CN116589567B - Potato virus X monoclonal antibody PVX-2 and its application - Google Patents

Abstract

The invention discloses a potato virus X monoclonal antibody PVX-2 and application thereof, wherein the heavy chain amino acid sequence of the monoclonal antibody PVX-2 is shown as SEQ ID NO.7, the light chain amino acid sequence is shown as SEQ ID NO.9, and the antibody can specifically react with PVX CP protein (PVX-CP) and PVX virus particles and does not react with PVY virus particles or healthy plants. Further detecting pairing condition by DAS-ELISA method, and if PVX-2 is coated antibody, it can be successfully paired with other antibodies, such as PVX-1, PVX-3 and PVX-6; the sensitivity detection result shows that PVX monoclonal antibodies can be diluted by 1:10240 (w/v, g/mL) times, and can be used as a potato X virus antibody detection kit.

Description

Potato virus X monoclonal antibody PVX-2 and its application

Technical field

The present invention relates to the field of biological detection, specifically to the potato virus X monoclonal antibody PVX-2, and also relates to the application of the monoclonal antibody PVX-2.

Background technique

Potato (Solanum tuberosum L.) originated in the Andes Mountains of South America. It is an annual herbaceous plant in the Solanaceae family. It reproduces vegetatively and its tubers can be used as both food and vegetables. It is one of the most important high-yielding crops in the world, second only to wheat, rice and corn. my country is the largest potato producer. Potato planting area and total output rank first in the world, but the yield per unit area is far lower than that of developed countries such as Europe and the United States.

Since my country implemented the potato staple food strategy in 2015, the strategic status of potatoes has been further enhanced, and improving the quality and popularity of virus-free seed potatoes has become an important guarantee for the healthy development of the industry. As a vegetatively propagated crop, potatoes are infected with the virus. Due to continuous planting, the virus will be passed on through tubers from generation to generation, resulting in an increase in the incidence rate year by year and a rapid decline in yield. In the field, potato viruses can infect non-toxic potatoes through juice friction, aphid transmission, etc., causing potato yield reduction and degradation. After potatoes are infected with the virus, they will carry the virus for life. The condition will worsen year by year with the planting generations, and finally they will lose their planting value.

Viral diseases have long troubled scientific researchers. Currently, there are more than 40 types of viruses found on potato crops, including a viroid. PVX is a member of the genus Potexvirus, also known as Potato common mosaic virus or Potato light mosaic virus. It has a wide host range and mainly infects Solanaceae crops. PVX is a major pest affecting the quality of potato seed potatoes. It is one of the key indicators to determine whether the quality of potato seeds is qualified.

After a single infection of a plant by PVX, the plant symptoms are slightly mosaic or latent, and sometimes the leaf surface is slightly wrinkled and the leaf margin is wavy, which will cause a small amount of yield reduction. However, when planted in the field, compound infection often occurs. When combined with PVY to infect potatoes, the symptoms are aggravated and the potato yield is greatly reduced. In severe cases, the yield can be reduced by 80%, leading to serious economic losses.

Therefore, it is necessary to establish a simple, fast, sensitive, economical and accurate antibody for potato virus disease detection to meet the needs of potato seed potato field quality testing services and provide technical support for the comprehensive advancement of potato seed potato quality testing and certification work.

Contents of the invention

In view of this, one object of the present invention is to provide a potato virus X monoclonal antibody PVX-2; the second object of the present invention is to provide hybridoma cells secreting the anti-potato virus X monoclonal antibody PVX-2; The third object of the present invention is to provide the application of the potato virus X monoclonal antibody PVX-2 in preparing a kit for detecting potato virus X; the fourth object of the invention is to provide the potato virus X monoclonal antibody PVX-2 2. Application in preparing antibodies for detecting potato virus X; The fifth object of the present invention is to provide a kit containing the potato virus X monoclonal antibody PVX-2.

In order to achieve the above objects, the present invention provides the following technical solutions:

1. Potato virus

Preferably, the nucleotide sequence encoding the heavy chain of the monoclonal antibody PVX-2 is shown in SEQ ID NO. 8, and the nucleotide sequence encoding the light chain of the monoclonal antibody PVX-2 is shown in SEQ ID NO. Shown as ID NO.10.

2. Hybridoma cells secreting the anti-Potato virus

3. Application of the potato virus X monoclonal antibody PVX-2 in preparing a kit for detecting potato virus X.

4. Application of the potato virus X monoclonal antibody PVX-2 in preparing antibodies for detecting potato virus X.

5. A kit containing the potato virus X monoclonal antibody PVX-2.

Preferably, the kit is an ELISA kit, an immunochemical kit, an immunofluorescence kit or a Western Blot detection kit.

The beneficial effects of the present invention are: the present invention discloses a potato virus This antibody can specifically react with the CP protein of PVX (PVX-CP) and PVX virus particles, but does not react with PVY virus particles or healthy plants. The pairing situation was further tested through the DAS-ELISA method. When PVX-2 is a coated antibody, it can be successfully paired with other antibodies, such as PVX-1, PVX-3 and PVX-6; the sensitivity test results show that the sensitivity of PVX monoclonal anti- All can be diluted 1:10240 (w/v, g/mL) and can be used as detection kits for detecting potato virus X antibodies.

Description of the drawings

In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

Figure 1 shows the amplification of PVX-CP gene fragment (M: 2K plusII marker; 1, 2: PVX-CP gene fragment (714bp));

Figure 2 shows the expression of PVX-CP (M: Marker; 1: protein precipitation; 2: protein supernatant; 3: total protein; 4: negative control);

Figure 3 shows the purification of PVX-CP (A: PVX-CP is eluted with different concentrations of imidazole, M: marker, 1:50mM imidazole is eluted, 2:100mM imidazole is eluted, 3: 300mM imidazole is eluted. B: PVX-CP purification Result, M: marker, 4: concentrated protein);

Figure 4 shows Western blot analysis of PVX monoclonal antibody specificity (M: protein marker, 1: healthy potato tissue culture seedlings, 2: potato tissue culture seedlings infected with PVX, 3: potato tissue culture seedlings infected with PVY);

Figure 5 shows the sensitivity analysis of PVX monoclonal antibodies.

Biological preservation:

The hybridoma cells secreting PVX-6 and PVX-2 antibodies will be sent to the China Type Culture Collection Center for preservation. The preservation address is Wuhan University, Wuhan, China, and are named 3D6D9B5 and 4C10F4F5 respectively; the preservation date of 3D6D9B5 is September 1, 2022. The number is CCTCC NO: C2022279, and the classification name is hybridoma cell line 3D6D9B5; the deposit date of 4C10F4F5 is September 1, 2022, the deposit number is CCTCC NO: C2022280, and the classification name is hybridoma cell line 4C10F4F5.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

Example 1. Expression of viral CP protein

(1)Construction of expression vector

Search the PVX gene sequence on the National Center for Biotechnology Information (NCBI), and design primers to amplify the full length of the CP protein gene based on the full-length sequence of the PVX CP protein gene (Table 1). The primers are listed on NCBI. After comparison, it was synthesized by Shanghai Sangon Bioengineering Technology Services Co., Ltd. The PVX-CP gene was amplified using the primers of PVX-CP-F and PVX-CP-R, and the amplification product was 714 bp.

Table 1. PVX-CP amplification primers

Use the extracted total plant virus RNA as a template and follow the instructions of the kit to synthesize cDNA. Use the synthesized cDNA as a template and use the primers in Table 1 for amplification. The reaction system is 50 μL, and the cDNA template is 2 μL. On the virus, 2 μL of each downstream primer (0.1 μmol·L ^-1 ), 25 μL of 2×taq enzyme, and ddH ₂ O were added to 50 μL. The reaction conditions were pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 35 cycles, and extension at 72°C for 10 min.

After PCR amplification of the PVX-CP gene, agarose gel electrophoresis analysis was used to detect a DNA fragment with a length of 714 bp (Figure 1). The PVX-CP gene fragment was obtained through gel recovery and connected to the pET28a vector, and Transfer to BL21 competent cells, invert the plate at 37°C for overnight culture, select single clone plaques and send them to Sangon for sequencing. After sequence comparison, the bacterial fluid with correct sequencing results is selected for protein expression.

(2) Expression and purification in E. coli

The method of expressing the target protein in E. coli is as follows:

a. Add the correctly sequenced Escherichia coli liquid into 5 mL of LB liquid culture medium, place it in a 37°C constant temperature shaking incubator, and culture it overnight at 250 rpm/min for 16-18 hours to serve as the seed liquid;

b. Transfer to fresh 200mL LB medium at a ratio of 1:100, culture at 37°C and 250rpm/min. When the bacterial solution OD600=0.6, add IPTG inducer and continue induction culture at 18°C;

c. 4℃, 5000rpm/min, 15min, collect the bacteria;

d. Resuspend the bacterial cells in lysis buffer (50mm Tris, 0.5M NaCl, pH 8.0) and conduct ultrasonic disruption. Ultrasonic conditions are: work 3s, off 2s, time 15min, repeat;

e. After sonication, the sample was run at 4°C, 5000rpm/min, for 15min. The supernatant and precipitate were taken and analyzed using SDS-PAGE gel for protein analysis. The results are shown in Figure 2. The results showed that the recombinant protein PVX-CP could be expressed.

The method for purifying viral CP protein is as follows:

a. Take the cultured cells, add lysis buffer (50mM Tris, 0.5M NaCl, pH 8.0), resuspend and conduct ultrasonic disruption; the ultrasonic conditions are: work 3s, off 2s, time 15min, repeat;

b. Centrifuge the ultrasonically broken bacterial solution in a low-temperature centrifuge at 4°C, 5000rpm/min for 15 minutes. Collect the supernatant. Since the supernatant is directly hung on the column, in order to prevent it from hanging on the column, a denaturant is added to the supernatant. Urea, the final concentration is 8M. After dissolving, let it stand for 1 hour at 4℃, centrifuge and take the supernatant;

c. Filter the supernatant obtained above with a 0.45 μm filter membrane, and pass it through a Ni affinity chromatography column for protein purification. Proceed as follows:

d. Wash with 5 column volumes of deionized water to remove air and 20% ethanol;

e. Equilibrate the column with 5 to 10 times the column volume of Buffer A (Buffer A: 50mM Tris, 0.15M NaCl, 8M urea, pH 8.0);

f. Flow the sample through the Ni column at a speed of 0.5mL/min;

g. Equilibrate the column with Buffer A;

h. Elute with 50mM imidazole, 100mM, and 300mM imidazole respectively;

i. Perform SDS-PAGE gel analysis on the eluted samples to see if there is target protein. The results are shown in A in Figure 3.

The results show that different concentrations of imidazole can elute the viral CP protein, and the purity of the viral CP protein eluted from 100mM and 300mM is higher. Therefore, the proteins eluted with 100mM and 300mM imidazole were collected, diluted, dialyzed and concentrated, and the purity of the target protein was detected using SDS-PAGE gel. The results are shown in B in Figure 3, and the results show that the purity is very high.

Example 2. Preparation of potato X virus monoclonal antibody serum

(1) Immunized mice

The recombinant viral CP protein prepared in Example 1 was used as the immunogen, and 3 healthy Balb/c mice were selected. For the first immunization, 50 μg of antigen was used, and each mouse was emulsified with an equal volume of Freund's complete adjuvant and then injected subcutaneously into multiple points in the abdomen to immunize the mice. After the first immunization, every 14 days, 50 μg of antigen each was emulsified with an equal volume of Freund's incomplete adjuvant, and the mice were immunized by subcutaneous injection at multiple points in the abdomen for 3 times. At the beginning of the third immunization, blood was collected from the orbital venous plexus (or tail vein) of the mice 7 days after each immunization, and the antibody titer of the mouse blood was determined by indirect ELISA. The detection method is as follows:

(1) Protein coating: The experimental group used ELISA coating solution to dilute the PVX and PVY antigen proteins to 5 μg/mL, and the control group added ELISA coating solution, 100 μL/well, and coated overnight at 4°C, and washed twice with PBST;

(2) Blocking: Prepare 3% skimmed milk powder, 380 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

(3) Add sample: dilute the serum to the specified concentration, 100 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

(4) Secondary antibody: rabbit anti-mouse IgG-HRP 1:1000, 100 μL/well, incubate at room temperature for 1 hour, wash 3 times with PBST;

(5) Color development: TMB chromogenic solution A: solution B = 1:1, 100 μL/well, react at room temperature for 20 minutes;

(6) Termination: ELISA stop solution, 50 μL/well;

(7) Reading value: The main wavelength of the microplate reader is 450nm and the secondary wavelength is 630nm. The results are shown in Table 2.

Table 2. Mouse serum titer

The results showed that mouse No. 2 had the highest immune titer. Therefore, mouse No. 2 was selected to be injected intraperitoneally with 50 μg of antigen to strengthen the immunity. The immunity was strengthened for 3 to 7 days and cell fusion experiments were conducted.

(2) Preparation of viral monoclonal hybridoma cells

Cell fusion:

a. In a biological safety cabinet, collect about 10 ⁷ vigorously growing and well-shaped myeloma cells (Sp2/0) in a 50mL centrifuge tube, and resuspend them in DMEM (Glu 4.5g/L) medium without adding serum. Preheat the incubator at 37°C;

b. From immune-qualified mice 3 to 7 days after the booster immunization, under sterile conditions, take the spleens, grind and sieve, and then centrifuge to collect spleen cells;

c. Spleen cells and Sp2/0 are mixed and centrifuged, chemically fused with the fusion agent PEG1450, and DMEM is added to terminate the reaction;

d. Collect the fused cells by centrifugation, culture and screen them with high-glucose DMEM supplemented with NBS (newborn bovine serum) and HAT. After about 8 days, use indirect ELISA for primary screening of fusion, and the positive cell holes will be screened for fusion again;

e. Select monoclonal hybridoma cells that stably express antibodies. After the cells are expanded and cultured, the cells are taken for ascites production and frozen.

Cell fusion screening

Use the BSA competition ELISA method for detection. The steps are as follows:

a. Protein coating: dilute the PVX antigen or X virus grinding solution to the specified concentration with ELISA coating solution, 100 μL/well, coat overnight at 4°C, and wash twice with PBST;

b. Blocking: Prepare 3% skimmed milk powder, 380 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

c. Add sample: add 80 μL of cell supernatant in original volume, incubate at room temperature for 1 hour, and wash twice with PBST;

d. Secondary antibody: rabbit anti-mouse IgG-HRP 1:1000, 100 μL/well, incubate at room temperature for 1 hour, wash 3 times with PBST;

e. Color development: TMB chromogenic solution A: solution B = 1:1, 100 μL/well, react at room temperature for 20 minutes;

f. Termination: ELISA stop solution, 50 μL/well;

g. Reading value: The main wavelength of the microplate reader is 450nm, and the secondary wavelength is 630nm.

h. Select cells with good viability and perform the first subcloning.

This cell fusion resulted in 20 positive cell lines, see Table 3 for details:

Table 3. No. 2 mouse fusion screening

Cell subclone screening:

Cell cloning was performed using the limiting dilution method. Resuspend the positive cells and count them. Use 1 cell per 200 μL of culture medium as a guideline. Dilute the positive cells according to the count results. Add 200 μL of each well into a 96-well plate. Observe under the microscope after 7 to 9 days. Single cells will be marked. Cluster wells and perform indirect ELISA to detect positive cells. The detection method is as follows:

a. Protein coating: The experimental group used ELISA coating solution to dilute the PVX or PVY antigen protein to 1 μg/mL, and the control group added ELISA coating solution, 100 μL/well, and coated overnight at 4°C, and washed twice with PBST;

b. Blocking: Prepare 3% skimmed milk powder, 380 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

c. Add sample: Take the original cell supernatant, 80 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

d. Secondary antibody: rabbit anti-mouse IgG-HRP 1:1000, 100 μL/well, incubate at room temperature for 1 hour, wash 3 times with PBST;

e. Color development: TMB chromogenic solution A: solution B = 1:1, 100 μL/well, react at room temperature for 20 minutes;

f. Termination: ELISA stop solution, 50 μL/well;

g. Reading value: The main wavelength of the microplate reader is 450nm, and the secondary wavelength is 630nm.

h. Select monoclonal hybridoma cells that stably express antibodies. After the cells are expanded and cultured, the cells are taken for ascites production and frozen.

After two cell subclonings, only monoclonal and secondary clones were selected for microscopic examination and 6 positive monoclonal hybridoma cell lines were obtained (Table 4). The cells were expanded and cultured, and the cells were harvested for ascites production and cryopreserved.

Table 4. Subcloning results

(3) Preparation of potato virus monoclonal antibodies

Ascites preparation:

0.5 mL of sensitizer liquid paraffin was intraperitoneally injected per mouse, and 7 days later, positive hybridoma cells were intraperitoneally injected, and one mouse was injected with each cell line. Each mouse was injected with 10 ⁵ to 10 ⁶ cells, and the cells collected by centrifugation were resuspended in 1×PBS buffer before injection. From the 8th day onwards, a slight bulge in the abdominal cavity of the mice could be observed, and the mice were kept until the abdominal cavity was so rounded that it became inconvenient to move. The ascites was collected multiple times by drainage, centrifuged and then frozen at -80°C.

Ascitic fluid purification: Take the ascites fluid, dilute it with PBS and filter (0.22μm). Take the filtered sample and perform protein purification through a Protein G column. Proceed as follows:

a) Wash with 5 column volumes of deionized water to remove air and 20% ethanol;

b) Equilibrate the column with 5 to 10 times the column volume buffer, buffer: PB buffer;

c) Flow the sample through the Protein G column at a speed of 0.5mL/min;

d) Equilibrate the column with the above buffer;

e) Elute with glycine and neutralize with Tris.

f) Collect the above glycine-eluted samples and dialyze (dialysis Buffer: PBS) at 4°C overnight.

g) Take the dialyzed sample, concentrate it using ultrafiltration (ultrafiltration tube), and use SDS-PAGE gel to detect the purity of the target protein.

h) Conduct performance testing on antibodies whose purity meets the requirements.

Monoclonal antibody performance testing:

a) Protein coating: Dilute PVX-CP, X virus grinding fluid, Y virus grinding fluid, and healthy tissue grinding fluid respectively with ELISA coating solution, 100 μL/well, coat overnight at 4°C, and wash twice with PBST;

b) Blocking: Prepare 3% skimmed milk powder, 380 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

c) Add sample: Dilute the PVX monoclonal antibody to the specified concentration, 100 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

d) Primary antibody: rabbit anti-mouse IgG-HRP 1:1000, 100 μL/well, incubate at room temperature for 1 hour, wash 3 times with PBST;

e) Color development: TMB chromogenic solution A: solution B = 1:1, 100 μL/well, react at room temperature for 20 minutes;

f) Termination: ELISA stop solution, 50 μL/well;

g) Reading value: The main wavelength of the microplate reader is 450nm and the secondary wavelength is 630nm. The results are shown in Table 5.

Table 5. PVX monoclonal antibody performance testing

The results showed that the absorbance of PVX-CP and X virus were both 2 times greater than the negative control, and the Y virus did not cross-react with the PVX monoclonal antibody, and the performance of the PVX monoclonal antibody was usable.

Monoclonal Antibody Specific Detection:

a) Total protein extraction: Take 0.1g of plant tissue respectively, grind it in liquid nitrogen, add 250 μL of total protein extraction solution and 5 μL of 50× protease inhibitor;

b) Centrifuge at 13000 rpm for 15 minutes at 4°C and absorb the supernatant;

c) Add the supernatant to the loading buffer and mix well, boil in boiling water for 10 minutes, cool for 5 minutes, and centrifuge at 13000 rpm at 4°C for 10 minutes;

d) Prepare 10% SDS-PAGE separation gel and 5% stacking gel, and electrophorese at 180V until the loading buffer runs out after adding the sample;

e) Soak the PVDF membrane in methanol for 15 seconds before the end of electrophoresis;

f) After electrophoresis, soak the gel in the transfer solution for 15 minutes to transfer the membrane. 100V film transfer 1~1.5h;

g) After the membrane is transferred, wash it once with TBST, stain it with Ponceau red and take pictures. After washing it several times with TBST, it is blocked with 5% skimmed milk powder prepared in TBST and blocked at room temperature for 1 hour;

h) After blocking, add primary antibody diluted at 1:5000 and incubate at 4°C overnight;

i) After the primary antibody reaction is complete, wash the membrane with TBST, 4 times, 15 minutes each time;

j) Add secondary antibody diluted at 1:5000 and incubate at room temperature for 1 hour;

k) After the secondary antibody reaction is complete, wash the membrane with TBST, 4 times, 10 minutes each time;

l) Add ECL chromogenic solution and take pictures. The results are shown in Figure 4.

The results showed that PVX-1, PVX-2, PVX-3, PVX-4, PVX-5 and PVX-6 monoclonal antibodies could all produce specific immune reactions with the protein extracts of potato tissue culture seedlings infected with PVX. It does not produce a specific immune reaction with the protein extract of potato tissue culture seedlings infected with PVY, nor does it produce a specific immune reaction with the protein extract of healthy potato tissue culture seedlings.

Monoclonal Antibody Paired Testing:

a) Protein coating: Dilute the PVX monoclonal antibody to 1 μg/mL with ELISA coating solution, 100 μL/well, coat overnight at 4°C, and wash twice with PBST;

b) Blocking: Prepare 3% skimmed milk powder, 380 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

c) Add samples: PVX tissue is diluted 500 times, negative tissue is diluted 500 times, 100 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

d) Primary antibody: dilute the biotin-labeled PVX and PVY antibodies to 1 μg/mL, 100 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

e) Secondary antibody: Avidin-HRP 1:10000, 100 μL/well, incubate at room temperature for 1 hour, wash 3 times with PBST;

f) Color development: TMB chromogenic solution A solution: B solution = 1:1, 100 μL/well, react at room temperature for 20 minutes;

g) Termination: ELISA stop solution, 50 μL/well;

h) Reading value: The main wavelength of the microplate reader is 450nm and the secondary wavelength is 630nm. The results are shown in Table 5.

Table 5. PVX monoclonal antibody pairing results

Note: * means pairing is successful

Monoclonal Antibody Sensitivity Test:

a) Protein coating: dilute the potato virus tissue culture seedling grinding solution with ELISA coating solution, 1:10~1:163840 times gradient dilution, add ELISA coating solution to the control group, 100 μL/well, coat overnight at 4°C, PBST Clean 2 times;

b) Blocking: Prepare 3% skimmed milk powder, 380 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

c) Add sample: Dilute the serum to the specified concentration, 100 μL/well, incubate at room temperature for 1 hour, and wash twice with PBST;

d) Secondary antibody: rabbit anti-mouse IgG-HRP 1:1000, 100 μL/well, incubate at room temperature for 1 hour, wash 3 times with PBST;

e) Color development: TMB chromogenic solution A: solution B = 1:1, 100 μL/well, react at room temperature for 20 minutes;

f) Termination: ELISA stop solution, 50 μL/well;

g) Reading value: The main wavelength of the microplate reader is 450nm and the secondary wavelength is 630nm. The results are shown in Figure 5.

The results show that when coated with PVX and detected using the direct ELISA method, the sensitivity of PVX monoclonal antibodies can reach 1:10240-fold dilution.

In order to determine the sensitivity of each monoclonal antibody, according to the above method, the difference is that different concentrations of PVX-1, PVX-2, PVX-3, PVX-4, PVX-5 and PVX-6 monoclonal antibodies are used for detection. The results are shown in Table 6.

Table 6. PVX monoclonal antibody sensitivity test

The results showed that the PVX-6 monoclonal antibody had the highest sensitivity, followed by the PVX-2 monoclonal antibody, and PVX-6 and PVX-2 were subsequently selected.

PVX-6 and PVX-2 were sequenced. The heavy chain amino acid sequence of PVX-6 is shown in SEQ ID NO.3, the nucleotide sequence encoding the amino acid is shown in SEQ ID NO.4, and the light chain amino acid sequence of PVX-6 is shown in SEQ ID NO.4. The amino acid sequence of the chain is shown in SEQ ID NO.5, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO.6. The heavy chain amino acid sequence of PVX-2 is shown in SEQ ID NO.7, the nucleotide sequence encoding this amino acid is shown in SEQ ID NO.8, and the light chain amino acid sequence of PVX-2 is shown in SEQ ID NO.9. The nucleotide sequence encoding this amino acid is shown in SEQ ID NO. 10.

Select the hybridoma cells that produce PVX-6 and PVX-2 antibodies and send them to the China Type Culture Collection Center. The deposit address is Wuhan University, Wuhan, China, and are named 3D6D9B5 and 4C10F4F5 respectively; the deposit date of 3D6D9B5 is September 1, 2022, and the deposit number It is CCTCC NO: C2022279, and the classification name is hybridoma cell line 3D6D9B5; the deposit date of 4C10F4F5 is September 1, 2022, the deposit number is CCTCC NO: C2022280, and the classification name is hybridoma cell line 4C10F4F5.

The above-described embodiments are only preferred embodiments to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be determined by the claims.

Claims (7)

1. Potato virus X monoclonal antibody PVX-2, characterized by: the heavy chain amino acid sequence of the monoclonal antibody PVX-2 is shown as SEQ ID NO.7, and the light chain amino acid sequence is shown as SEQ ID NO. 9.

2. A nucleic acid of potato virus X monoclonal antibody PVX-2, characterized in that: comprises monoclonal antibody PVX-2 heavy chain nucleic acid and light chain nucleic acid, wherein the heavy chain nucleic acid sequence is shown as SEQ ID NO.8, and the light chain nucleic acid sequence is shown as SEQ ID NO. 10.

3. Hybridoma cell secreting the potexvirus monoclonal antibody PVX-2 according to claim 1 or 2, characterized in that: the hybridoma cells are preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of NO: c2022280, classified as hybridoma cell line 4C10F4F5.

4. Use of the potato virus X monoclonal antibody PVX-2 of claim 1 in the preparation of a kit for detecting potato virus X.

5. Use of the potato virus X monoclonal antibody PVX-2 of claim 1 for the preparation of an antibody for detecting potato virus X.

6. A kit comprising the potexvirus monoclonal antibody PVX-2 of claim 1.

7. The kit of claim 6, wherein: the kit is ELISA kit, immunochemical kit, immunofluorescence kit or Western Blot detection kit.