Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein

•The p29 gene encoding for putative coat protein of Citrus leprosis virus C (CiLV-C) were optimized for the expression in Escherichia coli.•The optimized p29 gene was synthesized in in vitro, cloned and expressed the putative coat protein in E. coli.•The purified expressed putative coat protein of C...

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Bibliographic Details
Published in:Journal of virological methods 2013-11, Vol.193 (2), p.548-553
Main Authors: Choudhary, Nandlal, Roy, Avijit, Guillermo, Leon M., Picton, D.D., Wei, G., Nakhla, M.K., Levy, L., Brlansky, R.H.
Format: Article
Language:English
Subjects:
affinity chromatography
agarose
Animals
Antibodies, Viral
antibody formation
antigens
Antigens, Viral - genetics
Antigens, Viral - immunology
Antigens, Viral - isolation & purification
Capsid Proteins - genetics
Capsid Proteins - immunology
Capsid Proteins - isolation & purification
CiLV-C
Citrus
Citrus - virology
Citrus leprosis disease
Citrus leprosis virus C
coat proteins
Codon - genetics
Dot-blot immunoassay
ELISA
enzyme-linked immunosorbent assay
Enzyme-Linked Immunosorbent Assay - methods
Escherichia coli
Escherichia coli - genetics
Expressed coat protein
Gene Expression
genes
Immunologic Tests - methods
industry
Plant Diseases - virology
Plant Viruses - immunology
Plant Viruses - isolation & purification
polyclonal antibodies
Polyclonal antibody
Rabbits
Recombinant Proteins - genetics
Recombinant Proteins - immunology
Recombinant Proteins - isolation & purification
serodiagnosis
tissues
Western blotting
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Summary:•The p29 gene encoding for putative coat protein of Citrus leprosis virus C (CiLV-C) were optimized for the expression in Escherichia coli.•The optimized p29 gene was synthesized in in vitro, cloned and expressed the putative coat protein in E. coli.•The purified expressed putative coat protein of CiLV-C was used for polyclonal antibody production in rabbits.•The polyclonal antibody, CREC13, was used to detect the CiLV-C in the crude extract of symptomatic CiLV-C infected tissues. Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2013.07.035