1.Making and sterilizing lysogeny broth, preparing agar plates
2.PCR amplification of SUMO-IL-18BPa-Fc, SUMO-IL-18BPc-Fc

1. PCR amplification of SUMO-IL-18BPa-Fc, SUMO-IL-18BPc-Fc
2. pET28a blank plasmid extraction and PCR amplification
3. Enzyme digestion of pET28a and verification that it has been cut
4. Gel-electrophoresis verification of pET28a, SUMO-IL-18BPa-Fc,SUMO-IL-18BPc-Fc
5. DNA extraction of pET28a, SUMO-IL-18BPa-Fc, SUMO-IL-18BPc-Fc from gel

1. Restriction digestion and ligation to create recombinant plasmids
2. Transformation of recombinant plasmids into DH5a E. coli

1. Selection and verification of monoclonal colonies
2. Send monoclonal colonies' plasmids for gene sequencing for further verification
3. Amplification of DH5a with desired plasmids

1. Transformation into BL21 E. coli
2. Selection and verification of monoclonal colonies
3. cultured bacteria

1. IPTG induction of protein synthesis
2. Extraction and lysis of BL21 bacteria
3. His-tag purification

1.Cell incubation with proteins (stored at different temperatures) and inflammatory factors
2.ELISA of cell supernatant to detect IFN-gamma levels