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Published on: February 27, 2016
[Cloning and expression analysis of Sirt2 in HEK293 cells]
1Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China. wt78213@126.com
Aim:
To construct eukaryotic expression vector of Sirt2 and detect its expression in HEK293 cells.
Methods:
Total RNA was isolated from brain tissue of adult SD rat. A 1 130 bp fragment containing the coding region of Sirt2 was amplified by RT-PCR and the resulting PCR product was subcloned into PMD20-T vector and sequenced. Coding region of Sirt2 was generated with PCR by using the PMD20-T-Sirt2 as template, the amplified PCR fragment was inserted into the EcoR I and Hind III sites of the pcDNA3.1myc-his(-)A expression vector, and the sequence was confirmed by DNA sequencing. The expression of new construct pcDNA3.1 myc-his(-)A-Sirt2 in HEK293 cells was detected by immunofluorescence.
Results:
The full length coding region of Sirt2 was obtained and confirmed by sequencing, the expression of Sirt2 was detected successfully in HEK293 cells.
Conclusion:
The eukaryotic expression vector of Sirt2 has been successfully constructed, which will provide a useful tool for designing an in-depth investigation of the role of Sirt2.
