CN116589567A - Potato X virus monoclonal antibody PVX-2 and application thereof - Google Patents
Potato X virus monoclonal antibody PVX-2 and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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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
Technical Field
The invention relates to the field of biological detection, in particular to a potato virus X monoclonal antibody PVX-2 and also relates to application of the monoclonal antibody PVX-2.
Background
Potato (Solanum tuberosum l.) originates from the andes mountain in south america, the solanaceae, annual herbs, asexual reproduction, tuber-grain crops, one of the most important high-yield crops worldwide, next to wheat, rice and maize. China is the first country of potato production, the potato planting area and the total yield are all the first world, but the single yield level is far lower than that of developed countries such as Europe and America.
In China, after the potato staple food strategy is implemented in 2015, the potato strategic position is further improved, and the quality and the popularity of the detoxified seed potato are important guarantees for the industrial healthy development. The potato is used as a vegetative propagation crop, and after the potato is infected with the virus, the virus is transmitted through tuber generation due to annual planting, so that the incidence rate is increased year by year, and the yield is rapidly reduced. In the field, potato viruses can enable non-toxic potato viruses to infect in a mode of juice friction, aphid virus transmission and the like, so that yield reduction and degradation of potatoes are caused. The potato will take poison for life after being infected by virus, the disease condition aggravates year by year along with the planting algebra, and finally the planting value is lost.
For a long time, virus diseases have plagued scientific workers, and more than 40 virus types are currently found on potato crops, and one virus type is also included. PVX is a member of Potexvirus, also called potato common mosaic virus or potato light mosaic virus, has a wide host range, mainly infects solanaceae crops, and is one of key indexes for judging whether the quality of potato seeds is qualified or not as one of main pests affecting the quality of potato seeds.
After PVX single infects plants, the symptoms of the plants are slightly floral or hidden, and sometimes the leaves are slightly shrunken and the edges of the leaves are wavy, so that a small amount of yield reduction can be caused. However, in field planting, compound infection often occurs, and when the compound infection is carried out on potatoes with PVY, symptoms are aggravated, so that the yield of the potatoes is greatly reduced, and 80% of yield reduction can be caused when the compound infection is serious, so that serious economic loss is caused.
Therefore, the potato virus disease detection antibody is simply, conveniently, rapidly, sensitively, economically and accurately established, the requirements of potato seed field quality detection service are met, and technical support is provided for comprehensive promotion of potato seed quality detection and authentication work.
Disclosure of Invention
Accordingly, it is an object of the present invention to provide a potato virus X monoclonal antibody PVX-2; it is another object of the present invention to provide a hybridoma cell which secretes said anti-potato virus X monoclonal antibody PVX-2; the invention further aims at providing an 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 application of the potato virus X monoclonal antibody PVX-2 in preparing an antibody for detecting potato virus X; the fifth object of the present invention is to provide a kit comprising the potato virus X monoclonal antibody PVX-2.
In order to achieve the above purpose, the present invention provides the following technical solutions:
1. the potato virus X monoclonal antibody PVX-2, wherein 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.
Preferably, the nucleotide sequence of the heavy chain of the monoclonal antibody PVX-2 is shown as SEQ ID NO.8, and the nucleotide sequence of the light chain of the monoclonal antibody PVX-2 is shown as SEQ ID NO. 10.
2. Hybridoma cells secreting the anti-potato X virus monoclonal antibody PVX-2, wherein the hybridoma cells are preserved in China Center for Type Culture Collection (CCTCC) NO: C2022280.
3. the application of the potato virus X monoclonal antibody PVX-2 in preparing a kit for detecting potato virus X.
4. The application of the potato virus X monoclonal antibody PVX-2 in preparing an antibody for detecting potato virus X.
5. Kit containing said 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 invention has the beneficial effects that: the invention discloses a potato virus X monoclonal antibody PVX-2, wherein the heavy chain amino acid sequence of the coded antibody 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.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 shows PVX-CP gene fragment amplification (M: 2K plusII marker;1, 2: PVX-CP gene fragment (714 bp));
FIG. 2 shows PVX-CP expression (M: marker;1: protein precipitation; 2: protein supernatant; 3: total protein; 4: negative control);
FIG. 3 shows PVX-CP purification (A: different concentration imidazole elution PVX-CP, M: marker,1:50mM imidazole elution, 2:100mM imidazole elution, 3:300mM imidazole elution. B: PVX-CP purification results, M: marker,4: protein after concentration);
FIG. 4 shows the specificity of PVX monoclonal antibodies (M: protein marker,1: healthy potato tissue culture seedlings, 2: PVX-infected potato tissue culture seedlings, 3: PVY-infected potato tissue culture seedlings) by Western blot analysis;
FIG. 5 shows PVX monoclonal antibody sensitivity analysis.
Biological preservation:
the hybridoma cells secreting PVX-6 and PVX-2 antibodies are delivered to China center for type culture collection, and the collection addresses are 3D6D9B5 and 4C10F4F5 respectively; the 3D6D9B5 preservation date is 2022, 9 months and 1 day, and the preservation number is CCTCC NO: c2022279, designated as hybridoma cell line 3D6D9B5; the preservation date of 4C10F4F5 is 2022, 9 and 1, and the preservation number is CCTCC NO: c2022280, classified as hybridoma cell line 4C10F4F5.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.
Example 1 viral CP protein expression
(1) Expression vector construction
The gene sequence of PVX was searched at the center of biotechnology information (National Center for Biotechnology Information, NCBI), and primers for amplifying the full length of the CP protein gene were designed based on the full length sequence of the CP protein gene of PVX (Table 1), and the primers were aligned at NCBI and synthesized by Shanghai Biotechnology services, inc. Wherein PVX-CP gene is amplified by using PVX-CP-F and PVX-CP-R primer, and the amplified product is 714bp.
TABLE 1 PVX-CP amplification primers
cDNA was synthesized by the procedure described in the kit using total RNA of the plant virus as a template, the synthesized cDNA as a template, and the primers shown in Table 1 were used for amplification in a reaction system of 50. Mu.L, 2. Mu.L of cDNA template, and virus upstream and downstream primers (0.1. Mu. Mol. L -1 ) mu.L of each 2. Mu.L of 2×taq enzyme, 25. Mu.L of ddH 2 O was made up to 50. Mu.L. The reaction conditions were 94℃for 5min,94℃for 30s,55℃for 30s,72℃for 1min, 35 cycles, and 72℃for 10min.
After the PVX-CP gene is amplified by PCR, a DNA fragment (figure 1) with the length of 714bp is detected by agarose gel electrophoresis analysis, the PVX-CP gene fragment is obtained by glue recovery, the PVX-CP gene fragment is connected to a pET28a carrier and is transferred into BL21 competent cells, a flat plate is inverted at 37 ℃ for overnight culture, and then monoclonal bacterial plaques are selected to be sent to a worker for sequencing, and bacterial liquid with the correct sequencing result is selected for protein expression by sequence comparison.
(2) Expression and purification in E.coli
The method for expressing the target protein in the escherichia coli comprises the following steps:
a. adding the coliform bacteria liquid with correct sequencing into 5mL of LB liquid culture medium, putting into a constant-temperature shaking incubator at 37 ℃ and culturing at 250rpm/min for 16-18h at night to obtain seed liquid;
b. transferring the strain into fresh 200mL LB culture medium according to the ratio of 1:100, culturing at 37 ℃ under the condition of 250rpm/min, supplementing IPTG inducer when the bacterial liquid OD < 600 > = 0.6, and respectively continuing to induce and culture at 18 ℃;
c.4 ℃,5000rpm/min,15min, and collecting thalli;
d. the cells were resuspended in lysis buffer (50 mm Tris, 0.5M NaCl, pH 8.0) and sonicated. The ultrasonic conditions are as follows: work 3s, off 2s, time 15min, repeat one pass;
e. the sonicated samples were subjected to protein analysis at 4℃at 5000rpm/min for 15min, and the supernatant and pellet were taken and subjected to SDS-PAGE gel, respectively, and the results are shown in FIG. 2. The results show that the recombinant protein PVX-CP can be expressed.
The method for purifying the viral CP protein is as follows:
a. taking cultured thalli, adding a lysis buffer (50 mM Tris, 0.5M NaCl, pH 8.0) for resuspension, and performing ultrasonic disruption; the ultrasonic conditions are as follows: work 3s, off 2s, time 15min, repeat one pass;
b. centrifuging the bacteria liquid after ultrasonic crushing in a low-temperature centrifuge at 4 ℃ at 5000rpm/min for 15min, collecting supernatant, directly hanging the supernatant on a column, adding a denaturant urea into the supernatant to ensure that the supernatant is not hung on the column, wherein the final concentration is 8M, standing for 1h at 4 ℃ after dissolving, centrifuging, and taking the supernatant;
c. the supernatant obtained above was filtered through a 0.45 μm filter, and the protein was purified by a Ni affinity column. The method comprises the following steps:
d. washing with 5 times of deionized water to remove air and 20% ethanol;
e.5-10 column volumes Buffer A balance column, (Buffer A:50mM Tris, 0.15M NaCl, 8M urea, pH 8.0);
f. passing the sample through the Ni column at a speed of 0.5 mL/min;
g. equilibrate column with Buffer a;
h. eluting with 50mM imidazole, 100mM, 300mM imidazole, respectively;
i. the eluted samples were analyzed by SDS-PAGE gel for the presence of the target protein, and the results are shown in FIG. 3A.
The results show that different concentrations of imidazole can elute the viral CP proteins, and the purity of the eluted viral CP proteins is higher in 100mM and 300 mM. Thus, 100mM and 300mM imidazole-eluted proteins were collected, diluted, dialyzed and concentrated, and the purity of the target protein was measured by SDS-PAGE gel, and the results are shown in FIG. 3B, and the results show that the purity was high.
EXAMPLE 2 preparation of Potato X Virus monoclonal antibody serum
(1) Immunized mice
3 healthy Balb/c mice were selected using the recombinant viral CP protein prepared in example 1 as an immunogen. For the first immunization 50. Mu.g of antigen was used, and after each was emulsified with an equal volume of Freund's complete adjuvant, the mice were immunized by abdominal multipoint subcutaneous injection. After the first immunization, mice were immunized 3 times with 50 μg antigen, each emulsified with an equal volume of Freund's incomplete adjuvant, and injected subcutaneously in the abdomen multiple times. Starting the third immunization, taking blood from the orbital venous plexus (or tail vein) of the mouse 7d after each immunization, and measuring the antibody titer of the blood of the mouse by indirect ELISA, wherein the detection method is as follows:
(1) Protein coating: the experimental group is respectively diluted with ELISA coating liquid to 5 mug/mL, the control group is added with ELISA coating liquid to 100 mug/hole, the coating is carried out at 4 ℃ overnight, and PBST is washed for 2 times;
(2) Closing: preparing 3% skimmed milk powder, 380 mu L/hole, incubating for 1h at room temperature, and cleaning with PBST for 2 times;
(3) Sample adding: serum is taken and diluted to the specified concentration, 100 mu L/hole is incubated for 1h at room temperature, and PBST is washed for 2 times;
(4) And (2) secondary antibody: rabbit anti-mouse IgG-HRP 1:1000, 100. Mu.L/well, incubated for 1h at room temperature, PBST washed 3 times;
(5) Color development: TMB color development solution A: solution b=1:1, 100 μl/well, room temperature reaction for 20min;
(6) And (3) terminating: ELISA stop solution, 50. Mu.L/well;
(7) Reading: the measurement results of the main wavelength of 450nm and the auxiliary wavelength of 630nm of the enzyme-labeled instrument are shown in Table 2.
TABLE 2 serum titers of mice
The results show that the immune titer of the No. 2 mouse is highest, so that the No. 2 mouse is selected to be subjected to intraperitoneal injection of 50 mug of antigen for enhancing immunity for 3-7 days, and a cell fusion experiment is carried out.
(2) Preparation of virus monoclonal hybridoma cell
Cell fusion:
a. collecting well-grown myeloma cells (Sp 2/0) of about 10 in a biosafety cabinet 7 Re-suspending the culture medium of DMEM (Glu 4.5 g/L) without adding serum in a 50mL centrifuge tube, and preheating an incubator at 37 ℃;
b. taking spleen from immunized mice 3 to 7 days after the immunization, grinding and sieving, and centrifugally collecting spleen cells under aseptic conditions;
c. mixing spleen cells with Sp2/0, centrifuging, chemically fusing with a fusion agent PEG1450, and adding DMEM to terminate the reaction;
d. centrifuging to collect fusion cells, culturing and screening by using high-sugar DMEM added with NBS (neonatal bovine serum) and HAT, performing fusion primary screening by using indirect ELISA after about 8 days, and performing fusion secondary screening on positive cell holes;
e. selecting monoclonal hybridoma cells which stably express the antibody, performing cell expansion culture, taking the cells for ascites production, and freezing the cells.
Cell fusion screening
Detection by BSA competition ELISA method comprises the following steps:
a. protein coating: diluting PVX antigen or X virus grinding fluid to a specified concentration by ELISA coating fluid respectively, coating at 4 ℃ overnight, and cleaning PBST for 2 times;
b. closing: preparing 3% skimmed milk powder, 380 mu L/hole, incubating for 1h at room temperature, and cleaning with PBST for 2 times;
c. sample adding: adding cell supernatant in original times, incubating for 1h at room temperature by 80 mu L, and cleaning with PBST for 2 times;
d. and (2) secondary antibody: rabbit anti-mouse IgG-HRP 1:1000, 100. Mu.L/well, incubated for 1h at room temperature, PBST washed 3 times;
e. color development: TMB color development solution A: solution b=1:1, 100 μl/well, room temperature reaction for 20min;
f. and (3) terminating: ELISA stop solution, 50. Mu.L/well;
g. reading: the main wavelength of the enzyme-labeled instrument is 450nm, and the auxiliary wavelength is 630 nm;
h. the first subcloning was performed with good viability.
The cell fusion of this time obtained 20 positive cells, see in particular table 3:
table 3, no. 2 mouse fusion screen
Cell subcloning screening:
cell cloning was performed by limiting dilution. And (3) re-suspending the positive cells, counting the positive cells, diluting the positive cells according to the counting result on the basis of 1 cell per 200 mu L of culture medium, adding 200 mu L of the positive cells into a 96-well plate, performing microscopic examination after 7 to 9 days, marking single cell cluster holes, and detecting the positive cells by an indirect ELISA method. The detection method comprises the following steps:
a. protein coating: the experimental group is respectively coated with ELISA coating liquid and diluted PVX or PVY antigen protein to 1 mug/mL, the control group is added with ELISA coating liquid, 100 mug/hole, coated overnight at 4 ℃, and PBST is washed for 2 times;
b. closing: preparing 3% skimmed milk powder, 380 mu L/hole, incubating for 1h at room temperature, and cleaning with PBST for 2 times;
c. sample adding: taking the cell supernatant of the original times, 80 mu L/hole, incubating for 1h at room temperature, and cleaning the PBST for 2 times;
d. and (2) secondary antibody: rabbit anti-mouse IgG-HRP 1:1000, 100. Mu.L/well, incubated for 1h at room temperature, PBST washed 3 times;
e. color development: TMB color development solution A: solution b=1:1, 100 μl/well, room temperature reaction for 20min;
f. and (3) terminating: ELISA stop solution, 50. Mu.L/well;
g. reading: the main wavelength of the enzyme-labeled instrument is 450nm, and the auxiliary wavelength is 630 nm;
h. selecting monoclonal hybridoma cells which stably express the antibody, performing cell expansion culture, taking the cells for ascites production, and freezing the cells.
After 2 times of cell subcloning, only microscopic monoclonal and binary clone are selected for detection, 6 positive monoclonal hybridoma cell strains (table 4) are obtained, cell expansion culture is carried out, cells are taken for ascites production, and the cells are frozen.
TABLE 4 subcloning results
(3) Potato virus monoclonal antibody preparation
Preparing ascites:
the sensitizer liquid paraffin is injected into the abdominal cavity for 0.5 mL/mouse, and positive hybridoma cells are injected into the abdominal cavity after 7 days, and one mouse is knocked out for each cell. Injection 10 per mouse 5 ～10 6 Cells were collected by centrifugation, resuspended in 1 XPBS buffer and then injected. Until the day 8, the micro-protuberance of the abdominal cavity of the mouse can be observed, the mouse is continuously fed until the abdominal cavity is round and the movement is inconvenient, the ascites is collected for a plurality of times by a drainage method, and the mouse is frozen at the temperature of minus 80 ℃ after centrifugation.
Purifying ascites: the ascites was diluted with PBS and filtered (0.22 μm), and the filtered sample was subjected to Protein purification by Protein G column. The method comprises the following steps:
a) Washing with 5 times of deionized water to remove air and 20% ethanol;
b) 5-10 times of column volume buffer balance column, buffer: PB buffer;
c) Passing the sample through a Protein G column at a rate of 0.5 mL/min;
d) Balancing the column by using the buffer;
e) Elution with glycine and neutralization with Tris.
f) The glycine eluted samples were collected and dialyzed at 4 ℃ (dialysis Buffer: PBS) overnight.
g) The dialyzed sample was collected, concentrated by ultrafiltration (ultrafiltration tube), and the purity of the target protein was detected by SDS-PAGE gel.
h) And detecting the performance of the antibody with the purity meeting the requirement.
Monoclonal antibody performance detection:
a) Protein coating: respectively diluting PVX-CP, X virus grinding fluid, Y virus grinding fluid and healthy tissue grinding fluid with ELISA coating fluid, coating 100 mu L/hole at 4 ℃ overnight, and cleaning PBST for 2 times;
b) Closing: preparing 3% skimmed milk powder, 380 mu L/hole, incubating for 1h at room temperature, and cleaning with PBST for 2 times;
c) Sample adding: diluting PVX monoclonal antibody to a specified concentration of 100 mu L/hole, incubating for 1h at room temperature, and cleaning PBST for 2 times;
d) An antibody: rabbit anti-mouse IgG-HRP 1:1000, 100. Mu.L/well, incubated for 1h at room temperature, PBST washed 3 times;
e) Color development: TMB color development solution A: solution b=1:1, 100 μl/well, room temperature reaction for 20min;
f) And (3) terminating: ELISA stop solution, 50. Mu.L/well;
g) Reading: the measurement results of the main wavelength of 450nm and the auxiliary wavelength of 630nm of the enzyme-labeled instrument are shown in Table 5.
TABLE 5 detection of PVX monoclonal antibody Performance
The results show that PVX-CP and X virus absorbance are both 2-fold greater than negative control and that Y virus does not cross react with PVX monoclonal antibodies, which are available in their performance.
Monoclonal antibody specific detection:
a) Total protein extraction: respectively taking 0.1g of plant tissue, grinding with liquid nitrogen, and adding 250 μl of total protein extract and 5 μl of 50×protease inhibitor;
b) Centrifuging at 13000rpm at 4deg.C for 15min, and collecting supernatant;
c) Adding loading buffer into the supernatant, mixing, boiling in boiling water for 10min, cooling for 5min, and centrifuging at 13000rpm at 4deg.C for 10min;
d) Preparing 10% SDS-PAGE separating gel and 5% concentrating gel, and carrying out 180V electrophoresis until loading buffer is out after sample addition;
e) Soaking the PVDF film in methanol for 15s before electrophoresis is finished;
f) After electrophoresis, the gel is soaked in the film transfer liquid for 15min, and film transfer is carried out. Transferring the film at 100V for 1-1.5 h;
g) After transferring the film, cleaning the film once by using TBST, dyeing and photographing the film by using ponceau, cleaning the film for several times by using TBST, and sealing the film by using 5% skimmed milk powder prepared by using TBST at room temperature for 1h;
h) After blocking, adding primary antibody diluted by 1:5000, and incubating overnight at 4 ℃;
i) After the primary reaction is completed, washing the membrane by TBST for 4 times for 15min each time;
j) Adding secondary antibody diluted in a ratio of 1:5000, and incubating for 1h at room temperature;
k) After the secondary antibody reaction is completed, the membrane is washed by TBST for 4 times for 10min each time;
l) adding ECL color development liquid to take photos, and the result is shown in FIG. 4.
The results show that PVX-1, PVX-2, PVX-3, PVX-4, PVX-5 and PVX-6 monoclonal antibodies can specifically immunoreact with the protein extract of PVX-infected potato tissue culture seedlings, do not specifically immunoreact with the protein extract of PVY-infected potato tissue culture seedlings, and do not specifically immunoreact with the protein extract of healthy potato tissue culture seedlings.
Monoclonal antibody pairing detection:
a) Protein coating: diluting PVX monoclonal antibody to 1 mug/mL, 100 mug/hole with ELISA coating liquid, coating overnight at 4 ℃, and cleaning PBST for 2 times;
b) Closing: preparing 3% skimmed milk powder, 380 mu L/hole, incubating for 1h at room temperature, and cleaning with PBST for 2 times;
c) Sample adding: PVX tissue 500-fold dilution, negative tissue 500-fold dilution, 100. Mu.L/well, incubation for 1h at room temperature, PBST wash 2 times;
d) An antibody: respectively diluting PVX and PVY antibodies marked with biotin to 1 mug/mL, 100 mug/hole, incubating for 1h at room temperature, and cleaning PBST for 2 times;
e) And (2) secondary antibody: avidin-HRP 1:10000, 100. Mu.L/well, incubated for 1h at RT, PBST washed 3 times;
f) Color development: TMB color development solution A: solution b=1:1, 100 μl/well, room temperature reaction for 20min;
g) And (3) terminating: ELISA stop solution, 50. Mu.L/well;
h) Reading: the measurement results of the main wavelength of 450nm and the auxiliary wavelength of 630nm of the enzyme-labeled instrument are shown in Table 5.
TABLE 5 PVX monoclonal antibody pairing results
Note that: * To be successfully paired with
Monoclonal antibody sensitivity detection:
a) Protein coating: dilute potato virus tissue culture Miao Yanmo solution with ELISA coating solution, 1:10 to 1:163840 times of gradient dilution, adding ELISA coating liquid into a control group, 100 mu L/hole, coating overnight at 4 ℃, and cleaning PBST for 2 times;
b) Closing: preparing 3% skimmed milk powder, 380 mu L/hole, incubating for 1h at room temperature, and cleaning with PBST for 2 times;
c) Sample adding: serum is taken and diluted to the specified concentration, 100 mu L/hole is incubated for 1h at room temperature, and PBST is washed for 2 times;
d) And (2) secondary antibody: rabbit anti-mouse IgG-HRP 1:1000, 100. Mu.L/well, incubated for 1h at room temperature, PBST washed 3 times;
e) Color development: TMB color development solution A: solution b=1:1, 100 μl/well, room temperature reaction for 20min;
f) And (3) terminating: ELISA stop solution, 50. Mu.L/well;
g) Reading: the measurement result of the main wavelength of 450nm and the auxiliary wavelength of 630nm of the enzyme-labeled instrument is shown in FIG. 5.
The results show that PVX monoclonal antibody sensitivity can reach 1:10240 dilution when coated with PVX and detected by direct ELISA.
To determine the sensitivity of each monoclonal antibody, the detection was performed using PVX-1, PVX-2, PVX-3, PVX-4, PVX-5 and PVX-6 monoclonal antibodies at different concentrations, respectively, according to the method described above, and the results are shown in Table 6.
TABLE 6 sensitivity detection of PVX monoclonal antibodies
The results showed that PVX-6 monoclonal antibody had the highest sensitivity, followed by PVX-2 monoclonal antibody, followed by PVX-6 and PVX-2.
Sequencing PVX-6 and PVX-2, wherein the heavy chain amino acid sequence of PVX-6 is shown as SEQ ID NO.3, the nucleotide sequence for encoding the amino acid is shown as SEQ ID NO.4, the light chain amino acid sequence of PVX-6 is shown as SEQ ID NO.5, and the sum nucleotide sequence for encoding the amino acid is shown as SEQ ID NO. 6. The heavy chain amino acid sequence of PVX-2 is shown as SEQ ID NO.7, the nucleotide sequence for encoding the amino acid is shown as SEQ ID NO.8, the light chain amino acid sequence of PVX-2 is shown as SEQ ID NO.9, and the nucleotide sequence for encoding the amino acid is shown as SEQ ID NO. 10.
Selecting hybridoma cells producing PVX-6 and PVX-2 antibodies to be sent to China center for type culture collection (China university of Wuhan, china) with a preservation address of 3D6D9B5 and 4C10F4F5; the 3D6D9B5 preservation date is 2022, 9 months and 1 day, and the preservation number is CCTCC NO: c2022279, designated as hybridoma cell line 3D6D9B5; the preservation date of 4C10F4F5 is 2022, 9 and 1, and the preservation number is CCTCC NO: c2022280, classified as hybridoma cell line 4C10F4F5.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to 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. Potato virus X monoclonal antibody PVX-2, characterized by: the nucleotide sequence of the heavy chain of the monoclonal antibody PVX-2 is shown as SEQ ID NO.8, and the nucleotide sequence of the light chain of the monoclonal antibody PVX-2 is shown as SEQ ID NO. 10.
3. A hybridoma cell secreting anti-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.
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