Construction Of Rabbit Scfv Phage Library And Selection Of Estrogen Receptor ?(ER?) Antibodies

Objective:We aimed to construct a phage library,of rabbit single-chain antibody and select a high-affinity estrogen receptor(ERa)single-chain antibody(scFv)for immunohistochemistry(IHC),and to explore the feasibility of using phage display technology to select high affinity antibodies for IHC.Methods:ERa recombinant protein was used as an immunogen to immunize New Zealand white rabbits,from which lymphocytes total RNA were extracted followed by reverse transcription to synthesize cDNA.Then heavy chain variable region(VH)and light chain variable region(VL)fragments of antibody were amplified by PCR and spliced into scFv genes.We constructed pCANTAB5E-scFv recombinant plasmids and transformed them into E.coli TGI competent cells to construct phage library of antibody.Five rounds of affinity enrichment from the antibody library were performed using ERa-positive breast cancer tissue sections and ERa recombinant protein as solid phase respectively.Positive clones were future selected using phage-ELISA and immunohistochemistry of ERa-positive T47D cell.The scFv gene of positive cloned was sequenced,and recombinant plasmid Pet-27b(+)-scFv was constructed and transformed into E.coli Rosetta(DE3)to induced and express.The scFv recombinant protein expressed was purified through nickel column.The application value of this single-chain antibody was evaluated by immunohistochemistry.ResuLts:1.The rabbit lymphocytes total RNA were extracted and cDNA were reverse transcribed successfully,VH and VL fragments of antibody were amplified and spliced into the scFv genes.2.The pCANTAB5E-scFv recombinant plasmids were constructed successfully and electroporated into TG1competent cells.A phage single-chain antibody library with a capacity of 1.92 × 107,a recombinant phage titer of 1.57×1012 pfu/mL and a recombination rate of 95%was established.3.The phage antibody library was specifically enriched and a positive clone B19 was screened out successfully.The homology of the VH was 96.1%and the homology of the VL was 92.4%.4.B19-scFv was successfully cloned into the prokaryotic expression vector pET-27b(+),and transformed into E.coli Rosetta.After expression and purification the B19-scFv recombinant protein was obtained,which showed clear nuclear localization in IHC results,less background and non-specific staining in IHC results.Conclusions:In this study,we constructed and identified a rabbit phage antibody library,from which an anti-ER? antibody was selected out successfully,and showed good IHC staining results.The results suggested that phage display technology could be applied to select high affinity antibodies for immunohistochemistry.