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| 牛叠朊编码基因缺失突变体的构建及在大肠杆菌中的表达 | |
| 引用本文： | 杨生海,殷宏,刘永生,马艳平,丁耀忠,孙彩琴,丁小丽,张杰.牛叠朊编码基因缺失突变体的构建及在大肠杆菌中的表达[J].中国兽医学报,2011,31(10). |
| 作者姓名： | 杨生海 殷宏 刘永生 马艳平 丁耀忠 孙彩琴 丁小丽 张杰 |
| 作者单位： | 1. 中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室,甘肃兰州730046／中农威特生物科技股份有限公司,甘肃兰州730046 2. 中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室,甘肃兰州,730046 3. 中农威特生物科技股份有限公司,甘肃兰州,730046 4. 江苏畜牧兽医职业技术学院,江苏泰州,225300 |
| 基金项目： | 国家自然科学基金资助项目(30671563) |
| 摘 要： | 本实验室已制备出多株针对牛叠朊的单克隆抗体,为充分鉴定这些单克隆抗体针对的抗原表位,通过基因克隆表达的策略获取牛叠朊基因缺失突变体的重组蛋白,以期为单克隆抗体的表位鉴定提供物质材料。经过对牛成熟叠朊编码基因及预测蛋白结构特征的分析,设计表达9个缺失突变体的引物。以PCR扩增出叠朊基因的缺失突变体,与pET-30a（＋）表达载体连接后转入E.coli DH5α中,经双酶切和测序鉴定后将重组质粒转入E.coliJM109和E.coli BL21表达宿主菌中,经IPTG诱导表达后,以SDS-PAGE、Western blot和ELISA检测表达产物的相对分子质量、相对表达量和反应原性。结果表明,成功构建了9个牛叠朊编码基因的缺失突变体,通过IPTG的诱导表达,其中有6个缺失突变体被大肠杆菌高效表达,并且具有较好的反应原性,这为其单克隆抗体表位的进一步鉴定奠定了物质基础。 |
| 关 键 词： | 牛叠朊 缺失突变体 免疫印迹 酶联免疫吸附分析 |
Construction of truncated mutants of gene encoding bovine doppel and their expression in E.coli |
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| YANG Sheng-hai,YIN Hong,LIU Yong-sheng,MA Yan-ping,DING Yao-zhong,SUN Cai-qin,DING Xiao-li,ZHANG Jie.Construction of truncated mutants of gene encoding bovine doppel and their expression in E.coli[J].Chinese Journal of Veterinary Science,2011,31(10). | |
| Authors: | YANG Sheng-hai YIN Hong LIU Yong-sheng MA Yan-ping DING Yao-zhong SUN Cai-qin DING Xiao-li ZHANG Jie |
| Institution: | YANG Sheng-hai1,2,YIN Hong1,LIU Yong-sheng1,MA Yan-ping1,DING Yao-zhong1,SUN Cai-qin2,DING Xiao-li3,ZHANG Jie1*(1.Key Laboratory of Animal Virology of Ministry of Agriculture,State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China,2.China Agricultural Veterinarian Biology Science and Technology Co.Ltd.,3.Jiangsu Animal Husbandry and Veterinary College,Taizhou,Jiangsu 225300,China) |
| Abstract: | Several monoclonal antibody(Mab) against bovine doppel(DPL) have been prepared by our laboratory.In order to identify the epitopes recognized by these Mabs,truncated mutants of genes encoding bovine DPL were developed through gene cloning and the corresponding recombinant proteins were generated by their expression in E.coli.The prepared fusion proteins could provide materials for the analysis of epitopes of the Mabs.Primers for cloning 9 truncated mutants were designed.The truncated mutants were cloned by ... |
| Keywords: | bovine doppel truncated mutants Western blot ELISA |
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