百合斑驳病毒CP与CI基因的融合表达、多抗制备及其应用  

Production and application of antisera to expression product of CP and CI fusion gene of Lily mottle virus

作　　者：童勋章[1,2] 王亚军[1,2] 谢忠奎[1,2] 张玉宝[1,2] 安丽萍[1,2] 郭志鸿[1,2] 郭芳[1,2] 

机构地区：[1]中科院寒区旱区环境与工程研究所皋兰生态与农业综合试验站,兰州730000 [2]中科院寒区旱区环境与工程研究所极端环境生物抗逆特性与生物技术实验室,兰州730000

出　　处：《植物病理学报》2010年第5期475-481,共7页Acta Phytopathologica Sinica

基　　金：中国科学院科技支甘工程专项(KJZG-2008-3);中国科学院重要方向性项目(KSCX2-YW-N-44-07)

摘　　要：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 cDNA fragments of CP gene and the 3′-terminal of CI gene of Lily mottle virus（LMoV） were amplified by RT-PCR from imported cutflower lilies in Gansu Province.The amplified PCR fragments were then inserted into the vector pET-28a（＋）,and the recombinant plasmid pET-28a-CP-CI200 was constructed.After transformation of the recombinant pET-28a-CP-CI200 to E.coli BL21 strain,the fusion protein was induced expressed.The purified protein was obtained by Ni^2＋ affinity chromatography.After renatu-ration,the protein was used to raise polyclonal antibodies in rabbit.Western blot analysis showed that the antisera were specifically reacted to viral CP and CI of LmoV-infected lily leaf but not to healthy leaf.A DAS-ELISA method was developed with the polyclonal antibodies.Sixty lily leaf samples were detected by both DAS-ELISA and RT-PCR method respectively.The results revealed that compared with RT-PCR method,DAS-ELISA had a specifity of 93.3% and sensitivity of 93.3%.

关 键 词：百合斑驳病毒 外壳蛋白 细胞质内含体 抗体 DAS-ELISA 病毒检测 

分 类 号：S432.4[农业科学—植物病理学]