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用RT-PCR方法从长春感染芜菁花叶病毒(Turnip mosaic virus,TuMV)的十字花科蔬菜中扩增获得该病毒的p3基因,并对其序列进行了比较分析。结果表明,本研究所获得的10个TuM V分离物p3基因含1 065个核苷酸,其序列一致率为98.8%~99.6%,与GenBank中其他15个TuMV分离物核苷酸一致率为80.9%~99.4%。根据p3基因核苷酸序列构建的系统进化树显示:25个TuMV分离物可分为4个组,本研究得到的10个TuMV分离物均属于basal-BR组。将TuMV JCR06分离物p3基因N端663 by片段克隆至原核表达载体pET-28a(+)并在大肠杆菌BL21(DE3)pLysS中表达出分子量约为28 kDa的融合蛋白。以纯化的融合蛋白为抗原免疫家兔,制备了P3蛋白的特异性抗血清。以TuMV侵染的萝卜为抗原,间接ELISA测定抗血清的效价为1:2 048。Western blotting分析表明,制备的抗血清能与诱导表达的融合蛋白发生特异性反应。
The p3 gene of this virus was amplified by RT-PCR from cruciferous vegetables of Changchun-infected Turnip mosaic virus (TuMV), and its sequence was analyzed comparatively. The results showed that the pM gene of 10 TuMV isolates contained 1065 nucleotides in this study, and their sequence identities were 98.8% -99.6%. The nucleotide identities of the three TuMV isolates with other 15 TuMV isolates in GenBank were 80.9% ~ 99.4%. Phylogenetic tree based on the nucleotide sequence of p3 showed that 25 TuMV isolates could be divided into 4 groups. All 10 TuMV isolates in this study belonged to basal-BR group. The N-terminal 663 by fragment of the TuMV JCR06 isolate p3 gene was cloned into the prokaryotic expression vector pET-28a (+) and the fusion protein with a molecular weight of about 28 kDa was expressed in E. coli BL21 (DE3) pLysS. The purified fusion protein was used as antigen to immunize rabbits to prepare the specific antiserum of P3 protein. The TuMV-infected radish was used as the antigen and the titer of the indirect ELISA was 1: 2,048. Western blotting analysis showed that the prepared antiserum reacted specifically with the induced fusion protein.