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


CONCLUSIONS
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.
Zhao
Qing-tao
QT
Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Ma
Long-yang
LY
Xue
Guo-zhu
GZ
Zhao
Ai-zhi
AZ
Dou
Ke-feng
KF
D000912:Antibodies, Neoplasm / Q000235:genetics / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000276:immunology*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism
D000912:Antibodies, Neoplasm; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region