| Summary: | 碩士 === 國立陽明大學 === 微生物暨免疫學研究所 === 88 === A single-chain Fv (scFv) is composed of variable domains of heavy chain and light chain tethered together with an artificial polypeptide-linker. Because of several unique properties such as small size, being easy to engineer, and high stability at low concentration, scFvs may be useful in diagnosis, therapy, and research. It has been possible to express scFv antibody on the surface of M13 so that this approach becomes an alternative to hybridoma in generating antibody with high specificity.
To set up a phage-display system to display scFv on surface of M13, we used previously established hybridomas as models. We extracted mRNA of hybridoma HP6A1 and SC1D7 and prepared cDNA of variable heavy chains (VH) and variable light chains (VL). We linked cDNA of VH and VL together and constructed 6A1 scFv of HP6A1. This scFv cDNA is inserted into pMal-c2, to express an MBP (maltose-binding protein)-6A1 scFv fusion protein. We found that this MBP-6A1 scFv binds to Pin25L (the parental antigen) more specifically than Pin24L (an antigen variant) on an ELISA basis assay. We also inserted the scFv cDNA into phagemid pCGMT and transformed the plasmid into E. coli HB2151, to see the expression of scFv. HP6A1 scFv was found insoluble and aggregated in inclusion bodies. We then incorporated the skp chaperone and hoped that Skp could improve the solubility of scFv. No apparent effect was observed with the properties of the expressed HP6A1 scFv.
Changing bacterial strains may alter the expression pattern of recombinant scFv. Skp really improve the expression of HP6A1 scFv-gene III fusion protein in JM109. When recombinant phage was prepared from TG1, the phages did recognize Pin25L (the parental antigen) better than the variant. However, coexpression of Skp in the same bacteria neither enhances the phage binding toward the specific antigen.
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