摘要
目的构建SIK2cDNA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-△1（280-926）、SIK2-△2（400-926）、SIK2-△3（1-400）、SIK2-△4（700-926）五个cDNA片段，并将扩增的cDNA片段插入原核表达载体pGEX4T2，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、SIK2A1、SIK2-A2、SIK2-A3和SIK2A4五个截短体的原核表达，用考马斯亮蓝染色和Westernblot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Westernblot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。
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-△1 （280--926）, SIK2-△2 （400-926）, SIK2-△3 （1-400）, andSIK2-△4 （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 BL21 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 BL21, which lays expereimental foundation for further study on the function of each domain of SIK2.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2014年第3期290-294,共5页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.30960093
31160183
31270894)~~
