关键词: RNase A, 克隆, 表达, 鉴定

Abstract: 【Objective】The purpose of this study is to avoid the use of RNase A of animal origin in preparation of plasmid DNA for gene therapy and gene immunization.【Method】Total RNA was isolated from bovine pancreatic and the correct size of ribonuclease A gene was amplified by RT-PCR. The RT-PCR product was subcloned into pGEM-T vector for sequencing. Then the gene of correct sequence was subcloned into prokaryotic expression vector pEZZ18 for expression as an EcoRI /BamHI fragment.【Result】SDS-PAGE analysis of temperature-induced recombinant E.coli showed that a predicted 28 kD fusion protein was expressed in the periplasmic space. The products was added into the crude plasmid isolated by alkaline lysis, and incubated at 37℃ for half an hour, the agrose gel electrophoresis showed that the RNA was removed, and the plasmid, particularly the supercoiled plasmid was not degraded.【Conclusion】These results demonstrated that the recombinant protein can hydrolysis the RNA and could be used in purifying plasmid DNA as a substitute for the RNase A from animal origin.

Key words: Ribonuclease A, Cloning, Expression, Characterization