The Humanization Of FGF-1-mscFv And Its Soluble Expression

Single chain variable fragment (scFv) is a kind of micromolecule geneticallyengineered antibody which has many advantages such as low hetrotypic effect, smallmolecular mass, strong penetrating power and short cut synthetic method. Therefore,we cloned the variable regions of the light chain (VL) and heavy chain (VH) from aFGF-1specific hybridoma and assembled into single chain variable fragment with alinker peptide. But there were many papers have reported that scFv of murine origincan induce a kind of human anti-mouse antibody reaction (HAMA reaction) in thehuman body which will not only reduce the efficacy of the antibody drug but alsocause allergic reactions. So we designed a humanization plan to transformed theamino acid sequence of anti-FGF-1-mscFv into human origin. First, we found thehighest homology amino acid sequence of the murine scFv in the database of humanantibody to find best fit FR (Framework region) template. We then analysised theCDR (complementary-determining region) of the heavy and light chain according tothe kabat principles. Through a fixed position method, we analyzed the residues at theVL/VH domain interface, specific residues, vernier residues, Canonical residueswhich may sustain the construction of CDR. Using the principle of the homologymodeling we got the mscFv structure modeling of the three-dimensional space andanalyzed the embedded residues, the antigen-binding active sites, as well as the CDRneighboring residues in the spatial structure. The above mentioned residues mightplay a role in maintaining affinity of the antigen-binding activity of the parentantibody, so they should be retained in the humanized sequence. Another analysis ofthe surface residues in the spatial structure of mscFv was made to reduce the HAMAreaction of humanized antibody, and these residues should be replaced in thehumanized sequence. We obtained the amino acid sequence of the hscFv (humanizedscFv) from the CDR sequence of the murine scFv, the human FR template and the FRamino acid residues which should change back to the murine’s. Inorder to express thehscFv in Escherichia coli, we did an optimization of the codes of hscFv. Then weobtained the pET22b-hscFv vector through synthetizing and subcloning the optimizednucleotide sequence of hscFv into the soluble expression pET22b vector. We alsoacquired the recombinant plasmid pET22b-mscFv from the pET28a-mscFv throughthe same work. The E.coli produced soluble mscFv and hscFv in the periplasmthrough optimization of strains, vectors, temperature, culture medium, and inducingagent concentration. The immunoblotting experiment showed that the protein whichhas a molecular weight of about30kd contains the fusion His-tag. The ELISAexperiment shows that hscFv protein produced in vitro remains the binding activitywith FGF-1protein. In summary, these results show the potential application ofanti-FGF-1hscFv to study its function in euaryotic cells.