sprouts, and purification and characterization of the scfv using the expression system.The main results of this study are summarized as follows:(1) The p35S-30B-GFP vector was used to establish the transient expression system in injected tobacco plants. The GFP were accumulated at the highest level on the 8-10 th day of post-inoculation. The EHA105 harboring p35S-30B-sc Fv was used to infect tobacco plants, the transient expression of sc Fv has a strong antigen-binding capacity.(2) The p CAPE1 and p CAPE2-GFP were used to establish the transient expression system in injected pea plants, and the GFP were accumulated at the highest level on the 10-12 th day of post-inoculation. The p CAPE2-sc Fv and p CAPE2-GFP-sc Fv expression vector were built up, and to infect the pea by injecting. The expression of sc Fv gene in RNA and protein level, and ELISA showed that sc Fv and GFP-sc Fv had a fair antigen-binding capacity.(3) A large scale transient expression system of foreign protein by using vacuum infestation technology was established in pea sprouts, and the expression level of GFP up to 324 mg/kg.FW(The soluble protein content of plants account for 1.2%).(4) Anti-a FGF single chain antibody was expressed in pea sprouts, about 30 k Da sc Fv protein was obtained by Ni- affinity chromatography column, the dilution of the sc Fv protein had a fair antigen-binding capacity.In this study, we firstly expressed anti-a FGF sc Fv using plant virus vector in tobacco and pea plants. A safe, efficient foreign protein expression system was established in the pea sprouts using vacuum system.