Expression of rice dwarf phytoreovirus Pns6 and the specificity analysis of its monoclonal antibodies.
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 degrees C) 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 degrees C 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.
Ji
Xu
X
Peking-Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, 100871, China.
Wei
ChunHong
C
Li
Yi
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D007114:Immunization; D051379:Mice; D012275:Oryza / Q000821:virology*; D018515:Plant Leaves / Q000821:virology; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012087:Reoviridae / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins