The Roles Of The Single-chain Variable Fragment Specific For Human Acidic Fibroblast Growth Factor

Human acidic fibroblast growth factor(aFGF or FGF-1) is a member of the structurally related heparin-binding growth factor family,aFGF has a broad range of normal biological activities such as angiogenesis,wound healing,bone fracture healing,cell migration and cellular differentiation,aFGF can play also an important role in tumorgenesis and metastasis, indicating that aFGF could be a target for the cancer treatment.To explore the relationship between aFGF and tumors growth,a single-chain variable fragment(scFv) antibody specific for aFGF was generated.This scFv may block the activity of aFGF which leads to tumor cell growth inhibition.In order to study the activity of scFv,the cDNA of anti aFGF-scFv was subcloned into a mammalian expression vector pEGFP-C1 to fuse with GFP at the amino-terminus.In previous study we have found that the immunofluorescence results showed that the scFv could bind with endogenous aFGF as the parent monoclonal antibody and that the overexpression of scFv in cancer cells could make cells become smaller and rounder in MDA-MB-231 and HepG2 cells.In this study the analysis of Flow Cytometry(FCM),using 293T and MCF7 cells,revealed that the cell cycle was arrested at G1 phase due to the expression of scFv. These results showed that anti aFGF-scFv could inhibit the growth of the cells.Further,we report successful electro-gene therapy(EGT) by using plasmid DNA for tumor-bearing mice.Subcutaneously inoculated Lewis lung cancer(LLC) was subjected to EGT,which consists of intratumoral injection of a naked plasmid encoding a therapeutic gene, anti aFGF-scFv gene,followed by in vivo electroporation(EP).EGT,by using the anti aFGF-scFv gene significantly inhibited the growth of tumors.Based on these results,it appears that EGT can be used successfully for treating murine solid tumors and the anti aFGF-scFv antibody can inhibit the growth of the tumor cells overexpressing the aFGF.However,anti aFGF-scFv as a medicine might be harmful for the normal cells because the aFGF is not a tumor specific antigen.In other hand,we have found that the scFv interacts with its antigen in the cytoplasm but delivering proteins with therapeutic potential activity into cells is difficult because of their size and biochemical properties.Thus,it has been difficult to utilize such proteins as therapeutic drugs.Therefore,the therapeutic application of proteins could be achieved by the development of delivery vectors that are capable of the efficient delivery of various proteins into cells.In this work,we want to test a strategy for cancer targeting and intracellular delivery of anti aFGF-scFv by generating a fusion protein consisting of our protein of interest scFv,a protein translocation domain(Pseudomonas exotoxin A domainⅡ,PEⅡ) and a ligand which can bind with cancer cells(human epidermal growth factor receptor binding fragment,hEGFfr).Antibody engineering and advances in bacterial and yeast expression systems have enabled production of soluble and active heterologous proteins.However,the functional expression yields of these heterologous proteins vary widely.The results described in this thesis elucidate the factors influencing the expression of hEGFfr-PEⅡ-scFv fusion protein in Escherichia coli and in Pichia pastoris and the roles of this protein in cancer targeting and tumor growth inhibition.The recombinant gene(hEGFfr-PEⅡ-scFv) encoding for this fusion protein was cloned into a prokaryotic expression vector pET-28a(+) to fuse with 6 His*Tag at the amino-terminus.The recombinant plasmid was transformed into the E.coli BL21(DE3) cells.The expression of hEGFfr-PEⅡ-scFv was successfully induced by 1mM IPTG.The expressed hEGFfr-PEⅡ-scFv protein was purified by using the Ni-NTA His Bind Resins.But the E.coli expression system has led to the formation of inclusion bodies,so the fusion protein was refolded and we have successfully got a soluble active protein with a weak binding activity with its antigen and a fewer concentration so we have chosen the yeast expression system which offers several advantages and can solve the low expression and the formation of inclusion bodies problems found with E.coli expression system.For that,the fusion gene(6 His*Tag-hEGFfr-PEⅡ-scFv) derived from the pET-28a(+) recombinant plasmid was cloned into a P.pastoris expression vector pPIC9K to fuse withα-factor,a secretion signal.This part of work is going on and will be completed by further studies.These works will make some foundation for studying the functions of the anti aFGF-scFv antibody.