海南五指山美洲商陆抗病毒蛋白PAP-Ⅰ基因的原核表达及活性验证  

Expression and Identification of Pokeweed Antiviral Protein PAP-Ⅰ Gene in Escherichia coli in Wuzhi Mountain in Hainan Island

作　　者：黄超[1,2,3] 张丽丽[2] 邓柳红 张春发[2] 

机构地区：[1]海南大学农学院,海南海口570228 [2]中国热带农业科学院热带生物技术研究所,海南海口571101 [3]海南省蛋白质工程药物重点实验室/海口市生命科学药物工程技术研究开发中心,海南海口570311

出　　处：《热带生物学报》2016年第1期82-88,共7页Journal of Tropical Biology

基　　金：海南省重大科技项目(ZDZX2013008-04-03);海口市重点科技计划项目(2012-060);国家自然科学基金项目(31101200)

摘　　要：提取春季海南五指山美洲商陆叶片总RNA,利用RT-PCR技术获得c DNA,再根据Genbank上公布的PAP-Ⅰ基因序列设计引物扩增PAP-Ⅰ基因,并连接到p^(ET)-30a载体上进行表达,表达的产物以不溶的包涵体形式存在,经过变性、镍柱亲和层析纯化,纯化后的重组蛋白经过Western Blot验证表明该基因得到了正确表达,利用柱上循环复性后的重组PAP-Ⅰ蛋白经麦胚提取物转录/翻译偶联系统验证具有抑制蛋白质合成的活性。Total RNA was extracted from pokeweed leaves in spring at Wuzhi Mountain in Hainan,and c DNA was obtained using RT-PCR technology. After the gene PAP-Ⅰ was amplified according to the primers which were designed based on the sequence published on the Genbank. The PAP-Ⅰ gene was connected to the vector p（ET）-30a for expression. The product was in the form of insoluble inclusion body. This product was denatured and purified through nickel column chromatography. The purified recombinant protein was verified by Western Blot,which indicated that the gene was expressed correctly. The recombinant protein PAP-Ⅰ was proved to have the activity to inhibit the protein synthesis through wheat germ extract transcription / translation coupling system.

关 键 词：美洲商陆 抗病毒蛋白 载体构建 原核表达 海南五指山 

分 类 号：Q786[生物学—分子生物学]