P3m2e

p3M2e was synthesized by GenScript Co., Ltd. Each M2e sequence was linked with a GSG4 linker (Gly–Ser–Gly–Gly–Gly–Gly). The eukaryotic expression vector pVAX1 (Invitrogen, Carlsbad, CA, USA) was used to construct the DNA vaccine. For p-p3M2e, p3M2e was digested with EcoRI and XhoI and then cloned into pVAX1 to create p-p3M2e. To construct p-tPA-p3M2e, the tPA signal sequence was amplified by PCR using the following primers: 5′-CCCAAGCTTATGGATGCAATGAAGAG AGGGCTCTGCTGTGTGCTGCTGCTG-3′ and 5′-CCGGAATTCGCTGGGCGAA ACGAAGACTGCTCCACACAGCAGCAGCACACAGCAGAG-3′. The PCR product was then digested with HindIII and EcoRI, followed by ligation into p-p3M2e to create p-tPA-p3M2e. The single-copy M2e plasmids (p-tPA-pM2e and p-pM2e) were constructed using similar methods. The plasmids were propagated in Escherichia coli DH5α bacteria and purified using NucleoBond® Xtra (MACHEREY-NAGEL GmbH and Co. KG).
The M2e peptide SLLTEVETPIRNEWGCRCNGSSD was synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (>95% purity).

p3M2e was synthesized by GenScript Co., Ltd. Each M2e sequence was linked with a GSG4 linker (Gly–Ser–Gly–Gly–Gly–Gly). The eukaryotic expression vector pVAX1 (Invitrogen, Carlsbad, CA, USA) was used to construct the DNA vaccine. For p-p3M2e, p3M2e was digested with EcoRI and XhoI and then cloned into pVAX1 to create p-p3M2e. To construct p-tPA-p3M2e, the tPA signal sequence was amplified by PCR using the following primers: 5′-CCCAAGCTTATGGATGCAATGAAGAG AGGGCTCTGCTGTGTGCTGCTGCTG-3′ and 5′-CCGGAATTCGCTGGGCGAA ACGAAGACTGCTCCACACAGCAGCAGCACACAGCAGAG-3′. The PCR product was then digested with HindIII and EcoRI, followed by ligation into p-p3M2e to create p-tPA-p3M2e. The single-copy M2e plasmids (p-tPA-pM2e and p-pM2e) were constructed using similar methods. The plasmids were propagated in Escherichia coli DH5α bacteria and purified using NucleoBond® Xtra (MACHEREY-NAGEL GmbH and Co. KG).
The M2e peptide SLLTEVETPIRNEWGCRCNGSSD was synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (>95% purity).