Construction of High Content Nanobody Library in Mammalian Cells by Linear-double-stranded DNA Based Strategies

Here, we developed two novel methods to construct high content nanobody library in mammalian cells. For the first method, we employed a l inear-double-stranded DNA b ased A ND- g ate (LBAG) strategy. Upstream- and downstream-linear-double-stranded DNAs (ldsDNAs), containing identical overlapping sequences, were co-transfected into cultured cell lines to conduct AND-gate calculation to form intact nanobody expression cassette. For the second method, we generated full-length nanobody expression ldsDNA by in vitro ligation of restrict cut up- and down-stream ldsDNAs. Then the ldsDNAs were directly transfected into mammalian cells to express nanobody library. Both methods generated over a million different nanobody sequences as revealed by high-throughput sequencing (HTS).