Monoclonal antibody for resisting cucumber green mottle mosaic virus and application and product thereof

Abstract

The invention provides a monoclonal antibody for resisting a cucumber green mottle mosaic virus as well as application and a product of the monoclonal antibody, and relates to the technical field of biology. The monoclonal antibody for resisting the cucumber green mottle mosaic virus, provided by the invention, is M2b-2 or M2b-13; the M2b-2 has a heavy chain variable region VH with an amino acid sequence as shown in SEQ ID NO.1 and a light chain variable region VL with an amino acid sequence as shown in SEQ ID NO.2; the M2b-13 has a heavy chain variable region VH with an amino acid sequence shown as SEQ ID NO.3 and a light chain variable region VL with an amino acid sequence shown as SEQ ID NO.4. The invention also discloses a preparation method of the heavy chain variable region. The monoclonal antibody for resisting the cucumber green mottle mosaic virus shows relatively high affinity to the CGMMV, is good in sensitivity and strong in specificity, and can be used for a double-antibody sandwich enzyme-linked immunological detection technology and development of rapid detection products such as colloidal gold test strips.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a monoclonal antibody against cucumber green mottle mosaic virus and applications and products thereof. Background Art

[0002] Cucumber green mottle mosaic virus (CGMMV) is a virus that belongs to the family Bacillusviridae. Virgaviridae )Tobacco mosaic virus genus ( Tobamovirus CGMMV is a positive-sense single-stranded RNA virus with rod-shaped virions that are highly stable and highly infectious. Most crops in the Cucurbitaceae family are highly susceptible to it. Infection can cause leaf chlorosis and mottling, stunting, and the fruit of watermelons to become rotten and lose their value.

[0003] CGMMV is primarily transmitted through infected seeds and mechanical damage, with seed infection rates reaching 10% to 50%. Production relies primarily on seed health testing and dry heat treatment to prevent and reduce the risk of seed transmission. Therefore, accurate and rapid detection is crucial for controlling the spread of the virus. Traditional detection methods, such as biological identification, offer high accuracy but are time-consuming and labor-intensive. Molecular detection methods like RT-PCR offer high sensitivity and reliable results, but they rely on specialized equipment and personnel, are costly, and impractical for rapid field screening. Immunoassays offer robustness, high accuracy, and low cost. Crucially, they rely on the specific binding of antibodies to target antigens (such as viral proteins). Signal amplification can be achieved through PCR or labeling techniques (enzymes, fluorescein, colloidal gold, etc.) to tailor detection to specific needs. For example, immunocapture RT-PCR can enrich the virus to overcome missed detection in low-load samples. Colloidal gold test strips enable rapid, batch testing without specialized equipment and techniques, making them suitable for field testing. Therefore, the development of highly sensitive and specific monoclonal antibodies against CGMMV is crucial for effectively blocking the spread of the virus.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The first object of the present invention is to provide a monoclonal antibody against cucumber green mottle mosaic virus to solve the problem of detecting cucumber green mottle mosaic virus.

[0006] A second object of the present invention is to provide a biomaterial.

[0007] The third object of the present invention is to provide a method for preparing the above-mentioned monoclonal antibody against cucumber green mottle mosaic virus.

[0008] The fourth object of the present invention is to provide the use of the above-mentioned monoclonal antibody against cucumber green mottle mosaic virus in the preparation of a detection product for cucumber green mottle mosaic virus.

[0009] The fifth object of the present invention is to provide a marker for cucumber green mottle mosaic virus.

[0010] The sixth object of the present invention is to provide a kit for detecting cucumber green mottle mosaic virus.

[0011] In order to achieve the above objectives, the following technical solutions are adopted: In a first aspect, the present invention provides a monoclonal antibody against cucumber green mottle mosaic virus, wherein the monoclonal antibody is M2b-2 or M2b-13; The M2b-2 has a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.1, and a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.2; The M2b-13 has a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.3, and a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.4.

[0012] In a second aspect, the present invention provides a biomaterial, wherein the biomaterial is selected from any one of ac: a nucleotide, said nucleotide comprising a nucleotide sequence encoding said anti-cucumber green mottle mosaic virus monoclonal antibody; b vector, said vector carrying the nucleotides in a; c. A cell carrying the nucleotide in a, or containing the vector in b, or expressing the monoclonal antibody against cucumber green mottle mosaic virus.

[0013] In a third aspect, the present invention provides a method for preparing the above-mentioned monoclonal antibody against cucumber green mottle mosaic virus, which is obtained by culturing the above-mentioned cells.

[0014] In a fourth aspect, the present invention provides use of the above-mentioned monoclonal antibody against cucumber green mottle mosaic virus in the preparation of a detection product for cucumber green mottle mosaic virus.

[0015] In a fifth aspect, the present invention provides a marker for cucumber green mottle mosaic virus, comprising the monoclonal antibody against cucumber green mottle mosaic virus and a marker; The monoclonal antibody against cucumber green mottle mosaic virus is coupled with a label.

[0016] As a further technical solution, the marker includes an enzyme, a fluorescent molecule marker, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin or streptavidin.

[0017] In a sixth aspect, the present invention provides a kit for detecting cucumber green mottle mosaic virus, the kit comprising the monoclonal antibody against cucumber green mottle mosaic virus or the marker of cucumber green mottle mosaic virus.

[0018] As a further technical solution, the kit includes an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic particle detection kit, an immunofluorescence detection kit or an immunoblotting detection kit.

[0019] As a further technical solution, the ELISA detection kit includes an enzyme labeling plate, the M2b-2 and M2b-13, a washing solution, a blocking solution and a color developing solution; The M2b-2 and M2b-13 are respectively coated on an ELISA plate or used as a secondary antibody.

[0020] As a further technical solution, the immunochromatographic detection kit includes a base plate and a sample pad, a binding pad, a detection pad and a sample suction pad stacked on the base plate in sequence; The conjugate pad contains the cucumber green mottle mosaic virus marker; The detection pad is provided with a detection line and a quality control line, and the detection line is coated with the monoclonal antibody against cucumber green mottle mosaic virus.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The monoclonal antibody against cucumber green mottle mosaic virus provided by the present invention shows high affinity to CGMMV, good sensitivity and strong specificity, and can be used in double antibody sandwich enzyme-linked immunosorbent assay technology and the development of rapid detection products such as colloidal gold test strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Results of colloidal gold test strips testing leaves; Figure 2 The results of the colloidal gold test strip test on bottle gourd seed sample 2 are shown. DETAILED DESCRIPTION

[0024] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0025] Generally, the nomenclature used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein and its technology are those well-known and commonly used in this area.Unless otherwise indicated, the methods and techniques of the present invention are generally according to those well-known in the art, and are carried out as described in various general and more specific references, which are cited and discussed throughout this specification.Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly achieved in this area, or as described herein.The nomenclature used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein and its laboratory procedures and technology are those well-known and commonly used in this area.

[0026] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of an antibody's heavy or light chain that recognizes and binds to an antigen. The composition and arrangement of amino acids in this region determine the antibody's specificity for antigen recognition. The heavy chain variable region may be referred to as "VH." The light chain variable region may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding site.

[0027] The term "vector" refers to a nucleic acid delivery vehicle into which nucleotides can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is called an expression vector. A vector can be introduced into a host cell through transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell.

[0028] Such vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas. In some embodiments, the vectors described herein contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRESs), and other expression control elements (such as transcription termination signals, polyadenylation signals, and polyU sequences).

[0029] In a first aspect, the present invention provides a monoclonal antibody against cucumber green mottle mosaic virus, wherein the monoclonal antibody is M2b-2 or M2b-13; The M2b-2 has a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.1: QIQLVQSGPELVKPGASVKMSCKASGYIFTNYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFNGKATLTSDRFSTAYMELNSLTSEDSAVYYCAIRGSYGPSFPYWGQGTLVTVSA (SEQ ID NO. 1).

[0030] The M2b-2 has a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.2: EIVLTQSPAIMSASPGEKVTMTCSASSSVNYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSNNPWTFGGGTKLEIK (SEQ ID NO. 2).

[0031] The M2b-13 has a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.3: QVQLQQPGPAVIKPSQSLSLTCIVSGFSITSSSYCWHWIRQPPGKGLEWMGRICYEGSKYYSPSIKSRSTISRDTSLNKFFIQLSSVTYEDTAMYYCSRESYGTPFYYSMDYWGQGTSVTVSS (SEQ ID NO. 3).

[0032] The M2b-13 has a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.4: DIVMTQSTSSLSASLGDRVTISCRASQDIANYLNWYQQKPDGTVKLLIYYTSRLYLGVPSRFSGRGSGTDYSLTISNLEQEDFATYFCQQSNTFPYTFGGGTKLEIK (SEQ ID NO. 4).

[0033] In a second aspect, the present invention provides a biomaterial, wherein the biomaterial is selected from any one of ac: a nucleotide, said nucleotide comprising a nucleotide sequence encoding said anti-cucumber green mottle mosaic virus monoclonal antibody; b vector, said vector carrying the nucleotides in a; c. A cell carrying the nucleotide in a, or containing the vector in b, or expressing the monoclonal antibody against cucumber green mottle mosaic virus.

[0034] In a third aspect, the present invention provides a method for preparing the above-mentioned monoclonal antibody against cucumber green mottle mosaic virus, which is obtained by culturing the above-mentioned cells.

[0035] The preparation method is simple and convenient, and the prepared antibody can be used for detecting cucumber green mottle mosaic virus.

[0036] In a fourth aspect, the present invention provides use of the above-mentioned monoclonal antibody against cucumber green mottle mosaic virus in the preparation of a detection product for cucumber green mottle mosaic virus.

[0037] The monoclonal antibody against cucumber green mottle mosaic virus provided by the present invention can specifically identify cucumber green mottle mosaic virus, and thus can be used for detecting cucumber green mottle mosaic virus.

[0038] In a fifth aspect, the present invention provides a marker for cucumber green mottle mosaic virus, comprising the monoclonal antibody against cucumber green mottle mosaic virus and a marker; The monoclonal antibody against cucumber green mottle mosaic virus is coupled with a label.

[0039] The marker can be used as a specific marker for cucumber green mottle mosaic virus.

[0040] In some optional embodiments, the label includes but is not limited to enzymes, fluorescent molecular labels, fluorescent microspheres, colored microspheres, colloidal gold, biotin or streptavidin.

[0041] In a sixth aspect, the present invention provides a kit for detecting cucumber green mottle mosaic virus, the kit comprising the monoclonal antibody against cucumber green mottle mosaic virus or the marker of cucumber green mottle mosaic virus.

[0042] In some optional embodiments, the kit includes an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic particle detection kit, an immunofluorescence detection kit or an immunoblotting detection kit.

[0043] In some optional embodiments, the ELISA detection kit includes an ELISA plate, the M2b-2 and M2b-13, a washing solution, a blocking solution, and a color developing solution; The M2b-2 and M2b-13 are respectively coated on an ELISA plate or used as a secondary antibody.

[0044] In some optional embodiments, the immunochromatographic detection kit includes a base plate and a sample pad, a conjugation pad, a detection pad and a sample suction pad stacked on the base plate in sequence; The conjugate pad contains the cucumber green mottle mosaic virus marker; The detection pad is provided with a detection line and a quality control line, and the detection line is coated with the monoclonal antibody against cucumber green mottle mosaic virus.

[0045] In some optional embodiments, the antibody in the cucumber green mottle mosaic virus marker and the monoclonal antibody coated on the detection line are M2b-2 or M2b-13, respectively.

[0046] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0047] Example 1 Screening of monoclonal antibodies against cucumber green mottle mosaic virus 1 Antigen Purified CGMMV virions.

[0048] 2 Animals and Immunization Animals: Several 5-8 week old Balb / C mice were selected.

[0049] Immune adjuvant: Freund's complete adjuvant is used for the first main injection, and Freund's incomplete adjuvant is used for subsequent booster injections. Both are thoroughly mixed with an equal volume of antigen before injection.

[0050] Immunization method: multiple injections on the back.

[0051] Immunization dose: Main injection of 100ug antigen / experimental mouse, booster injection of 50ug antigen / experimental mouse.

[0052] The immunization cycle is as follows:

[0053] 3. Antiserum Detection A small amount of blood was taken from the tail vein of the mouse to prepare antiserum, and then the antiserum titer was detected by indirect ELISA method to screen mice with high titer.

[0054] 4. Cell Fusion and Subcloning 4.1 Myeloma Cell Preparation One week before fusion, SP2 / 0 cells were revived and cultured normally to the logarithmic phase.

[0055] 4.2 Splenocyte Preparation The mice to be fused were selected and sacrificed by cervical dislocation on the day of fusion. The spleens were removed and spleen cells were collected and counted according to standard procedures.

[0056] 4.3 Cell fusion Myeloma cells and spleen cells were mixed at a ratio of 1:3-1:10 and fused using standard procedures. Hybridoma cells were then cultured in HAT complete DMEM medium. Hybridoma cells were visible three days after fusion. On the seventh day, the medium was replaced with 1 / 2 HAT complete medium, and on the eighth day, the medium was replaced with 1 / 2 HT medium. Screening began approximately 10 days after fusion.

[0057] Cell fusion results: After fusion, the cells were cultured in HAT selective medium and observed under a microscope. Multiple growing hybridoma cells were seen, proving that the fusion operation was successful.

[0058] 4.4 Fusion screening Pipette 100 μl of cell supernatant per well for indirect ELISA testing. Determine the positive wells based on the ELISA results. Use a single-channel pipette to inspect the entire plate for positive wells, performing a second retest to further confirm the positive wells.

[0059] 4.5 Subcloning Two rounds of subcloning were performed on the cells in the rescreened positive wells. The first subcloning was done by limiting dilution of the cells in the positive wells and transferring them to multiple wells. The cells were cultured in HT DMEM medium and observed under a microscope for about 7 days. The wells with cloned growth were detected by indirect ELISA. The wells with the highest OD value were selected as positive wells. The cells in the positive wells were selected for the second subcloning. Stable positive hybridoma cell lines were detected and used as the cells for the final preparation of monoclonal antibodies and expanded in culture.

[0060] 4.6 Monoclonal Antibody Subtype Identification The subtype of each supernatant was determined using a Southern Biotech monoclonal antibody subtyping kit. Prepare a strip coated with the immunogenic protein (50 ng / well). Collect 600 μl of supernatant from each clone and add 100 μl / well to six enzyme-labeled wells containing the corresponding protein. Incubate at 37°C for 1 hour, wash three times with PBST, and then add diluted secondary antibodies against IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 to each well. Incubate at 37°C for 1 hour, wash three times with PBST, and develop with TMB. Wells showing a signal corresponding to the subtype identified by the secondary antibody are the subtype of the antibody.

[0061] 4.7 After two rounds of subcloning and retesting, a total of 14 established cell lines were obtained.

[0062] 4.8 Final positive cell line number and subtype: Confirm that cell lines #2 and #13 are available positive cell lines.

[0063] 5. Ascites Preparation and Antibody Purification 5.1 Ascites Preparation Expand the positive cells and inject them into the peritoneal cavity of Balb / C mice (sensitized with incomplete Freund's adjuvant). Generally, a bulge in the abdomen will be seen 7-10 days later, indicating the development of ascites. When obvious ascites develops in the mouse, remove the ascites promptly.

[0064] 5.2 Ascites purification The ascites of the above cells was purified, and the purity of the antibody after purification was greater than 90%.

[0065] 5.2.1. Centrifuge the ascites, aspirate the light yellow liquid and calculate the volume.

[0066] 5.2.2. Load the Protein A / G filler into a gravity purification column and wash three times with PBS, each time using 10x column volume of PBS.

[0067] 5.2.3. Load the ascites into the column and mix gently at 4°C for 2-4 hours.

[0068] 5.2.4. Release the flow-through liquid and set aside.

[0069] 5.2.5. Wash the Protein A / G filler three times with PBS, elute the antibody with pre-cooled pH 3.0 HCL-Glycine eluent, and immediately neutralize the collected antibody with 10x PBS neutralizing solution.

[0070] 5.2.6. Detect the antibody concentration and combine the high-concentration collection tubes.

[0071] 5.2.7. Load the eluted antibody with higher concentration.

[0072] 5.2.8. Dialyze against PBS at 4°C overnight.

[0073] 5.3 Purity detection by SDS-PAGE.

[0074] 6. Antibody Sequencing 6.1 Cell recovery and culture: 6.1.1 Preheat complete culture medium in a 37°C water bath. Remove cells from liquid nitrogen and place in a 42°C water bath. Shake rapidly until the cryotube is completely liquefied. Centrifuge the liquefied cryotube at 1200 rpm for 5 minutes.

[0075] 6.1.2 Discard the supernatant from the cryopreservation tube, take 1 ml of complete culture medium, mix the cell pellet, and add it to one well of a 6-well cell culture plate. Then add 2 ml of complete culture medium and place the plate in a CO2 incubator. Observe the cell status regularly and passage the cells according to their growth rate.

[0076] 6.2 RNA extraction After reviving the hybridoma cell line, culture it until the cell count is about 1×10 8 The cells were collected and total RNA was extracted using a kit and stored at -80°C.

[0077] 6.3 cDNA cloning: Using cDNA as a template, upstream and downstream primers were designed and synthesized to amplify the heavy and light chain genes through PCR. The PCR products of the heavy and light chain genes were subjected to electrophoresis and the target fragments were recovered by gel excision.

[0078] 6.4 Antibody gene sequencing: Insert the target gene fragment into the vector, transform the constructed plasmid into competent DH5a, spread on the plate, and pick a single clone for sequencing.

[0079] 6.5 Bioinformatics Analysis The sequences were compared and analyzed to determine the gene sequence and encoded protein sequence of the antibody.

[0080] After sequencing, the amino acid sequences of the heavy chain variable region and light chain variable region of monoclonal antibody M2b-2 are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively; the amino acid sequences of the heavy chain variable region and light chain variable region of monoclonal antibody M2b-13 are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0081] Example 2 Monoclonal antibodies M2b-2 and M2b-13 against cucumber green mottle mosaic virus were purified and prepared, respectively. The amino acid sequences of the heavy chain variable region and light chain variable region of M2b-2 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the amino acid sequences of the heavy chain variable region and light chain variable region of M2b-13 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0082] 1. Double antibody sandwich ELISA test: The coated antibody M2b-13 was coated at a concentration of 2 μg / mL at 100 μL / well at 4°C overnight (antibody diluent: CB buffer). After washing, 250 μL / well of blocking solution was added and blocked at 37°C for 1 hour (or blocked at 4°C overnight); the plate was washed, and 100 μl / well of the sample to be tested, positive and negative controls were added, respectively, and incubated at 37°C for 1 hour; the plate was washed, and the detection antibody M2b-2 was labeled with biotin and diluted with experimental buffer to 2 μg / mL, 100 μl / well, and incubated at 37°C for 1 hour; the plate was washed, and 100 μl of horseradish peroxidase-labeled streptavidin SA-HRP (diluted 20,000 times) was added to each well and incubated at 37 degrees for 1 hour; the plate was washed, and 100 μl of Use 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution and allow color development to proceed for approximately 5 minutes (this time can be extended or shortened depending on the specific situation). Add 50 μl of stop solution to each well and read the result at a wavelength of 450 nm. Determine the results by calculating the I / H ratio: I / H = (average absorbance of the test sample - average absorbance of the blank control) / (average absorbance of the negative control - average absorbance of the blank control). If the I / H ratio is ≥ 3, the sample is considered positive.

[0083] The purified virus particles were quantified and then diluted into different concentrations. The repeatability of the antibodies was detected by double antibody sandwich ELISA. The test was repeated three times. The results are shown in Table 1. It can be seen from Table 1 that the consistency of this detection method is good.

[0084] Table 1 Repeatability results

[0085] Afterwards, the sensitivity of the antibody was tested using positive leaves and seeds as samples according to the above method, and compared with a commercial antibody (Agdia, SRA: 45702 / 1000). The specific method was as follows: 0.1g of watermelon leaf tissue with typical CGMMV symptoms was taken, 1mL of extraction buffer was added, and ground into a homogenate. The mixture was allowed to stand at room temperature for 10 minutes, and the supernatant was diluted with dilution buffer (PBS + 2% BSA) to different concentration gradient solutions: 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10-6 Take 300 seeds of poisonous gourd, add 50mL of extraction buffer and soak for 10 minutes, then dilute the supernatant to 10 -2 , 10 -3 , 10 -4 , 10 -5 The results showed that the sensitivity of the antibodies provided by the present invention was 1 order of magnitude higher than that of commercial antibodies for leaf tissues, and 2 orders of magnitude higher than that of commercial antibodies for seed samples (Table 2).

[0086] Table 2 Sensitivity of double antibody sandwich assay

[0087] Antibody specificity testing The ELISA method was used to test leaf samples infected with CGMMV, zucchini yellow mosaic virus (ZYMV), watermelon silver mottle virus (WMSoV), and cucumber mosaic virus (CMV). The results showed that the antibody was positive only for CGMMV-infected samples and negative for the other infected samples (Table 3).

[0088] Table 3 Antibody specificity detection

[0089] 2. Colloidal gold test strips: The detection antibody M2b-2 was mixed with a gold nanoparticle solution to form a colloidal gold-antibody complex, which blocked the unbound gold nanoparticle surface. After purification, the complex was sprayed onto the conjugate pad. The capture antibody M2b-13 was sprayed onto the detection line (T line) of the NC membrane. The control line (C line) was coated with mouse anti-IgG to prepare a test strip for later use.

[0090] Take 0.1 g of watermelon leaf tissue with typical CGMMV symptoms, add 1 mL of extraction buffer and grind into a homogenate. Let it stand at room temperature for 10 minutes. Take the supernatant and dilute it with dilution buffer (PBS + 2% BSA) to different concentration gradient solutions: 10 -2 , 10 -3 , 10 -4 , 10 -5 ; Take 300 seeds of poisonous gourd, add 50mL of extraction buffer and soak for 10 minutes, then take the supernatant and dilute it 10 times, 20 times, 40 times and 100 times respectively. Use the prepared colloidal gold test strips to test the above samples, repeat 2 times for each sample, and read the results in 10-15 minutes. The results are shown in Table 4. Figure 1 and Figure 2 shown. Table 4 Colloidal gold test strip sensitivity test

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody against cucumber green mottle mosaic virus, characterized in that The monoclonal antibody is M2b-2 or M2b-13; The M2b-2 has a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.1, and a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.2; The M2b-13 has a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.3, and a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.

4.

2. Biomaterial, characterized in that The biological material is selected from any one of ac: a nucleotide, said nucleotide comprising a nucleotide sequence encoding the anti-cucumber green mottle mosaic virus monoclonal antibody according to claim 1; b vector, said vector carrying the nucleotides in a; c. A cell carrying the nucleotide in a, or containing the vector in b, or expressing the monoclonal antibody against cucumber green mottle mosaic virus according to claim 1.

3. The method for preparing the monoclonal antibody against cucumber green mottle mosaic virus according to claim 1, wherein Obtained by culturing the cells as claimed in claim 2.

4. Use of the monoclonal antibody against cucumber green mottle mosaic virus according to claim 1 in preparing a detection product for cucumber green mottle mosaic virus.

5. A marker for cucumber green mottle mosaic virus, characterized in that The invention comprises the monoclonal antibody against cucumber green mottle mosaic virus and a marker according to claim 1; The monoclonal antibody against cucumber green mottle mosaic virus is coupled with a label.

6. The marker of cucumber green mottle mosaic virus according to claim 5, characterized in that The markers include enzymes, fluorescent molecular markers, fluorescent microspheres, colored microspheres, colloidal gold, biotin or streptavidin.

7. A kit for detecting cucumber green mottle mosaic virus, characterized in that: The kit comprises the monoclonal antibody against cucumber green mottle mosaic virus according to claim 1 or the marker of cucumber green mottle mosaic virus according to claim 5 or 6.

8. The kit according to claim 7, characterized in that The kit includes an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic particle detection kit, an immunofluorescence detection kit or an immunoblotting detection kit.

9. The kit according to claim 8, characterized in that The ELISA detection kit comprises an enzyme labeling plate, M2b-2 and M2b-13 as claimed in claim 1, a washing solution, a blocking solution and a color developing solution; The M2b-2 and M2b-13 are respectively coated on an ELISA plate or used as a secondary antibody.

10. The kit according to claim 8, characterized in that The immunochromatographic detection kit comprises a base plate and a sample pad, a binding pad, a detection pad and a sample suction pad stacked on the base plate in sequence; The conjugate pad contains the marker of cucumber green mottle mosaic virus according to claim 5 or 6; The detection pad is provided with a detection line and a quality control line, and the detection line is coated with the monoclonal antibody against cucumber green mottle mosaic virus according to claim 1.

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