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[In vitro cleavage action of c-erbB-2 oncogene-specific ribozyme]
1Institute of Digestive Diseases of PLA, Fourth Military Medical University, Xian 710032.
Objective:
To construct in vitro transcription vectors of genes of c-erbB-2 specific ribozyme and its substrate and to probe its in vitro cleavage action.
Methods:
According to the computer design, a specific restriction site EcoR V was added to the 3' end of the ribozyme gene (RZ1). Then, the RZ1 gene and its substrate gene were cloned into the in vitro transcription vector pGEM3Zf(-) separately. The recombinants containing RZ1 gene were first screened by agrose ge1 electrophoresis through EcoR V digestion and was identified by automatic sequencing. The products of in vitro transcription were labeled with 32P. In vitro cleavage reaction was performed at 37 degrees C for 1 h under the presence of Mg++. The cleavage product was analyzed by polyacrilamide gel electrophoresis. After autoradiography, the cleavage rate was counted by image analysis.
Results:
The recombinants containing the RZ1 gene were successfully selected by the EcoR V digestion and were designated as pGM3Z-RZ1. The automatic sequence analysis proved that the synthesized RZ1 gene was correct. The target gene was also cloned into the pGEM3Zf(-) under SP6 promoter. After in vitro transcription, the cleavage reaction was shown to have cut off 79.3% target RNA in 1 h.
Conclusion:
The c-erbB-2 oncogene-specific ribozyme has a high activity in vitro. It lays a foundation for the study of the therapeutic use of ribozyme in gene therapy of cancer.