Expression,Purification And Activity Detection Of Human Interleukin 18 In Escherichia Coli

Interleukin-18(IL-18)is an important member of the interleukin family.As a unique cytokine,similar to IL-1 ?,IL-18 participates in immune response and plays an important role in immune regulation of human being.IL-18 is a synthesis of a precursor with no activity,usually to be processed into the mature precursor with the help of Caspase 1,which performs different biological functions:IL-18 is involved in Thl and Th2 immune regulation and induces different kinds of cells to produce IFN ?;IL-18 can stimulate NK cells to produce IL-3,IL-9 as well as enhance the cytotoxic effects induced by Fas/FasL;Also,IL-18 can stimulate mast cells and basophilic cells to produce IL-4,IL-13 histamine and various chemical substances.It is clear that IL-18 is one of the pleiotropic cytokines with a variety of cellular biological functions.IL-18 exhibits unique biological functions by binding into specific IL-18 receptor on different cell types.At the same time,the activity of IL-18 is usually balanced by IL-18 binding protein(IL-18 BP).The severity of some diseases of human being is associated with an imbalance of IL-18 and IL-18 BP of patients,resulting in a sharp increase of free IL-18 in serum.A growing number of studies have confirmed that IL-18 plays an important role in regulating acute inflammation of animals.IL-18 is related closely to various diseases,for instance,myocardial function,emphysema,metabolic syndrome,psoriasis,inflammatory bowel disease,syndrome phagocytic cells,macrophage activation syndrome,sepsis,acute kidney injury,and other autoimmune diseases.In particular,IL-18 can activate NK cells to produce IFN ?,by synergistic effect with IL-12,thus improving the cytotoxictiy to tumor cells.Therefore,IL-18 can be used as an anti-tumor therapy against tumor spread.In all,IL-18 has a great value in both medical research and application.But judging from the studies of recent years,IL-18 expressed in labs is features with low production and activity.Therefore,this study starts from gene reformation according to the codon preference,and screen for appropriate plasmid and e.coli to conduct protein expression.Also,the most suitable conditions for both industrial production and purification is observed.In this study,optimized hIL-18 is connected to the plasmid pET28b to form a recombinant expression vector,which is transferred into e coli BL21(DE3)for protein expression and searching for the most suitable conditions for industrial utilization.At the same time,affinity chromatography was used to isolate and purify the expression products,and a final concentration of 0.7 mg/ml recombinant protein is obtained.What's more,the activity of the final protein is gauged by PBMC cell lines rapidly.The specific results of this study are shown as follow:? The hIL-18 gene is genetically modified and His tag is added at the end of the sequence in order to facilitate purification.?The expression vector is constructed via double enzyme digestion and the recombinant plasmid is transferred into E coli BL21(DE3)by chemical transformation,confirmed by DNA sequencing.?After the expression test of combination of different conditions,the most suitable induction condition for industrial production are obtained:the final concentration of IPTG is 1 mM,the temperature is 16?,and the induced expression time remains 16 hours;and the final concentration of expression test is 0.7 mg/ml.? With the help of the synergistic effect of PHA,the recombinant protein can promote the proliferation of PMBC lines.It' s clear that recombinant protein is of activation.In conclusion,this study realize the soluble expression of IL-18 in e coli via the construction of recombinant vector,fundamentally identified the activity of protein products and effectively improved the yield of target protein,providing a basis for the industrial mass production of IL-18 and laying a solid foundation for the basic medical research and clinical application of IL-18.