Freund s complete or incomplete adjuvant

The anti-CsMF6p/HDM was produced by immunizing BALB/c mice with the purified rCsMF6p/HDM. The protein (100 μg) was mixed with Freund’s complete or incomplete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) and injected into mice intraperitoneally three times at 2-week intervals. Two weeks after the final booster, the mice were sacrificed and the sera were collected. The immunoglobulin G (IgG) fraction was further purified with the Protein G agarose (Pierce, Rockford, lL, USA) according to the manufacturer’s instructions. The specificity of the anti-CsMF6p/HDM was confirmed by immunoblot analysis.

The NP gene of SFTSV was amplified by reverse transcription PCR (RT-PCR) with the Access RT-PCR System Kit (Promega, Madison, WI). The sequences of the forward primer and reverse primer were: GAG GTA CCA TGT CAG AGT GGT CC and AAT CTC GAG TTA CAG GTT CCT GTA AG, respectively. The amplification conditions and parameters were as follows: one cycles at 45 °C for 45 min, 94 °C for 2 min, 40 cycles at 94 °C for 30s, 60 °C for 1 min, 68 °C for 2 min, one cycles at 68 °C for 7 min. The PCR product was cloned into pMD19 (Simple) T-Vectors (Clontech Laboratories, CA). The cloned insert was excised from the recombinant vector by double enzyme digestion and sub-cloned into pet-32a vector to express the NP. The recombinant NP was purified with the pET Express & Purify Kit—His60 (Clontech Laboratories, CA).
The purified recombinant NP was mixed with equal volume complete or incomplete Freund’s adjuvant (Sigma-Aldrich, USA) and injected subcutaneously into six 6- to 8-weekold Kunming mice (The Animal Experiment Center of Shandong University, Jinan City, China) to make polyclonal antibody. Each mouse was injected with 100 μg recombinant protein at multiple sites at one-week interval for 4 times. Mice were sacrificed 15 days after the last immunization to obtain sera. Sera were frozen at − 80 °C until use as the primary antibody.