Pgex 6p 1 plasmid

The sequence coding for 6×His tag-labelled ΔMKD1 (ΔMKD1) was amplified by RT-PCR using specific primers (see Supplementary Table S1). The amplified PCR products were introduced into SgfI and PmeI sites of the pF3KWG (BYDV) Flexi plasmid (Promega). The ΔMKD1 protein was synthesized using the TNT SP6 High-Yield Wheat Germ Protein Expression System (Promega). In vitro transcription/translation was performed according to the manufacturer’s instructions. The ΔMKD1 protein was purified using a Ni Sepharose High-Performance column (GE Healthcare). MKK1, MKK2, and MKK5 were amplified from cDNA by PCR using specific primers (Supplementary Table S1). Amplified fragments of MKK1, MKK2, and MKK5 were cloned into SmaI and NotI (blunt ended) sites of the pGEX6p-1 plasmid (GE Healthcare). MKK1, MKK2, and MKK5 plasmids were transformed into E. coli BL21-CodonPlus (DE3)-RIL (Agilent Technologies). The recombinant proteins glutathione S-transferase (GST)–MKK1, GST–MKK2, and GST–MKK5 were digested by PreScission Protease (GE Healthcare). The resulting MKK1, MKK2, and MKK5 proteins were purified using a glutathione Sepharose 4 Fast Flow column (GE Healthcare).

The gene sequences of ureB, hspA, and ureB-hspA were amplified by PCR from the vaccine SH02, cloned into an expression vector pGEX-6P-1(+) plasmid (GE Healthcare, Pittsburgh, USA), and placed between BamHI and NotI restriction sites (UreB-F: 5′-CGCGGATCCAAAAAGATTAGCAGAAAAG-3′, UreB-R: 5′-AAATATGCGGCCGCCTAGAAAATGCTAAAGAG-3′; HspA-F: 5′-CGCGGATCCAAGTTTCAACCATTAG-3′, HspA-R: 5′-AAATATGCGGCCGCTTAGTGTTTTTTGTGATC-3′; UreB-HspA-F: 5′- CGCGGATCCAAAAAGATTAGCAGAAAAG-3′, UreB-HspA-R: 5′- AAATATGCGGCCGCTTAGTGTTTTTTGTGATC-3′). The recombinant plasmid was transformed into Escherichia coli (E. coli) BL21 (DE3) cells (Cwbio, Suzhou, China), and protein expression was subsequently induced with 1 mM IPTG at 20°C before being harvested after 18 hours by centrifugation. The bacterial pellets were resuspended in PBS and sonicated. Any insoluble cellular components were removed by centrifugation, and the rGST-UreB, rGST-HspA, and rGST-UreB-HspA fusion proteins were purified by Price Glutathione Superflow Agarose (Thermo, Rockford, USA) according to the manufacturer’s protocol. Moreover, the GST tag was excised with PreScission Protease (Beyotime, Nanjing, China) and removed with GST-tag Purification resin (Beyotime, Nanjing, China). Finally, the purity and concentration of the recombinant protein rUreB, rHspA, and rUreB-HspA were determined via SDS-PAGE, Western Blot, and BCA assay.