[Cloning and expression analysis of Sirt2 in HEK293 cells].
OBJECTIVE
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.


CONCLUSIONS
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.
Wang
Tao
T
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China. wt78213@126.com
Wang
Wei
W
Xu
Qing
Q
Guan
Lu-yuan
LY
Zhang
Bin
B
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008297:Male; D010957:Plasmids / Q000235:genetics; D051381:Rats; D056565:Sirtuin 2 / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; C097475:Sirt2 protein, rat; D056565:Sirtuin 2