Production of polyclonal antibodies against Pelargonium zonate spot virus coat protein expressed in Escherichia coli and application for immunodiagnosis.
Pelargonium zonate spot virus (PZSV) is identified recently in tomato plants in the United States. To develop serological diagnostic tools for the detection of this virus, the production of good quality antibodies is a necessity. The coat protein (CP) gene of a California isolate of PZSV was cloned into a bacterial expression vector (pTriEX-4 Ek/LIC). The plasmid pTriEX-4-PZSV-CP was transformed into Escherichia coli Rosetta 2(DE3)pLacI and the recombinant PZSV-CP was expressed as a fusion protein containing N-terminal hexa-histidine and S tags. Expressed PZSV-CP was purified under denaturing conditions by affinity chromatography yielding 3mg refolded protein per 200mL of bacterial culture, and used as an antigen for raising PZSV-CP antiserum in rabbits. Specificity of the antiserum to PZSV was shown by Western blot and ELISA. When used in Western blot analysis, the antiserum was able to detect the recombinant protein, the PZSV coat protein and PZSV infected plant samples. The antiserum was successfully used in indirect-ELISA at dilutions of up to 1:16,000 to detect PZSV in infected leaf samples. Direct ELISA was successful only with denatured antigens. This is the first report on production of polyclonal antiserum against recombinant coat protein of PZSV and its use for detection and diagnosis of virus using serological methods.
Gulati-Sakhuja
Anju
A
USDA-ARS, Salinas, CA 93905, USA.
Sears
John L
JL
Nuñez
Alberto
A
Liu
Hsing-Yeh
HY
D000818:Animals; D000914:Antibodies, Viral; D015153:Blotting, Western / Q000379:methods; D019177:Bromoviridae / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007159:Immunologic Tests / Q000379:methods*; D018551:Solanum lycopersicum / Q000821:virology*; D010935:Plant Diseases / Q000821:virology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D014481:United States
D000914:Antibodies, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins