Generation and characterization of H5 HA gene recombinant C-KCE-HA.
(A) The organization of the 158-kbp attenuated commercial DEV vaccine strain (C-KCE). (B) A portion of the genome C-KCE expanded to show the gB, UL26 gene and inter-genic region is depicted. (C) The organization of transfer vector pBlue-lox-gB-UL26-Amp digested by PacI contains an enhanced red fluorescent protein gene and its expression cassette, two copies of the direct orientation 34-bp Loxp and two copies of the reverse complement 18-bp I-sceI. (D) After homologous recombination, pBlue-lox-gB-UL26 was inserted into the genome of C-KCE with the red fluorescent protein as a selection marker. (E) After MAGIC, the red fluorescent gene, CMV promoter, and ploy A were entirely replaced by the HA gene and its cassette. (F) After Cre/Loxp–mediate recombination, the BAC backbone was excised only 34-bp Loxp sequence positioned in C-KCE-HA genome.