原核质粒pGEX-4T-2-PLEKHQ1及真核质粒pCMV-Myc-PLEKHQ1的构建与蛋白表达  

Construction of pGEX-4T-2-PLEKHQ1 prokaryotic plasmid and pCMV-Myc-PLEKHQ1 eukaryotic plasmid and their protein expression

作　　者：陆琤[1] 周晨辰[1] 张鹏飞[1] 张硌[1] 

机构地区：[1]军事医学科学院附属医院医学工程科,北京100071

出　　处：《中国医学装备》2015年第5期5-8,共4页China Medical Equipment

基　　金：国家自然科学基金(31400739)"PH结构域蛋白PLEKHQ1协调巨噬细胞迁移与激活的机制研究";北京市自然科学基金(5144033)"PH结构域蛋白PLEKHQ1调控巨噬细胞激活及迁移的机制研究"

摘　　要：目的:构建基因PLEKHQ1的原核质粒及真核质粒。方法:全长编码基因PLEKHQ1的聚合酶链反应(PCR)产物经Eco RⅠ和Kpn1双酶切后,分别与双酶切后的载体pGEX-4T-2及载体pCMV-Myc相连,在pGEX-4T-2-PLEKHQ1转化E.coli DH5α提取质粒后,转化E.coli BL21中诱导GST-Q1融合蛋白表达,利用谷胱甘肽琼脂糖4B纯化诱导的融合蛋白;而pCMVMyc-PLEKHQ1则瞬转至293TX细胞;用蛋白质印迹法(Western blot)检测GST-Q1和Myc-Q1融合蛋白表达。结果:将获得的重组质粒进行双酶切鉴定,得到约1500 bp的目的片段,符合预计大小。质粒经测序分析正确后,Western blot检测到诱导及纯化后的GST-Q1和转染293TX后的Myc-Q1蛋白。结论:成功构建的pGEX-4T-2-PLEKHQ1和pCMV-Myc-PLEKHQ1重组质粒,为深入研究PLEKHQ1功能奠定良好的基础。Objective：To construct prokaryotic plasmid and eukaryotic plasmid of PLEKHQ1 gene. Methods： The PCR product of the PLEKHQ1 coding sequence, which was digested with EcoR1 and Kpn1 restriction enzymes, was taped into the plasmid pGEX-4T-2 and pCMV-Myc. Then pGEX-4T-2-PLEKHQ1 was transformed into E.coli DH5α and plasmid DNA was extracted. After that, the expression of GST-Q1 fusion protein was induced in BL21 and was purified by Glutathione Sepharose 4B. While the pCMV-Myc-PLEKHQ1 was transfected into TX cells. The expression of both GST-Q1 and Myc-Q1 was detected by Western blot. Results：The restriction enzyme digestion showed target fragment of 1500bp, which was as expected. The recombinant plasmid was sequenced right and GST-Q1 protein and Myc-Q1 protein could be detected by Western blot. Conclusion：The recombinant prokaryotic plasmid and eukaryotic expression plasmid were successfully constructed, which laid a foundation for further research for PLEKHQ1.

关 键 词：基因PLEKHQ1 pGEX-4T-2载体 pCMV-Myc载体 重组质粒 表达 

分 类 号：R392.1[医药卫生—免疫学]