Aim: To express secretively chimeric Fab antibody HAb18 (cFab) against human hepatocellular carcinoma in Pichia pastoris.
Methods: Genes encoding CL chain and Fd fragment of cFab antibody HAb18 were subcloned into vectors pPIC9K and pPICZalphaA, respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/CL and pPICZalphaA/Fd were transformed into the genome of Pichia pastoris GS115. Mut(+) multiple insert transformants were screened by G418 and Zeocin and then induced with 5 mL/L methanol to express cFab.
Results: 4 days after methanol induction, 26 mg/L of the cFab fragment was detected in the culture supernatant. Western blot proved that the expressed protein could specifically bind with HAb18GEF antigen.
Conclusion: The successful expression of cFab/HAb18 in Pichia pastoris lays the foundation for large-scale production and further application of the antibody.