Production of polyclonal antibodies to the coat protein gene of Indian isolate of Apple stem grooving virus expressed through heterologous expression and its use in immunodiagnosis

Apple stem grooving virus (ASGV) is one of the most important viruses infecting fruit trees. This study aimed at the molecular characterization of ASGV infecting apple (Malus domestica) plants in Santa Catarina (SC). RNA extracted from plants infected with isolate UV01 was used as a template for RT-PCR using specific primers. An amplified DNA fragment of 755 bp was sequenced. The coat protein gene of ASGV isolate UV01 contains 714 nucleotides, coding for a protein of 237 amino acids with a predicted Mr of approximately 27 kDa. The nucleotide and the deduced amino acid sequences of the coat protein gene showed identities of 90.9% and 97.9%, respectively, with a Japanese isolate of ASGV. Very high amino acid homologies (98.7%) were also found with Citrus tatter leaf capillovirus (CTLV), a very close relative of ASGV. These results indicate low coat protein gene variability among Capillovirus isolates from distinct regions. In a restricted survey, mother stocks in orchards and plants introduced into the country for large scale fruit production were indexed and shown to be infected by ASGV (20%), usually in a complex with other (latent) apple viruses (80%).

Download Full-text

Analysis of the Coat Protein Gene of Indian Strain of Apple Stem Grooving Virus

Journal of Plant Biochemistry and Biotechnology ◽

10.1007/bf03323442 ◽

2009 ◽

Vol 19 (1) ◽

pp. 91-94 ◽

Cited By ~ 14

Author(s):

Anuradha Negi ◽

Tanuja Rana ◽

Yogesh Kumar ◽

Raja Ram ◽

Vipin Hallan ◽

...

Keyword(s):

Coat Protein ◽

Coat Protein Gene ◽

Protein Gene ◽

Apple Stem Grooving Virus ◽

Indian Strain ◽

Apple Stem

Download Full-text

Sequence diversity and potential recombination events in the coat protein gene of Apple stem pitting virus

Virus Research ◽

10.1016/j.virusres.2011.03.003 ◽

2011 ◽

Vol 158 (1-2) ◽

pp. 263-267 ◽

Cited By ~ 15

Author(s):

Beata Komorowska ◽

Paweł Siedlecki ◽

Szymon Kaczanowski ◽

Beata Hasiów-Jaroszewska ◽

Tadeusz Malinowski

Keyword(s):

Coat Protein ◽

Coat Protein Gene ◽

Protein Gene ◽

Sequence Diversity ◽

Apple Stem ◽

Apple Stem Pitting Virus ◽

Stem Pitting

Download Full-text

Nucleotide sequences of apple stem pitting virus and of the coat protein gene of a similar virus from pear associated with vein yellows disease and their relationship with potex- and carlaviruses

Journal of General Virology ◽

10.1099/0022-1317-75-7-1535 ◽

1994 ◽

Vol 75 (7) ◽

pp. 1535-1542 ◽

Cited By ~ 49

Author(s):

W. Jelkmann

Keyword(s):

Coat Protein ◽

Coat Protein Gene ◽

Protein Gene ◽

Nucleotide Sequences ◽

Apple Stem ◽

Apple Stem Pitting Virus ◽

Stem Pitting

Download Full-text

Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

Fitopatologia Brasileira ◽

10.1590/s0100-41582007000600007 ◽

2007 ◽

Vol 32 (6) ◽

pp. 496-500 ◽

Cited By ~ 10

Author(s):

Thor V.M. Fajardo ◽

Danielle R. Barros ◽

Osmar Nickel ◽

Gilmar B. Kuhn ◽

F. Murilo Zerbini

Keyword(s):

Escherichia Coli ◽

Coat Protein ◽

Western Blot ◽

Coat Protein Gene ◽

Protein Gene ◽

Polyclonal Antibodies ◽

Yield And Quality ◽

Sds Page ◽

Grapevine Leaves

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.

Download Full-text

Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis

Fitopatologia Brasileira ◽

10.1590/s0100-41582004000500017 ◽

2004 ◽

Vol 29 (5) ◽

pp. 558-562 ◽

Cited By ~ 18

Author(s):

Osmar Nickel ◽

Maria L.P.N. Targon ◽

Thor V.M. Fajardo ◽

Marcos A. Machado ◽

Ana P. Trivilin

Keyword(s):

Escherichia Coli ◽

Coat Protein ◽

Fusion Protein ◽

Polyclonal Antibodies ◽

Molecular Techniques ◽

Bacterial Cells ◽

Additional Tool ◽

Apple Stem Grooving Virus ◽

Apple Stem ◽

Specificity And Sensitivity

The coat protein gene of Apple stem grooving virus (ASGV) was amplified by RT-PCR, cloned, sequenced and subcloned in the expression vector pMal-c2. This plasmid was used to transform Escherichia coli BL21c+ competent cells. The ASGV coat protein (cp) was expressed as a fusion protein containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP fusion protein was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fusion protein gave specific reactions to ASGV from infected apple (Malus domestica) cv. Fuji Irradiada and Chenopodium quinoa at dilutions of up to 1:1,000 and 1:2,000, respectively, in plate trapped ELISA. The ASGVcp/MBP fusion protein reacted to a commercial antiserum against ASGV in immunoblotting assay. The IgG against ASGVcp/MBP performed favorably in specificity and sensitivity to the virus. This method represents an additional tool for the efficient ASGV-indexing of apple propagative and mother stock materials, and for use in support of biological and molecular techniques.

Download Full-text