Purification and serological analyses of tospoviral nucleocapsid proteins expressed by Zucchini yellow mosaic virus vector in squash.
A plant viral vector engineered from an in vivo infectious clone of Zucchini yellow mosaic virus (ZYMV) was used to express the nucleocapsid proteins (NPs) of tospoviruses in planta. The open reading frames (ORFs) of NPs of different serogroups of tospoviruses, including Tomato spotted wilt virus, Impatiens necrotic spot virus, Watermelon silver mottle virus, Peanut bud necrosis virus, and Watermelon bud necrosis virus (WBNV), were in frame inserted in between the P1 and HC-Pro genes of the ZYMV vector. Six histidine residues and an NIa protease cleavage site were added at the C-terminal region of the inserts to facilitate purification and process of free form of the expressed NPs, respectively. Approximately 1.2-2.5 mg/NPs 100 g tissues were purified from leaf extracts of zucchini squash. The expressed WBNV NP was used as an immunogen for the production of highly specific polyclonal antisera and monoclonal antibodies. The procedure provides a convenient and fast way for production of large quantities of pure NPs of tospoviruses in planta. The system also has a potential for production of any proteins of interest in cucurbits.
Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan.
Tsung-Chi
TC
Chen
Hei-Ti
HT
Hsu
Rekesh K
RK
Jain
Ching-Wen
CW
Huang
Chen-Hsuan
CH
Lin
Fang-Lin
FL
Liu
Shyi-Dong
SD
Yeh
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D002498:Centrifugation; D002846:Chromatography, Affinity; D028464:Cucurbita / Q000821:virology*; D005814:Genes, Viral; D005822:Genetic Vectors; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D016366:Open Reading Frames; D010936:Plant Extracts; D018515:Plant Leaves / Q000821:virology; D017800:Potyvirus / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D017865:Tospovirus / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007106:Immune Sera; D019590:Nucleocapsid Proteins; D010936:Plant Extracts; D011994:Recombinant Proteins