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作 者 ：蒋磊¹,邓竹根¹,谢朝阳¹,杨友志¹,李祥宇¹,丁菲² ,江彤¹

期 刊 ：植物病理学报 2013年 43卷 1期 页码：20-26

关键词：草莓镶脉病毒；ORF Ⅱ基因；克隆；原核表达；多抗血清；

Keywords:Strawberry vein banding virus, gene ORF Ⅱ, clone, prokaryotic expression, antiserum,

摘 要 ：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ORF Ⅱ基因,克隆并测序。结果表明,SVBV中国分离物ORF Ⅱ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ORF Ⅱ基因相比较,结果表明,SVBV中国分离物 ORF Ⅱ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 ORF Ⅱ基因插入原核表达载体pET32a（+）,重组质粒pET-ORF Ⅱ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 ORF Ⅱ基因表达的蛋白。

Abstract:The total DNA was extracted from strawberry leaves infected with Strawberry vein banding virus (SVBV) by CTAB method. Specific primer pair was designed to amplify the gene ORF Ⅱ of SVBV-China isolate, and then the fragment was cloned and sequenced. The result showed that the full length of gene ORF Ⅱ of SVBV-China isolate was 486 nts, encoding 161 amino acids. Comparing the sequence of gene ORF Ⅱ of SVBV-China isolate with those of SVBV-USA isolate and other members of Caulimovirus, the result indicated that gene ORF Ⅱ of SVBV-China isolate shared the highest sequence similarity (91.2%) with that of SVBV-USA isolate. The SVBV gene ORF Ⅱ was inserted into the prokaryotic expression vector pET-32a(+), and the recombinant plasmid was transformed into E. coli BL21 (DE3). The fusion protein with an approximate molecular weight of 38 kDa was obtained with IPTG induction and Ni²⁺-NTA affinity column purification. The purified recombinant fusion protein was used to immunize rabbits and obtained the antiserum of a high titer. Western blot analysis indicated that the prepared antiserum could specifically bind to purified recombinant fusion protein. ELISA was conducted with antiserum, and the protein expressed by SVBV gene ORF Ⅱ could be detected in the sap of infected strawberry leaves.

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