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中国小麦花叶病毒(Chinese wheat mosaic virus,CWMV)是引起我国小麦花叶病的重要病原之一,其基因组由2条单链正义RNA片段(RNA1-2)组成。本研究根据发表的基因组序列设计特异性引物,扩增了CWMV复制酶基因的部分片段(nt102~1101),并克隆至原核表达载体pGEX-6P1,然后导入大肠杆菌E.coli BL21(DE3)pLysS中诱导表达。重组的复制酶蛋白经亲和层析纯化后免疫家兔制备多克隆抗体。Western-blot分析表明该抗体具有高度的特异性,能用于病株体内CWMV复制酶蛋白的检测。检测分析显示在病株体内,CWMV基因组RNA1可直接充当其复制酶基因的mRNA;在感染的细胞中,其复制酶组分主要是RNA1 ORF1编码的蛋白,分子量约153 kDa,且特异性地定位于膜结构上。
Chinese wheat mosaic virus (CWMV) is one of the important pathogens causing wheat mosaic disease in China. Its genome consists of two single-stranded sense RNA fragments (RNA1-2). In this study, partial primers of CWMV replicase gene (nt102 ~ 1101) were amplified and cloned into prokaryotic expression vector pGEX-6P1 and then introduced into E.coli BL21 (DE3) pLysS Inducible expression. The recombinant replicase protein was purified by affinity chromatography and immunized rabbits to prepare polyclonal antibodies. Western-blot analysis showed that the antibody was highly specific and could be used for the detection of CWMV replicase protein in diseased plants. The results showed that the CWMV genomic RNA1 could directly act as the mRNA of the replicase gene in the diseased plants. In the infected cells, the replicase components were mainly RNA1 ORF1-encoding proteins with a molecular weight of about 153 kDa and were specifically located On the membrane structure.