Cloning and expression of single chain variable fragment of monoclonal antibody against norfloxacin in Pichia pastoris

Abstract

Currently, recombinant antibody technology has provided an alternative approach for engineering low-cost antibodies with desirable affinities and specificities. In this research, a single chain variable fragment (scFv) that recognizes norfloxacin antibiotic was successfully designed and constructed. The cDNA was synthesized from total RNA of antinorfloxacin-producing hybridoma clone (Nor155). DNA encoding VH and VL regions of this hybridoma were amplified and sequenced. DNA fragments of VH and VL regions were approximately 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by addition of (Gly4Ser)3 as a linker between VH and VL regions and subcloned into pPICZαA, an expression vector of Pichia pastoris, to generate pPICZαA-scFv (pJM01). After sequencing analysis, the scFv antibody gene cassette revealed an open reading frame of 1161 bp with the potential to encode a polypeptide of 386 amino acids and a predicted molecular weight of 42.5 kDa (intracellular) and 32.67 kDa (secreted), respectively. The pJM01 was incorporated into the Pichia chromosome at AOX1 promoter and transformants were confirmed by Southern blot analysis, PCR and sequencing analysis. Southern blot analysis was carried out in 9 transformants, which showed that the scFv gene was integrated into the Pichia chromosome. By performing the scFv expression in shaker culture, transformant O5 showed the highest scFv expression, among 9 transformants. The mRNA transcript level of transformant O5 showed highest relative normalized expression at 24 h, 688 times compared to the control. The recombinant scFv antibody was purified by immobilized metal affinity chromatography (IMAC). SDS-PAGE and Western blot revealed a predominant protein at 32.67 kDa of the purified scFv, thus indicating the scFv antibody was successfully expressed by P. pastoris. The specific binding and affinity in ELISA and surface plasmon resonance (SPR) indicated that the purified scFv still functional in term of antigen binding. From all results, it could be concluded that scFv of monoclonal antibody Nor155 could be produced in yeast. In addition, the binding ability of the obtained scFv to the corresponding antigen remains functional.