Cloning and expression analysis of Sirt2 in HEK293 cells / 细胞与分子免疫学杂志

ABSTRACT

AIM:To construct eukaryotic expression vector of Sirt2 and detect its expression in HEK293 cells. METHODS: Total RNA was isolated from brain tissue of a-dult 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 se-quenced. 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.