Construction Of Human Nature High-capacity Liver Cancer Ribosome Display Single-chain Fv Library

OBJECTIVE:The aim of this study is to construct human nature high-capacity and good-diversity ribosome display single-chain Fv library of liver cancer.METHODS:We isolated human lymphocytes from peripheral blood (10 liver cancers,26 healthy adult volunteers, each 2ml and two two-year-old children, two newborn, each 2ml) and extracted RNA for amplifying human V genes and CK gene as the spacer by RT-PCR. By designing the appropriate primers,5 VH germ line genes,10 VL germ line genes and one CK gene were successfully amplified, which could basically suffice for the human antibody diversity. Genes encoding single-chain Fv fragments VH-VL were assembled by improved two-fragment SOE PCR with a linker DNA encoding the peptide (Gly4Ser)3. Finally, the elements for ribosme display such as a T7 promoter, a ribosome binding site, initiation codon ATG and CK gene were introduced after the SOEing products were amplified. Moreover, ribosome display templates were connected to T-Vector into E.coli DH5a, were verified by blue/white screening and further colony PCR analysis and sequencing.RESULTS:1,We constructed natural human liver cancer ribosome display scFv library with a volume of 2.65×1013.2,According to PCR analysis of recombination clones and sequencing, We could find that the results indicated the library had a good diversity.CONCLUSION:In this study, we used a modified overlap extension PCR (SOE-PCR) method to construct high storage capacity and great diversity of a good human-derived liver cancer ribosome display single chain antibody library, it improved the efficiency of library construction. The antibody library was playing a great role in antibodies screening and modification for liver cancer therapy.