Bl21 de3 cells

A Cg-SOHLH1 polyclonal antibody was prepared against our recombinant protein. The sequence of ORF was amplified using enzyme site-specific primers (Table 1) and was cloned into the pET-28a vector. The recombinant vector was transformed into DH5α cells (TaKaRa, Japan) for amplification and then transformed into BL21 (DE3) cells (TaKaRa, Japan) for protein expression. The expressed protein was purified by a Ni-NTA column using Ni-NTA Sefinose^TM Resin (BBI, United States) and was sent to Sangon Bio Company for antibody preparation. The purified protein was injected into rabbits for polyclonal antibody production, and the serum of rabbits was tested by ELISA each week during immunization. After 6 weeks of immunization, the antiserum was harvested from the rabbits, and the antibody was purified and stored at -20°C after western blot testing.

MmedCSP3 was PCR-amplified using gene-specific primers (Table 1). The sample cDNA of the antennae was used as the template. The PCR product was first cloned into a pEASY-T3 Vector (Takara, Dalian, China) and then excised and cloned into an expression vector pET-30a (+) for expression in prokaryotic BL21 (DE3) cells (Takara, Dalian, China). The transformation of the strain with pET-30a (+) / MmedCSP3 was incubated in 500 mL of LB medium with 100 μg / ml kanamycin at 37°C. When the OD of the culture reached 0.4–0.6, the protein was induced with 1 mM isopropylthio-β-galactoside (IPTG) at 16°C and vibrated for 16 h at 200 rpm. The cultures were harvested by centrifugation at 12000 × g for 25 min at 4°C. The supernatant was obtained by sonication, purified by Ni ion affinity chromatography (ÄKTA avant 25, GE Healthcare, USA). Soon after the His-tag was removed with recombinant enterokinase (Novoprotein, Shanghai, China), followed by a second purification mentioned above. A 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was conducted to check the size and purity of recombinant MmedCSP3.

BL21 (DE3) cells (Takara, Japan) were transformed with the pGEX-ANX A1 expression vectors for full-length or mutants, and the bacteria were incubated in LB with ampicillin at 37 °C until the optical density at 600 nm (OD₆₀₀ nm) reached 0.8. Protein expression was induced by adding 1 mmol L⁻¹ isopropyl-β-thiogalactopyranoside for 4 h at 37 °C. Cell pellets were resuspended in buffer containing 20 mmol L⁻¹ Tris-HCl (pH 7.5), 100 mmol L⁻¹ NaCl, and 0.1% Tween-20, sonicated twice for 5 min at 4 °C and centrifuged at 20,000 × g for 30 min. The supernatant was incubated with glutathione Sepharose 4B (GE Healthcare) for 1 h at 4°C. The resin was washed five times with the same buffer, the glutathione S-transferase tag was cleaved by adding Precision Protease (GE Healthcare) and the sample was further incubated for 16 h at 4 °C. Cleaved proteins were recovered from the supernatants and stored at −80 °C.