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[Construction, expression and location of eukaryotic expression vector CMV-FLAG-RB in human prostate cancer PC-3
Bin Lu1, Xian-lu Song, Fang-li Song
1Department of Urinary Surgery, Nanfang Hospital1, Southern Medical University, Guangzhou, China. lub180@163.com
Objective:
To construct an eukaryotic recombinant expression vector for retinoblastoma 1 gene (RB-1) and investigate the role of RB-1 in prostate cancer.
Methods:
The coding sequence of RB-1 gene tagged with FLAG was amplified from the plasmid CMV-RB by PCR method. The fragment was cloned into CMV expression vector and identified by restriction enzyme digestion and sequence analysis. Western Blotting was used to detect RB-1 expression and immunofluorescence was used to observe RB-1 distribution in PC-3 cells transfected with the recombinant.
Results:
The expression vector CMV-FLAG-RB was successfully constructed as confirmed by PCR, endonuclease digestion and DNA sequence analysis. RB-1 protein was highly expressed and showed a nuclear distribution in PC-3 cells transfected with the recombinant.
Conclusions:
The eukaryotic expression vector for RB-1 has been successfully constructed and can be efficiently expressed in PC-3 cells. The expression of RB-1 is located in the cell nuclei.
