原核质粒pGEX-4T-2-PLEKHQ1及真核质粒pCMV-Myc-PLEKHQ1的构建与蛋白表达
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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 Eco R1 and Kpn1 restriction enzymes, was taped into the plasmid pGEX-4T-2 and pCMVMyc. 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 1500 bp, 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.
Objective: To construct prokaryotic plasmid and eukaryotic plasmid of PLEKHQ1 gene. Methods: The PCR product of the PLEKHQ1 coding sequence, which was digested with Eco R1 and Kpn1 restriction enzymes, was taped into the plasmid pGEX-4T-2 and pCMVMyc. 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 1500 bp, 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.
引文
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