The genome of rice dwarf phytoreovirus(RDV)is composed of 12 double-stranded RNA segments,of which segment S6 encodes a non-structural protein Pns6 identified as the movement protein.In this report,Pns6 with a 6-histi...The genome of rice dwarf phytoreovirus(RDV)is composed of 12 double-stranded RNA segments,of which segment S6 encodes a non-structural protein Pns6 identified as the movement protein.In this report,Pns6 with a 6-histidine tag at the N-terminal was expressed in E.coli after induction under low temperature(18℃)and low concentration(0.4 mmol/L and 0.2 mmol/L)of IPTG,and then purified by Ni-chelated affinity chromatography.Stability analysis indicated that the expressed HisPns6 protein was stable at 37℃after 24 h treatment.This recombinant protein was then used to make monoclonal antibody.Total 18 hybridoma clones were obtained.The specificity of antibodies was tested by Western blot using native Pns6 extracted from RDV-infected rice leaves,and 15 positive clones were confirmed.Mapping of the antigenic sites of Pns6 using antibodies showed that the most sensitive antigen determinant is located in the C-terminal region(the 296th-509th amino acids)of Pns6,which is confirms bioinformatics analysis.展开更多
基金Supported by the Special Commonweal Industry Research Program of Agriculture(Grant no.nyhyzx07-051)the National High Technology Research and Development Program of China(Grant No.2007AA021505)to Prof.LI Yi
文摘The genome of rice dwarf phytoreovirus(RDV)is composed of 12 double-stranded RNA segments,of which segment S6 encodes a non-structural protein Pns6 identified as the movement protein.In this report,Pns6 with a 6-histidine tag at the N-terminal was expressed in E.coli after induction under low temperature(18℃)and low concentration(0.4 mmol/L and 0.2 mmol/L)of IPTG,and then purified by Ni-chelated affinity chromatography.Stability analysis indicated that the expressed HisPns6 protein was stable at 37℃after 24 h treatment.This recombinant protein was then used to make monoclonal antibody.Total 18 hybridoma clones were obtained.The specificity of antibodies was tested by Western blot using native Pns6 extracted from RDV-infected rice leaves,and 15 positive clones were confirmed.Mapping of the antigenic sites of Pns6 using antibodies showed that the most sensitive antigen determinant is located in the C-terminal region(the 296th-509th amino acids)of Pns6,which is confirms bioinformatics analysis.
