桑脉带相关病毒N蛋白多克隆抗体的制备

 桑脉带相关病毒(Mulberry vein banding associated virus,MVBaV)属于番茄斑萎病毒科(Tospoviridae)正番茄斑萎病毒属(Orthotospovirus)的新成员,是广西桑树病毒病的主要病原病毒。本研究将MVBaV核外壳蛋白(nucleocapsid protein, N protein)基因连接到pET-30a原核表达载体上,获得重组表达载体pET-30a-MVBaV-N并转化大肠杆菌菌株BL21(DE3),经IPTG诱导可表达产生一个含His标签、分子量约36.0 kDa的融合蛋白。用 Ni^{2 +} -NTA树脂纯化融合蛋白,将其免疫日本大耳兔制备多克隆抗体。间接ELISA测定抗体的效价为1∶256 000。Western blot 检测结果显示,该多克隆抗体在稀释倍数1∶1 000时与细菌表达的融合N蛋白及感病桑叶中的N蛋白产生特异性识别,但不与寄主蛋白产生交叉反应,表明具有良好的特异性和较高的灵敏度。将抗体按1∶2 500稀释后进行间接ELISA,可有效检出桑叶中的MVBaV。MVBaV N蛋白抗体的成功研制,为桑脉带病毒病的诊断及MVBaV与寄主相互作用机制的研究提供了重要的试剂。

Preparation of polyclonal antibodies against nucleocapsid protein of mulberry vein banding associated virus

Mulberry vein banding associated virus (MVBaV), the main pathogenic virus of mulberry viral disease in Guangxi Zhuang Autonomous Region, belongs to the genus Orthotospovirus in the family Tospoviridae. The nucleocapsid protein (N) gene of MVBaV was cloned into prokaryotic expression vector pET-30a and the construct was used to transform Escherichia coli strain BL21 (DE3). The His-tagged fusion protein with an apparent molecular weight of 36 kDa was expressed after induction by IPTG. The Ni-NTA resin purified fusion protein was used to immunize Japanese big-ear rabbits. The prepared polyclonal antibody had a titer of 1∶256 000 as tested with the indirect enzyme-linked immunosorbent assay (ELISA). Western blot analysis showed that the antibody in dilution of 1∶1 000 reacted specifically with prokaryotic expressed fusion protein and protein sample prepared from the MVBaV-infected mulberry leaves, indicating that the antibody had a relatively high efficiency and specificity. In ELISA assays with the antibodies in dilution of 1∶2 500, MVBaV infected mulberry could be detected. The successful development of polyclonal antibody against the N protein of MVBaV provides an important reagent for diagnosis and surveillance of MVBaV in the field, and also for the study of interaction between MVBaV and mulberry.