Construction of a natural phage antibody library of human anaplastic thyroid carcinoma.
The objective of this study was to identify and construct a human natural phage single-chain antibody (scFv) library of human anaplastic thyroid carcinoma (ATC) using phage display technology. Total RNA was extracted from lymphatic tissue near an ATC and used to amplify variable heavy chain (VH) and variable light chain (VL) fragments with added linker sequences using reverse transcription-polymerase chain reaction (RT-PCR). After purification, the VH and VL amplicons were used to produce scFv fragments with added SfiI and NotI restriction enzyme recognition sites using splicing-overlap-extension PCR. Following digestion, the scFv gene was cloned in the pCANTAB-5E plasmid, and the recombinant phagemids were transformed into the susceptible Escherichia coli TG1 strain. After infection by the helper phage M13K07, a human ATC phage antibody library was successfully constructed. Clear 28 S and 18 S bands could be seen in the total RNA from the library, and the sizes of the VH, VL, and scFv genes contained therein were approximately 370, 350, and 750 bp, respectively. In addition, the conversion efficiency as measured by the pUC19 standard plasmid was 10(8) CFU/μg, and the positive insert ratio was 86.4% (19/22). These results demonstrated the successful construction of a human ATC scFv antibody gene library, and might provide the experimental basis for the further screening and identification of a phage single-chain antibody with ATC cell-specificity.
Department of Nuclear Medicine, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China.
J M
JM
Xi
Department of Nuclear Medicine, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China.
H
H
Pang
Department of Nuclear Medicine, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China.
X L
XL
Hu
Department of Nuclear Medicine, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China.
Z J
ZJ
Wang
D001435:Bacteriophages / Q000235:genetics; D061505:Cell Surface Display Techniques; D004926:Escherichia coli; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology*; D008221:Lymphoid Tissue / Q000276:immunology; D012313:RNA / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology; D065646:Thyroid Carcinoma, Anaplastic / Q000235:genetics / Q000276:immunology* / Q000473:pathology
D007143:Immunoglobulin Heavy Chains; D057127:Single-Chain Antibodies; D012313:RNA