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采用RT-PCR方法从甘肃地区引种的切花百合上克隆了百合斑驳病毒(Lily mottle virus,LMoV)的外壳蛋白(coatprotein,CP)基因与细胞质内含体(cytoplasmic inclusions,CI)基因的3′端600 bp片段,连接到原核表达载体pET-28a(+)上,构建了融合表达的重组质粒pET-28a-CP-CI200。重组质粒转化大肠杆菌BL21成功表达了融合蛋白CP-CI200。经镍柱亲和层析获得纯化的融合蛋白,进一步用其免疫家兔制备了多克隆抗体。Western Blot结果显示,该多抗与感染LMoV的百合叶片中的病毒CP与CI均发生特异性反应,而对健康百合叶片无反应。用该多抗建立双抗夹心酶联免疫吸附法(DAS-ELISA)检测百合叶片样品,相对于RT-PCR方法其灵敏度和特异性均达到了93.3%。研究结果为快速、准确检测百合斑驳病毒的试剂盒研制奠定了基础。
The coat protein (CP) gene of Lily mottle virus (LMoV) and the 3 ’end of the cytoplasmic inclusions (CI) gene were cloned by RT-PCR from cut flowers lily introduced from Gansu. 600 bp fragment was ligated to the prokaryotic expression vector pET-28a (+) to construct the recombinant plasmid pET-28a-CP-CI200. The recombinant plasmid was transformed into E. coli BL21 and successfully expressed the fusion protein CP-CI200. The purified fusion protein was obtained by nickel column affinity chromatography, and the polyclonal antibody was further prepared by immunizing rabbits. The results of Western Blot showed that the polyclonal antibodies specifically reacted with the virus CP and CI in the lily leaves infected with LMoV, but did not respond to healthy lily leaves. The sensitivity and specificity of this polyclonal antibody assayed by DAS-ELISA to detect lily leaf samples were 93.3% compared with RT-PCR. The results laid the foundation for the rapid and accurate detection of lily mottle virus kit.