水稻黑条矮缩病毒基因组S7编码的2个非结构蛋白在病株中的表达检测

 水稻黑条矮缩病毒（RBSDV）基因组片段S7含有2个非重叠的阅读框，所编码的蛋白分别为p7a和p7b。根据RBSDV S7序列（EU111804）设计特异性引物分别扩增编码p7a和p7b蛋白的基因片段，并亚克隆至原核表达载体pET 32a（+）和pSBET上,再以大肠杆菌BL21(DE3)pLysS或BL21(DE3)pLysE为宿主菌进行高水平表达,利用纯化的蛋白免疫小鼠，制备了p7a和p7b蛋白的特异性抗血清。蛋白质印迹分析表明p7a和p7b蛋白均在水稻病株中表达，但p7a含量较为丰富，易于在病株中检测到，而p7b在病株中含量则很低；在病毒粒子中，两者均未检测到任何信号，表明两者均为RBSDV的非结构蛋白。

Detection of Two Nonstructural Proteins Encoded by Genome Segment S7 of Rice Black-Streaked Dwarf Virus in Infected Rice Plants

The genome segment S7 of Rice black-streaked dwarf virus(RBSDV) contains two non-overlapping major open reading frames(ORFs),which encoded two proteins,p7a and p7b,respectively.Based on the sequence of S7(EU111804),two pairs of S7-specific primers were designed and respectively used to amplify the fragments encoding the p7a and p7b proteins.The expected products were sub-cloned into the expression vector pET-32a(+) or pSBET and expressed at high level in E.coli BL21(DE3)pLysS or BL21(DE3)pLysE,respectively.An antiserum specifically against p7a or p7b proteins was prepared using the purified expression proteins to immunize mice.The results of Western blot revealed that both p7a and p7b proteins were expressed in infected plants,and p7a protein was more abundant and easily detected than p7b protein.However,both have not been detected in the viral particles,indicating that both proteins encoded by RBSDV S7 were nonstructural proteins.