Generation Of Rad1 Antibody And CcFv Antibody Display System

Objectives 1 To generate antibodies for the DNA damage response protein Rad1 using hybridoma technology; to conduct cell biology experiments using the generated Rad1 antibody F10. 2 Display antibody VH and VL domain on the inner membrane of E. coli via a coiled-coiled heterodimeric variable domain(cc Fv), to construct a large antibody display library, to screen for the antibody mutant with high affinity using FACS(Fluorescence activated cell sorter); to optimaze the cell sorting technique.Methods 1 Construct recombinant m Rad1 protein prokaryotic expression vector and purify the m Rad1 protein via GST resin,using the purified protein to immune BALB/c mice, then carry out hybridoma technology to generate Rad1 monoclonal antibody; Verify the antibodies via Western blotting, ELISA, immunoprecipitation and immunofluorescence, detect antibody’s affinity through SPR, investigate the Rad1 expression level after HU treatment. 2 Construct the plasmids of cc Fv antibody display system, construct antibody mutant library via Error-prone PCR, display the antibody on the inner membrane of E. coli, screen the good mutant via FACS, rescue the good antibody genes, sequencing and expressing the selected antibody, confirm it’s affinity via SPR.Results 1 A high quality of Rad1 antibody was generated via the optimazed hybridoma technology; Using this antibody, we found Rad1 is under the regulation of another DNA damaging reponse protein Rad9 after HU treatment. 2 Successfully construct a anti-TNFα antibody’s display library in the type of cc Fv format and increased it’s affinity using this display system, successfully display the Rad1 antibody’s Fv genes on the inner membrane of E. coli.Conclusions 1 Prolonging the immunization time and increasing the antigen purity are critical for generating a high quality’s anti-Rad1 antibody and the other antibodies in general. 2 The generated Rad1 antibody F10 m Ab has hight affinity and specificity that can be used in Western blotting, ELISA, immunoprecipitation and immunofluorescence. 3 Rad1 is under the regulation of Rad9 s after HU treatment;our data indicate that the previously established working model for DNA repair should be modified. 4 cc Fv display on the inner membrane of E. coli has been successfully used for antibodies’ maturation; precise sorting is critical for antibody’s evolution.