桑花叶卷叶病相关病毒外壳蛋白抗体的制备及利用

对桑花叶卷叶病相关病毒外壳蛋白抗原性较好的核心结构域进行克隆和序列多样性分析,结果表明50个克隆中有19个克隆株,CP16为优势株。构建重组表达载体pET-28a-SUMO-CP16,成功在大肠杆菌Rosetta中诱导表达该蛋白,利用亲和层析纯化重组蛋白并制备多克隆抗体。经Western杂交和ID-ELISA检测合格的抗体偶联Tosylactivated磁珠,用于桑花叶卷叶病相关病毒的纯化。经qPCR技术检测显示,纯化后的病毒样本中的背景RNA得到极大的减少,获得了高纯度的病毒样品。

Preparation and Utilization of Polyclonal Antibody against Coat Protein from Mulberry Mosaic Leaf Roll-associated Virus

The core domain with good antigenicity of coat protein of Mulberry mosaic leaf roll virus (MMLRaV) was cloned and the sequence diversity was analyzed. The results showed that there were 19 clonal stains out of the 50 clones, and the strain CP16 was dominant. The recombinant expression vector pET-28a-SUMO-CP16 was constructed and the CP protein was successfully expressed in E.coli Rosetta. The recombinant protein was purified by affinity chromatography, and the polyclonal antibody was prepared and detected by Western blot and ID-ELISA,and then, the qualified antibody was conjugated with Tosylactivated magnetic beads, which would be applied in the purification of MMLRaV. The background RNA and virus RNA from extraction and the purified virus sample were then detected by qPCR. The results showed that the background RNA greatly reduced, and highly-purified virus samples were obtained.