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

Abstract

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.

Preparation of Polyclonal Antibodies to Grapevine fanleaf Virus Coat Protein Expressed in Escherichia coli

“…Antibodies against recombinant proteins of a number of plant viruses have been prepared successfully (Iracheta-Cardenas et al, 2008;Lee and Chang, 2008;Raikhy et al, 2007;Jain et al, 2005;Abou-Jawdah et al, 2004;Nickel et al, 2004;Cerovska et al, 2003;Hourani and Abou-Jawdah, 2003;Korimbocus et al, 2002;Kumari et al, 2001). By the use of different IPTG concentrations it was concluded that the optimal expression was achieved, when transformed E. coli Rosetta cells was induced with 1 mM IPTG at OD = 0.5 and grown at 37°C for 4 h. Different IPTG concentrations including 1 mM (Bragard et al, 2000;Kadkhodayan et al, 2000;Saini and Vrati, 2003;Thomas and Baneyx, 1996) and 0.4 mM (Jacob and Usha, 2002) have been reported as the optimal concentration with various genes of interest for expression of Cardamom mosaic virus CP and 0.1 mM (Petrzik et al, 2001) for that of Prunus necrotic Ring Spot virus (PNRSV) CP in E. coli.…”

“…Preparation of antibodies to plant viruses by expressing virus Coat Protein (CP) gene in bacteria has been boosted by the advent of recombinant DNA technology in recent years (Ling et al, 2007;Nickel et al, 2004;Minafra et al, 2000). This method bears advantages as virus purification, a crucial step in conventional antibody preparation is not required.…”

“…Molecular methods such as RT-PCR are generally not suitable tests for indexing large numbers of samples due to the costs and relative complexity of execution (Fajardo et al, 2007). Indexing on woody indicators as an alternative, takes a long time for symptoms to appear (Nickel et al, 2004). Therefore, serological tests particularly Enzyme-Linked Immune Sorbent Assay (ELISA) has been used commonly for screening a large number of samples (Zimmermann et al, 1990).…”

Development of polyclonal antibodies against nucleocapsid protein of watermelon silver mottle virus and their application to diagnostic

“…The resulting polyclonal antiserum against NP can also be successfully applied for efficient detection of field samples infected by WSMoV. However, not all antibodies raised against recombinant antigens are generally functional when used with non-denatured materials (plant extracts) (Jelkmann and Keim-Konrad, 1997;Nickel et al, 2004). Although WSMoV is an enveloped quasi-spherical particle, the produced polyclonal antibodies are still effective at recognizing the virus by indirect ELISA.…”

Highly efficient immunodiagnosis of Large cardamom chirke virus using the polyclonal antiserum against Escherichia coli expressed recombinant coat protein

“…Polyclonal antiserum to the purified preparation of LCCV from large cardamom leaf tissues was raised but the quality was not adequate for efficient detection of the virus by enzyme linked immunosorbent assay (ELISA) [10]. Prokaryotic (E. coli) expression of plant viral antigen provides several advantages in antigen preparation over the traditional methods and the recombinant antigen has utilized to develop immunodiagnosis of several plant viruses [2,4,6,12,13,18]. However there is no report of expression of recombinant CP in bacteria and its application in immunodiagnosis of LCCV.…”