[Cloning and expression of heavy chain variable region genes against telomerase protein hTERT].
Single domain antibodies against telomerase protein hTERT were prepared by technique of displayed on the surface of recombinant bacterio phages. Total RNA of spleen lymphocytes were extracted from mice immunized recombinant hTERT and transcripted to cDNA. First-strand cDNA was used as a template, heavy chain variable region genes against hTERT were amplified with VHfor and VHback primers by PCR technique. Amplification reaction yielded a fragment about 350 base pairs in length. Amplified cDNA were cloned into the vehide of bacteriophage PCANTAB 5E, the phagemid containing VH gene transformed into competent E. coli TG1, in the presence of helper phage M13K07, VH-g3 fusion proteins were display on the surface of recombinant phages. The phage carrying VH genes that encode binding activities could be detected directly with DOT BLOT. Single domain antibodies were generated successfully and had binding activities with hTERT. Results suggest that phage display technique be a new way of making antibodies. VH genes were cloned successfully, which could provide possibility for futher preparing single-chain antibodies(ScFv) anti-hTERT.
Zhang
Hui
H
Department of Pathology, Chongqing University of Medical Sciences, Chongqing 400016.
Zhang
Bo
B
Han
Jisheng
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D005260:Female; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D019098:Telomerase / Q000276:immunology*
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019098:Telomerase