Abstract: In order to construct a high-capacity ribosome display anti-clenbuterol single chain Fv (CBLscFv) library for the selection of high affinity scFv antibody, CBL-BSA immunogen was immunized to Balb/c mice. Spleen cells were isolated and total RNA was extracted by the Trizol method for cloning heavy chain and light chain gene by RT-PCR. VH and VL were rearranged randomly by SOEing (splicing by overlap extension). Finally, the elements T7 promoter, 3＇ stem loop structure, ribosome binding sites, spacers and 5＇ stem loop structure were screened in vitro. The constructed library was verified by blue/white screening and further sequencing. The results showed that the titer of antiserum from immunized mice for library construction was 1:128000. The heavy and light chain variable region genes were successfully amplified to be approximately 360 bp and 330 bp, respectively. The fragment of the constructed ribosome display library was approximately 1200 bp with a capacity of 1.75 × 1013. Meanwhile, sequence analysis was carried out using IMGT/V-QUEST database. The results showed that the heavy chain and light chain variable region genes were rearranged by mouse IG set, and the homology rates were more than 84%. Amino acid sequence alignment revealed a homology rate of 37.82% for heavy chain amino acid sequences and 39.09% for light chain amino acid sequences. The diversity was observed in VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3. Therefore, a high-capacity ribosome display library with the function for antibody screening was successfully constructed.

Key words: ribosome display, clenbuterol, single-chain fragment variant (scFv)