Purification and serological analyses of tospoviral nucleocapsid proteins expressed by Zucchini yellow mosaic virus vector in squash

Purification and serological analyses of tospoviral nucleocapsid proteins expressed by Zucchini... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Virological Methods Elsevier

Purification and serological analyses of tospoviral nucleocapsid proteins expressed by Zucchini yellow mosaic virus vector in squash

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Publisher
Elsevier
Copyright
Copyright © 2005 Elsevier B.V.
ISSN
0166-0934
eISSN
1879-0984
D.O.I.
10.1016/j.jviromet.2005.05.014
Publisher site
See Article on Publisher Site

Abstract

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.

Journal

Journal of Virological MethodsElsevier

Published: Nov 1, 2005

References

  • Engineering Zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits
    Arazi, T.; Slutsky, S.G.; Shiboleth, Y.M.; Wang, Y.; Rubinstein, M.; Barak, S.; Yang, J.; Gal-On, A.
  • Potato virus X as a vector for gene expression in plants
    Chapman, S.; Kavanagh, T.; Baulcombe, D.C.
  • A plant virus vector for systemic expression of foreign genes in cereals
    Choi, I.R.; Stenger, D.R.; Morris, T.J.; French, R.
  • Characterisation of genetically modified Cucumber mosaic virus expressing histidine-tagged 1a and 2a proteins
    Gal-On, A.; Canto, T.; Palukaitis, P.
  • Susceptibility to recombination rearrangements of a chimeric Plum pox potyvirus genome after insertion of a foreign gene
    Guo, H.S.; Lopez-Moya, J.J.; Garcia, J.A.
  • Watermelon bud necrosis tospovirus is a distinct virus species belonging to serogroup IV
    Jain, R.K.; Pappu, H.R.; Pappu, S.S.; Reddy, M.K.; Vani, A.
  • Development of a plant-derived subunit vaccine candidate against Hepatitis C virus
    Nemchinov, L.G.; Liang, T.J.; Rifaat, M.M.; Mazyad, H.M.; Hadidi, A.; Keith, J.M.
  • Technical advance. Tobacco rattle virus as a vector for analysis of gene function by silencing
    Ratcliff, F.; Martin-Hernandez, A.M.; Baulcombe, D.C.
  • The expression of a single-chain Fv antibody fragment in different plant hosts and tissues by using Potato Virus X as a vector
    Roggero, P.; Ciuffo, M.; Benvenuto, E.; Franconi, R.
  • Peanut yellow spot virus is a member of a new serogroup of Tospovirus genus based on small (S) RNA sequence and organization
    Satyanarayana, T.; Gowda, S.; Reddy, K.L.; Mitchell, S.E.; Dawson, W.O.; Reddy, D.V.R.

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