Development of immunodiagnostics for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3) in grapevine using in vitro expression and purification of its coat protein gene

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

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Translational in vitro activity of the 3a gene and the coat protein gene derived from brome mosaic virus RNA 3 by site-specific cleavage with RNase H

FEBS Letters ◽

10.1016/0014-5793(89)81010-5 ◽

1989 ◽

Vol 254 (1-2) ◽

pp. 66-68 ◽

Cited By ~ 3

Author(s):

E.V. Smirnyagina ◽

O.V. Karpova ◽

N.A. Miroshnichenko ◽

N.P. Rodionova ◽

J.G. Atabekov

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Coat Protein Gene ◽

Protein Gene ◽

Brome Mosaic Virus ◽

Rnase H ◽

In Vitro Activity ◽

Site Specific ◽

Virus Rna

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GENETIC ENGINEERING OF THE ZUCCHINI YELLOW MOSAIC VIRUS COAT PROTEIN GENE FOR EXPRESSION IN PLANTS.

HortScience ◽

10.21273/hortsci.25.9.1130d ◽

1990 ◽

Vol 25 (9) ◽

pp. 1130d-1130

Author(s):

Guowei Fang ◽

Rebecca Grumet

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Coat Protein Gene ◽

Protein Gene ◽

Zucchini Yellow Mosaic Virus ◽

In Vitro Transcription ◽

Yellow Mosaic Virus ◽

35S Promoter ◽

Plant Expression Vector

Zucchini yellow mosaic virus (ZYMV), a potyvirus, can cause major losses in cucurbit crops. With the goal of genetically engineering resistance to this disease we have engineered the ZYMV coat protein gene into a plant expression vector. The complete coat protein coding sequence, or the conserved core portion of the capsid gene, was attached to the 5' untranslated region of tobacco etch virus (TEV) in the pTL37 vector (Carrington et al., 1987, Nucl. Acid Res. 15:10066) The capsid constructs were successfully expressed by in vitro transcription and translation systems as verified by SDS-PAGE and ZYMV coat protein antibody. The constructs were then subcloned using polymerase chain reaction and attached to the CaMV 35 S transcriptional promoter on the CIBA-GEIGY pCIB710 plasmid. The constructs containing the CaMV 35S promoter, the 5' untranslated leader of TEV, and ZYMV coat protein sequences were then put between the Agrobacterium tumefaciens left and right borders in the pCIB10 vector and transferred to A. tumefaciens strain LBA4404 by triparental mating. These vectors are now being used to transform muskmelon and cucumber; resultant transgenic plants will be tested for ZYMV coat protein expression.

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Advances in development of transgenic resistance to beet necrotic yellow vein virus (BNYVV) in sugar beet

Genetika ◽

10.2298/gensr0503181n ◽

2005 ◽

Vol 37 (3) ◽

pp. 181-189

Author(s):

Nevena Nagl ◽

Ivan Atanasov ◽

Krasimir Rusanov ◽

Svetlana Paunovic ◽

Lazar Kovacev ◽

...

Keyword(s):

Coat Protein ◽

Sugar Beet ◽

Coat Protein Gene ◽

Protein Gene ◽

Transformation Method ◽

Yellow Vein ◽

Bar Gene ◽

Axillary Shoots ◽

Viral Cdna

Fragments of viral cDNA containing the coat protein gene of beet necrotic yellow vein virus were cloned in plant transformation vector pCAMBIA3301M with the bar gene as selectable marker. Vector pC3301MCPL carrying coat protein gene with leader sequence, and pC3301MCPS with coat protein gene, were used in Agrobacterium - mediated transformation of sugar beet. The transformation method used was based on the fact that sugar beet develops axillary shoots in in vitro conditions, when placed on media with citokinins. Since this ability is not genotype or ploidy dependant it is widely used for sugar beet vegetative multiplication. Sterile seedlings, with removed cotyledons and lower half of hypocotyl, were used as starting material. After transformation ex-plants were put on micropropagation medium with cephotaxime and phosphinotricyn (ppt), where axillary shoots started to develop. Since concentration of ppt was not selective enough, after two subcultivations it was increased twofold. Only one sample, transformed with pC3301MCPS preserved morphogenetic potential for micropropagatio, and it was tested for presence of COS fragment and bar gene bz PCR with soecific primers.

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