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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。
The tissue culture plantlets of ’Vitis vinifera’ Crimson Seedless’ and the resistant plantlet ’1103P’ which were susceptible to Phyllostachys nauplius were used as explants. The fluorescence quantitative PCR We selected the VvEXPA1 family member of the expansin gene whose expression was up-regulated after phylloxera infection. The VvEXPA1 gene fragment was cloned by RT-PCR and ligated into the prokaryotic expression vector pET-32a. The recombinant plasmid was transformed into E. coli DE3. The recombinant plasmid pET32a-VvEXPA1 was verified by open reading frame. The fusion protein was induced by IPTG. The protein was in the form of inclusion body. After purification, New Zealand rabbits were immunized to prepare the polyclonal antibody against expansin. The specificity and titer of the antibody were detected by Western-blot and ELISA. The results show that the prepared antibodies have high specificity and the ELISA shows a titer of 1:51 200.