[Construction of camelidae natural nanobody phage dispaly library and screening, production and identification of anti-CD19 nanobody].

Objective To construct and verify camelidae natural nanobody phage display library for selection of nanobodies against various antigens, and to obtain and identify the nanobody targeting CD19. Methods The total RNA of spleen of Bactrian camel was reverse transcribed and the variable region gene fragment of its heavy chain was obtained by nested PCR. It was constructed into the pCANTAB5e phagemid vector and electrotransformed into TG1 E. coli to develop the natural nanobody phage display library. After rescued by the KM13 helper phage, its capacity and diversity were analyzed and identified. Nanobody against CD19 was screened using biotinylated antigen combined with streptavidin magnetic beads, followed by ELISA, sequencing, exogenous expression and verification. Results The constructed natural phage nanobody display library had great diversity, and its fragment insertion rate was about 100%. The amino acid homology of 20 randomly selected clones was 65.85%, and the titer of the display library rescued by the helper phage was 9.0×1013 CFU/mL. After panning with CD19 as the antigen, the positive clones were sequenced and analyzed, and finally anti-CD19 nanobody sequences were obtained. The exogenously expressed anti-CD19 nanobody based on the sequences was verified having the ability to bind to CD19. Conclusion A camelidae natural nanobody phage display library with high titer and great diversity has been successfully constructed. Three anti-CD19 nanobody sequences have been obtained by panning with CD19. In addition, this study provides technical support for researching and developing diagnostic kits and antibody drugs targeting CD19, and it is a novel direction to improve CAR-T cells targeting CD19.