Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both...Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both in the supernatant and inside the bacterial cell,but the insoluble protein had no biological activity and could not be refolded. The rotative speed of the shaker and the temperature of induction were optimized to maximize the expression of the soluble fusion protein. From the supernatant of the cell sonicates Glutathion Sephrose 4B affinity column chromatography was employed to isolate the fusion protein which could be purified to >80 0 0 in a single step. The yield of soluble GST IL 6 was about 10 mg per liter culture. The GST was site specifically cloven by 6 hours of treatment with thrombin and from the thrombin digest mixture IL 6 was purified by Q high performance ion exchange chromatography. From 1 liter of E.coli culture 2 mg refined IL 6 was obtained. The purified IL 6 had a purity of more than 95 0 0 and a biological activity of 1.02×10 8 IU/mg.展开更多
Radix Adenophorae, a traditional Chinese medicine, has been reported to have a variety of biological functions. In the present study, a polysaccharide component, Radix Adenophorae Polysaccharide （RAPS）, was purified...Radix Adenophorae, a traditional Chinese medicine, has been reported to have a variety of biological functions. In the present study, a polysaccharide component, Radix Adenophorae Polysaccharide （RAPS）, was purified from Radix Adenophorae by decoloring with ADS-7 macroporous adsorption resin, DEAE-52 cellulose ion-exchange chromatography, and Sephacryl S-300HR gel chromatography, with the purity of 98.3% and a molecular weight of 1.8 × 104 Da. The cell viability assay and microscopic examination revealed that RAPS promoted the proliferation and activation of macrophages. At 400 μg·mL-1, RAPS stimulated RAW264.7 cell proliferation by 1.91-fold compared with the control. Meanwhile, RAPS significantly increased the secretion of pro-inflammatory cytokines （TNF-a and IL-6） in a dose-dependent manner in the supernatant of RAW264.7 cell culture as determined by ELISA. At 400 μg·mL-1, the production of TNF-a was 20.8-fold higher than that of the control. Simultaneously, the production of nitric oxide （NO） and the expression of inducible nitric oxide synthase （iNOS） were increased in RAW264.7 cells incubated with RAPS, as measured by Griess assay and Western blot analysis. The NO production of cells treated with RAPS （400 μg·mL-1） reached 15.8 μmol·L-L, which was 30.4-fold higher than that of the control （0.53 μmol·L-1）. These data suggested that RAPS may enhance the immune function and protect against exogenous pathogens by activating macrophages.展开更多
文摘Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both in the supernatant and inside the bacterial cell,but the insoluble protein had no biological activity and could not be refolded. The rotative speed of the shaker and the temperature of induction were optimized to maximize the expression of the soluble fusion protein. From the supernatant of the cell sonicates Glutathion Sephrose 4B affinity column chromatography was employed to isolate the fusion protein which could be purified to >80 0 0 in a single step. The yield of soluble GST IL 6 was about 10 mg per liter culture. The GST was site specifically cloven by 6 hours of treatment with thrombin and from the thrombin digest mixture IL 6 was purified by Q high performance ion exchange chromatography. From 1 liter of E.coli culture 2 mg refined IL 6 was obtained. The purified IL 6 had a purity of more than 95 0 0 and a biological activity of 1.02×10 8 IU/mg.
基金supported by the National Science and Technology Major Project Foundation of China(No.2012ZX09102301-003)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)
文摘Radix Adenophorae, a traditional Chinese medicine, has been reported to have a variety of biological functions. In the present study, a polysaccharide component, Radix Adenophorae Polysaccharide （RAPS）, was purified from Radix Adenophorae by decoloring with ADS-7 macroporous adsorption resin, DEAE-52 cellulose ion-exchange chromatography, and Sephacryl S-300HR gel chromatography, with the purity of 98.3% and a molecular weight of 1.8 × 104 Da. The cell viability assay and microscopic examination revealed that RAPS promoted the proliferation and activation of macrophages. At 400 μg·mL-1, RAPS stimulated RAW264.7 cell proliferation by 1.91-fold compared with the control. Meanwhile, RAPS significantly increased the secretion of pro-inflammatory cytokines （TNF-a and IL-6） in a dose-dependent manner in the supernatant of RAW264.7 cell culture as determined by ELISA. At 400 μg·mL-1, the production of TNF-a was 20.8-fold higher than that of the control. Simultaneously, the production of nitric oxide （NO） and the expression of inducible nitric oxide synthase （iNOS） were increased in RAW264.7 cells incubated with RAPS, as measured by Griess assay and Western blot analysis. The NO production of cells treated with RAPS （400 μg·mL-1） reached 15.8 μmol·L-L, which was 30.4-fold higher than that of the control （0.53 μmol·L-1）. These data suggested that RAPS may enhance the immune function and protect against exogenous pathogens by activating macrophages.
