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Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly
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[Construction of eukaryotic expression vector of EGFRi-IL-24 recombinant gene]
Jianling Wang1, Xinying Fan, Leyuan Bao
1Tianjin Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.
Abstract:
The epithelial growth factor receptor interference (EGFRi) was obtained by synthetic primers. Overlapping PCR was used to produce EGFRi-IL-24 fusion gene, which is linked by Gly4Ser3. After sequence analysis, EGFRi-IL-24 was cloned into expression vector pPIC9k; EGFRi-IL-24/pPIC9k was linearized with SacI,and then transformed to electroporated pastoris GS115. Subsequently, positive clone was selected by G418 and PCR, and its phenotype was determined by SDS-PAGE and MTT assay. The results demonstrated that EGFRi-IL-24 protein was expressed and shown to have the potential for use in researches of its biological function and in clinical application.
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