大麦黄矮病毒PAV青海分离物运动蛋白抗血清制备及其与同属BYDVs的血清学关系

Antiserum preparation of the movement protein of Barley yellow dwarf virus PAV Qinghai isolate and its serological relationship with other BYDVs

大麦黄矮病毒(Barley yellow dwarf viruses, BYDVs)属于黄症病毒科,主要以蚜虫为介体进行传播,引发多种作物减产或绝收。本文将BYDV-PAV青海分离物的运动蛋白(MP)克隆到原核表达载体pDB-MBP-His上,转化大肠杆菌Rosetta(DE3),在IPTG诱导下表达蛋白分子量约为61 kDa的融合蛋白。将纯化后的蛋白作为抗原免疫‘新西兰’大白兔制备抗血清,Western blot检测本生烟瞬时表达带标签的MP蛋白,结果显示该抗血清效价为1∶32 000,灵敏度达1∶256,且该抗血清可与相差34个氨基酸的PAV015株系以及隶属于黄症病毒属其他的BYDVs(PAS、MAV、KerⅡ和KerⅢ)MP均能发生强烈的血清学反应,具有血清学相关性。本研究制备的大麦黄矮病毒PAV青海分离物运动蛋白多克隆抗体为探索BYDV-PAV运动蛋白的功能以及作用机制打下了基础。

Barley yellow dwarf viruses(BYDVs) belong to the family Luteoviridae, which are mainly transmitted by aphids, causing crop losses or failures in many crops. In this study, movement protein(MP) gene of Barley yellow dwarf virus(BYDV)-PAV Qinghai isolate was integrated into a prokaryotic expression vector pDB-MBP-His, and transformed to the Escherichia coli strain Rosetta(DE3). The fusion MP(61 kDa) was induced by IPTG, and the ‘New Zealand’ rabbits were used to prepare the specific antiserum by immunization. The PAV-MP-3 Flag fusion protein which was transient expressed in Nicotiana benthamiana. Western blot results showed that the titer of antiserum reached 1∶32 000 and the detection sensitivity was 1∶256. Moreover, the antiserum can produce strong serological reaction with the MP of PAV015 isolate and other BYDVs(PAS、MAV、Ker Ⅱ and Ker Ⅲ) belonging to the genus Luteovirus. The polyclonal antibody against the movement protein of BYDV-PAV Qinghai isolate prepared in this work provides the basis for exploring the function and mechanism of BYDV-PAV movement protein.