A. tumefaciens-mediated transient expression as a tool for antigen production for cucurbit yellow stunting disorder virus.
The emerging importance of criniviruses, together with their limited characterisation, necessitates the development of simple tools to enable rapid detection and monitoring in case of an outbreak. While serological tools would be ideal, criniviruses are notoriously difficult to purify and traditional methods of antibody production, requiring purified virus particles, are extremely challenging. The development of a novel molecular strategy for in planta viral antigen preparation to bypass particle purification and allow antibody production are described. An A. tumefaciens-mediated transient expression system, coupled with a green fluorescent protein (GFP) purification method was employed to generate CYSDV coat protein (CP) in whole plant leaves. The CYSDV CP gene was ligated into a GFP construct, transformed into A. tumefaciens and agroinfiltrated into N. benthamiana. Expression levels of the recombinant protein were increased by co-infiltration with the viral gene-silencing suppressor P19 from TBSV. The recombinant protein, purified from plant leaves was used to immunise rats for the preparation of polyclonal antisera.
Steel
E
E
The Food and Environment Research Agency Fera, Sand Hutton, York YO41 1LZ, UK. elspeth.steel@fera.gsi.gov.uk
Barker
I
I
Danks
C
C
Coates
D
D
Boonham
N
N
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D029383:Crinivirus / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D018515:Plant Leaves / Q000235:genetics; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014026:Nicotiana / Q000235:genetics; D014170:Transformation, Genetic
D000914:Antibodies, Viral; D000956:Antigens, Viral; D011994:Recombinant Proteins