Hort. Environ. Biotechnol. 56(3):316-323. 2015. DOI 10.1007/s13580-015-0139-5
ISSN (p rint) : 2211-3452 ISSN (online) : 2211-3460
Research Report
Improved Detection of Cucumber Mosaic Virus Coat Protein (CMV-CP) in Genetically Modified Pepper (Capsicum annuum) Using a Polyclonal Antibody to a Synthetic CP Peptide 1ಲ
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Jongmin Choi , Chanvorleak Phat , Eunji Kim , Minji Kim , Gung Pyo Lee , 3 1* Ki-Hyun Ryu , and Chan Lee 1
School of Food Science and Technology, Chung-Ang University, Anseong, Gyeonggi-Do 456-756, Korea Department of Integrative Plant Science, Chung-Ang University, Anseong, Gyeonggi-Do 456-756, Korea 3 Department of Horticultural Science, Seoul Women’s University, Seoul 139-774, Korea
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*Corresponding author:
[email protected] These author contributed equally to this work.
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Received November 12, 2014 / Revised March 27, 2015 / Accepted May 25, 2015 GKorean Society for Horticultural Science and Springer 2015
Abstract. Enormous economic losses caused by infection of peppers with Cucumber mosaic virus (CMV) have spurred the development of CMV-resistant peppers using genetic techniques. However, growing concern from the general public over genetically modified (GM) crops has focused attention on safety and labeling issues. In this study, we developed and optimized a qualitative and quantitative enzyme-linked immunosorbent assay (ELISA) to detect CMV-resistant GM peppers. Two types of antibodies, virion-derived polyclonal antibody (VPAb) prepared from a CMV-Fny virion-immunized rabbit and peptide-based polyclonal antibody (PPAb) prepared from rabbits immunized with a peptide fragment of CMVcoat protein (CMV-CP) (LPDSVTEYDKKLVSR) predicted from the nucleotide sequence were prepared, and their affinities were compared. Optimized ELISAs using VPAb and PPAb as the primary antibodies, respectively, were carried out to measure the level of CMV-CP in GM peppers cultivated at an officially approved facility in Korea. Color development (reflecting CMV-CP levels) was 2.5-fold higher when PPAb was used as the primary antibody compared to VPAb as the primary antibody. No statistical differences were observed among GM pepper cultivars. These results imply that PPAb has higher specificity for CMV-CP than VPAb does, and that the analytical system presented in this study can be used to evaluate the level of CMV-CP in genetically modified peppers. Additional key words: CMV-resistant pepper, Cucumber mosaic virus (CMV), enzyme-linked immunosorbent assay (ELISA), peptide-based polyclonal antibody
Introduction Agricultural biotechnology has opened new avenues in the development of virus-resistant plants. The initial objective for developing biotech crops was to increase crop yield through the introduction of resistance against plant diseases caused by insects or viruses. Insertion of viral coat proteins (CP) of specific viruses has been widely used to enhance resistance against viral diseases (Holst-Jensen et al., 2012). Various pepper plants were successfully developed by insertion of cucumber mosaic virus coat protein (CMV-CP) genes using transgenic techniques, including CMV-resistant sweet peppers (Chen et al., 2003), CMV- and tomato mosaic virus (ToMV)-resistant sweet peppers (Lanfermeijer et al., 2003), and CMV- and tobacco mosaic virus (TMV)-resistant chili peppers (Cai et
al., 2003). The increase in the number of viral-resistant genetically modified (GM) crops has also led to an increase in public awareness of GM food and feed. The safety and stability of GM organisms and crops is still a heated topic of debate worldwide (Brent et al., 2003). Some consumer groups want GM crops to be managed and separated from natural crops. Safety assessment and labeling information are in high demand, and techniques to reliably assess the presence and or amount of inserted and expressed recombinant protein is becoming an important part of safety assessment and management of genetically modified organisms (GMOs). Qualitative and quantitative techniques to detect viral coat proteins are therefore in great demand. The DNA-based polymerase chain reaction (PCR) techniques
Hort. Environ. Biotechnol. 56(3):316-323. 2015.
have been successfully developed to identify plant viruses (Cunha et al., 2009; Mekuria et al., 2003) with high sensitivity. To address the drawbacks of this method, namely the fact that it is time-consuming, difficult, and costly (Yu et al., 2005), protein-based immunological methods such as immunocapture RT-PCR for detection of seed-borne viruses (Lee et al., 2010), and dot blot immunobinding assay for detection of pepper yellow leaf curl Indonesia virus (Hidayat et al., 2009) have been developed. Enzyme-linked immunosorbent assays (ELISAs) are low-cost, give rapid results, and are not labor intensive, and may therefore be more favorable than other conventional methods, particularly for serial measurements of a large numbers of samples (Holdenrieder et al., 2005; Kazutoyo et al., 2008). Although ELISA is not as sensitive as PCR, it allows rapid detection and accurate quantification of a specific target protein. It has already been fully applied for diagnosis of viruses; for example, an ELISA were developed for detection of avian influenza virus (Jensen et al., 2013) and apple chlorotic leaf spot virus (Blystad, accessed 10 April 2014). In this paper, we present an optimized ELISA for detecting the coat protein of CMV. This method allows identification of GM peppers generated by insertion of CP and detection of wild-type virus in peppers in the field. To develop this ELISA, we evaluated two types of antibodies, virion polyclonal antibody (VPAb) and peptide polyclonal antibody (PPAb), and compared their reactivity to CMV-CP to investigate specificity.
Fig. 2. DNA sequence of CMV-CP gene and amino acids.
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Materials and Methods &DSVLFXP VSHFLHV The CMV-resistant GM pepper H15 (Capsicum annuum var. Longunt) and its corresponding control line P2377 were used in this study. Seeds of these peppers were provided by the Biotechnology Institute of Nongwoo Bio, Korea. Transgenic peppers (H15) were prepared by transfection of Agrobacterium with the coat protein gene of CMVP0 (Lee et al., 2009) into control line (p2377). A cloning vector, pCAMBIA 2300 (Fig. 1), encoding the CMVP0-CP (CMV-Fny coat protein, Fig. 2) gene was inserted into Agrobacterium strain EHA105 or LBA4404. Agrobacterium culture was then inoculated into explants prepared from seeds of inbred lines (Lee et al., 2004). Samples of transgenic peppers (T3; second generation by selfcross) grown in a GM field along with the corresponding
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Fig. 1. Vector used for genetic transformation n ; A coat protein gene was cloned from the CMV-P0 pathogen and subcloned into a pCAMBIA 2300 vector.
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Jongmin Choi, Chanvorleak Phat, Eunji Kim, Minji Kim, Gung Pyo Lee, Ki-Hyun Ryu, and Chan Lee o
control lines were harvested and stored at -70 C until analysis. ([SUHVVLRQ DQG SXULILFDWLRQ RI U&09&3 IURP ( FROL To express recombinant CMV-CP (rCMV-CP) in bacteria, an expression plasmid was constructed, and the recombinant protein was induced and expressed sequentially in two competent Escherichia coli cell types, namely E. coli JM109 and BL21. Polymerase chain reaction (PCR) was carried out using CMVCP-pCAMBIA-2300 described above as the template DNA. Primers were designed based on the nucleotide sequence of the coat protein published in GenBank (National Center for Biotechnology Information). Amplification products were then digested with NdeI (5’ end) and XhoI (3’ end) and ligated into pET-22b(+) vector (Novagen, Whitehouse Station, NJ., USA) digested with the same restriction enzymes. The vector was then inserted into E. coli JM109 cells (Takara, Shiga, Japan), and plasmid DNA was extracted using a Miniprep kit (Qiagen, Valencia, CA, USA) after cells had grown sufficiently in LB medium containing 5% ampicillin (Sigma-Aldrich, St. Louis, MO, USA). Extracted plasmid was transformed into E. coli BL21-Gold (DE3) pLys cells (Stratagene, La Jolla, CA, USA), and the cells were grown by vigorous shaking in LB medium containing 5% ampicillin at 37°C until the cell optical density at 600 nm reached 0.5. Treatment of cells with 1 mM isopropyl-1-thio-ȕ-D-galactopyranoside (IPTG) (Takara, Shiga, Japan) was followed by shaking at 20°C for 4 h to induce expression of the recombinant protein. Cells were harvested by centrifugation for 30 min at 8,988 × g and resuspended in 30 mL of imidazole (20 mM) containing 0.1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). After cells were lysed by a French pressure cell press (SLM Aminco, Waltham, MA, USA), cell debris was removed by centrifugation (1 h, 15,729 × g) for the subsequent chromatography step. rCMV-CP was purified by passing cell-free extract through Ni-NTA resin (Qiagen, Valencia, CA, USA) previously equilibrated with 20 mM imidazole for affinity purification of the hexahistidine-tagged rCMV-CP (Loughran and Walls, 2011; Meredith et al., 2004). Bound protein was gradually eluted in a stepwise gradient using 20, 100, 112.5, 125, and 200 mM imidazole in elution buffer. Presence of purified rCMV-CP was determined by electrophoresis and Western blot analysis. 3UHSDUDWLRQ RI DQWLERGLHV IRU &09&3 LQ SHSSHU We evaluated two different types of antibodies, namely virion-derived polyclonal antibodies (VPAb) and peptide polyclonal antibodies (PPAb) specific to CMV-CP, as the primary antibodies in quantitative ELISA assays for CMV-CP (Agindotan et al., 2003). Virion-derived polyclonal antibody: VPAb was kindly
provided by Seoul Women’s University. A rabbit was injected subcutaneously with 1 mg of CMV-Fny virion in 1.5 mL saline solutions mixed with an equal volume of complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Two more injections were administered at one-week intervals. One week after the third injection, the rabbit was sacrificed, and VPAb was purified from its blood. Both ELISA and Western blotting were used to examine clones that successfully secreted antibodies specific to CMV. Peptide polyclonal antibody: Polyclonal antibody specific to a peptide fragment of CMV-CP was prepared by Abfrontier (Seoul, Korea). One milligram of synthesized peptide fragment based on the CMV-CP protein sequence was injected into two rabbits (R1 and R2) in saline solution emulsified in complete Freund’s adjuvant. Two more injections were administered at two-week intervals. Three weeks after the third injection, the rabbits were sacrificed, and PPAbs were purified from their blood. Both ELISA and Western blotting were used to examine clones that successfully secreted antibodies specific to CMV-CP. Antisera containing antibodies for CMV-CP were then kept at -70°C until analysis. 6'63$*( DQG :HVWHUQ EORW DQDO\VLV SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Manns (2011), using 12% acrylamide gels with a molecular weight standard marker (Bio-Rad, Hercules, CA, USA). A fixed amount of sample (SDS-buffered solution of unknown samples) was loaded on SDS polyacrylamide gels and subjected to electrophoresis, after which protein bands were visualized by staining with Coomassie brilliant blue R250. Isolated proteins in the acrylamide gel were transferred onto PVDF membranes (Millipore Corp., Bedford, MA, USA) at a current of 100 V for 1 h using a Trans-blot SD semi-dry transfer cell (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. After exposing the PVDF membranes to 5% skim milk/TBS (Trisbuffered saline, including 10 mM Tris-HCl pH 7.5 and 150 mM NaCl) for 1 h, the membranes were probed with the designed peptide polyclonal antibodies in 3% skim milk/TBS for 1 h. Unbound antibody was removed by washing the membranes twice with TBS for 10 min. The membranes were then treated with secondary antibodies and anti-IgG alkaline phosphatase conjugate (Sigma-Aldrich, St. Louis, MO, USA) and washed three times for 30 min. Membranes were stained using nitroblue tetrazolium (Promega, Madison, WI, USA) and 5bromo-4-chloro-3-indoyl phosphate (Promega, Madison, WI, USA) as substrates in alkaline phosphatase buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) for less than 10 min, and the reaction was stopped when the desired color density was obtained.
Hort. Environ. Biotechnol. 56(3):316-323. 2015.
([WUDFWLRQ RI VROXEOH SURWHLQV Soluble proteins from GM and non-GM peppers cultivated in Korea University fields were extracted as described previously (Bang et al., 2011). Frozen peppers (20 g) were homogenized in a pre-chilled blender with chilled 0.05 M potassium phosphate buffer, pH 8.0, containing 1.0 mM ethylenediaminetetraacetic acid (EDTA), 1.0 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (SigmaAldrich, St. Louis, MO, USA) (buffer A). To remove cell debris, centrifugation was performed for 30 min at 8,000 rpm, and then polyethyleneimine (PEI, 0.2%) was added to the supernatant to eliminate nucleic acids and negatively-charged proteins. After centrifugation, crude protein solution was stored at -70°C for subsequent steps. All procedures were performed at 4°C. 'HWHUPLQDWLRQ RI VROXEOH SURWHLQ FRQFHQWUDWLRQ Protein concentrations were routinely measured using Bradford’s method (Bradford, 1976) with bovine serum albumin (BSA) as the protein standard and a dye-binding kit purchased from Bio-Rad. Absorbance of samples was measured at 590 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). (Q]\PHOLQNHG LPPXQRVRUEHQW DVVD\ (/,6$ The ELISA was carried out as described originally by Clark and Adams (1977). To optimize the procedures, details of the assessment were slightly modified according to the recommendations of Talukder et al. (2005) and Ryu et al. (2010). The following ELISA protocol for detection of CMVCP was standardized after optimization. Optimum conditions are listed in Table 1. Before the experiment to quantify CMVCP in GM peppers, primary antibody dilutions of 1:1,000; 1:3,000; 1:10,000; 1:30,000; 1:50,000; and 1:100,000 were evaluated to determine the optimal antibody titer. Incubation time and temperature (Table 1) were determined in pilot experiments as described previously in the text.
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In brief, a 96-well microplate (Nunc, Roskilde, Denmark) was coated with 200 ȝL per well of the antigen diluted in phosphate buffered saline (PBS) to a concentration of 3.5 mg total protein per mL. At the same time, rCMV-CP purified from E. coli BL21 was loaded on the same plate at protein concentrations of 5, 10, 50, 100, 200, and 300 nM as the known standard substrate. The plate was sealed and incubated at 37°C for 3 h. Between each step, the plate was manually washed four times with PBST (PBS containing 0.05% Tween20, pH 7.4) to remove any proteins or antibodies that were not specifically bound. Next, a solution containing non-reacting protein, namely 3% skim milk dissolved in PBST, was added to block any plastic surfaces in the well that remained uncoated by antigen. Incubation was performed for 2 h at 37°C. Each antibody was diluted in 1% skim milk in PBST (1:3,000 dilution for VPAb and 1:1,000 for PPAb) and then independently added to antigen wells and control antigen wells (in the same diluent). The plate was then incubated for 3 h at 37°C. After washing, goat anti-rabbit IgG alkaline phosphatase conjugate (secondary antibody, Sigma-Aldrich, St. Louis, MO, USA) was diluted in 1% skim milk in PBST and added to all wells. The plate was then incubated for another 2 h at 37°C. After unbound antibody was removed by washing, the plate was washed again with 10 mM diethanolamine (containing 0.5 mM MgCl2, pH 9.5). Color development was initiated by adding 0.1% 4-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO, USA) as a substrate in PBS for 20 min at room temperature. The substrate changed color upon reaction with alkaline phosphatase, and this color change was measured by recording absorbance at 405 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The optical density of samples was compared to that of the standard curve to obtain quantitative results. 6WDWLVWLFDO PHWKRGV WR FRPSDUH JURXS PHDQV RI *0 DQG 1*0 SHSSHUV The SAS 9.1 software (TS Level 1M3, SAS Institute, USA) was used for statistical analyses to compare group means.
Table 1. Optimum conditions of ELISA for the detection of CMV-CP. 3DUDPHWHU
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Twelve samples each of soluble protein extracts from H15 and P2377 were measured to assess the concentration of CMV-CP. Statistical significance was determined by Dunnett’s t-test. A probability (p) value less than 0.05 was considered to indicate statistical significance.
Results and Discussion 2YHUH[SUHVVLRQ RI U&09&3 LQ ( FROL %/ Transformants coding for CMV-CP were grown in LB medium containing ampicillin. Recombinant CMV-CP (rCMVCP) expression was then induced from E. coli BL21 using a pET-22b (+) expression system through the addition of 1 mM IPTG. Overexpressed hexahistidine (His6)-tagged rCMV-CP was partially purified by Ni-NTA affinity column chromatography. Afterward, all fractions eluted by an imidazole gradient from 20 to 200 mM were subjected to SDS-PAGE to detect the presence of rCMV-CP. The His6-tagged protein was observed in the 100 and 125 mM imidazole fractions (data not shown). Fractions eluted at 100 mM imidazole seemed to contain a large amount of rCMV-CP, but non-specific proteins were present as well. The His6-tagged rCMV-CP of higher purity was eluted in the 112.5 and 125 mM imidazole fractions. These fractions were therefore collected and enriched using Amicon Ultra columns (10 K, Millipore, USA) and subjected to Western blot analysis. Purified rCMV-CP exhibited a single band in Western blot analysis and was therefore used in further experiments as CMV-CP. A similar approach was used by Seo et al. (2009) to express the human papillomavirus (HPV) 18 L1 gene, which encodes the L1 major capsid protein, in E. coli BL21 as a fusion protein with a glutathione-Stransferase (GST) tag. The gene was cloned into the pGEX-4T-1 vector, and the expression plasmid was transformed into E. coli. After induction of expression of the HPV-L1 protein in E. coli by IPTG induction, the GST-tagged protein was purified by glutathione-Sepharose resin from the cell lysate. The purity of the isolated protein was confirmed by Western blot analysis using rabbit anti-PV polyclonal antibody. Janknecht et al. (1991) used Ni-NTA agarose resin to purify denatured SRF-His6-tagged protein expressed from vaccinia recombination vector pMS-56. The SRF-His6-tagged protein eluted over a broad range of
imidazole concentrations. Elution with 0.8-40 mM imidazole resulted in a large proportion of contaminants, but also the elution of some SRF-His6-tagged protein. The vast majority of the tagged protein was eluted at 80 mM imidazole. 3UHSDUDWLRQ RI SRO\FORQDO DQWLERGLHV IRU &09&3 We evaluated two different types of polyclonal antibodies for qualitative and quantitative detection of CMV-CP. The VPAb was produced by immunization of rabbits with the CMV-Fny virion, while PPAb was obtained after immunization of rabbits with a synthetic peptide fragment based on the nucleotide sequence of CMV-CP. For PPAb production, peptide sequences of CMV-CP that exhibited high homology to phosphorylation, glycosylation, and myristoylation sites in other proteins were excluded to minimize the possibility of non-specific binding of the antibody. Transmembrane sites were excluded for the same reason. Peptide sequences exhibiting low hydrophobicity and high antigenicity near the N-terminus and C-terminus were selected to produce peptide antibodies (Table 2). Of seven candidates, one (no. 4) had structural features more suitable for producing a peptide antibody than the others and was therefore selected. The PPAbs were obtained from two rabbits (R1 and R2) immunized using the selected synthetic peptide. The PPAbs produced in R1 and R2 were compared to select the antibodies that were more specific for CMV-CP. No significant difference was observed between the reactivity of R1 and R2; R2 was therefore arbitrarily chosen for further study. Zein et al. (2009a) reported successful development of an immunological analysis method with monoclonal antibodies (MAbs) to CMV secreted in mouse hybridoma-cell lines that reacted with Pepo-CMVCP. These MAbs were successfully applied in an immunological assay to detect CMV from infected tobacco plants. Plagemann (2004) provided monoclonal antibodies produced from epitopes of the N-protein of porcine reproductive and respiratory syndrome virus (PRRSV). Several epitopes were determined from the amino acid sequence of the N-protein of PRRSV isolates, and monoclonal antibodies were produced from pigs. The characteristics of produced MAbs were investigated by indirect and competition ELISAs with synthetic peptides. PPAbs from R2 and VPAb produced corresponding bands when rCMV-CP
Table 2. Sequence of 7 candidates for peptide polyclonal antibody production. 3HSWLGH UHJLRQ ]
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