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Updated: Aug 19, 2026
Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly
Published on: February 9, 2015
[Construction of the eukaryotic expression vector containing rat transforming growth factor beta type II receptor and
Zi-yang Yu1, Li-huang Zhang, Hang-ping Yao
1Institute of Immunolgy, College of Medicine, Zhejiang University, Hangzhou 310031, China.
Objective:
To express a bioactive fusion protein comprising rat soluble transforming growth factor beta type II receptor and interferon gamma(rsTGFbetaR II-IFN gamma) in mammalian cells.
Methods:
mRNA was extracted from rat liver and the sTGFbetaR II-IFN gamma genes amplified by RT-PCR, then the two gene segments were cloned into the same pSecTag2A expression vector, and pSecTag2A/rsTGFbetaR II-IFN gamma recombinant plasmid was obtained, which was later transfected into CHO cells using liposomes. The expression of pSecTag2A/rsTGFbetaR II-IFN gamma in the supernatant was detected by ELISA and Western blotting. The bioactivities of the fusion protein were tested by sTGF betaR II-IFN gamma and IFN gamma bioassays.
Results:
pSecTag2A/rsTGFbetaR II-IFN gamma transfectants expressed rsTGF betaR II-IFN gamma fusion protein. The purified fusion protein exhibited anti-viral activity and antagonized the proliferation-inhibitive effect of TGFbeta1 on CCL-64 cells. It inhibits the HSC activation in vitro.
Conclusion:
The pSecTag2A/rsTGFbetaR II-IFN gamma recombinant plasmid constructed in this study can express bioactive rsTGFbetaR II-IFN gamma fusion protein.
