牛胰腺核糖核酸酶A的基因克隆、表达及初步鉴定

Cloning and Expression of Bovine Pancreatic Ribonuclease A Gene and Preliminary Characterization of the Recombinant Enzyme

目的为了避免在制备基因治疗或基因免疫的质粒时使用动物源性的核糖核酸酶A(RNaseA)。方法提取牛胰腺总RNA,利用RT-PCR扩增出牛核糖核酸酶AcDNA,RT-PCR产物克隆到pGEM-T载体测序验证后,将此cDNA亚克隆到大肠杆菌分泌型表达载体pEZZ18中。结果SDS-PAGE电泳显示其转化菌经温度诱导后能表达预计的大小为28kD重组蛋白。用渗透法释放出表达产物,将其加入到经碱裂解粗提的质粒DNA中并孵育0.5h,琼脂糖凝胶结果表明表达产物能很好地去除细菌的RNA并且不降解超螺旋质粒DNA。结论在质粒的纯化过程中,重组牛核糖核酸酶可以替代动物源性的RNaseA。

[Objective] The purpose of this study is to avoid the use ofRNase A of animal origin in preparation ofplasmid DNA for gene therapy and gene immunization. [Method] Total RNA was isolated from bovine pancreatic and the correct size of fibonuclease 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 pEZZ 18 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,