Expression, Purification And Characterization Of Human ScFv And Diabody Against BFGF In Pichia Pastoris

To construct and high level express the human anti-bFGF ScFv and Diabody inPichia pastoris and study theirs biological functions.The gene of human anti-bFGFScFv was subcloned from plasmid pCOM3X-1A/ScFv, the variable light (VL) andheavy (VH) genes were respectively cloned from ScFv, The V genes were fused byoverlap PCR to produce Diabody fragments with a linker of five amino acids (G4S),The genes of ScFv and Diabody were respectively constructed into expression vectorpPICZαA. The constructed expression vector pPICZαA-ScFv and pPICZαA-Diabodywere linearised and transformed to Pichia pastoris by electroporation. Thetransformants were induced by methanol, and the anti-bFGF ScFv and Diabody wereexpressed. The expression products were purified by affinity chromatography of Ni-Seproase6FF and ion exchange chromatography of DEAE Sepharose FF. The resultsof SDS-PAGE and Western blotting showed that anti-bFGF ScFv and Diabody werehigh level expressed successfully, The target protein was purified from the expressionproducts and the purity was more than95%. The results of ELISA showed that thepurified recombinant ScFv and Diabody could combine with bFGF specifically. Theresults of CCK8showed that the purified recombinant ScFv and Diabody couldinhibit the proliferation of A549cells in a dose-dependent manner in vitro. The resultsof Annexin V/PI and Hoechst33258showed that Diabody can induce the apoptosis ofA549obviously. The results demonstrated that the anti-bFGF ScFv and Diabody canbe high level expressed in Pichia pastoris with good biological activity.