Pmd19 t plasmid vector

The cDNA sequence of AwMyD88 was obtained by reverse transcription - polymerase chain reaction (RT-PCR). Gene specific primers (Supplementary Table S1) were designed using Primer Premier 5.0 software according to the sequences of the AwMyD88 gene from our constructed A. woodiana cDNA library. The open reading frame (ORF) sequence of AwMyD88 was amplified using LA Taq DNA polymerase (Takara) in a 25 μl reaction volume containing 15.25 µl of dH₂O, 4 µl of dNTP Mixture (2.5 mM each), 2.5 µl of 10×LA Taq Buffer II (Mg²⁺ Plus), 1 µl of each primer (10 μM), 0.25 µl of LA Taq and 1 µl of cDNA template. The PCR amplification program was performed according to the manufacturer’s protocol. Briefly, 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 2 min were conducted for amplification. The PCR amplification products were separated using 1.0% agarose gel electrophoresis with Goldview nucleic acid stain, purified with a TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 and subsequently cloned into the pMD19-T plasmid vector (Takara, Japan) according to the manufacturer’s instructions. Positive colonies were screened and further confirmed by DNA sequencing.

DNA template of VP2 gene was extracted from MDCK cell pellets infected with CPV using MiniBEST Viral DNA/RNA Extraction Kit Ver.5.0 (Takara, Japan). A pair of primers with Xho I and Hind III was designed according to DNA sequences on Genbank (DQ354068.1) and used to amplify the whole sequence of VP2 gene (Table 1). PCR was performed using Taq Plus DNA Polymerase (Vazyme, Nanjing, China) or PCR Hero™ (Foregene, Chengdu, China) in a Bio-Rad cycler (United States). The PCR procedure was as follows: denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing at 65°C for 15 s; and polymerization at 72°C for 130 s with 30 cycles; extension at 72°C for 10 min. PCR products were detected in 1% agarose gel and visualized under UV light. The VP2 gene was amplified and cloned into pMD19-T plasmid vector (TaKaRa, Japan) to generate a recombinant plasmid of pMD19-T VP2.

The β-catenin was amplified by PCR from the cDNA of sheep skin, which contains SacII and BamHI sites, with the primer shown in Table 1. The purified products were inserted into PMD19T plasmid vector (TAKARA, Japan) and sequenced at SinoGenoMas Co., Ltd. (Beijing, China). The 2.0 kb K14 promoter with AseI and AfeI sites obtained by PCR was cloned into PMD-19T vector and sequenced as above. In order to remove the CMV promoter of pIRES-EGFP, the restriction enzyme AfeⅠ and AseⅠ were used to digest and the human K14 promoter was inserted into the AseⅠand AseⅠ site to produce the skin-specific expression plasmid K14-IRES-EGFP. Afterwards, the ovine β-catenin gene was inserted into K14-IRES-EGFP plasmid by digesting with restriction enzyme BamHⅠ and SacⅡ. The resultant plasmid K14-β-catenin-IRES-EGFP was then sequenced (SinoGenoMax Co., Ltd., Beijing, China).