摘要
为了进行水稻草状矮缩病毒(Rice grassy stunt virus,RGSV)诊断方法和蛋白功能研究。通过RT-PCR方法从感染RGSV的水稻中克隆该病毒的P2基因,并将此基因片段重组到原核表达载体pDEST17上。将重组载体转化E.coli Rosetta,经IPTG诱导后获得分子量约为29kDa含HIS标签的融合蛋白。以诱导的融合蛋白为抗原,免疫新西兰大耳白兔获得多克隆抗体,经酶联免疫检测发现其效价达到l:8192。并用制备的抗体建立了特异、灵敏的检测RGSV的IC-RT-PCR和Dot-blot ELISA方法,为该病毒的检测、诊断提供了保障,同时也为P2蛋白的结构和功能研究奠定了基础。
P2 gene of Rice grassy stunt virus was amplified by RT-PCR from the infected rice leaves, and it was subcloned into prokaryote expression vector pDEST17 to construct recombinant expression plasmid, which then was transformed into E. coil Rosetta. The 29 kDa HIS6-tag fusion protein was obtained with induction of IPTG from E. coil Rosetta cells, which was used to immunize the healthy rabbits as an antigen. The antiserum of P2 protein was obtained after five times injections and the ELISA analyses showed that the potency of antiserum was 1：8192. Then, the antiserum was used to establish the Immunocapture RT-PCR and Dot-blot ELISA method to detect the RGSV. Therefore, the P2 protein and antiserum provided the technical support for the diagnosis of RGSV disease and protein function study.
出处
《中国农学通报》
CSCD
2012年第30期1-5,共5页
Chinese Agricultural Science Bulletin
基金
国家重点基础研究发展规划"(973"计划)项目"稻飞虱成灾机理与可持续治理的基础研究"(2010CB126203)
转基因生物新品种培育重大专项"水稻重要细菌和病毒病害抗性调控基因及其元件的发掘和功能分析"(2009ZX08009-044B)
国家"十二五"科技支撑计划项目"农林生物灾害防控关键技术研究与示范"(2012BAD19B03)
福建省教育厅科技项目"RGSV基因沉默抑制子对水稻miRNA代谢的影响"(JA11080)
国家自然科学基金项目"水稻条纹病毒基因沉默抑制子与水稻OsSGS3互作机理"(31171821)
福建省自然科学基金项目"WRKY基因在水稻抗条纹叶枯病中作用的研究"(2011J05051)
