1. [Recombinant prokaryotic plasmid construction and high expression of FUS1 gene].
- Author
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Zhang B, Huo X, Peng L, Qi ZL, Xu XJ, Li Y, Qiu B, and Zheng LK
- Subjects
- Blotting, Western, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Escherichia coli genetics, Humans, Mesenchymal Stem Cells cytology, Mesenchymal Stem Cells metabolism, Transformation, Genetic, Tumor Suppressor Proteins genetics, Umbilical Cord cytology, Plasmids genetics, Recombinant Proteins biosynthesis, Tumor Suppressor Proteins biosynthesis
- Abstract
Objective: To construct the prokaryotic plasmid of FUS1 gene for efficient FUS1 expression in E.coli strain Rosetta(DE3)2plys., Methods: The full-length FUS1 gene was amplified by PCR from the total RNA of umbilical mesenchymal stem cells and cloned into pET-32a(+) vector followed by identification with PCR and sequencing. The recombinant plasmid pET-32a(+)-FUS1 was transformed into the E.coli strain Rosetta(DE3)2plys and the target protein expression was induced by IPTG., Results: The plasmid pET-32a(+)-FUS1 was obtained successfully as verified by PCR and sequence analysis. High expression of the fused FUS1 protein was achieved after induction by low-concentration IPTG (25 micromol/L) for 3 h, and the recombinant FUS1 protein accounted for 40% of the total bacterial protein of Rosetta(DE3)2plys., Conclusion: The recombinant FUS1 plasmid has been successfully cloned, which allows highly efficient FUS1 expression in Rosetta (DE3)2 plys.
- Published
- 2007