The gene coding for the full HA protein of A/California/04/ 2009(H1N1) virus was amplified from plasmid CMV-R-Cali-04-09 with the forward primer 59-CGCCACCATGAAGGC-TATCC-39 and reverse primer 59-TTAGATGCAGATTCTG-CACTG-39. Detailed procedures for fragmentation and reassembly were described previously, except that PhusionH high-fidelity polymerase (NEB) was used in reassembly [24] . The reassembled DNA samples were purified and modified to add 59 A overhangs by an A-Tailing Kit (Takara). The backbone vector pCTCON-T was digested with Xcm I and purified with a Tiangen gel purification kit to reduce self-ligation. The gene fragments and backbone vector were ligated at 16uC for 16 h and then electroporated into E. coli DH5a competent cells for propagation. The plasmid library was then isolated and used to transform yeast EBY100 cells by the Li-Ac method [40] . Expression of peptide libraries on yeast EBY100 cells was performed as reported by Wittrup's group [23] . Transformed yeast cells were grown for 24 h at 30uC with shaking in SDCAA medium (yeast nitrogen-based casamino acid medium containing 20 g?L 21 glucose) and passed one time into fresh medium to eliminate dead cells. The library cells were then centrifuged, resuspended in SGCAA medium (yeast nitrogen-based casamino acid medium containing 20 g?L 21 galactose) to an OD 600 value of 0.5-1 and induced at 20uC for 36-48 h with shaking.