重组双链RNA依赖的Caspase募集蛋白的表达、纯化及其活性

Expression,purification and activity of recombinant double-stranded RNA-dependent caspase recruiter

目的表表达并纯化重组双链RNA依赖的Caspase募集蛋白,并鉴定其生物学活性。方法将重组质粒pRSET-dsCARE转化大肠杆菌BL21(DE3),分别于37、28℃表达目的蛋白,Ni-NTA亲和层析纯化,纯化产物经SDS-PAGE、Western blot、HPLC鉴定目的蛋白,MTT法检测dsCARE对转染poly(I∶C)细胞的增殖抑制作用。结果dsCARE重组蛋白在大肠杆菌BL21(DE3)中呈组成性稳定表达,平均每升培养基可收获湿菌体约10 g;纯化产物的SDS-PAGE纯度达95.8%;纯化的目的蛋白能与抗-His多抗发生特异性反应;HPLC纯度达95.1%;每10 g湿菌体可纯化dsCARE重组蛋白约7.5 mg,浓度约220μg/ml;纯化的dsCARE重组蛋白与转染poly(I∶C)的HeLa细胞共同孵育,可剂量依赖性抑制HeLa细胞的增殖活性。结论成功建立了重组dsCARE蛋白的表达、纯化工艺及活性鉴定方法,为其应用和后续研究奠定了基础。

Objective To express and purify recombinant double-stranded RNA-dependent caspase recruiter（dsCARE） and determine its biological activity.Methods E.coli BL21（DE3） was transformed with recombinant plasmid pRSET-dsCARE and incubated at 37 and 28℃ respectively.Expressed protein was purified by Ni-NTA affinity chromatography,and identified by SDS-PAGE,Western blot and HPLC.The inhibitory effect of dsCARE on proliferation of poly（I ∶ C）-transfected HeLa cells was determined by MTT method.Results Stable constitutive expression of dsCARE was observed in E.coli BL21（DE3）.About 10 g of wet bacteria were harvested from 1 L of culture medium in average.The purified dsCARE,at a concentration of about 220 μg / ml,reached a SDS-PAGE purity of 95.8% and a HPLC purity of 95.1%,which showed specific reaction with anti-His polyclonal antibody and dose-dependent inhibitory activity to proliferation of poly（I ∶ C）-transfected HeLa cells.About 7.5 mg of recombinant dsCARE was purified from 10 g of wet bacteria.The purified dsCARE co-incubated with HeLa cells transfected with poly（I ∶ C） showed dose-dependent inhibitory effect on proliferation of HeLa cells.Conclusion The procedure for expression and purification and the method for determination of activity of dsCARE was successfully developed,which laid a foundation of application and further study of dsCARE.