响应葡萄根瘤蚜侵染的扩展蛋白基因筛选及其多克隆抗体的制备  

Identification of VvEXP Gene from Grape Roots Infected by Phylloxera and Preparation of Polyclonal Antibody to Grape Expansin

作　　者：季兴龙 韩宁[2] 翟衡[1] 杜远鹏[1] 

机构地区：[1]山东农业大学园艺科学与工程学院,作物生物学国家重点实验室,山东泰安271018 [2]齐鲁工业大学生物工程学院,济南250353

出　　处：《园艺学报》2016年第1期15-24,共10页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31201609);山东省中青年科学家基金项目(BS2012NY007);山东省自然科学基金项目(ZR2013CQ022);高等学校博士学科点专项科研基金项目(20113702120007)

摘　　要：以对葡萄根瘤蚜敏感的‘克瑞森无核’葡萄(Vitis vinifera ‘Crimson Seedless’)组培苗和抗性砧木‘1103P’组培苗为试材,通过荧光定量PCR技术从扩展蛋白基因家族中筛选到经根瘤蚜侵染后表达上调的扩展蛋白基因家族成员VvEXPA1。采用RT-PCR方法克隆到VvEXPA1基因片段,连接至原核表达载体pET-32a中,转化大肠杆菌DE3菌株,测序确定构建的重组载体pET32a-VvEXPA1开放阅读框正确,并经IPTG诱导表达了融合蛋白。该蛋白以包涵体的形式存在,纯化后免疫新西兰兔,制备扩展蛋白多克隆抗体,通过Western-blot和ELISA检测抗体的特异性及效价。结果显示制备的抗体具有较高的特异性,ELISA显示效价为1:51 200。Based on qRT-PCR method,an expansin（VvEXPA1）was acquired and investigated from infected Crimson Seedless grape and 1103 P grape rootstock in present study. VvEXPA1 was cloned into prokaryotic expression vector pET32 a after identification by enzymatic digestion and sequencing,and then the recombinant plasmid with VvEXPA1 was transformed into E. coliDE3. The results showed that the protein was highly expressed in E. coli and the molecular weight of the recombinant protein was 41 kD. The specific antibody was prepared by immunizing rabbit with the purified protein,and then detected by Western-blotting and ELISA. Finally, the high specific antibody was obtained and the titers of anti-VvEXPA1 antibody were 1︰51 200.

关 键 词：葡萄 根瘤蚜 扩展蛋白 VvEXPA1 多克隆抗体制备 

分 类 号：S663.1[农业科学—果树学]