中国小麦花叶病毒CP和CRP蛋白原核表达抗血清制备及RNA2侵染性克隆构建.docVIP

中国小麦花叶病毒CP和CRP蛋白的原核表达、抗血清制备及RNA2侵染性克隆构建 　　摘要：中国小麦花叶病毒（Chinese wheat mosaic virus，CWMV）引起的小麦土传花叶病在山东烟台、威海等地危害严重。本研究克隆了CWMV烟台分离物的衣壳蛋白（Coat protein，CP）及富含半胱氨酸蛋白（Cysteine-rich protein，CRP）基因，并将其连接到原核表达载体pEHISTEV，转化大肠杆菌Rosetta。经IPTG诱导，表达出分子量均为19 kD的CP和CRP。将二者从凝胶中切下，乳化后免疫新西兰大耳兔4次，获得了两种蛋白的多克隆抗体。ELISA检测表明，CWMV CP和 CRP抗血清的效价分别为1∶4096和1∶2048。Western blot分析证明该抗血清只与感染CWMV的小麦有特异性反应，而与健康或感染小麦黄花叶病毒的小麦无反应。利用含T3启动子的引物通过RT-PCR扩增出CWMV RNA2全长片段，经T/A克隆连接到pMD18-T，获得质粒pMD18-T-CWMV-RNA2。该质粒经Xba Ⅰ线性化后，利用T3 RNA聚合酶进行体外转录，转录产物摩擦接种本氏烟，15℃培养3天后，利用Western blot可从接种叶片中检测到瞬时表达的CWMV CP蛋白。 　　关键词：中国小麦花叶病毒；衣壳蛋白（CP）；富含半胱氨酸蛋白（CRP）；抗血清制备；侵染性克隆 　　中图分类号：S188+S435.121.4 文献标识号：A 文章编号：1001-4942（2015）08-0006-05 　　Abstract Chinese wheat mosaic virus （CWMV） is a furovirus transmitted by Polymyxa graminis and induces soil-borne wheat mosaic disease. It has caused severe damage to winter wheat production in Yantai and Weihai of Shandong Province. The coat protein （CP） and cysteine-rich protein （CRP） genes of CWMV Yantai isolate were amplified by RT-PCR and cloned to prokaryotic expression vector pEHISTEV. The resultant plasmids were transformed into the competent cells of E. coil Rosetta. After induced with IPTG， both plasmids expressed protein with molecular weight of 19 kD. The New Zealand rabbits were immunized four times with CP and CRP cut from SDSrespectively， and the polyclonal antibody of the two proteins were obtained. The titers of antisera against CWMV CP and CRP were 1∶4096 and 1∶2048， respectively. In the Western blot assay， the antisera showed specific positive reaction only with wheat plants infected by CWMV， but not with those infected by WYMV or healthy ones. The full-length RNA2 of CWMV was amplified via RT-PCR with primer containing T3 promoter， and was linked to pMD18-T to produce the plasmid of pMD18-T-CWMV-RNA2. This plasmid was linearized with Xba Ⅰ and used as template for in vitro transcription with T3 RNA polymerase. The in vitro transcription product was mechanically inoculated to Nic