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用CTAB法从感染草莓镶脉病毒(Strawberry vein banding virus,SVBV)的草莓叶片中提取总DNA,设计特异性引物扩增SVBV中国分离物ORFⅡ基因,克隆并测序。结果表明,SVBV中国分离物ORFⅡ基因全长486 nts,编码161个氨基酸。将其与SVBV美国分离物以及花椰菜花叶病毒属其他成员的ORFⅡ基因相比较,结果表明,SVBV中国分离物ORFⅡ基因与SVBV美国分离物的核苷酸序列相似性最高,达91.2%。将SVBV ORFⅡ基因插入原核表达载体pET32a(+),重组质粒pET-ORFⅡ转化大肠杆菌BL21(DE3)。经IPTG诱导及Ni2+-NTA亲和柱纯化,获得分子质量约为38 kDa的融合蛋白。以此纯化的融合蛋白为抗原免疫家兔,可以制备出高效价的抗血清。Western blot分析表明,制备的抗血清能与重组融合蛋白发生特异性反应。利用抗血清进行ELISA测定,能够检测出感病草莓植株病汁液中SVBV ORFⅡ基因表达的蛋白。
Total DNA was extracted from strawberries infected with Strawberry vein banding virus (SVBV) by CTAB method. Specific primers were designed to amplify the ORF Ⅱ gene of SVBV isolates in China and cloned and sequenced. The results showed that the ORF Ⅱ gene of SVBV isolates from China was 486 nts in length and encoded 161 amino acids. The results showed that the ORFⅡ gene of SVBV isolates from China shared the highest similarity with the SVBV isolates, which was 91.2%. The SVBV ORF Ⅱ gene was inserted into the prokaryotic expression vector pET32a (+), and the recombinant plasmid pET-ORFⅡ was transformed into E. coli BL21 (DE3). After IPTG induction and Ni2 + -NTA affinity column purification, the molecular weight of about 38 kDa fusion protein was obtained. Using this purified fusion protein as antigen to immunize rabbits, high titer antiserum can be prepared. Western blot analysis showed that the prepared antiserum reacted specifically with the recombinant fusion protein. Using antiserum for ELISA assay, the protein of SVBV ORF Ⅱ gene in strawberry juice could be detected.