Abstract:

Objective: To construct a single-domain heavy chain antibody phage display library for food safety detection. Methods: Total RNA was purified from peripheral lymphocytes of two healthy non-immune alpaca (Lama pacos) and directly used for complementary DNA (cDNA) synthesis. The repertoire of VHH encoding DNAs was amplified by semi-nested PCR, and the PCR products were cloned into the phagemid vector pHEN1. Through the electroporation of E. coli TG1, a primary library was established and then rescued by the helper phage KM13 to generate a phage display library. The generated phage display library was used to pan three different artificial antigens by solid phage biopanning. Results: The VHH encoding DNAs was amplified. After electroporating for 10 times, the primary library named as SNAL was generated containing more than 107 independent clones. The cloning efficiency was up to 87%. The titter of the phage display library obtained after the rescue with KM13 named as SNA-PDL was up to 1013 CFU/mL. All antigen screening data exhibited a significant enrichment in phage particles. Conclusion: A naive single-domain antibody phage display library has been successfully constructed. This library exhibits good diversity and can be used to the recovery of binders.

Key words: single-domain heavy chain antibody, VHHs, phage display library, alpaca, hapten