2.3 Expression and purification of recombinant NTD of S proteins
According to the sequence of the S gene of PEDV G1 strain CV777 (GenBank accession No. AF353511.1) and G2 strain CH/ZMDZY/11 (GenBank accession No. KC196276.1), the N-terminal (aa 1-380) domain (NTD) gene of CV777 and CH/ZMDZY/11 S gene, which was designated as G1-NTD and G2-NTD was synthesized based on the codon usage of Escherichia coli . Restriction endonucleases sites Nco I and Bam HI were introduced at the 5’ and 3’ end of the synthesized gene, respectively. The synthesized NTD genes were cloned into same sites of pET28a (+). The resulted plasmid was designated as pET-G1-NTD and pET-G2-NTD.
The expression vector pET-G1-NTD and pET-G2-NTD were introduced intoEscherichia coli. BL21 (DE3) bacterial host. BL21 (DE3) strain harboring expression vector was pre-cultured in 5 mL of Luria-broth (LB) medium containing 50 μg/mL kanamycin at 37°C overnight and was then transferred into 1 L of LB medium containing 50 μg/mL kanamycin. After the optical density at 600 nm reached 0.8, 1 mL of 1M isopropyl-D-thiogalactoside (IPTG) was added to the medium, and the cells were further cultured at 25°C overnight. The induced cells were collected by centrifugation. The cells (typically 5-6 g wet weight of cells from one liter of culture) were resuspended in 100 mL lysis buffer (10 mM sodium phosphate, 0.15 M NaCl, 1 mM EDTA, pH 7.2 PBS, containing 1% Nonidet P-40, 1mM phenylmethylsulfonyl fluoride, 1mM 2-mercaptoethanol, and proteinase inhibitor mixture), and sonicated at 0 °C. The insoluble fraction was removed by centrifugation at 10,000 x g for 30 min at 4 °C. The supernatants were used to analyze the expression of G1-NTD and G2-NTD in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Purification of recombinant G1-NTD and G2-NTD proteins was carried out by using the HisPur Ni-NTA Spin Purification Kit (Thermo Fisher Scientific, MA, USA) according to the instructions of manufacture.