EFFECT OF VL AND VH CONSENSUS SEQUENCE-SPECIFIC PRIMERS ON THE BINDING AND EXPRESSION OF A MINI- MOLECULE ANTIBODY DIRECTED TOWARDS HUMAN GASTRIC CANCER
- Author:
Jing LI
1
;
Yan WANG
;
Zhuozhi WANG
;
Zhiwei DONG
Author Information
1. Navy General Hospital
- Keywords:
gastric cancer;
Fab;
ScFv;
PCR
- From:
Chinese Medical Sciences Journal
2000;15(3):133-139
- CountryChina
- Language:Chinese
-
Abstract:
Objective. To construct ScFv and Fab from murine anti-gastric cancer monoclonal antibody( mAb)3H11. Methods. At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FRI. The bacterial expressed 3H11 Ab fragments showed no antigen binding activity. Then, phage antibody library andrandom mutated library were constructed from 3H1 1 hybridoma cells and panning selection was performed. Again the i-dentification of positive clone was failed. Finally the N-terminal sequences of V regions were resumed to 3H1 1 original sequences by site-directed mutagenesis via PCR. Results. Binding activity to gastric cancer cells was detected only from N-terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fiagments was not affected. Correction of either VL or VH N-terminal se-quences could partially resume the antigen binding activity. Conclusion. Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody. Received for publication Oct. 26,1998.
