Construction, Selection And Expression Of The ScFv Binding Specifically To Hepatocyte Carcinoma Cells

In this study, phage display was used to construct a scFv library in order to select antibody binding specifically to hepotocyte carcinoma cells.The total RNA was isolated from spleens of BALB/C mice immunized with Hep-G2 cells. A set of oligonucleotide primers were designed and used to amplified VH and VL genes which were then joined into single chain variable fragments(scFv) by a DNA linker encoding peptide(Gly4Ser)j. The scFv fragment was cloned into a phagemid (pCANTABSE) and expressed as a fusion protein with E tag and phage M13 p3 protein in E. coli TGI .The recombinant phage were rescued by being cocu-Itured with helper phage M13KO7 to construct phage display antibody library. The library was screened with HepG2 cells by binding-elution-enrichment procedure. The positive recombinant phages were identified by ELISA. After five rounds of panning and screening, a phage-scFv named scFv-14 binding HepG2 cells specifically was selected from the library.ScFv-14 Was then cloned and infected E.coli toplO.Induced with IPTG,scFv-I4 scFv-14-E-tag fusion protein secreted into the periplasm of host bacterisa of host bacteria?E.coli top 10. The expressed scFv was detected by SDS-PAGE and Western blot.By constructing the expression plasmid pET24a- scFv-14, scFv-14 can be expressed highly efficient in E.coli .the yield of purified protein was up to 150 mg/L cultures.The His-tag at the C-terminus allowed the protein to be detected by cell ELISA by using anti-His-tag antibodyThe selecting and studying of binding specifically to hepatocyte carcinoma cell line HepG2 provide us the basis for further study of the targeting therapeuty of liver cancer.