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Construction of ouabain single chain Fv phage antibody library by phage display

AIM To construct a ouabain ScFv(single chain fragment variable) antibody library by phage display. METHODS Mice were immunized by OUA OVA(ouabain ovalbumin). mRNA was isolated from the spleen lymphocyte and reversely transcripted. The heavy chain and kappa light chain variable region genes (V H and V L) repertoirs of immunoglobin were amplified individually and assembled into ScFv by a linker with overlap extension PCR. ScFv was reamplified with RS primer, meantime S fi I and NotI restriction sites were added to the ScFv different ends. ScFv was digested by S fi I and NotI, and cloned into the phagemid pCANTAB 5E and introduced into E.coli TG1 by electroporation. Phagemid containing bacterial colonies were infected with M13KO7 and the ScFv fusion protein was displayed on the surface of recombinant phage. RESULTS Titer of rat serum was 1.2×10 6 after being immunized with OUA OVA. The V H, V L fragment was 340 bp and 325 bp, respectively. After assembly, the ScFv was about 720 bp. The library capacity was 1.2×10 8. Ten clones of twelve clones contained 2.1 kb fragment after EcoRI and Hin dⅢ digestion. Transfection test indicated that the titer of the culture supernatant was about 9×10 14 pfu·L -1 . CONCLUSION A phage display library of ouabain ScFv antibody has been constructed successfully.