KR101294758B1 - Detecting method of CMV or GMO that tolerant to CMV - Google Patents

Abstract

The present invention relates to antigens and antibodies comprising peptides specifically selected from the envelope protein amino acid sequence of a cucumber mosaic virus (CMV). More specifically, the present invention specifically selects a specific peptide sequence of the envelope protein of CMV for effective detection of CMV and detection of CMV resistant transformants incorporating the envelope protein of CMV, an antibody comprising the same A composition comprising an antibody, a detection kit, and a detection method.
The antibody disclosed in the present invention, a composition comprising the antibody, a detection kit, and a detection method have a very high detection amount and a markedly low cross-reactivity with other proteins, so that the detection method can accurately and effectively analyze CMV-CP in a sample. Can be provided.

Description

Detecting method of CMV or GMO that tolerant to CMV}

The present invention relates to an antigen comprising a peptide specifically selected from the envelope protein amino acid sequence of a cucumber mosaic virus (CMV) and immunospecific antibodies thereof. More specifically, the present invention specifically selects a specific peptide sequence of the envelope protein of CMV for the effective detection of CMV or the introduction of envelope protein gene of CMV to detect transformants resistant to CMV, and the antigen The present invention relates to a polyclonal antibody, a detection composition comprising the antibody, a detection kit, and a detection method using the same.

Pepper is the 7th largest vegetable growing area in the world, and it is a very important vegetable crop that is used by various people in the form of food, dyes and medicines by about 5 billion people on earth. Red pepper is a very important crop in the domestic seed market. Domestic pepper seed sales account for about 20% (30 billion won) of the whole vegetable seed market, and the total industrial sales related to pepper production are annually worth 2 trillion won. In addition, domestic pepper production totals 5% of the world's pepper production, so the international status is high. The reason is because Korean pepper breeders have excellent breeding technology, secure a lot of excellent genetic resources, and have infrastructure of cultivation, production, processing and sales network of pepper. Therefore, pepper is one of the few domestic crops with global competitiveness.

According to a recent study, peppers showed the highest rate of 51.6% of the crops in which the virus was detected in agricultural fields in 2009, which is a big blow to the vegetable crop industry in Korea. There are 66 kinds of viruses in peppers, of which 29 are known to cause damage worldwide, and the remaining viruses are caused locally. In Korea, about 10 kinds of viruses have been reported in pepper crops. If you look at the representative types of viruses infected with Korean pepper, Broad bean wilt virus 2 (BBWV2), Cucumber mosaic virus (CMV), Impatience necrotic spot virus (INSV), Pepper mild mottle virus (PMMoV), Tomato spotted wilt virus (TSWV) are all five species (Choi et al ., 2009).

CMV (cucumber mosaic virus) belongs to the genus cucumovirus (cucumovirus) and is a RNA plant virus with a tripartite genome of (+) sense single-strand of 28-30 nm diameter co-shaped particles. The genome consists of three types of RNA and often contains satellite RNAs with 330 to 380 sequences. CMV is distributed all over the world and covers the crops and weeds among plant infectious viruses. It has the widest host range (infected with over 1000 plant species) and has important global economy. Symptoms are extremely diverse, and one of the most common symptoms is that the growth of the plant is severely inhibited, resulting in loss of productivity, with apparent mosaic symptoms on the leaves. Yellow spots or gangrene spots appear on the fruit, sometimes the skin is rough and crumbled with an unclear color.

The pepper's CMV (cucumber mosaic virus) is a virus that causes a lot of damage to farmers' pepper harvest worldwide, including Korea and China. In particular, CMV, which has been rampant in Korean farms since the mid-1980s, caused a lot of damage to the yield.

However, in the mid 90's, the CMVP0 (Fny-CMV) virus-resistant gene source was used to breed new varieties that were resistant to CMV. Since then, several seed companies have developed similar CMV-resistant varieties. As a result, CMVP0 virus resistant varieties were commercialized without any problem until 2000, but unfortunately these resistant varieties are recently infected with the new virus (Ca-P1-CMV). Ca-P1-CMV is a terrifying virus that infects all domestic pepper varieties, which is causing a lot of damage to pepper farmers. In addition, CMV is widespread in China and India. Therefore, it is significant to develop new peppers that are resistant to Ca-P1-CMV and CMVP0 in terms of preoccupation of red pepper seeds.

Thus, in a recent research and registration patent (Registration No. 10-0804766) is characterized by having a complex resistance to CMVP0 and Ca-P1-CMV infection by introducing the CMVP0-CP gene using Agrobacterium (Agrobacterium) The inventors of the present application have developed a transformed pepper, and the inventors of the present application have developed a peptide polyclonal antibody specific for the envelope protein of CMV inserted into a cucumber mosaic virus (CMV) and a transgenic new strain that is resistant to the CMV and its Detection compositions, detection kits, and detection methods have been developed to enable rapid analysis of new resistant varieties.

An object of the present invention is to select an amino acid sequence of the envelope protein of the cucumber mosaic virus to provide a certain sequence as an antigen, and to provide an immunospecific peptide polyclonal antibody of this antigen.

Still another object of the present invention is to provide a detection composition, a detection kit, and a detection method of a plant transformed with the envelope protein gene of cucumber mosaic virus or CMV using the antibody.

The present invention comprises a cucumber mosaic virus (CMV) envelope protein antigen consisting of the amino acid sequence of SEQ ID NO: 5, preferably the cysteine (SEQ ID NO: 9) added to the N-terminal of the amino acid sequence of SEQ ID NO: Cucumber mosaic virus coat protein antigen fused to a carrier protein.

Most proteins are likely to undergo post-synthesis processing, sugar chain addition, phosphorylation, and post-translational modifications. In addition, since the protein has a part which is easy to become an antigen and a part which is difficult to be due to a steric structure, etc., comprehensive consideration and judgment are required to select an antigen.

In one embodiment of the present invention, the amino acid sequence of the envelope protein of the cucumber mosaic virus was partially excluded by domain exclusion assay and membrane protein domain investigation. In the present invention, the ubiquitous domain, phosphorylation site, myristoylation site, and glucosyl site, which are found everywhere, were excluded in order to detect a specific subject in the domain exclusion assay, and there was no membrane protein. In addition, seven candidate sequences with low hydrophobicity and high antigenicity were selected through hydrophobicity and antigenicity assay, and homologous search was performed. In addition, considering the tertiary structure, the amino acid sequence of SEQ ID No. 5 having the highest antigen suitability was selected.

In addition, the present invention is a cucumber mosaic virus coat protein fused to the carrier protein or a cysteine (SEQ ID NO: 9) added to the N-terminal of the amino acid sequence of SEQ ID NO: 5 or the envelope protein consisting of the amino acid sequence of SEQ ID NO: 5 And antibodies against protein antigens, preferably the antibody is a polyclonal antibody.

The term 'polyclonal antibody' used in the present invention is an antibody obtained through the blood by inducing a humoral immune reaction by injecting an external substance such as a protein antigen into an organism, and responding to various antigenic determinants of the antigen. It is a complex of different antibodies.

In one embodiment of the present invention, the antigen synthesized through the selected amino acid sequence was subjected to immune response to two rabbits, and the resulting peptide polyclonal antibody was isolated. Reactivity and specificity between the produced antibody and CMV envelope protein were confirmed by Western blot (FIG. 6), and the coat protein (CP) of the synthesized cucumber mosaic virus (CMV) and CMV-CP expressed in bacteria were expressed. Peptide fragments were subjected to enzyme-linked immunosorbent adsorption (ELISA) analysis with antigens. As a result, both antibodies responded to both antigens and there was no significant difference between the two antibodies (FIG. 7).

The present invention includes a composition for detecting a transformant into which a cucumber mosaic virus or CMV envelope protein containing the antibody is introduced, and a detection kit comprising the antibody, a detection method comprising using the composition or the detection kit, and preferably Preferably, the composition, the detection kit, and the detection method are for enzyme-linked immunosorbent detection (ELISA).

Detection methods using antibodies include, but are not limited to, radioimmunoassay (RIA), fluorescent antibodies, enzyme-linked immunosorbent assay (ELISA), and the like. Radioimmunoassay is a method of labeling a radioisotope to either an antigen or an antibody and quantitatively tracking the antigen-antibody reaction to measure the amount of the antigen or antibody. Fluorescent antibody method is a method of fluorescence microscopy of the partial distribution of specific antigen molecules in tissues or cells using a fluorescently labeled antibody.

In addition, the term 'enzyme-linked immunosorbent assay' used in the present invention is a method of labeling an enzyme to an antibody or an antibody and confirming the intensity of the antigen-antibody reaction using the enzyme activity as an index and quantitatively measuring the antigen or the antibody. to be.

The present invention relates to antigens and antibodies comprising peptides specifically selected from the envelope protein amino acid sequence of a cucumber mosaic virus (CMV). More specifically, the present invention specifically selects a specific peptide sequence of the envelope protein of CMV for effective detection of CMV and detection of CMV resistant transformants incorporating the envelope protein of CMV, an antibody comprising the same A composition comprising an antibody, a detection kit, and a detection method.

The antibody disclosed in the present invention, a composition comprising the antibody, a detection kit, and a detection method have a very high detection amount and have a significantly low cross-reactivity with other proteins, thereby providing a detection method that can effectively analyze CMV-CP in a sample. can do.

1 is a cloning vector pCAMBIA 2300 encoding the gene of CMV-CP.
Figure 2 is a condition of enzyme-linked immunosorbent assay for CMV-CP analysis extracted from GM and Non-GM pepper.
Figure 3 is a membrane protein domain test results in the amino acid sequence of CMV-CP.
4 shows hydrophobicity and antigenicity for the entire sequence of CMV-CP.
5 is an amino acid sequence that is a candidate for peptide polyclonal antibodies. * Cys added to the C-terminus or N-terminus are fused to a carrier protein.
Fig. 6 shows the results of Western blot using E. coli expressed and purified CMV-CP as antigen. (A) shows the result when using the existing polyclonal antibody, (B) the peptide polyclonal antibody produced as the primary antibody.
7 is a standard curve in ELISA for comparing the specificity of two peptide polyclonal antibodies
(A) and (B) use peptide fragments of synthesized CMV-CP as antigens, and (C) and (D) use CMV-CP expressed in E. coli as antigens.
8 is a standard curve according to the concentration of the antibody. (A) is a peptide polyclonal antibody (B) is a conventional polyclonal antibody
9 is a result of screening CMV-CP extracted from GM plants and non-GM plants by performing ELISA using two antibodies. (A) is a polyclonal antibody (B) is a peptide polyclonal antibody
10 is a T-test showing the quantification of CMV envelope protein extracted from GM pepper and detectable CMV envelope protein content based on this. The existing polyclonal antibody was calculated by subtracting the response to non-GM pepper from the response to GM pepper. (A) The Korea University (B) The pepper was grown and studied in the packaging of Chung-Ang University.

Hereinafter, the present invention will be described in more detail to more specifically describe the present invention. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

< Example 1  >

1-1: Sample

The pepper samples used were transformed CMVP0-CP (CMV coat protein) genes against CMV, GM pepper (H15), a transformant resistant to CMVP0 and CMVP1, and a corresponding non-GM pepper (P2377, control). line). CMV-CP was transformed using Agrobacterium and pCAMBIA-2300 was used as the expression vector of CMV-CP gene (FIG. 1). The transformation process was performed in Nongwoo Bio Co., Ltd. (Lee et al ., 2009), and the crops were separately grown on Korea University pavement and Chung-Ang University pavement, harvested and stored at -70 ℃.

1-2: Polyclonality  Antibody ( Polycolnal antibody )

Dacron antibodies used in this experiment were produced and provided by Seoul Women's University. CMVP0 virions were used as immunogens, and primary inoculations were performed on experimental rabbits by emulsification in 1 mg CMVP0 virions / 1.5 ml saline and 1.5 ml complete Freund's Adjuvant (Sigma, USA). Weeks 2 and 3 were further inoculated in experimental rabbits by emulsification in 1 mg CMVP0 virion / 1.5 ml saline and 1.5 ml incomplete Freund's Adjuvant (Sigma, USA). One week after the third inoculation, blood was collected to confirm antibody production.

1-3: Pretreatment of sample (virus resistance GM Isolation of Water Soluble Protein from Pepper)

Viral-resistant GM pepper (H15) and control non-GM pepper (P2377, control line) were harvested and frozen and stored at -70 ° C for the extraction of CMV-CP protein, a viral pathogenic gene product. Take some of the frozen stored pepper and precool (4 ° C) sample grinding buffer (1 mM phenylmethyl-sulfonyl fluoride (PMSF), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% proteolysis) 50 mM potassium phosphate buffer at pH 8.0 containing enzyme inhibitor mix (Sigma P27214) was added. Grinding using a high speed homogenizer (homogenizer) and centrifugation (8,000 rpm, 4 ℃, 25 minutes) to remove the pulverized pepper tissue and debris. Polyethylenimine (PEI) was added 0.2% to the supernatant to precipitate DNA and centrifugation (8,000 rpm, 4 ° C., 25 minutes) to remove the precipitated DNA. Protein was precipitated by adding 70% ammonium sulfate to the supernatant, and the protein content was measured after dissolving the precipitated protein in a small amount of buffer solution (50 mM potassium phosphate buffer, pH 8.0). All procedures were performed at 4 ° C.

1-4: CMV - CP Standard of

E. coli BL21 strain transformed with CMV-CP gene was inoculated into a flask containing LB broth (Bacto, 25 g / L containing 0.1% ampicillin) and cultured (37 ° C., 150 rpm). After incubation until the OD ₆₀₀ was 0.5 or higher, 0.01% isopropylthiogalacttoside (IPTG) was added and induction was performed at 25 ° C. and 150 rpm for 4 hours. Centrifuge the culture broth (8,000 rpm, 4 ° C, 20 minutes) to dissolve the supernatant-free pellets in Lysis buffer (10 mM imidazole), and then break the cell wall with French press and centrifuge (12,000 rpm, 4 ° C, 1). Time) to remove the pulverized tissue to extract the water-soluble protein of the cultured strain. The extracted solution was subjected to affinity chromatography using a polypeptide chromatography column (Bio-Rad, 731-1550) to partially purify the CMV-CP. As the elution buffer, imidazole was used at a concentration gradient of 10 mM to 250 mM, and the collected portion was confirmed by SDS-PAGE and Western blot to confirm the presence of CMV-CP. Purified CMV-CP was used as standard for enzyme-linked immunosorbent assay (ELISA) and Western blot of samples.

1-5: Peptides Polyclonality  Antibody production

An attempt was made to produce peptide polyclonal antibodies through peptides synthesized using the amino acid sequence of the immunogen CMV-CP (SEQ ID NO: 1, 218 amino acids).

Part of the amino acid sequence of CMV-CP, which is considered to be highly likely to be produced for antibody production, is domain exclusion analysis, transmembrane domain search, hydrophobicity and antigenicity. The test was performed using the test and homology search. Specific domain sites with high similarity are excluded from the ubiquitous domain region because the specificity of the similar domains is different from other proteins. Therefore, the glucosylation site and the myristoylation site ( Myristoylation site) was excluded because the synthesized peptide and the expressed protein is not the same. Sites with low hydrophobicity and high antigenicity were generally considered because of high possibility of producing specific antibodies and high possibility of being exposed to the outside in the tertiary structure. The homologous sequence search (NCBI) was used to examine how the selected peptide moieties matched with other proteins.

Immune antigen synthesized via the selected amino acid sequence was first immunized to two rabbits (R1, R2) by emulsification with complete freund's adjuvant (Sigma Chemical Co., St. Louis, MO, USA). (1 mg / rabbit) was performed and additional immunization (500 μg / rabbit) was performed by mixing the same immunogen with incomplete freund's adjuvant 4 and 6 weeks after the first immunization. Serum was collected after 1 week of each additional immunization at week 4 and 6 and final serum was collected at week 9 to confirm antibody production.

Reactivity and specificity between the produced antibody and CMV-CP were confirmed by Western blot, and titer was measured by enzyme-linked immunosorbent assay.

1-6: Peptides Polyclonality  Confirming the reactivity of the antibody ( Western  Blot)

In order to confirm whether the produced peptide polyclonal antibody is responsive to the expressed and purified CMV-CP (from E. coli ) protein, the same polyclonal antibody as that of the existing polyclonal antibody (provided by Seoul National University) Western blot was performed using the prepared peptide polyclonal antibodies R1 and R2.

The same amount of purified CMV-CP protein was first treated with two sets of 12% acrylamide gels followed by Laemmli (Laemmli et al ., 1970) . SDS-PAGE was carried out and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA) for 90 minutes at 100 V current.

The transferred PVDF membrane was treated with 5% skim milk / TBS for 1 hour, and the produced peptide polyclonal antibody and the existing polyclonal antibodies as primary antibodies, respectively, as a primary antibody, had a ratio of 1: 1,000 for 3% skim milk / TBS. Diluted with and treated for 2 hours. The remaining unbound antibodies were washed with TBS, followed by dilution of the same secondary antibody (anti-rabbit IgG alkaline phosphatase, Sigma Aldrich, St. Louis, MO, USA) at a ratio of 1: 1,000. The membrane was treated for 1 hour. Similarly the remaining antibodies were washed with TBS and then nitrobluetetrazolium (NBT) (Promega, Madison, WI, USA) and 5-bromo-4-chloro-3-indoyl phosphate (Promega, Madison, WI, USA) Treatment of the mixed reagents confirmed the color reaction on the membrane.

1-7: Establishment of enzyme-linked immunosorbent assay Enzime linked immunosorbant  assay; ELISA )

An optimal enzyme-linked immunosorbent assay (ELISA) method for CMV-CP analysis extracted from GM pepper was established under the conditions as described in FIG.

CMV-CP expressed in pretreated GM and Non-GM pepper samples and bacteria was dissolved in 200 μl in 96-well microplates (Nunc, Roskilde, Denmark) dissolved in 50 mM potassium phosphate buffer (pH 8.0). Aliquot, coated at 37 ° C. for 3 hours, washed twice with PBST (PBS containing 0.05% Tween 20, pH 7.4), and 200 μl of 3% skim milk (dissolved in PBST) to enhance the specificity of the reaction. After 2 hours of blocking at 37 ° C., the plate was washed twice with PBST. Peptide polyclonal antibodies and polyclonal antibodies were diluted 1 / 1,000 and 1 / 3,000 times in the washed wells, and 200 μl were dispensed into each well. The reaction was carried out at 37 ° C. for 3 hours, and then twice with PBST. Washed. In order to compare the results of CMV-CP analysis of peptide polyclonal and polyclonal antibodies, two antibodies were treated separately in the same sample, and the concentrations of the antibodies were selected through optimization experiments. Into the washed wells, 200 μl of goat anti-rabbit IgG (alkaline phosphatase, Sigma, A3687) was used as a secondary antibody and reacted at 37 ° C. for 2 hours, followed by washing twice with PBS. 200 μl of nitrophenyl phosphate (PNPP, Sigma, N4645) was added to each well at a concentration of 1 mg / ml, and reacted at room temperature. The absorbance of the sample at 405 nm was measured at 10 minute intervals until the absorbance was 0.1 or more.

< Example 2 > Peptides Polyclonality  Antibody Construction

In order to prepare a peptide polyclonal antibody that specifically reacts to CMV-CP, a suitable part for antibody production was searched based on the amino acid sequence of CMV-CP.

The domain exclusion test did not identify the ubiquitous domains, and the phosphorylation site (26-29 amino acid; SsaD, 74-76 amino acid; TlK, 124-126 amino acid; TvR), myristoylation site (159-164). amino acid (GVqaNN) and glucosylation sites (43-46 amino acid; NKTL) were identified and excluded from antibody production. In addition, membrane proteins are generally excluded from antibody production, but it was confirmed that there was no domain predicted by membrane protein domain test (FIG. 3). As a result of confirming the hydrophobicity and antigenicity of the entire CMV-CP sequence is shown in FIG. In general, the regions with low hydrophobicity and high antigenicity have high antibody specificity and are more likely to be exposed to the outside in the tertiary structure. The seven candidates predicted are shown in FIG. Considering the tertiary structure among the seven sites predicted by the above results, it is thought that the sites 92-106 belonging to SEQ ID NO: 5 are more likely to produce antibodies than the other candidates. Antibodies were produced.

< Example 3 > Peptides Polyclonality  Confirming the reactivity of the antibody ( Western Blot )

Reactivity of the antibody produced by Western blot using the newly produced peptide polyclonal antibody R2 and the existing polyclonal antibody (provided by Seoul National University) using CMV-CP expressed and purified in E. coli as an antigen. Confirmed. First, as shown in FIG. 6, CMV-CP protein extracted from E. coli was expressed and purified through the colored bands. The reactivity of the produced peptide polyclonal antibody (FIG. 6B) and the CMV-CP expressed in bacteria was confirmed through color development, and similarly high levels were developed when compared with the conventional polyclonal antibody (FIG. 6A). It can be seen that the peptide polyclonal antibody recognizes the CMV-CP protein well enough, and thus the antibody has sufficient binding capacity with the CMV-CP. CMV-CP has 218 amino acids and theoretically has a molecular weight of 24,090 Da (FIG. 1), but it is about 25,000 Da (BMS, according to the type of size marker used in the SDS-PAGE and western blot experiments). 161-0373), 24,000 Da (BMS, 161-0318), 27,000 Da (BMS, 161-0374) and the band appeared to have some differences.

< Example  4> antibody selection and Potency  compare

Peptide polyclonal antibodies were generated and isolated from two rabbits through the same process (peptide polyclonal antibody; R1 and R2). ELISA was performed using the peptide fragment of the synthesized CMV-CP as an antigen to select the high specificity among the two peptide polyclonal antibodies obtained, and to confirm the reactivity with CMV-CP other than the synthesized peptide fragment. ELISA was performed using the expressed CMV-CP as an antigen. As shown in FIG. 7, the peptide polyclonal antibodies R1 and R2 responded to both antigens, and there was no significant difference between the two antibodies. Although the reactivity with CMV-CP expressed in bacteria was relatively weak, it showed sufficient reactivity to detect. Since there was no significant difference between the two antibodies, a randomly selected peptide polyclonal antibody R2 was used in subsequent comparisons with the polyclonal antibody.

< Example  5> Determination of antibody concentration

In order to establish the concentrations of peptide polyclonal and polyclonal antibodies, the concentration of each antibody was 1 / 1,000, 1 / 3,000, 1 / 5,000, 1 / 10,000, 1 / 30,000, 1 / 50,000, 1 / 100,000. Reactivity was compared for the same antigens that were diluted and expressed in E. coli (FIG. 8). As expected, both the peptide polyclonal antibody and polyclonal antibody showed higher reactivity with higher concentrations, and the reactivity of 1 / 1,000 titer of the peptide polyclonal antibody and 1 / 3,000 of the polyclonal antibody was clear. Suitable for comparing results.

< Example  6> In samples using enzyme immunoassay CMV -CP detection

CMV-CP detection of red pepper samples was attempted by ELISA using two different antibodies. The same process was performed using GM pepper with the CMV-CP gene inserted as a test group and Non-GM pepper without the gene inserted as a control group. The standard curve was the standard curve using CMV-CP produced through bacterial expression. Was written. The pepper samples used were harvested after cultivation in Korea University pavement and Chung-Ang University pavement. As a result, both the peptide polyclonal antibody and the polyclonal antibody showed strong reactivity with the GM pepper extract with the CMV-CP gene inserted, and the polyclonal antibody could be visually confirmed even in the non-GM pepper extract as a control. It was confirmed that it showed a non-specific reaction with other proteins besides CMV-CP. On the other hand, in the case of the peptide polyclonal antibody produced, only reaction with GM pepper extract was confirmed (FIG. 9).

CMV-CP in pepper samples was quantified using the standard curve of CMV-CP produced by bacterial expression.In the case of polyclonal antibodies, non-GM was considered as nonspecific reaction other than the target protein in the degree of reaction with GM pepper extract. Calculated by subtracting the degree of reaction with pepper extract (Fig. 10). The CMV-CP in GM peppers of Korea University packaging showed 0.173 μg / g (polyclonal antibody, per g raw pepper) and 0.460 μg / g (peptide polyclonal antibody, per g fresh pepper). CMV-CP in GM capsicum of the university packaging showed 0.135 μg / g (polyclonal antibody, per g fresh pepper) and 0.272 μg / g (peptide polyclonal antibody, per g fresh pepper). Peptide polyclonal antibodies result in higher reactivity since there is no cross-reaction with non-GM pepper. Based on these results, the difference in CMV-CP content that can be detected by polyclonal and peptide polyclonal antibodies was analyzed by ANOVA using the SAS 9.1 program (TS Level 1M3, SAS Institute Inc., USA). Significance was verified and compared through post hoc analysis by multiple tests of Dunnett's T-test at p <0.05 level (FIG. 10). Enzyme-immunoassay using peptide polyclonal antibody showed that the detectable CMV-CP content was about 2.01 times higher than that of Chung-Ang University red pepper sample to 2.66 (Korea University red pepper sample). It was confirmed that there is a significant difference in reactivity between the two antibodies and CMV-CP for.

Attach an electronic file to a sequence list

Claims (10)

Cucumber mosaic virus (CMV) envelope protein antigen, consisting of the amino acid sequence of SEQ ID 5; A cucumber mosaic virus (CMV) envelope protein antigen wherein the cysteine of the amino acid sequence of SEQ ID NO: 9 is fused to a carrier protein. delete A polyclonal antibody against the antigen of claim 1. Cucumber mosaic virus (CMV) or CMV envelope protein containing a polyclonal antibody of claim 4 wherein the transformant detection composition is introduced. The method of claim 5, wherein the detection composition is a detection composition, characterized in that for enzyme-linked immunosorbent assay (ELISA). A transformant detection kit in which a cucumber mosaic virus (CMV) or CMV envelope protein is introduced, comprising the polyclonal antibody of claim 4. 8. The kit according to claim 7, wherein the kit is for enzyme-linked immunosorbent assay (ELISA). A method for detecting a transformant having a CMV or CMV envelope protein introduced therein using the composition according to claim 5. 10. The detection method according to claim 9, wherein the detection method uses an enzyme-linked immunosorbent assay (ELISA).

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