[Expression, purification and activity analysis of human single-chain antibody against hepatocellular cancer]

Abstract

Aim: To express and characterize an active form of a single-chain antibody (scFv) from the gene of human phage antibodies which is specific for hepatocellular cancer.

Methods: The complementary DNAs encoding the variable regions of the light chain (V(L)) and heavy chain (V(H)) were connected by a (Gly(4)Ser)(3) linker using a splicing by overlap extension polymerase chain reaction. The resultant scFv gene was cloned to the pET28a(+) vector and expressed in E.coli as inclusion bodies. Then the inclusion bodies were solubilized, denatured and renatured. Finally, the affinity constant of scFv was determined by noncompetitive enzyme immunoassay.

Results: The target protein amounted 26% of the total protein in the condition of A(600) at 0.8 for 6 hours. After renatured, the purity of target protein was 95% and the affinity constant of the scFv was 3.6x10(7) mol/L.

Conclusion: An active form of scFv which is specific for hepatocellular cancer can bind selectively with hepatocellular cancer cells, which provides a theoretical basis for immunological detection and clinical use of scFv.