| Objective：Using phage display technology,constructing the naturalhuman medullary thyroid carcinoma of phage antibody library.Methods：Total RNA was extracted from the lymphatic tissue near themedullary thyroid carcinoma.VH and VL fragments were amplified withRT-PCR.Using them as templates to amplify VH-Linker and VL-Linker,then restricted enzyme cutting sites sfi I and Not I after scFv genefragments was assembled with splicing-overlap-extensionPCR(SOE-PCR).The scFv gene was cloned in pCANTAB-5E Phasmid,then transferred to Ecoli TG1. After the helper phage M13K07infection, ahuman phage antibody library was constructed.The insertion ratio of scFvgene was identified with PCR, the double enzyme digestion products ofpositive clones were analyzed with1.0%agarose gel electrophoresis.Result：Clear28S and18S strips were found in total RNA, The sizeof VH gene is about370bp, VL gene is about350bp,the size of scFv geneis about750bp. Determination of conversion efficiency reached108cfu/μgwith pUC19standard plasmid, and the positive insert ratio was87.5%(21/24).Conclusion： Human medullary thyroid carcinoma phage display library was constructed successfully. It could provide experimental basisfor further screening of people with medullary thyroid cancer cell-specificphage single-chain antibody. |
