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

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.