Construction of Pichia pastoris expression vector for production of scFv-Fc fusion antibody against 40 000 adipocyte-specific plasma membrane protein

The Fc fragment DNA sequence of the immune gamma globulins (IgG1) was amplified from the human peripheral blood lymphocyte by reverse transcriptase-polymerase chain reaction (RT-PCR). The nucleotide sequence analysis revealed that this 699 bp DNA fragment possessed the complete hinge area, CH2 and CH3 structural region, and showed 100% identity to the Fc fragment DNA sequence of human IgG1 heavy chain which had been deposited in the GenBank database. Subsequently this Fc fragment DNA was inserted into pPICZαA vector of Pichia pastoris to construct the pPIgG1 plasmid. Next, scFv antibody gene of Pichia pastoris whose protein product was against the 40 000 adipocyte-specific plasma membrane protein was inserted into the pPIgG1 plasmid and constructed the pPIgG1-scFv Pichia pastoris expression vector. Validation analysis showed that ligation direction of the scFv gene of Pichia pastoris and the Fc fragment DNA was correct and was in agreement with the correct reading frame of scFv-Fc. These results suggested that the scFv-Fc Pichia pastoris expression vector had been successfully constructed and this established the primary foundation to the express a great quantity of scFv-Fc fusion antibody protein which had the similar structure to IgG and also possessed the desirable affinity and bioactivity through this vector.