2.3. Plasmids

The M2 epitope (5’-AGTCTTCTAACCGAGGTCGAAACGCCTATCAGAAACGAATGGGGG-3’) was introduced into the Ca2 or putative Ca2 antigenic sites of the HAs through PCR to generate plasmids expressing the PR8 Ca2 M2, cH5/1 Ca2 M2, cH8/1 Ca2 M2 and the cH11/1 Ca2 M2 hemagglutinin genes. For example, to generate PR8 Ca2 M2 HA, the nucleotide sequence of the M2e insertion was split into two primers—the M2e insert reverse primer (5’-GATAGGCGTTTCGACCTCGGTTAGAAGACTCTCATGGGAGCATGCTGCCG-3’) and the M2e insert forward primer (5’-GTCGAAACGCCTATCAGAAACGAATGGGGGGGGAAAAGCAGTTTTTACAG-3’), which have 15 nucleotides of overlap for In-Fusion cloning (Takara Bio). Two segments were amplified for each HA gene, the 5’ segment using the PR8 HA NCR forward (5’-CCGAAGTTGGGGGGGAGCAAAAGCAGGGGAAAATA-3’) and M2e insert reverse primers, and the 3’ segment using the M2e insert forward and the PR8 HA NCR reverse (5’-GGCCGCCGGGTTATTAGTAGAAACAAGGGTGTTTTTC-3’) primers. Both PR8 HA NCR forward and PR8 HA NCR reverse primers contain 15 nucleotides of overlap with the end sequences of the linearized vector, which was generated by SapI restriction enzyme (New England Biolabs, Inc., Ipswich, MA, USA) digestion of the pDZ ambisense plasmid [63]. The two modified HA segments were subsequently cloned into the pDZ vector through In-Fusion cloning (Takara Bio, Kusatsu, Shiga Prefecture, Japan). The recombination products were transformed into Escherichia coli DH5α competent cells (Thermo Fisher Scientific) and plasmids were purified using QIAprep Spin Miniprep kit (Qiagen). All the other cHA Ca2 M2 plasmids were generated using the same approach. The pCAGGS PR8 M2 plasmid was constructed by amplifying the M2 opening reading frame (ORF) sequence from the PR8 M segment through PCR and subcloning the M2 ORF into a mammalian expression vector-pCAGGS. Sequences of HA or M2 gene were confirmed by Sanger sequencing (Macrogen). The pRS PR8 7 segment plasmid used to rescue recombinant influenza viruses has been described previously [64].