[In vitro cleavage action of c-erbB-2 oncogene-specific ribozyme].
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.


CONCLUSIONS
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.
Bi
F
F
Institute of Digestive Diseases of PLA, Fourth Military Medical University, Xian 710032.
Zhang
X
X
Fan
D
D
D018734:Genes, erbB-2; D015316:Genetic Therapy; D016337:RNA, Catalytic / Q000494:pharmacology* / Q000627:therapeutic use
D016337:RNA, Catalytic