牛胰腺核糖核酸酶A的基因克隆表达及初步鉴定-中国农业科学.PDFVIP

中国农业科学 2006,39(6):1248-1252 Scientia Agricultura Sinica 牛胰腺核糖核酸酶A的基因克隆、表达及初步鉴定 1 1 2 1 1 张 泉 ，袁 燕 ，朱鸿飞 ，张鑫宇 ，孙怀昌 1 2 （扬州大学兽医学院，扬州 225009 ； 中国农业科学院生物制品工程技术中心，北京 100081 ） 摘要：【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（RNase A）。【方 法】提取牛胰腺总RNA，利用RT-PCR扩增出牛核糖核酸酶A cDNA，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电泳显示其转化菌经温度诱导后能表 达预计的大小为28 kD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0.5 h， 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RNase A。 关键词：RNase A ; 克隆; 表达; 鉴定 Cloning and Expression of Bovine Pancreatic Ribonuclease A Gene and Preliminary Characterization of the Recombinant Enzyme 1 1 2 1 1 ZHANG Quan , YUAN Yan , ZHU Hong-fei , ZHANG Xin-yu , SUN Huai- chang (1 College of Veterinary Medicine , Yangzhou University, Yangzhou 225009; 2 Bioproducts Engineering Center of Chinese Academy of Agricultural Sciences,Beijing 100081) 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 】SDSanalysis of temperature-induced recombinant E.coli showed that a predicted 28 kD fusion protein was expressed in the periplasmic space. The