SIK2 cDNA及其截短体原核表达重组体的构建及表达

摘要

Objective To construct recombinant plasmids of SIK2 cDNA and its truncated mutants and induce its expression in E.coil.Methods We designed primers of SIK2 and its truncated mutants.The gene fragments of SIK2,SIK2-Δ1 (280-926),SIK2-Δ2 (400-926),SIK2-Δ3 (1-400),and SIK2-Δ4 (700-926)were amplified by polymerase chain reaction (PCR)and cloned into pGEX-4T-2 vector to construct recombinant plasmids with GST. The plasmids were transformed into E.coil BL2 1 respectively,and induced with IPTG to express fusion protein. The results were confirmed by Coomassie blue staining and Western blot.Results We successfully constructed recombinant plasmid of SIK2 cDNA and its truncated mutants.Coomassie blue staining and Western blot resutls showed that these plasmids were induced to be expressed in E.coil BL21.Conclusion SIK2 cDNA and its truncated mutants were overexpressed in E.coil BL2 1 ,which lays expereimental foundation for further study on the function of each domain of SIK2 .%目的：构建 SIK2 cDNA及其截短体的原核表达重组质粒,在大肠杆菌中诱导表达。方法分别设计 SIK2及其截短体引物,采用PCR方法分别扩增 SIK2、SIK2-Δ1(280~926)、SIK2-Δ2(400~926)、SIK2-Δ3(1~400)、SIK2-Δ4(700~926)五个 cDNA片段,并将扩增的 cDNA片段插入原核表达载体 pGEX-4T-2,构建 GST标签的重组质粒。将构建好的重组质粒转化到大肠杆菌BL21中,用IPTG诱导SIK2、SIK2-Δ1、SIK2-Δ2、SIK2-Δ3和SIK2-Δ4五个截短体的原核表达,用考马斯亮蓝染色和Western blot进行鉴定。结果成功构建了SIK2全长及其截短体cDNA的重组质粒,用考马斯亮蓝染色及 Western blot鉴定显示,SIK2全长及其截短体重组质粒均在大肠杆菌中诱导表达。结论在大肠杆菌中成功地诱导表达了 SIK2重组蛋白及其截短体,为进一步研究 SIK2各结构域的功能提供了实验基础。