Aim: To screen the antibodies against transferrin receptor(TfR) from a full humanized phage antibody library.
Methods: Total RNA was extracted from HeLa cells and then the extracellular domain of TfR gene was amplified by RT-PCR in two parts. A full-length gene of extracellular domain was obtained by two rounds of PCR. The gene was inserted into the plasmid pPROEX(TM) HTa and transformed into E.coli BL21(DE3). Transformant was expressed under 1 mmol/L IPTG induction. The target protein was purified and refolded by one-step affinity chromatography with Ni(+)-NTA agarose. The antibodies against TfR were screened by proteins coupled to immunotubes from a whole-hog humanized phage single chain antibody library with sink size of 10(13) cfu/L.
Results: The extracellular domain of TfR gene with about 1.9 kb was obtained. DNA sequencing of TfR gene proved to be identical to the published sequence. Eight single chain antibodies specifically binding to human TfR have been screened.
Conclusion: Antibodies specifically binding to natural TfR can be obtained by screening the library with refolded protein, which lays the foundation for treatment of tumor and central nervous system diseases.
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