The Expression,Purification And Activity Detection Of FGF-1-hscFv

From murine antibody,chimeric antibody,humanized antibody and human antibody,scientist managed to reduce the murine and human anti-mouse antibody(HAMA),which facilitates the clinical use of antibody.Single-chain fragment variable(sc Fv)is the smallest immunoglobulin molecule with full antigen-binding activities.sc Fv only consists by the variable regions of a heavy(V_H)chain and a light(V_L)chain,which are joined together by a flexible peptide linker.Due to the small size and specific targeting property,sc Fv is proposed to be a potential biological material for cancer targeted therapy,such as directed therapy,gene therapy,tumor imaging analysis and so on.Fibroblast growth factor 1(FGF-1),a member of FGF family,plays a variety of biological functions.Research evidence showed FGF-1 is overexpressed in several kinds of tumors and is related to tumorigenesis and metastasis.In consideration the important biological function of FGF-1 in tumorigenesis,anti-FGF-1 sc Fv(sc Fv1C9)was constructed and verified to have antineoplastic activity in our previous work.On this basis,we further humanized anti-FGF-1 sc Fv(sc Fv1C9),named FGF-1-hsc Fv.In this thesis,we optimized the expression conditions of p ET22b-hsc Fv engineered bacteria.Resaults showed the best inducing consitions are 0.2m M IPTG at 28℃for 6h.Then,we did a lab scale fermentation to express FGF-1-hsc Fv.Protein product was purified by affinity chromatography.Coomassie brilliant blue staining and Western blot proved a successful expression of soluble FGF-1-hsc Fv.Using purified FGF-1-hsc Fv,we confirmed its antigen-binding activity to FGF-1 by an indirect ELISA.Morever,we verified the antineoplastic activity of FGF-1-hsc Fv on MDA-MB-231 human breast cancer cell line.Flow cytometry analysis also showed that FGF-1-hsc Fv induced G1 arrest.FGF-1-msc Fv was used as positive control in all aforementioned experiments.Decrease of biological activity in our humanized antibody was not detected.To obtain higher yield,we performed pilot scale fermentation.Finally,in order to investigate the therapeutic efficacy of FGF-1-hsc Fv in vivo,we established tumor models in nude mice and carried out a preliminary experiment with 25mg/kg,50mg/kg FGF-1-hsc Fv.Results from this thesis offer a new cancer treatment targeting FGF-1,and also increase the reservoir of sc Fv in order to meet more demand for cancer treatment.