| OBJECTIVE To construct the recombinant expression vector containing the transcriptional regulation sequence of DF3/MUC1 promoter on human breast carcinoma and diphtheria toxin A-chain gene. To explore the influence of diphtheria toxin A-chain (DTA) targeting DF3/MUC1 on the biological behavior of breast carcinoma cell.METHODS Transcriptional regulation sequence of DF3/MUC1 gene was amplified from human MCF-7 breast cancer cells by PCR and inserted into the pGL3-Basic vector to construct recombinant plasmid pGL3-DF3,and it was transient transfected into breast carcinoma cells of DF3 positive and negative. The activity of DF3 promoter was expressed by analyzing the relative luciferase activities. The DTA gene fragment was cut out from pIBI30-DTA plasmid with restriction enzymes and replaced the luciferase gene of recombinant plasmid pGL3-DF3 to construct recombinant plasmid pGL3-DF3-DTA. The constructed pGL3-DF3-DTA eukaryotic plasmid was transient transfected into MCF-7 cells. Successful transfection was confirmed by RT-PCR. After 24 hours, the biological behaviors of the DTA gene transfected breast carcinoma cells were observed.RESULTS The expression vector containing transcriptional regulation sequence of human breast cancer DF3/MUC1 promoter and diphtheria toxin A-chain gene has been successfully constructed and identified by restriction endonuclease and sequencing. The MCF-7 cells transfected with positive recombinant plasmid demonstrated significantly inhibited cell growth in vitro. Obvious apoptosis was observed in morphnogy. The apoptosis ratio compared to control groups was raised by flow cytometry.CONCLUSIONS The expression vector pGL3-DF3-DTA was constructed successfully, and DTA targeting DF3/MUC1 gene has selective killing effect on breast carcinoma cell line and can inhibit its growth. It has potential value in gene therapy of breast carcinoma.
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