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  

Construction of truncated mutants of gene encoding bovine doppel and their expression in E.coli

作　　者：杨生海[1,2] 殷宏[1] 刘永生[1] 马艳平[1] 丁耀忠[1] 孙彩琴 丁小丽[3] 张杰[1] 

机构地区：[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 [2]中农威特生物科技股份有限公司,甘肃兰州730046 [3]江苏畜牧兽医职业技术学院,江苏泰州225300

出　　处：《中国兽医学报》2011年第10期1442-1448,共7页Chinese Journal of Veterinary Science

基　　金：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。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 polymerase chain reaction（PCR） and then transformed into E.coli DH5α after ligation with prokaryotic expression vector pET-30a（＋）.The recombinant plasmids identified by double-enzyme digestion and sequencing were introduced into host strain E.coli JM109 and E.coli BL21 and then induced to express with isoproplyl-β-D-thiogalactoside（IPTG）.The molecular weight,relative expression level and reaction to polyclonal antibody against DPL of the fusion proteins were detected by SDS-PAGE,Western blot and indirect ELISA.9 truncated mutants of gene encoding bovine DPL were successfully constructed.6 out of 9 mutants were expressed at high level in E.coli with the induction of IPTG and could well react to the specific antibody againt DPL.The work would lay a material foundation for the analysis of epitopes recognized by the Mab prepared by our laboratory.

关 键 词：牛叠朊 缺失突变体 免疫印迹 酶联免疫吸附分析 

分 类 号：S852.65[农业科学—基础兽医学] R535[农业科学—兽医学]