Screening Of High Affinity Rabbit HER2 Antibodies Based On Phage Display Technology

Objective:To obtain the heavy chain and light chain genes of rabbit HER2 antibody and synthesize single-chain antibody ScFv,and construct a complete phage single-chain antibody library by phage infection.High-affinity HER2 antibodies were screened by using two methods:immunoplates with ELISA plate antigen and HER2 positive breast cancer tissue sections.The screening effect was compared from the enrichment factor and positive rate of specific antibody library.The use of tissue sections as a solid phase vector for the screening of phage libraries.Methods:New Zealand white rabbits were immunized with HER2 protein,total RNA of spleen was extracted,and cDNA was synthesized by reverse transcription.Degenerate rabbit primers were designed and the variable region sequences of heavy chain and light chain of HER2 antibody were amplified by PCR using a cDNA as a template.The heavy chain and light chain of the HER2 antibody were amplified using a Linker to assemble the ScFv fragment.The recombinant pCANTAB5e-ScFv plasmid was obtained by double enzyme digestion and directional cloning.The competent cell TG1 was prepared,and pCANTAB5E-ScFv was transformed into TG1 competent cells;the transformed host strain TGI was infected with filamentous helper phage M13K07,thereby constructing rabbits.Anti-HER2 single chain antibody phage library.ELISA plates were coated with antigen and HER2 positive breast cancer tissue sections were immunohistochemically stained according to the“Adsorption-Elution-Amplification”protocol.After 5 rounds of screening,the final antibody library was obtained.After cloning,the crude extracts induced by pCANTAB5e-ScFv positive strains were detected by Phage-Elisa,and the best-selected pCANTAB5e-ScFv strain was selected for sequencing.The homologous alignment of the obtained heavy chain and light chain gene sequences was performed by the immunoglobulin(IG)and Nucleotide BLAST analysis software.Results:1.Total RNA of spleen has been extracted successfully,VH,VL spliced to obtain complete ScFv gene.2.The constructed primary antibody library of HER2 has a storage capacity of 1.68x106 and the recombination rate is 83.3%.3.Using two methods,after 5 rounds of panning,the yield increased gradually,the specific phage was effectively enriched,and the specificity of the antibody library screened by the ELISA plate as a solid-phase carrier increased by about 105 fold.HER2 positive mammary gland The specificity of the antibody library screened by the cancer tissue section as a solid-phase carrier increased 4180-fold.4.The positive rate of the specific antibody library screened out by HER2 tissue section was 60%,which was also higher than the positive rate of 25%of the specific antibody library screened out by the ELISA plate.Compared with the enrichment multiple and positive rate,the tissue section was better as a solid-phase carrier for the phage library.5.Two positive clones with good response were selected and sequenced.The homology of the gene of the heavy chain variable region and rabbit-derived antibody was 95.4%,and the homology of the light chain variable region was 91.4%.The V1 gene family has a complete sequence structure.Conclusion:The large-capacity rabbit HER2 single-chain antibody library was successfully constructed and specific antibody with high affinity was selected.The tissue section was used as a solid-phase vector for phage display library and the immunohistochemical screening method was feasible.The screening effect was better than the microtiter plate.