Construction of PR domain eukaryotic expression vector and its inhibitory effect on esophageal cancer cells
Abstract
Objective: PR domain is responsible for the tumor suppressing activity of RIZ1. The study aimed to construct human PR domain eukaryotic expression vectors, transfect human esophageal cancer cells (TE13), and evaluate the anticancer activity of PR domain on human esophageal cancer TE13 cells.
Methods: First, mRNA was extracted from human esophageal cancer tissue by RT-PCR, then reverse-transcribed to cDNA. After amplifying from the DNA template, PR domain was linked to T vector. Second, after extraction, PR domain was cut using enzyme and linked to pcDNA3.1(+). Then, the plasmid was transfered to Trans1-T1 phage resistant competent cells, following by extracting the ultrapure plasmid, and transfecting into TE13 cells. In the end, the protein expression of pcDNA3.1(+)/PR domain in TE13 was detected by Western blot, and the apoptosis of TE13 by technique of flow cytometry.
Results: More than 5,000 bp purposed band of pcDNA3.1(+)/PR domain plasmid was found by agarose gel electrophoresis. After transfection, the PR domain (molecular weight of about 28 Da) was found only in 3, 4 and 5 groups by Western blot. Flow cytometry assay showed apoptosis in experimental group was significantly more than that in the control group (P<0.05).
Conclusions: The PR domain eukaryotic expression vector was constructed successfully. The protein of the PR domain could be expressed in esophageal cancer TE13 cells firmly after transfection, and a single PR domain could promote apoptosis of TE13 cells.
Methods: First, mRNA was extracted from human esophageal cancer tissue by RT-PCR, then reverse-transcribed to cDNA. After amplifying from the DNA template, PR domain was linked to T vector. Second, after extraction, PR domain was cut using enzyme and linked to pcDNA3.1(+). Then, the plasmid was transfered to Trans1-T1 phage resistant competent cells, following by extracting the ultrapure plasmid, and transfecting into TE13 cells. In the end, the protein expression of pcDNA3.1(+)/PR domain in TE13 was detected by Western blot, and the apoptosis of TE13 by technique of flow cytometry.
Results: More than 5,000 bp purposed band of pcDNA3.1(+)/PR domain plasmid was found by agarose gel electrophoresis. After transfection, the PR domain (molecular weight of about 28 Da) was found only in 3, 4 and 5 groups by Western blot. Flow cytometry assay showed apoptosis in experimental group was significantly more than that in the control group (P<0.05).
Conclusions: The PR domain eukaryotic expression vector was constructed successfully. The protein of the PR domain could be expressed in esophageal cancer TE13 cells firmly after transfection, and a single PR domain could promote apoptosis of TE13 cells.