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Polyclonal antibodies against the recombinantly expressed coat protein of the Citrus psorosis virus.

ABSTRACT: Psorosis is a damaging disease of citrus that is widespread in many parts of the world. Citrus psorosis virus (CPsV), the type species of the genus Ophiovirus, is the putative causal agent of psorosis. Detection of CPsV by laboratory methods, serology in particular is a primary requirement for large-scale surveys but their production has been impaired by the difficulty of obtaining sufficient clean antigen for immunization. Specific PAbs against coat protein were produced in E. coli using recombinant DNA approach. The full length CP gene fragment was amplified by RT-PCR using total RNA extracted from CPsV infected citrus leaves and CP specific primers. The obtained product (1320bp) was cloned, sequenced and sub-cloned into pET-30(+) expression vector. Expression was induced and screened in different bacterial clones by the presence of the expressed protein (48kDa) and optimized in one clone. Expressed CP was purified using batch chromatography under denaturing conditions. Specificity of expressed protein was demonstrated by ELISA before used as antigen for raising PAbs in mice. Specificity of the raised PAbs to CPsV was verified by ELISA and western blotting. The raised PAbs were showed highly effectiveness in screening by ELISA comparing with the commercial antibodies purchased from Agritest, Valanzano, Italy. The expression of CPsV CP gene in E. coli, production of PAbs using recombinant protein as an antigen, the suitability of these antibodies for use in immunodiagnostics against the CPsV Egyptian isolate have been accomplished in this work.

SUBMITTER: Salem R

PROVIDER: S-EPMC5936883 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Polyclonal antibodies against the recombinantly expressed coat protein of the <i>Citrus psorosis virus</i>.

Salem Reda R Arif Ibrahim A IA Salama Mohamed M Osman Gamal E H GEH

Saudi journal of biological sciences 20171016 4

Psorosis is a damaging disease of citrus that is widespread in many parts of the world. Citrus psorosis virus (CPsV), the type species of the genus Ophiovirus, is the putative causal agent of psorosis. Detection of CPsV by laboratory methods, serology in particular is a primary requirement for large-scale surveys but their production has been impaired by the difficulty of obtaining sufficient clean antigen for immunization. Specific PAbs against coat protein were produced in <i>E. coli</i> using ...[more]

PMID: 29740238

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Project description:BackgroundReduced height-1 dwarfing alleles affect DELLA proteins belonging to a family of putative transcriptional regulators that modulate plant growth and development. The Arabidopsis thaliana genome encodes five DELLA proteins, whereas monocot plants, such as rice, barley, and wheat, each have a single DELLA protein. In wheat, wild-type Rht-B1a and Rht-D1a genes encode DELLA proteins and have many alleles that contain lesions. Among them, Rht-B1b and Rht-D1b are the most common mutant dwarfing alleles, which have played a key part in the creation of high-yielding wheat varieties. Despite their fundamental roles in plant biology, until now, DELLA proteins in wheat have been mainly researched regarding the phenotypic effect of defective Rht mutants on yield-related traits, without studies on the underlying mechanisms. The RHT-1 protein has yet to be detected in wheat tissues, owing to a lack of appropriate molecular tools for characterization of RHT function and protein interactions in signal transduction. This study is focused on the production of a polyclonal antibody to the wheat RHT-D1A protein.ResultsTo generate the anti-RHT-D1A antibody, we expressed and purified soluble 6xHis-tagged RHT-D1A. The purified recombinant RHT-D1A was injected into New Zealand white rabbits to generate polyclonal antiserum. The polyclonal anti-RHT-D1A antibody was purified by ammonium sulfate precipitation, followed by affinity chromatography on protein A-agarose beads. The purified polyclonal antibody was demonstrated to be effective in immunoblotting, western blot hybridization, and immunoprecipitation. In wheat seedling extracts, the polyclonal antibody recognized a protein with a molecular mass close to the predicted molecular weight of the endogenous RHT-D1A protein. We also demonstrated that RHT-D1A disappears in response to exogenous and endogenous gibberellic acid.ConclusionThe purified polyclonal antibody raised against the recombinant RHT-D1A protein is sufficiently specific and sensitive and could be a useful tool for future insights into upstream and downstream components of DELLA-regulatory mechanisms in wheat plants.

Project description:BackgroundExpression of virus coat protein (CP) in Escherichia coli often leads to production of partially folded aggregated proteins which are called inclusion bodies. Grapevine fanleaf virus (GFLV) is one of the most serious and widespread grapevine virus diseases around the world and in Iran.ObjectiveThe main objective of this study was to find a simple and brief method for producing polyclonal antibodies (PAbs) to be used for immunodiagnosis of GFLV.Material and methodsAn antigenic determinant in GFLV CP gene was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain BL21 (DE3).The recombinant coat protein of GFLV (CP42) was expressed and characterized by SDS-PAGE and western blot analysis using commercial anti-GFLV antibody. Expression of the CP was detected in the form of inclusion bodies in insoluble cytoplasmic fraction. Then, the inclusion bodies were isolated from the bacterial cells and injected into rabbits for PAbs production. The reaction of the antiserum was checked by ELISA assay. In order to analyze efficiency of the produced PAbs, first the infected and uninfected grapevine samples were confirmed based on morphological symptoms then the indirect plate- trapped antigen Enzyme-linked Immunosorbent Assay (IPTA-ELISA) was applied using the commercial anti GFLV antibody. In the next ELISA assay, efficiency of the raised polyclonal antibody was compared with commercial one.ResultsThe expression of recombinant CP42 induced by IPTG was confirmed by the band of 42 kDa in SDS-PAGE and western blot. The antiserum of purified inclusion body immunized rabbit was reacted with CP42 and GFLV infected Grapevine samples. The results revealed an acceptable efficacy for prepared antibodies compared to that of commercial antibody.ConclusionsIt was evident that the recombinant coat protein in the form of inclusion bodies can be prepared and used as the antigen for immunizing animals in order to produce PAbs.

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Polyclonal antibodies against the recombinantly expressed coat protein of the Citrus psorosis virus.

Publications

Polyclonal antibodies against the recombinantly expressed coat protein of the <i>Citrus psorosis virus</i>.

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