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期 刊 ：植物保护学报 2005年

关键词：烟草蚀纹病毒；外壳蛋白基因；原核表达；抗血清；

Keywords:Tobacco etch virus, coat protein gene, prokaryotic expression, viral-specific antiserum,

摘 要 ：从山东省感病的烟草上分离获得了一烟草蚀纹病毒(TEV)分离物,命名为TEV-SD1。根据已报道烟草蚀纹病毒外壳蛋白(coat protein CP)基因序列设计并合成2条引物,通过RT-PCR扩增得到长度约800 bp的目的片段 。将目的片段与质粒pET-22b( + )连接,构建了包含TEV CP基因的原核表达载体pETEV-CP,并导入大肠杆菌BL21中。序列分析表明: TEV-SD1的CP基因全长789 bp,编码263个氨基酸;与GenBank中已报道的TEV 5个分离物CP基因相比,核苷酸及推导的氨基酸序列同源性为94.1%~98.6%。37 ℃培养条件下,BL21/pETEV-CP经IPTG诱导表达,SDS-PAGE结果显示,表达的TEV CP融合蛋白的相对分子质量约为33 kDa。以表达的融合蛋白为抗原,免疫家兔,制备的抗血清的效价为1/2 048,且具有良好的特异性。

Abstract:An isolate of Tobacco etch virus (TEV) was obtained from tobacco plants in Shandong Province, which was named as TEV-SD1. The coat protein (CP) gene of TEV-SD1 was cloned by reverse transcription-polymerase chain reaction (RT-PCR), and was subcloned into pET-22b ( + ) prokaryotic expression vector. The recombinant vector pETEV-CP was transformed into BL21 host strain of E.coli. Sequence analysis revealed that the CP genes was 789 nucleotides in length, which encodes the coat protein of 263 amino acids, and the CP genes shared 94.1% -98.6% nucleotides and amino acid homology with TEV CP genes of five strains registered in GenBank. The target fusion peptide with molecular weight of 33kDa was expressed under the condition of 37℃ and induced by IPTG at the final concentration of 1mmol/L. Rabbit was immunized using the expressed target peptide as antigen, and the antiserum was obtained. The antiserum gave a titer of 1/2 048 with high specificity to TEV.

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