Single domain antibody to human telomerase catalytic subunit: preparation and characterization.
OBJECTIVE
To develop a recombinant single domain antibody against hTERT, human telomerase catalytic subunit.


METHODS
A previously prepared His-tagged hTERT fusion protein was used as the antigen, and the variable regions in heavy chain (VH) of immunized mice were RT-PCR amplified and cloned into the pCANTAB 5E, a phagemid vector. By transfection, the display library of mouse VH was developed. The candidate clones were selected by affinity panning, and soluble VH were obtained after expression in E. coli, HB2151. The resultant single VH antibodies were characterized on their binding potentials by western blotting.


RESULTS
An about 350 bp VH fragment was amplified from spleen cells of mice immunized by His-tagged hTERT and expressed by phage displayed as VH library. The size of the library was 8 x 10(4). After three rounds of affinity panning, 4 independent clones were chosen and consequently expressed as soluble single domain antibodies (Mr = 16 000). In Western blot analysis, the single domain antibody from 2 of 4 clones proved to react with the His-tagged hTERT fusion protein (Mr = 167 000) without dependence of His-tags and also detect the native hTERT (Mr = 127 000) extracted from the human HeLa cancer cell line. DNA sequencing showed both of the single domain antibodies were encoded by the heavy chain variable region of the mouse.


CONCLUSIONS
The single domain antibodies developed were hTERT recognizable and hTERT specific, thus providing a basis for application of recombinant single domain antibody in inhibition of telomerase activity and anticancer therapy.
Zhang
Hui
H
Department of Pathology, The Health Science Center of Peking University, Beijing 100083, China. zhangbo@bjmu.edu.cn
Zhang
Bo
B
Wang
Junmei
J
Liu
Cheng
C
Han
Jisheng
J
Yang
Shaomin
S
Hou
Lin
L
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000235:genetics; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D019098:Telomerase / Q000276:immunology*
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019098:Telomerase