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

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。

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

Based on qRT-PCR method，an expansin（VvEXPA1）was acquired and investigated from infected Crimson Seedless grape and 1103P grape rootstock in present study. VvEXPA1 was cloned into prokaryotic expression vector pET32a after identification by enzymatic digestion and sequencing，and then the recombinant plasmid with VvEXPA1 was transformed into E. coli DE3. 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.