Figure 2

Construction of the synthetic library. (A) Schematic flow chart for the assembly of synthetic diversity in CDR3 based on an identified universal VHH framework of cAbBCII10. Synthetic VHH genes were generated by overlap PCR extension. Final PCR products were digested with Pst I and Not I, gel-purified sequentially and cloned into the phagemid vector pMECS. (B) Left. F-1 and R-1 were used to amplify a fragment of ~ 300 bp with a determined DNA sequence of cAbBCII10 FR1-FR3 as the template; Middle. Agarose gel electrophoresis of the PCR products using F-2 and R-2 as the amplification primers; Right. Final products assembled and amplified by overlapping PCR extension. (C) The capacity of the library measurement. Scraped colonies were diluted into 5 mL of PBS and 50 μL were used to do gradient dilution. (D) 24 clones were randomly picked to estimate the correct insertion rate by performing PCR. (E) 30 clones were randomly chosen for sequencing to detect the diversity of CDR3.