Monoclonal antibody PVM-4G8C4B6F2 capable of specifically recognizing potato virus M and application of monoclonal antibody PVM-4G8C4B6F2

Abstract

The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody for specifically recognizing potato virus M as well as a preparation method and application of the monoclonal antibody. The invention discloses a monoclonal antibody PVM-4G8C4B6F2 capable of specifically recognizing a potato virus M. The amino acid sequence of a heavy chain variable region of the monoclonal antibody PVM-4G8C4B6F2 is as shown in SEQ ID No.1, a light chain of the monoclonal antibody PVM-4G8C4B6F2 is a K-type light chain, and the amino acid sequence of a light chain variable region of the monoclonal antibody PVM-4G8C4B6F2 is as shown in SEQ ID No.2. The invention further discloses a preparation method of the monoclonal antibody PVM-4G8C4B6F2 capable of specifically recognizing the potato virus M. The monoclonal antibody PVM-4G8C4B6F2 capable of specifically recognizing the potato virus M. The monoclonal antibody PVM-4G8C4B6F2 capable of specifically recognizing the potato virus M can specifically recognize PVM, can be used for developing immunochromatography detection kits, immunochromatography detection kits and the like so as to meet the detection requirements in different scenes, and particularly has important application value in field rapid detection and large-scale screening.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a monoclonal antibody PVM-4G8C4B6F2 specifically recognizing potato virus M and application thereof. BACKGROUND

[0002] Potato (Solanum tuberosum L.) is one of the most important food crops in the world and plays a vital role in food production. However, during the cultivation of potato, the occurrence of viral diseases poses a serious threat to the yield and quality of potato. Among them, potato virus M (PVM) is particularly concerned due to its high incidence. PVM is mainly transmitted by non-persistent aphids and rubbing inoculation of infected leaf sap. Once infected, the virus will accumulate with the generations of potato vegetative propagation, leading to significant decline in potato quality and yield, and causing huge economic losses to the potato industry.

[0003] Currently, the detection and control of PVM mainly rely on traditional molecular biology methods such as reverse transcription polymerase chain reaction (RT-PCR), which have high accuracy but are complex, time-consuming, and require professional equipment, making it difficult to meet the demand for rapid detection of PVM.

[0004] Serological methods show broad application prospects in the field of plant virus detection due to their simple operation, rapid detection, and low cost. However, the establishment of serological methods depends on high-quality virus antibodies with high specificity. Currently, there is a lack of efficient and specific antibodies against potato M virus on the market, which limits the application of serological methods in PVM detection. Therefore, developing a monoclonal antibody that can specifically recognize potato M virus is of great significance for realizing rapid and accurate detection of PVM. SUMMARY

[0005] The application amplifies the PVM-CP protein gene sequence, constructs a prokaryotic expression vector, transforms E. coli, and after positive clone screening and identification, induces expression of the recombinant protein and performs purification. The purified PVM-CP recombinant protein is used as an antigen to immunize BALB / c mice, and mice with qualified serum titers are selected for booster immunization. Spleen cells of the mice are fused with myeloma cells, and antigen-specific hybridoma cells are screened. The positive hybridoma cell strain is obtained through subcloning screening. The hybridoma cell strain obtained through screening is injected into mice, and ascites is collected. The ascites is purified through Protein G, and the monoclonal antibody PVM-4G8C4B6F2 is screened.

[0006] Based on this, the purpose of the application is to provide a monoclonal antibody PVM-4G8C4B6F2 capable of specifically recognizing potato virus M and application thereof.

[0007] In order to achieve the above purpose, the application provides the following technical scheme.

[0008] The application provides a monoclonal antibody PVM-4G8C4B6F2 capable of specifically recognizing potato virus M, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody PVM-4G8C4B6F2 is shown in SEQ ID No. 1, and the light chain of the monoclonal antibody PVM-4G8C4B6F2 is a K-type light chain, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2.

[0009] Further, the coding nucleotide sequence of the heavy chain variable region of the monoclonal antibody PVM-4G8C4B6F2 is shown in SEQ ID No. 3, and the coding nucleotide sequence of the light chain variable region of the monoclonal antibody PVM-4G8C4B6F2 is shown in SEQ ID No. 4.

[0010] The application also provides application of the above monoclonal antibody PVM-44G8C4B6F2 in any one of the following aspects:

[0011] (1) detecting potato virus M;

[0012] (2) preparing a product for detecting potato virus M.

[0013] Further, the product for detecting potato virus M includes an immunochromatographic detection kit, a Western Blot detection kit, a colloidal gold detection kit and an immunochromatographic detection kit.

[0014] Preferably, the product for detecting potato virus M is an immunochromatographic detection kit.

[0015] The application further provides a product for detecting potato virus M, which comprises the monoclonal antibody PVM-4G8C4B6F2 according to any one of claims 1 to 2.

[0016] Further, the product is an immunochromatographic detection kit, a Western Blot detection kit, a colloidal gold detection kit or an immunochromatographic detection kit.

[0017] Preferably, the product is an immunochromatographic detection kit.

[0018] The application has the beneficial effect that the application discloses a monoclonal antibody PVM-4G8C4B6F2 capable of specifically recognizing potato virus M, the amino acid sequence of the heavy chain variable region of the monoclonal antibody PVM-4G8C4B6F2 is shown as SEQ ID No. 1, the light chain of the monoclonal antibody PVM-4G8C4B6F2 is K-type light chain, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2. The monoclonal antibody PVM-4G8C4B6F2 capable of specifically recognizing potato virus M according to the application can specifically recognize PVM and can be used for developing immunochromatographic detection kits, immunochromatographic detection kits and the like, so as to meet the detection requirements in different scenarios and has important application value in rapid field detection and large-scale screening. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 PVM-CP protein expression and purification; wherein, A: PVM-CP gene fragment amplification (M: DNA Marker); B: PVM-CP protein eluted by different concentrations of imidazole (M: Protein Marker, 1: induced bacterial body precipitation, 2-4: 50mM imidazole elution, 5-7: 200mM imidazole elution, 8-10: 500mM imidazole elution, 11: flow-through liquid); C: PVM-CP protein purification (M: Protein Marker).

[0020] Figure 2 Mouse serum titer determination results, wherein, 1 and 2 represent the numbers of immunized mice.

[0021] Figure 3 Specific detection of monoclonal antibodies, wherein, H: healthy sample, PVM: PVM-infected sample, PVS: PVS-infected sample, PVY: PVY-infected sample, PVX: PVX-infected sample, PLRV: PLRV-infected sample, +: positive control (plant juice infected with potato virus M as positive control), -: negative control (healthy potato plant juice as negative control). DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. It should be noted that, unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental reagents used in the embodiments are commercially available. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods.

[0023] Example 1: Preparation of Monoclonal Antibodies

[0024] 1. Expression and purification of PVM-CP protein

[0025] To obtain monoclonal antibodies against the potato virus M coat protein (PVM-CP), this invention uses a prokaryotic expression system to prepare the PVM-CP antigen. First, specific primers for the PVM-CP gene were designed for PCR amplification. Electrophoresis results showed a specific band of approximately 1000 bp. Figure 1 A) The fragment matched the expected target fragment (915 bp). After gel extraction and purification, the target fragment was cloned into the pET28a-PVM-CP recombinant expression vector via enzyme digestion and ligation.

[0026] The obtained recombinant expression vector was transformed into BL21 competent cells. Positive clones were selected and cultured overnight at 37℃. Induction with 0.5 mM IPTG at 18℃ for 16–18 h was performed. The bacterial pellet and supernatant were collected after induction and analyzed by SDS-PAGE. The results showed a clear band at 36 kDa in the bacterial pellet, indicating that PVM-CP protein could be expressed in vitro. The target protein was purified by elution with 50 mM, 200 mM, and 500 mM imidazole, respectively. The results showed that 200 mM and 500 mM imidazole had the highest elution efficiency. Figure 1 B). Collect the protein eluted with the above-mentioned concentration of imidazole, concentrate it using ultrafiltration, and determine the purity of the target protein using SDS-PAGE gel chromatography. Figure 1 C) The results showed that the obtained protein bands were clear and single, and the protein purity was high, which could be used for subsequent immunization of BALB / c mice.

[0027] The specific experimental procedures and methods for PCR amplification of the PVM gene, construction of the recombinant expression vector, and preparation of the PVM-CP protein are as follows:

[0028] 1.1 CP gene amplification in PVM

[0029] 1.1.1 Primer Design and Synthesis

[0030] The specific primers of PVM-CP were designed according to the gene sequence of PVM coat protein region provided on NCBI (Table 1), which were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd. The primer PVM-CP-F and PVM-CP-R amplified the CP gene of PVM, and the length of the amplification product was 915 bp.

[0031] Table 1 Cloning primers of CP gene

[0032]

[0033] 1.1.2 Extraction of viral genome

[0034] 1). The leaves of potato plants infected with PVM were ground into powder in liquid nitrogen and transferred to an enzyme-free 1.5 mL centrifuge tube;

[0035] 2). The extraction of RNA was completed according to the instructions of the Novozyme RNA extraction kit;

[0036] 3). The concentration of RNA was determined.

[0037] 1.1.3 Amplification of CP gene

[0038] 1). The synthesis of cDNA was completed with the extracted plant viral genome as the template and according to the instructions of the Novozyme reverse transcription kit;

[0039] 2). The amplification was carried out with the cDNA as the template and the primers in the table, and the reaction system was as shown in the table, and the PCR reaction program was as shown in Tables 2 and 3;

[0040] Table 2 PCR reaction system

[0041]

[0042] Table 3 PCR reaction program

[0043]

[0044]

[0045] 3). Agarose gel electrophoresis detection was carried out, and after the end, the gel imaging instrument was used to observe the size of the target band, and the target band was cut off for subsequent recovery and purification;

[0046] 4). The recovery and purification of PCR product was completed according to the instructions of the full-style gold DNA gel rapid purification kit.

[0047] 1.2 Construction of recombinant expression vector

[0048] 1.2.1 Construction of CP protein induction vector of PVM

[0049] The CP gene fragment of the purified PVM was constructed into the pET28a vector, and the reaction system was CP gene fragment 4 μL, pET28a vector 1 μL, ddH2O 5 μL, 37 °C for 30 min.

[0050] 1.2.2 Transformation of E. coli

[0051] 1). The BL21 competent cells were taken out from the -80 °C refrigerator and immediately placed on ice;

[0052] 2). 10 μL of the ligation system was added, mixed gently, and placed on ice for 30 min;

[0053] 3). 42 °C metal bath heat shock for 90 s, and then quickly placed on ice for cooling for 3-5 min;

[0054] 4). 1 mL of LB liquid medium (without antibiotics) was added to the tube, mixed, and then cultured at 37 °C, 200 rpm for 1 h;

[0055] 5). The above bacterial solution was centrifuged, 800 μL of supernatant was removed, the mixed medium was taken, and 100 μL was spread on a screening plate containing kanamycin;

[0056] 6). The inverted dish was cultured at 37 °C for 16-24 h;

[0057] 7). The transformed single colony bacteria were picked, and the cloned colonies were sent to GenScript Biotech (Shanghai) Co., Ltd. for sequencing.

[0058] 1.3 Preparation of PVM-CP protein

[0059] 1.3.1 In vitro expression of PVM-CP protein

[0060] 1). The E. coli bacterial solution containing pET28a-PVM-CP with correct sequencing was added to 5 mL of LB liquid medium, and cultured at 37 °C, 200 rpm for 16-18 h as a seed solution;

[0061] 2). The seed solution was transferred to a new LB medium at a ratio of 1:100, and cultured at 37 °C, 250 rpm. When the bacterial solution OD600=0.6, IPTG inducer was added to 0.5 mM, and the culture was continued at 18 °C for 16-18 h;

[0062] 3). 4 °C, 5000 rpm, centrifugation for 15 min, and the supernatant was removed to collect the bacteria;

[0063] 4). The bacteria were resuspended with lysis buffer (50 mM Tris, 0.5 M NaCl, pH 8.0) and ultrasonically broken;

[0064] 5). After ultrasonic, centrifuge the sample at 4°C, 5000rpm for 15min, take the supernatant and the precipitate respectively, and analyze the protein by SDS-PAGE.

[0065] 1.3.2 Purification of the expressed protein in vitro

[0066] 1). Take the cultured bacteria, add lysis buffer (50mM Tris, 0.5M NaCl, pH 8.0) to resuspend, and perform ultrasonic disruption under the following conditions: work 3s, off 2s, time 15min, repeat once;

[0067] 2). Centrifuge the ultrasonic disrupted bacteria in a low temperature centrifuge, collect the precipitate, dissolve the precipitate with denaturing buffer (50mM Tris, 0.15M NaCl, 8M urea, pH 8.0), and centrifuge to take the supernatant after 4°C standing for 1h;

[0068] 3). Filter the obtained supernatant with 0.45μm filter membrane, and purify the protein by Ni affinity chromatography column, and the purification steps are as follows:

[0069] 3.1). Wash with 5 times column volume of deionized water to remove air and 20% ethanol;

[0070] 3.2). Equilibrate the column with 5-10 times column volume of Buffer A (Buffer A: 50mM Tris, 0.15M NaCl, 8M urea, pH 8.0);

[0071] 3.3). Flow the sample through the Ni column at a speed of 0.5mL / min;

[0072] 3.4). Equilibrate the column with the above Buffer A;

[0073] 3.5). Elute with 50mM, 200mM and 500mM imidazole respectively;

[0074] 3.6). Analyze the eluted sample by SDS-PAGE;

[0075] 3.7). Collect the protein eluted by 50mM to 500mM imidazole, dilute with PBS to reduce the concentration of denaturant to 2M, dialyze at 4°C for 4h (dialysis buffer: PBS solution containing 2M urea), concentrate by ultrafiltration (ultrafiltration tube), and detect the purity of the target protein by SDS-PAGE.

[0076] 2. Preparation of PVM antibody serum

[0077] The PVM-CP protein obtained above is used as an antigen to immunize BALB / c mice. After 3 immunizations, the serum titer of the mice is determined by indirect ELISA method. When the serum titer of the mice reaches 81000, the immunization is qualified and can be used for preparation of monoclonal antibodies. The results show that the serum titer of the No. 2 mouse can reach 729000, and the performance is better, so that the mouse is subjected to a booster immunization for cell fusion screening. Figure 2

[0078] The specific experimental process and method of the mouse immunization and the mouse serum titer determination are as follows:

[0079] 2.1 Mouse immunization

[0080] 1). The PVM-CP protein expressed in vitro is used as an antigen, and healthy BALB / c female mice are selected. The first immunization uses 50 μg per mouse and an equal volume of Freund's complete adjuvant for emulsification, and the mice are subjected to abdominal multi-point subcutaneous injection;

[0081] 2). The mice are subjected to booster immunization every 14 days using 50 μg per mouse and an equal volume of Freund's complete adjuvant after emulsification;

[0082] 3). The serum titer of the mice is determined after the third immunization. When the serum titer of the mice reaches 81000, the immunization is determined to be qualified.

[0083] 2.2 Mouse serum titer determination

[0084] The mouse serum titer determination is determined by indirect ELISA method, and the steps are as follows:

[0085] 1). The antigen is diluted to 5 μg / mL with PBS coating solution in the experimental group, and the control group uses PBS, 100 μL per well, 4°C coating overnight, and PBST washing 2 times;

[0086] 2). Prepare 3% skimmed milk powder, 380 μL per well, room temperature shaking incubation for 1 h, and PBST washing 2 times;

[0087] 3). The serum is diluted to the specified concentration, 100 μL per well, room temperature shaking incubation for 1 h, and PBST washing 2 times;

[0088] 4). The goat anti-mouse IgG-HRP antibody is diluted by 1:5000, 100 μL per well, room temperature shaking incubation for 1 h, and PBST washing 2 times;

[0089] 5). Add TMB color developing liquid, 100 μL per well, room temperature reaction for 20 min;

[0090] 6). 2M sulfuric acid, 50 μl per well, room temperature reaction for 5-10 min; ​

[0091] 7). Read the readings of the microplate reader at 450nm and 630nm.

[0092] 3. Preparation of PVM hybridoma cells

[0093] The well-grown myeloma cells and the spleen cells of the No. 2 mice after 3 days of booster immunization were chemically fused, and after three days of culture, direct ELISA detection was performed. The plant juice infected with PVM was used as an antigen for coating, and the fusion cells were detected by preliminary screening. The readings of the microplate reader were recorded. When the positive readings were more than twice the negative readings, it was considered that the fusion cells could specifically recognize the antigen. Positive fusion cells were obtained after preliminary screening, as shown in Table 4.

[0094] Table 4 Fusion screening results

[0095]

[0096] The positive fusion cells obtained by screening were further subcloned by three times of limiting dilution method, and the hybridoma cell strain 4G8C4B6F2 capable of stably secreting PVM-CP monoclonal antibody was screened (Table 5).

[0097] Table 5 Subcloning screening results

[0098]

[0099] The specific experimental methods and processes of cell fusion screening and cell subcloning screening are as follows:

[0100] 3.1 Cell fusion

[0101] 1). Prepare well-grown myeloma cells (Sp2 / 0) with 10% FBS culture;

[0102] 2). Confirm the state of myeloma cells (Sp2 / 0) and feeder cells on the same day, and perform eye blood collection on qualified immunized mice. Centrifuge the serum for standby use. Sterile environment to take mouse spleen cells in serum-free medium for standby use;

[0103] 3). After washing the spleen, grind it evenly, sieve and centrifuge to collect the spleen cells, resuspend in serum-free medium and count;

[0104] 4). Mix the two according to the ratio of spleen cells: myeloma cells = 3:1, centrifuge after mixing, and use fusion agent PEG1450 for fusion. DMEM terminates the reaction and centrifuges;

[0105] 5). After centrifugation, the fused cells were resuspended with 60 ml of 15% FBS (with 2% HAT) at 100 μL / well, and then added to the 96-well plate with the feeder layer. After marking the project name, mouse number and date, the plate was placed in a carbon dioxide incubator for culture.

[0106] 3.2 Cell fusion screening

[0107] The cell fusion screening was performed by indirect ELISA. The specific steps were as follows:

[0108] 1). The experimental group was diluted with PBS coating solution to 1 μg / mL, and the control group was diluted with PBS. Each well was 100 μL, and was coated at 4°C overnight. PBST was used for washing twice;

[0109] 2). Prepare 3% skimmed milk powder, 380 μL / well, and incubate at room temperature for 1 h. PBST was used for washing twice;

[0110] 3). Dilute the serum to the specified concentration, 100 μL / well, and incubate at room temperature for 1 h. PBST was used for washing twice;

[0111] 4). Dilute the goat anti-mouse IgG-HRP antibody at 1:5000, 100 μL / well, and incubate at room temperature for 1 h. PBST was used for washing twice;

[0112] 5). Add TMB color developing solution, 100 μL / well, and react at room temperature for 20 min;

[0113] 6). 2M sulfuric acid, 50 μL / well, and react at room temperature for 5-10 min;

[0114] 7). Read the readings at 450 nm and 630 nm of the enzyme label.

[0115] 3.3 Cell subclone screening

[0116] After cell fusion screening, hybridoma cell strains were obtained, and cell subclone screening was continued. The hybridoma cells were cloned by limited dilution method. After 7 days, the single clone cell wells were observed by electron microscope, and screened in combination with ELISA results. The specific steps of ELISA were the same as above. After 3 times of cell subclone screening, the hybridoma cell strains meeting the standard were screened.

[0117] 4. Preparation of PVM monoclonal antibody

[0118] The hybridoma cell strain 4G8C4B6F2 obtained by screening was expanded and injected into the abdominal cavity of mice, and ascites was collected and purified to obtain the clone antibody PVM-4G8C4B6F2. The sequencing results show that the amino acid sequence of the heavy chain variable region of the monoclonal antibody PVM-4G8C4B6F2 is shown as SEQ ID No. 1, the coding nucleotide sequence of the heavy chain variable region of the clone antibody PVM-4G8C4B6F2 is shown as SEQ ID No. 3, the light chain of the monoclonal antibody PVM-4G8C4B6F2 is K type light chain, the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2, and the coding nucleotide sequence of the light chain variable region of the monoclonal antibody PVM-4G8C4B6F2 is shown as SEQ ID No. 4.

[0119] The specific experimental methods and processes of ascites preparation and purification are as follows:

[0120] 4.1 Preparation of ascites

[0121] The experimental mice were pretreated by intraperitoneal injection of 0.5 mL liquid paraffin (sensitizer), and 7 days later, each mouse was inoculated with single positive hybridoma cells (10 5 ~10 6 cells per mouse, intraperitoneally). On the 8th day after inoculation, the mouse abdominal swelling was observed, and when the abdominal cavity was significantly swollen and the activity was limited, sterile drainage technology was used to collect ascites in several times. The collected ascites was centrifuged and aliquoted, and stored at -80℃ ultra-low temperature environment for standby.

[0122] 4.2 Ascites purification

[0123] 1). Take the ascites and dilute it with PBS, filter it with a 0.2 μm filter membrane, and use Protein G purification in this experiment, the specific steps are as follows:

[0124] 2). Wash with deionized water to remove air and ethanol;

[0125] 3). PB buffer equilibration column;

[0126] 4). Flow the sample through the Protein G column at a flow rate of 0.5 mL / min;

[0127] 5). Elute with glycine and neutralize with Tris;

[0128] 6). Collect the glycine eluted sample, dialyze overnight at 4℃, and analyze the protein purity of the obtained sample.

[0129] Example 2 Monoclonal antibody recognition detection

[0130] To confirm that the monoclonal antibody PVM-4G8C4B6F2 can specifically recognize PVM antigen, the recognition performance of the antibody was detected by ELISA. The plant juice infected with potato virus M was used as a positive control, the healthy potato plant juice was used as a negative control, and PBS was used as a blank control. The results showed that the absorbance value of the positive control was higher than 2 times of the blank control, and the absorbance value of the healthy potato plant juice as an antigen was significantly lower than that of PVM (Table 6), indicating that the monoclonal antibody PVM-4G8C4B6F2 can recognize PVM without cross reaction with potato itself. Therefore, the performance of the monoclonal antibody PVM-4G8C4B6F2 in recognizing PVM is good, and it can be used for detecting PVM.

[0131] Table 6 ELISA detection of PVM monoclonal antibody PVM-4G8C4B6F2 specificity

[0132]

[0133] To further verify the specificity of the PVM monoclonal antibody PVM-4G8C4B6F2, Western Blot was used for analysis. The total protein of potato tissue culture seedlings infected with PVM and other viruses and healthy potato tissue culture seedlings was extracted, and the monoclonal antibody PVM-4G8C4B6F2 was used as a primary antibody for hybridization. The experimental results showed that the monoclonal antibody PVM-4G8C4B6F2 only detected a specific band of expected size (36 kDa) in the PVM infected sample, and had no cross reaction with other virus infected proteins and healthy controls Figure 3 ). The results showed that: (1) the monoclonal antibody PVM-4G8C4B6F2 has high specificity to PVM coat protein; (2) the antigen epitope recognition has no false positive reaction; (3) it meets the specificity requirements of the antibody for the development of immunochromatographic test strips, and can be used for the construction of immunochromatographic test strip system.

[0134] The specific experimental methods and processes of ELISA experiment and Western Blot experiment are as follows:

[0135] 1. ELISA experiment

[0136] 1). The PVM infected tissue juice was used as a positive control, the healthy potato plant juice was used as a negative control, and PBS was used as a blank control. The dilution was 100 μL / well, and the room temperature shaking bed incubation was 1 h. PBST was used for washing 2 times;

[0137] 2). 3% skimmed milk powder was prepared, 380 μL / well, room temperature shaking bed incubation was 1 h, and PBST was used for washing 2 times;

[0138] 3). The prepared PVM monoclonal antibody PVM-4G8C4B6F2 is diluted, 100 muL / hole, incubated at room temperature for 1h, and washed with PBST for 2 times;

[0139] 4). The goat anti-mouse IgG-HRP antibody is diluted at 1:5000, 100 muL / hole, incubated at room temperature for 1h, and washed with PBST for 2 times;

[0140] 5). TMB color developing liquid is added, 100 muL / hole, and reacted at room temperature for 20min;

[0141] 6). 2M sulfuric acid, 50 muL / hole, and reacted at room temperature for 5-10min;

[0142] 7). Read the readings at 450nm and 630nm of the enzyme label instrument.

[0143] 2. Western Blot experiment

[0144] The specificity of the monoclonal antibody is verified by Western Blot experiment, and the experimental process and method are as follows:

[0145] 1). Refer to the gel preparation kit of Yaenzyme to complete SDS-PAGE gel preparation, fix the protein gel glass plate in the electrophoresis tank, ensure that the glass gel plate is tightly attached to the rubber pad, and add ultrapure water to the electrophoresis tank to observe whether water leaks, and then add electrophoresis liquid to the electrophoresis tank after confirming that there is no water leakage;

[0146] 2). Take 0.5g sample in a 1.5mL centrifuge tube, pre-cool in liquid nitrogen, break for 30s at 1200rpm, add 500 muL-1mL of lysis buffer, vortex thoroughly, and lyse on ice for 10min, centrifuge at 12000rpm for 10min at 4 DEG C after lysis, take 100 muL supernatant, add 50 muL 3xloading buffer, mix well, and heat at 95 DEG C metal bath for 10min, open the cover to release gas every 2-3min during the period, and balance the pressure;

[0147] 3). Add protein Marker and sample to be tested to the sample hole in sequence;

[0148] 4). Connect the electrophoresis tank with the electrophoresis instrument, run at 80V until the sample reaches the junction of the concentrated gel and the separation gel, adjust the voltage to 120V, run until the protein Marker is completely separated, and stop electrophoresis;

[0149] 5). Take off the protein glue, put the cut PVDF membrane into methanol for activation for 15s, and put the cut filter paper and sponge into the Trans Buffer for full wetting, according to the order of negative-positive, according to the order of sponge-filter paper-protein glue-PVDF membrane-filter paper-sponge, place them in turn, avoid air bubbles during the process, clamp the clamp plate and put it into the transfer tank, transfer the membrane at 100V for 1h, and place the transfer instrument in the ice box to avoid high temperature and burn the membrane;

[0150] 6). Transfer the PVDF membrane to the WB incubation box, add the ponceau red dye, fully cover the PVDF membrane, incubate on the shaker for 3min, observe the protein band transfer, record the results, wash the PVDF membrane with TBST for 3 times, each for 5min, to ensure that there is no ponceau red dye residue on the PVDF membrane;

[0151] 7). Add an appropriate amount of blocking solution (5% skimmed milk powder), fully cover the PVDF membrane, and incubate on the shaker at room temperature for 1h;

[0152] 8). After the completion of blocking, wash the PVDF membrane with TBST for 3 times, each for 5min, add an appropriate amount of antibody to be tested, and incubate on the shaker at room temperature for 1h;

[0153] 9). Wash the PVDF membrane with TBST for 3 times, each for 5min, add the second antibody diluted according to 1:5000, and incubate on the shaker at room temperature for 1h, then wash with TBST for 3 times, each for 5min;

[0154] 10). Mix ECL electrochemical luminescence developing solution A and B according to 1:1, evenly spread the PVDF membrane, and develop with electrochemical luminescence imaging instrument.

[0155] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A monoclonal antibody PVM-4G8C4B6F2 that specifically recognizes potato virus M, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody PVM-4G8C4B6F2 is shown in SEQ ID No. 1, and the light chain of the monoclonal antibody PVM-4G8C4B6F2 is a K-type light chain, the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

2. The monoclonal antibody PVM-4G8C4B6F2 according to claim 1, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody PVM-4G8C4B6F2 is shown in SEQ ID No. 3, and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody PVM-4G8C4B6F2 is shown in SEQ ID No.

4.

3. The use of the monoclonal antibody PVM-44G8C4B6F2 according to any one of claims 1 to 2 in any of the following: (1) Detection of potato virus M; (2) Prepare products for detecting potato M virus.

4. The application according to claim 3, characterized in that, The products for detecting Potato M virus include: immunochromatographic assay kits, Western blotting assay kits, colloidal gold assay kits, and immunochromatographic assay kits.

5. The application according to claim 4, characterized in that, The product used to detect potato virus M is an immunochromatographic assay kit.

6. A product for detecting potato virus M, characterized in that, The product comprises the monoclonal antibody PVM-4G8C4B6F2 as described in any one of claims 1 to 2.

7. The product according to claim 6, characterized in that, The products mentioned are immunochromatographic assay kits, Western blotting assay kits, colloidal gold assay kits, and immunochromatographic assay kits.

8. The product according to claim 7, characterized in that, The product is an immunochromatographic assay kit.

Images