A pair of BTV1 VP2 primers containing EcoRI and XhoI sites were designed according to the complete gene sequence of BTV1 VP2 (GenBank accession number KF664124) and used to amplify the full length VP2 gene (Synthesized by Sangon Biotech, China) The sense primer was 5′-ATGGACGAGCTGGGTAT-3′ and the antisense primer was 5′-TTAAACGTTGAGGAGCTTAGT-3′. The PCR amplification was carried out using PrimeSTAR DNA polymerase kits (TaKaRa, China) with the following reaction conditions: a predenaturation at 98 °C for 10 sec, 32 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, and extension at 68 °C for 30 s; and an extension at 68 °C for 5 min. The PCR product was visualised in a 1% agarose gel and then purified using DNA Gel Extraction Kit (OMEGA, US). Then, the extracted VP2 gene was inserted between the EcoR I and Xho I sites of the vector pET-28a. The recombinant vector pET-28a-VP2 was identified by restriction enzyme digestion and the BTV1 VP2 insert was verified by sequencing. The recombinant expression vector was confirmed by restriction enzyme digestion and sequencing. E.coli DH5α was used for amplification of the recombinant plasmid. Then, pET28a-VP2 was transformed into E.coli BL21(DE3) and E.coli Rosetta(DE3) competent cell lines to induce the expression of the His-tagged BTV1 VP2 protein.

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