Construction and next-generation sequencing analysis of a large phage-displayed V(NAR) single-domain antibody library from six naïve nurse sharks

Feng, Mingqian; Bian, Hejiao; Wu, Xiaolin; Fu, Tianyun; Fu, Ying; Hong, Jessica; Fleming, Bryan D; Flajnik, Martin F; Ho, Mitchell

To construct a large shark V NAR library, we developed a method called EASeL. As illustrated in Figure 1A , we isolated peripheral blood leukocytes from six adult nurse sharks (G. cirratum) and amplified shark V NAR sequences with PCR primers. The reverse primers contain a 19-nucleotide sequence on the pComb3x backbone. Extension overlap PCR was conducted to combine the V NAR and phagemid backbone (Fig. 1B) . Finally, after gel purification we performed a self-ligation with T4 ligase to circularize the assembled V NAR pComb3x plasmids. Our shark V NAR library size (∼1.2 × 10 10 ) was determined by titration based on the number of individual TG1 bacteria colonies on agar plates. This high-efficiency method is a significant improvement over the conventional phage library construction method, which usually requires many rounds of restriction enzyme digestion and ligation, making the previous method labor intensive and time consuming. Moreover, the sequence diversity of the shark V NAR is preserved and represented in our method to a maximum degree due to highly efficient PCR-based amplification and ligation.