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[The expression and localization of EGFP-Sam68 fusion protein in HeLa cells]
Hongwei Cao1, Fengqi Li, Hua Zhang
1College of Biological Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China.
Objective:
To clone the full-length cDNA of Src-associated protein p68 in mitosis (Sam68), construct the eukaryotic green fluorescent protein expression vector of Sam68 and further investigate its expression and cellular localization in HeLa cells.
Methods:
Total Sam68 RNA was extracted from HeLa cells, and then Sam68 gene fragment was amplified by RT-PCR. The PCR product was analyzed by endonuclease digestion and sequencing, and the correct fragment was inserted into pcDNA3.0-EGFP fluorescent expression vector. The constructed expression vector was then transfected into HeLa cells. Western blotting was performed to detect Sam68 expression and immunofluorescence cytochemistry was used to observe its cellular localization.
Results:
Restriction endonuclease digestion and sequence analysis confirmed that Sam68 was inserted into pcDNA3.0-EGFP, and the vector expressed the EGFP-Sam68 fusion protein correctly. Sam68 was localized in the nucleus of HeLa cells.
Conclusion:
Eukaryotic expression vector pcDNA3.0-EGFP-Sam68 has been constructed successfully.
