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

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 Hort. Environ. Biotechnol. 56(3):316-323. 2015.

Identification and Safety Assessment of Cucumber Mosaic Virus Coat Protein in Genetically Modified Pepper (Capsicum annuum)

“…2). Choi et al (2015) observed that, in immunoblot analysis, the expression of CMV-CP in GM peppers appeared as a major band at 26 kDa. Also in immunoblot analysis, used specific monoclonal antibodies (mAbs) for Pepo-and M2-CMV to detect CMV-CP in infected tobacco plants, and revealed the presence of 26 and 52 kDa viral proteins.…”

“…In previous studies, Choi et al (2015) developed antibodies that were designed from the peptide sequence of CMV-CP. Using peptide polyclonal antibodies, an immunoaffinity column was produced to purify CMV-CP.…”

“…However, virus infection of peppers has caused heavy losses in the rural economy. According to Choi et al (2015), chili peppers were infected at the highest level (51.6%) of all vegetablesfruit cultivated in outfield systems in Korea. Several studies have reported that over 60 species of viruses can infect peppers (Kenyon et al, 2014).…”

“…In the same way, optimization of qualification and quantification methods for viral CPs in the wild-type virus is required to assess the safety of these crops. In this study, we applied recently established analytical methods to detect viral CPs in virus-resistant GM crops using a peptide polyclonal antibody (Choi et al, 2015). An enzyme-linked immunosorbent assay (ELISA) was used to quantify the inserted protein in GM crops.…”

Asymmetric Synthesis and Bioselective Activities of α-Amino-phosphonates Based on the Dufulin Motif

“…29,30 Purification of CMV-CP. 31,32 Genetically engineered CMV-CP was expressed, into which hexahistidine (His) tags were incorporated. To express the protein, we transformed each plasmid into E. coli strain BL21 (DE3) RIL.…”