Construction of human medullary thyroid carcinoma phage antibody library and preliminary identification

Abstract:

Objective：To construct natural human medullary thyroid carcinoma phage antibody library using phage display technology. Methods：Total RNA was extracted from the lymphatic tissue around medullary thyroid carcinoma. VH and VL fragments were amplified with RT-PCR，which were used as templates to amplify VH-Linker and VL-Linker. Then restricted enzyme cutting sites sfi Ⅰ and NotⅠ after single chain fragment variable（scFv） gene fragments was assembled with splicing-overlap-extension PCR. The scFv gene was cloned in pCANTAB-5E plasmid then transferred to Ecoli TG1. After the helper phage M13K07 infection，a human phage antibody li－brary was constructed. The insertion ratio of scFv gene was identified with PCR and the double enzyme digestion products of positive clones were analyzed with 1.0% agarose gel electrophoresis. Results：Clear 28 S and 18 S strips were found in total RNA；size of VH gene was about 370 bp，of VL gene was about 350 bp and of scFv gene was about 750 bp. Determination of conversion efficiency reached 108 cfu/μg with pUC19 standard plasmid and the positive insert ratio was 87.5%（21/24）. Conclusions：Human medullary thyroid carcinoma phage display library is constructed successfully. It could provide experimental basis for further screening of people with medullary thyroid cancer cell-specific phage single-chain antibody.