【摘要】 将牛白细胞介素18(bovine interleukin-18,BoIL-18)的成熟蛋白基因亚克隆到原核表达载体pGEX6p-1中,并转化至大肠杆菌(Escherichia coli)BL21(DE3)获得重组菌株。该重组菌经0.3mmol/L IPTG诱导8h,SDS-PAGE电泳表明,BoIL-18融合蛋白获得了高效表达,分子量约为44kD,凝胶薄层扫描分析显示,融合蛋白占工程菌菌体总蛋白的31.8%;用亲和层析法对表达产物进行纯化,检测纯化产物的生物学活性。对外周血单核细胞(PBMC)的增殖实验证明,BoIL-18融合蛋白浓度大于0.10mg/L时,对PBMC有明显的增殖作用;对IFN-γ的诱生实验显示,BoIL-18融合蛋白浓度大于0.20mg/L时,能促进IFN-γ的产生,且随着BoIL-18浓度的增加,对IFN-γ的诱生作用增强。更多还原

【Abstract】 The cDNA of bovine interleukin-18(BoIL-18)was subcloned into pGEX-6P-1 vector and transformed into Escherichia coli BL21(DE3).The recombinant protein was expressed successfully in Escherichia coli by induced with 0.3 mmol/L IPTG for 8 h.SDS-PAGE results indicated that engineering bacteria could express a 44 kD product.And densitometric scanning showed the expressed fusion protein was about 31.8% of total bacterial protein of BL21.The fusion protein was purified by affinity chromatography.Then the biological activity of purified product was assayed.The PBMC(peripheral blood mononuclear cells)proliferation indicated that the BoIL-18 fusion protein could enhance proliferation of PBMC when it was more than 0.10 mg/L.And the ELISA method showed that the BoIL-18 fusion protein could induce IFN-γ production in spleen lymphocyte when it was more than 0.20 mg/L,and the amount of BoIL-18 fusion protein and its inducing effect on IFN-γ had a direct proportion.It can be concluded that the purified BoIL-18 fusion protein has functional activity and can be applied for further studies.更多还原