Prokaryotic Expression and Antiserum Production of Chinese Wheat Mosaic Virus (CWMV)CP and CRP and Construction of Their RNA2 Infection Clones
Kong Fanhui
Tuo Jianbo
Wei Jiao
Tang Wei
Li Xiangdong
Chi Shengqi
Tian Yanping
Yu Jinfeng
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.
Keywords：Chinese wheat mosaic virus (CWMV)Coat protein (CP)Cysteine -rich protein (CRP)Antiserum preparationInfectious clone
Publication Date：2015-08-01
Online Publishing Date：2026-05-22(First online date of this platform, not the publication date of the document)
Pages：5( 6-10 )