After one passage of library cells into fresh medium, heterogeneous plasmid DNA was isolated from the library and transformed into E. coli DH5a cells. For each library, a total of 100 colonies were randomly picked from the plates and sequenced using a pair of primers (pCTCON2-Seq-For: 59-GTTCCAGAC-TACGCTCTGCAGG-39 and pCTCON2-Seq-Rev: 59-GTTC-CAGACTACGCTCTGCAGG-39). The plasmids carrying gene fragments inserted in-frame with the original HA gene were retransformed into yeast EBY100 cells, induced, and labeled as described for FCM detection on a BD-FACS Calibur cytometer (Shanghai, China). The ratio of recombinant antigen expressed on the yeast surface (RAYS) was determined by dividing the mean fluorescence intensity of peptide expressing yeast cells by the mean fluorescence intensity of the non-expressing (pCTCON-2) yeast cells. Clones with a RAYS ratio $ 2 were considered positive and the corresponding peptide sequences were selected for further analysis [26] .