Abstract
Nanobody is one special type of single-domain antibody fragment with multiple advantages over traditional antibody. Our previous work established linear-double-stranded DNA (ldsDNA, or PCR amplicon) as novel biological parts for building AND gate circuits in mammalian cells. The construction process of the ldsDNA-based AND gate genetic circuits is similar to that of V(D)J recombination, which serves as the genetic principle for the generation of antigen-receptor (antibody and T-cell receptor) diversity. Here, we employed this ldsDNA-based AND-gate strategy to construct nanobody library in mammalian cells. The sequence complexity of complementary determining regions (CDRs) was introduced into ldsDNA by PCR amplification. After being co-transfected into mammalian cells, the up- and down-stream ldsDNAs undergo AND gate linkage and form full nanobody coding regions, containing CDR1-3. High throughput sequencing identified 22,173 unique oligonucleotide sequences in total generated by this strategy. Thus, we developed a novel method to construct nanobody library in mammalian cells, which has a great potential in the development of nanobody-therapeutics.