Abstract :
Objective: To construct prokaryotic expression vector of Amuc_1100 gene, the ELP (ELSTIN-LIKE
PROTEIN) is used as purification label to express fusion with the target protein and detect the biological activity of recombinant protein at cell level and animal level. Methods: the recombinant plasmid pET28a-Amuc_1100-ELP is designed and synthesized, and transformed into e. coli competent cell bl21 (de3) to induce expression. The recombinant egg was isolated and purified by the reversible phase change cycling (ITC) characteristic of ELP10 white. At the cellular level, PBMCs cells from mice were extracted to detect the expression level of IL-10. At the animal level, obese mice induced by gastric gavage with high fat detected the improvement effect of the protein on mouse metabolism.
Results: The recombinant protein with the expected molecular weight of 60 kDa was expressed by E.coli expression system. After circulating through ITC, the recombinant protein with high purity was obtained. The protein increased the expression level of IL-10 in PBMCs cells, and significantly improved the obesity and metabolic disorders induced by high-fat diet in mice. Conclusion: The recombinant protein expression plasmid pET28a-Amuc_1100-ELP was successfully constructed. After expression and purification, the recombinant protein expression plasmid has high purity and has 15 biologically active recombinant protein. This study laid a foundation for the industrial production and clinical application of Amuc_1100.
Keyword :
Molecular Biology; Escherichia Coli; Recombinant Protein; ITC cycle; Metabolic Syndrome