马铃薯M病毒衣壳蛋白原核表达和多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of the Potato Virus M Coat Protein

本研究利用RT-PCR技术从发病马铃薯植株叶片中克隆马铃薯M病毒(Potato virus M,PVM)的衣壳蛋白全长基因,该基因由912个碱基组成。将其亚克隆到表达载体pET-28b(+)中后,转化大肠杆菌菌株Rosetta。经IPTG诱导处理后,获得的重组蛋白大小约为33 kDa,与预期大小一致。经镍离子亲和层析纯化后免疫日本大耳白兔,制备多克隆抗体,免疫4次后多抗血清效价为1∶64 000。经ELISA和Western blot分析表明,多克隆抗体能够特异性的识别PVM。PVM多克隆抗体的制备为PVM快速检测方法的建立奠定了基础。

Coat Protein coding gene of Potato Virus M（PVM） was cloned from leaf of infected potato plant by RT-PCR, and it was composed of 912 base pairs. It was subcloned to pET-28b（＋）, the construct was then transformedinto E.coli Rosetta. Induced by IPTG, coat protein was expressed as a 33 kDa protein. After the purification by Ni-NTA resin, the purified protein was used as the antigen to immunize rabbits. The serum titer reached to 1：64 000 af-ter the forth immunization. These anti-serums can recognize specifically natural PVM that was proved by ELISAand Western blot analysis. The preparation of polyclonal antibody against PVM laid a foundation for the establish-ment of rapid detection method for PVM.