山东农业科学Issue(8)：6-10,5.DOI:10.14083/j.issn.1001-4942.2015.08.002
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
Prokaryotic Expression and Antiserum Production of Chinese Wheat Mosaic Virus (CWMV)CP and CRP and Construction of Their RNA2 Infection Clones
摘要
Abstract
Chinese wheat mosaic virus (CWMV)is a furovirus transmitted by Polymyxa graminis and in-duces 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 result-ant plasmids were transformed into the competent cells of E.coil Rosetta.After induced with IPTG,both plas-mids expressed protein with molecular weight of 19 kD.The New Zealand rabbits were immunized four times with CP and CRP cut from SDS -PAGE respectively,and the polyclonal antibody of the two proteins were ob-tained.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 tran-scription with T3 RNA polymerase.The in vitro transcription product was mechanically inoculated to Nicotiana benthamiana leaves,and a band specific for CWMV CP was detected from the inoculated N.benthamiana leaves after three days.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Key words
Chinese wheat mosaic virus (CWMV)/Coat protein (CP)/Cysteine -rich protein (CRP)/Antiserum preparation/Infectious clone分类
农业科技引用本文复制引用
孔凡惠,脱建波,魏娇,唐伟,李向东,迟胜起,田延平,于金凤..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[J].山东农业科学,2015,(8):6-10,5.基金项目
国家公益性行业（农业）科研项目（201303021）；山东省现代农业产业技术体系 （）
