Secreted anti-prunus necrotic ring spot virus monoclonal antibody hybridoma cell line and its monoclonal antibody application
A highly efficient serological detection method was established using the 9E1 cell line, which secretes monoclonal antibodies against plum necrosis ringspot virus, prepared through hybridoma technology. This method solves the problem of detecting plum necrosis ringspot virus in existing technologies and achieves rapid, specific, and sensitive detection results, suitable for port inspection and field testing.
Patent Information
- Application Number
- CN202211440502.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-17
- Publication Date
- 2025-11-28
- Estimated Expiration
- 2042-11-17
AI Technical Summary
Current technologies lack effective detection methods for rapid and accurate detection of plum necrosis ringspot virus (PNRSV), which affects the monitoring and control of plant viral diseases.
Hybridoma cell line 9E1, which secretes monoclonal antibodies against Prunus necrotic ringspot virus, was prepared using hybridoma technology. ACP-ELISA, dot-ELISA, and tissueprint-ELISA methods were established using this monoclonal antibody, and corresponding detection kits were developed.
It provides a rapid, specific, sensitive, and accurate detection method, reducing reliance on equipment and is suitable for port inspection and quarantine, field testing, and scientific prevention and control of viral diseases.
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Figure CN115960841B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, and particularly relates to a hybridoma cell strain secreting anti-Prunus necrotic ring-spot virus monoclonal antibody and application of the monoclonal antibody. BACKGROUND
[0002] Prunus necrotic ring-spot virus (PNRSV) belongs to the Bromoviridae family and the Ilarvirus genus. The particle morphology of PNRSV is an isometric polyhedron with a diameter of about 22-23 nm, and there are three sizes of particles. The viral genome is composed of three positive single-stranded RNAs, RNA1 and RNA2 encode replication proteins P1 and P2, respectively, and RNA3 encodes a motor protein and a coat protein.
[0003] PNRSV is a viral pathogen that occurs widely around the world and can harm many plants. The natural hosts mainly include cherry, peach, apricot, plum, apple, hops, and rose, and other hosts include tobacco, watermelon, melon, pumpkin, zucchini, kidney bean, cowpea, pea, lettuce, sunflower, etc. PNRSV is currently listed as a Class II dangerous harmful organism in the List of Inbound Plant Quarantine Dangerous Diseases, Insects, and Weeds of the People's Republic of China. PNRSV was first discovered on plum and peach trees and mainly distributed in temperate regions of Europe, America, and Oceania. The first report of PNRSV in China was on naturally infected begonia in Xi'an, Shaanxi Province. Subsequently, PNRSV has been reported in many other regions, such as Shaanxi, Shandong, Liaoning, Sichuan, Xinjiang, and Yunnan.
[0004] Peach trees infected with PNRSV show some symptoms of chlorotic ring spots and necrotic spots on young leaves in spring, but the symptoms are not easily identified in summer. Some do not show obvious symptoms in spring, and some show small cracks at the suture of the fruit. PNRSV can cause plants to exhibit a variety of different symptoms, which are related to the virus strain, the susceptibility of the host variety, and the environmental conditions. Common symptoms include leaf fragmentation, zonate leaves, linear lines, necrotic ring spots, coarse mottle, and leaf drop, and even whole plant death. PNRSV can also infect fruit trees mixedly with apple mosaic virus and apple chlorotic leaf spot virus.
[0005] So far, there is no effective control agent for plant virus disease, and simple, rapid, sensitive, specific, accurate and practical detection technology is the key to virus disease monitoring, early warning and green prevention and control. In order to strengthen the inspection and quarantine technology of PNRSV in China, it is urgent to establish a simple, rapid, economical, effective and high-throughput practical detection technology for detecting PNRSV. Compared with conventional methods such as indicator plant inoculation, electron microscopy observation and molecular detection, serological method has the advantages of simplicity, economy, easy operation and high-throughput detection, and is the most practical method for plant virus detection and investigation. However, the establishment of serological method depends on high-quality virus antibodies with high specificity. The present application uses prokaryotic expressed PNRSV coat protein (CP) as antigen, and prepares a hybridoma cell strain secreting PNRSV specific monoclonal antibody through hybridoma technology. The serological method and kit for detecting PNRSV are established by using the secreted monoclonal antibody as detection antibody, so as to provide reagent and technical support for the inspection and quarantine of PNRSV at ports, the investigation of epidemiology, the analysis of virus genome function, the breeding of resistance, the establishment of scientific prevention and control, etc. SUMMARY
[0006] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a hybridoma cell strain secreting anti-Prunus necrotic ring spot virus monoclonal antibody and application of the monoclonal antibody.
[0007] The specific technical solutions adopted by the present application are as follows:
[0008] In a first aspect, the present application provides a hybridoma cell strain 9E1 secreting anti-Prunus necrotic ring spot virus monoclonal antibody, which can secrete specific monoclonal antibody against Prunus necrotic ring spot virus. The hybridoma cell strain 9E1 was deposited with the China General Microbiological Culture Collection Center on November 7, 2022, and the deposit number is CGMCC No. 45329.
[0009] In a second aspect, the present application provides a hybridoma cell strain secreting anti-Prunus necrotic ring spot virus monoclonal antibody. The indirect ELISA titer of the monoclonal antibody in ascites is 10 -7 , the antibody type and subclass are IgG2b and kappa light chain, and the monoclonal antibody has specific immunoreaction with the 30kDa coat protein of Prunus necrotic ring spot virus. The sensitivity of ACP-ELISA and dot-ELISA methods established by using the monoclonal antibody for detecting crude extract of PNRSV infected leaf tissue is 1:10240 and 1:1280 dilution (w / v, g / mL) respectively.
[0010] The anti-Prunus necrotic ring spot virus monoclonal antibody has specific immunoreaction with Prunus necrotic ring spot virus, and does not have immunoreaction with Prunus pustule virus, cucumber green mottle mosaic virus, apple stem grooving virus, apple necrotic mosaic virus and healthy plant tissue.
[0011] In a third aspect, the present application provides the use of the anti-Prunus necrotic ring spot virus monoclonal antibody as described in the above second aspect in the detection of the virus, which is various immunological detection methods and detection kits established by using the monoclonal antibody as a detection antibody.
[0012] Compared with the prior art, the present application has the following beneficial effects: 1) The hybridoma cell strain provided secretes a large amount of anti-PNRSV specific monoclonal antibody, and the ACP-ELISA, dot-ELISA and Tissueprint-ELISA serological detection methods and detection kits established by using the monoclonal antibody as a detection antibody can quickly, specifically, sensitively and accurately detect PNRSV; 2) The use of the monoclonal antibody prepared by the present application for detecting PNRSV virus has low technical requirements for the operator and does not require expensive equipment such as electron microscopes and PCR instruments; 3) The use of the monoclonal antibody prepared by the present application can effectively be used for the detection and diagnosis of PNRSV in the field, the inspection and quarantine at ports, and the epidemiological investigation of the virus disease, the functional analysis of the virus genome, the breeding of resistance, the scientific prevention and control, etc. BRIEF DESCRIPTION OF DRAWINGS
[0013] Figure 1 Results of RT-PCR cloning of PNRSV CP gene (A) and expression and purification of recombinant protein (B);
[0014] (1B) M: Protein Marker; 1-3: Recombinant proteins eluted by 100 mM imidazole, 200 mM imidazole and 50 mM imidazole.
[0015] Figure 2 Results of Western blot experiment for analyzing the specificity of 9E1 monoclonal antibody
[0016] Figure 3 Results of specificity (A) and sensitivity (B) analysis of dot-ELISA method
[0017] Figure 4 Results of specificity analysis of Tissue print-ELISA method
[0018] BIOLOGICAL DEPOSIT
[0019] The hybridoma cell strain 9E1 secreting the anti-Prunus necrotic ring spot virus monoclonal antibody was preserved in the China General Microbiological Culture Collection Center on November 7, 2022, and the address of the center is No. 1, Beichen West Road, Haidian District, Beijing, China, and the postal code is 100101, and the preservation number is CGMCC No. 45329. DETAILED DESCRIPTION
[0020] The hybridoma cell strain 9E1 secreting the anti-Prunus necrotic ring spot virus monoclonal antibody was preserved in the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences on November 7, 2022, and the preservation number is CGMCC No. 45329, and it can secrete the anti-Prunus necrotic ring spot virus monoclonal antibody.
[0021] The anti-Prunus necrotic ring spot virus monoclonal antibody secreted by the hybridoma cell strain 9E1 has an indirect ELISA titer of 10 -7 , the antibody type and subclass are IgG2b and kappa light chain, and the monoclonal antibody has specific immunoreaction with the 30kDa shell protein of the Prunus necrotic ring spot virus, and the sensitivity of the ACP-ELISA and dot-ELISA methods established by using the monoclonal antibody for detecting the crude extract of the PNRSV infected leaf tissue reaches 1:10240 and 1:1280 times dilution (w / v, g / mL) respectively.
[0022] The anti-Prunus necrotic ring spot virus monoclonal antibody has specific immunoreaction with the Prunus necrotic ring spot virus, and does not have immunoreaction with Prunus pustule virus, cucumber green mottle mosaic virus, apple stem grooving virus, apple necrotic mosaic virus and healthy plant tissues.
[0023] The anti-Prunus necrotic ring spot virus monoclonal antibody is used for the application of the virus detection, and various immunological detection methods and immunological detection kits established by using the monoclonal antibody as a detection antibody.
[0024] The hybridoma cell strain provided by the application can secrete a large amount of anti-Prunus necrotic ring spot virus monoclonal antibody, and the monoclonal antibody secreted by the hybridoma cell strain has high specificity, good sensitivity, high titer and good stability. The high-throughput serological method for detecting PNRSV established by using the monoclonal antibody as a detection antibody can be applied to the detection and diagnosis of PNRSV in port inspection and quarantine and field plant samples, so as to provide detection reagents and technical support for the detection and diagnosis of PNRSV in port inspection and quarantine and field samples and the scientific prevention and control of the virus disease.
[0025] The application will be further described in combination with the embodiments and the drawings.
[0026] I. Preparation of hybridoma cells and preparation of monoclonal antibodies thereof
[0027] 1. Preparation of immunogen and detection antigen
[0028] 1.1. Cloning of PNRSV CP gene and construction of prokaryotic expression vector thereof:
[0029] 1.1.1. Extraction of total RNA from PNRSV infected plants by Trizol method
[0030] 1) After the 0.1 g of PNRSV infected leaf was ground into powder with liquid nitrogen in a mortar, it was transferred into a centrifuge tube, 1 mL Trizol extraction solution was added, vortexed, and placed in a fume hood for 5-10 min;
[0031] 2) 0.2 mL chloroform was added, vortexed for 15 s, and placed at room temperature for 5-10 min;
[0032] 3) Centrifuged at 12,000 rpm for 15 min at 4°C, then the supernatant was taken out in small amounts for several times, the same volume of isopropanol was added, vortexed, and precipitated at -20°C for 20-60 min;
[0033] 4) Centrifuged at 12,000 rpm for 10 min at 4°C, the supernatant was carefully removed with a pipette, and the precipitate was left;
[0034] 5) 1 mL 75% ethanol was added to the obtained white feather-like precipitate, the centrifuge tube was tapped to wash the precipitate, centrifuged at 12,000 rpm for 5 min, and the supernatant was discarded;
[0035] 6) Step 5 was repeated once;
[0036] 7) After centrifugation at 12,000 rpm for 5 min at 4°C, the supernatant was removed, dried in a fume hood, and the RNA was dissolved with 30 μL DEPC water. 1 μL of the RNA sample was used to determine the content and purity of the RNA on a Nanodrop ultraviolet spectrophotometer, and stored at -80°C for standby.
[0037] 1.1.2 RT-PCR
[0038] A pair of specific primers was designed according to the reported sequence of the PNRSV encoded coat protein gene:
[0039] PNRSV
[0040] CP-F: 5'-CAGCAAATGGGTCGC GGATCC ATGGTTTGCCGAATTTGCAA-3' and
[0041] PNRSV CP-R:
[0042] 5'-TGCGGCCGCAAGCTT GTCGAC CTAGATCTCAAGCAGGTCTTC-3'. The BamH I enzyme cutting site is underlined in PNRSV CP-F, and the Sal I enzyme cutting site is underlined in PNRSV CP-R. The primer synthesis was performed by Genesee Biotech Co., Ltd.
[0043] Reverse transcription was performed with HiScript II Reverse Transcriptase kit produced by Nanjing Novozyme Company, and PCR reaction was performed with KOD One PCR Master Mix produced by Japan Toyo Spinning Company. The extracted RNA was heated in a 65°C metal bath for 5-10 min, taken out and placed on ice for 5 min, and then added to the reverse transcription system, which was 10 μL. TM
[0044] The reverse transcription amplification system was as follows:
[0045]
[0046] The reverse transcription PCR instrument program settings were as follows:
[0047]
[0048] The cDNA obtained by reverse transcription was stored in a -30°C refrigerator for standby use. Then, KOD One PCR Master Mix was used for PCR reaction, and bands with a size of about 723 bp were amplified. TM Figure 1 A).
[0049] The PCR reaction system was as follows:
[0050]
[0051] The PCR instrument program settings were as follows:
[0052]
[0053] 1.1.3 Connection of target fragment to vector
[0054] After the PCR product was purified with the Axygen gel cutting and recovery kit, the pET-28a plasmid was extracted with the Axygen plasmid extraction kit, and then the target fragment was connected to the pET-28a vector through homologous recombination cloning with the enzyme-cut plasmid, and stored in a 4°C refrigerator.
[0055] 1.1.4 Transformation of E. coli competent DH5a with connection product
[0056] 1) One tube of low-temperature stored E. coli competent DH5a was gradually thawed on ice, 10 μL of pET-28a-PNRSV CP recombinant plasmid was added, the bottom of the centrifuge tube was lightly flicked with fingers to mix it, and it was placed in an ice box for 20-40 min.
[0057] 2) 42°C heat shock E. coli competent DH5a mixed with pET-28a-PNRSV CP for 90 s, then put on ice or 4°C for 5-7 min;
[0058] 3) Incubate the heat-shocked bacteria at 37°C for 1 h; then centrifuge at 8,000 rpm for 2 min to remove most of the culture medium, and suspend the bacteria with the remaining culture medium;
[0059] 4) Spread the bacteria on solid LB medium (containing 100 μg / L kanamycin) and incubate at 37°C overnight.
[0060] 1.1.5 Colony PCR identification and preservation of positive clones
[0061] After overnight incubation, gently pick a single colony with a toothpick and perform colony PCR.
[0062] Reaction system:
[0063]
[0064]
[0065] After agarose gel electrophoresis analysis of the PCR product, the band size of the target gene of the positive clone was about 723 bp; a small amount of the positive clone was picked and expanded in LB medium containing kanamycin for 12 h, and the plasmid was extracted. One part was sent to a biological company for sequencing, and the other was stored in the refrigerator for later use. After the sequencing results were blast sequence alignment, it was verified that the PNRSV CP gene sequence in the expression vector was correct and the reading frame was accurate. The previously saved recombinant plasmid was heat-shocked to transform competent cells BL21 (DE3), and recovery culture, plate culture and single colony PCR verification were performed. The positive colonies were induced for gene expression. At the same time, the positive bacterial liquid with an OD value of 0.8-1.0 was added with 15% glycerol and stored in the refrigerator at 80°C for preservation and backup. 600
[0066] 1.2 Prokaryotic expression and purification of PNRSV CP protein
[0067] 1.2.1 Induced expression of PNRSV CP
[0068] 1) Inoculate the expression strains containing pET-28a-PNRSV CP and pET-28a empty vector, respectively, in 2 tubes of 5 mL liquid LB containing kanamycin (100 μg / mL) and incubate at 37°C overnight;
[0069] 2) The overnight culture was diluted 1:100 into 200 mL liquid LB containing kanamycin (100 μg / mL) and incubated at 37°C with shaking until the OD 600 = 0.4-0.6;
[0070] 3) 1 mL of the bacterial solution of the expression strain containing pET-28a-PNRSV CP was centrifuged, the bacterial cells were suspended in ddH2O and mixed with an appropriate amount of 5x SDS sample buffer, and denatured at 100°C for 10 min;
[0071] 4) IPTG was added to the bacterial solution of step 2) to a concentration of 0.5 mmol / L, and the expression was induced at 16°C for 12 h; 1 mL of the induced bacterial solution was centrifuged, the bacterial cells were suspended in ddH2O and mixed with an appropriate amount of 5x SDS sample buffer, and denatured at 100°C for 10 min;
[0072] 5) The denatured sample was centrifuged at 4°C and 8,000 rpm for 3 min, and the supernatant was subjected to SDS-PAGE electrophoresis to examine the expression of PNRSV CP. It was found that PNRSV CP was successfully expressed.
[0073] 1.2.2 Purification of PNRSV CP by guanidine hydrochloride
[0074] 1) Prepare buffer 1: 50 mM PBS buffer at pH 7.4. Preparation: 0.5M NaH2PO4 19 mL, 0.5M Na2HPO4 81 mL, 6M guanidine hydrochloride, NaCl 29.3g, dissolve in water and make up to 1000 mL.
[0075] Buffer 2: 50 mM phosphate buffer, pH 7.4, i.e. PBS solution at pH 7.4. Preparation: 0.5M NaH2PO4 19 mL, 0.5M Na2HPO4 81 mL, 6M guanidine hydrochloride, NaCl 29.3g and imidazole 34g, dissolve in water and make up to 1000 mL;
[0076] Buffer 3: Buffer B with different imidazole concentrations:
[0077] Correspondence:
[0078]
[0079] 2) After 12 h of IPTG induction at 16°C, the 200 mL LB culture solution of the expression strain was centrifuged at 8,000 rpm and the supernatant was discarded, and the bacterial cells were collected;
[0080] 3) Resuspend the bacteria pellet with 30 mL PBS buffer; sonicate the bacteria suspension with an ultrasonic disrupter for 30 min until the bacteria suspension is clear, then centrifuge at 8,000 rpm for 10 min to obtain the supernatant; resuspend the pellet with PBS buffer, and take a small amount of the resuspended pellet to be added with SDS-PAGE protein loading buffer (indicated by the arrow) for SDS-PAGE detection;
[0081] 4) Add 500 μl Ni2+-NTA Agarose to the supernatant, and incubate at 4°C for 2 h with slow rotation;
[0082] 5) Pour the mixture of Ni2+-NTA Agarose and the supernatant into a special plastic purification column, and allow the mixture to slowly flow out by gravity; pour the eluate back into the purification column, and allow the eluate to slowly flow out by gravity; take a small amount of the eluate (indicated by the arrow) to be added with protein loading buffer for SDS-PAGE detection;
[0083] 6) Equilibrate the column with buffer 1 for 1-2 column volumes until no liquid flows out;
[0084] 7) Perform stage elution with buffer B containing 10, 20, 50, 100, 200, 300, and 400 mM imidazole, respectively, for at least 1 column volume (as appropriate) for each stage, and collect the elution peaks of each stage;
[0085] 8) After elution, wash the column with buffer 1 for 2 column volumes, and then wash the column with distilled water for 5 column volumes; store the column at 4°C, and repeat the use.
[0086] 9) Perform SDS-PAGE electrophoresis on the eluate of each concentration to check the protein purification, as shown in Fig. B, the PNRSV CP recombinant protein is successfully purified. Figure 1
[0087] 2 Preparation of PNRSV CP monoclonal antibody
[0088] 2.1 Immunization of mice
[0089] Immunize 8-week-old BALB / c female mice with the purified and dialyzed expression protein: dilute the purified protein with physiological saline, take 100 μL (containing 50 μg of protein antigen) per mouse, mix with an equal volume of Freund’s complete adjuvant (FCA), emulsify thoroughly, and inject 200 μL per mouse intraperitoneally and subcutaneously, with an interval of 3 weeks; take an equal amount of antigen and an equal volume of Freund’s incomplete adjuvant, emulsify thoroughly, and inject 200 μL per mouse intraperitoneally and subcutaneously for the second time, with an interval of 3 weeks; then inject the mice intraperitoneally with a doubled dose of antigen, and take the spleen cells for fusion 3 days later.
[0090] 2.2 Cell fusion
[0091] The spleen cells of the immunized mice and mouse myeloma cells (SP2 / 0) were mixed at a ratio of 8:1 in serum-free RPMI 1640 (Gibco) medium, and after centrifugation at 1,500 rpm for 5 min, the medium was removed. The centrifuge tube containing the cells was placed in a 37°C water bath and 1 mL of 50% PEG (molecular weight 1500) fusion agent was added. After 2 min of fusion, the fusion was terminated with serum-free RPMI 1640 medium, and after centrifugation at 1,500 rpm for 5 min, the supernatant was removed and the precipitate was suspended in RPMI 1640 medium containing HAT, fetal bovine serum, and ampicillin. The suspension was then dispensed into 96-well cell plates, which were then incubated in a 37°C, 5% CO2 cell incubator.
[0092] 2.4 Screening of hybridoma cells and cell cloning
[0093] After 7 days of incubation in the cell incubator, the medium was replaced with RPMI 1640 medium containing HT, fetal bovine serum, and ampicillin. When the fused cells covered more than 15% of the bottom of the wells, the wells were coated with the appropriate concentration of PNRSV CP recombinant protein, and positive wells were screened using indirect ELISA. After detection, the positive wells were added with an appropriate amount of RPMI 1640 medium containing HT, fetal bovine serum, and ampicillin. After 1-2 days, the specificity of the antibodies secreted by the positive cell strains was analyzed using dot-ELISA, and hybridoma cells with good specificity were identified.
[0094] The hybridoma cells specific to the protein and leaf tissue fluid were subjected to limited dilution cell cloning. When cloning the cell strains, the hybridoma cells with good specificity were aspirated from the positive wells and subjected to gradient dilution in cell culture plates. Under a microscope, wells with approximately 100 cells were found, and the cells were suspended in medium and dispensed into 96 wells of a cell plate after blowing. After 5-6 days of incubation, wells with only single cell clusters were selected and added with an appropriate amount of medium. After 3-4 days, the wells containing monoclonal cells were subjected to antibody positive detection and antibody specificity detection. The cloning was continued for 3-4 times until each monoclonal cell well of the cloned cells secreted antibodies.
[0095] Finally, one hybridoma cell strain 9E1 capable of secreting PNRSV highly specific and sensitive monoclonal antibodies was obtained, which is the aforementioned hybridoma cell strain with the preservation number CGMCC No. 45329. After more than 3 months of in vitro passage and multiple freeze-thaw recovery, the cell strain grew well and stably secreted antibodies. After expansion culture, it was used for ascites preparation and cell freezing.
[0096] 2.6 Preparation of monoclonal antibody ascites and purification
[0097] Take 14 weeks old BALB / c male mice, intraperitoneal injection of 0.3-0.5 mL pristane, 7-10 days after intraperitoneal injection of about 7 x 10 5
[0098] Take 1 volume of ascites and add 2 volumes of 0.85% physiological saline to dilute, room temperature, stirring, dropwise add saturated ammonium sulfate solution (pH 7.0), 4°C overnight, 12,000 rpm centrifugation for 10 min, 2 mL 0.85% physiological saline suspension of the precipitate, after dialysis in 0.85% physiological saline at 4°C for 24 h, the purified monoclonal antibody is obtained, -80°C storage.
[0099] 2.6 Subclass identification and ascites titer determination of monoclonal antibody
[0100] The type and subclass of monoclonal antibody were identified using Sigma's immunoglobulin standard subclass identification kit, and the specific steps were carried out according to the instructions. The results showed that the subclass of 9E1 monoclonal antibody was IgG2b, κ light chain. The ascites titer of monoclonal antibody was detected by indirect ELISA method using PNRSV CP recombinant protein as antigen, and the analysis results showed that the ascites titer of monoclonal antibody reached 10 -7 .
[0101] 3 Western blot specificity analysis of monoclonal antibody
[0102] 1) Sample preparation: 0.1 g of healthy leaves and infected PNRSV disease leaves were respectively taken and quickly frozen with liquid nitrogen in a mortar, and then 1 mL of 2xSDA-PAGE protein loading buffer was added after grinding, mixed and transferred to a 1.5 mL centrifuge tube, and then placed in a 100°C metal bath for 10 min to denature the protein, then quickly placed on ice for 5 min, and then centrifuged at 12,000 rpm for 5 min. The supernatant is the protein sample; take an appropriate amount of PNRSV CP recombinant protein 20 μL, add 5 μL of 5x protein buffer, and denature the protein in a 100°C metal bath for 10 min.
[0103] 2) Sample loading and SDS-PAGE electrophoresis: the prepared SDS-PAGE gel was placed on the gel holder, then placed in the corresponding position of the positive and negative electrodes, and then added with sufficient protein electrophoresis buffer, 20 μL of each protein sample was loaded, and protein Marker was added, and constant voltage method was used for protein electrophoresis, first 80V electrophoresis for 0.5h, then 110V electrophoresis for 1.5h;
[0104] 3) Wet transfer: After the electrophoresis, the SDS-PAGE gel in the gel plate was taken out and soaked in wet transfer buffer. The SDS-PAGE gel was tightly combined with a NC membrane with the same size as the SDS-PAGE gel. Air bubbles were carefully removed. The two sides of the gel and the membrane were combined with filter paper and sponge pad. The transfer clamp was clamped. Then, the gel was placed in the electrophoresis tank, and the gel was placed on the cathode side. The constant current was 300 mA, and the transfer time was 50 min.
[0105] 4) Western blot: After the protein transfer, the NC membrane was taken out and washed with PBST buffer to remove the wet transfer buffer. The membrane was placed in PBST containing 5% skim milk, and then placed in a 37°C incubator for 30 min or in a 4°C refrigerator overnight for blocking. Then, the blocked membrane was taken out and placed in 5,000-fold diluted monoclonal antibody (primary antibody) at 37°C for 1 h. The membrane was washed with PBST for 4 times, each time for 2 min. Then, the membrane was placed in 8,000-fold diluted enzyme-labeled secondary antibody at 37°C for 1 h. The membrane was washed with PBST for 5 times, each time for 2 min, and then washed with PBS once. 10 mL of AP enzyme substrate buffer was added with 33 μL of BCIP and 66 μL of NBT to form the color developing solution. The treated NC membrane was placed in the color developing solution at room temperature, and the membrane was completely submerged in the color developing solution. Then, the membrane was covered with tin foil to block the light. When the specific band gradually appeared and became clear, the membrane was taken out. Then, the excess color developing solution was washed away with PBS. After the membrane was dried with filter paper, it was photographed and recorded. The results are shown in FIG. 9E1 monoclonal antibody specifically recognized the recombinant PNRSV CP and PNRSV CP protein in the diseased leaves. Figure 2
[0106] 4) Analysis of the specificity of monoclonal antibodies by ACP-ELISA method
[0107] The specificity of the monoclonal antibody was analyzed by ACP-ELISA method. The steps of ACP-ELISA method were as follows: the diseased leaves and healthy leaves infected by the above-mentioned viruses were ground in a mortar, and were homogenized with ELISA coating solution at a ratio of 1:20 (w / v, g / mL). After centrifugation at 8,000 rpm for 3 min, 100 μL of supernatant was added to each well of the ELISA plate, and the plate was coated at 4°C overnight or at 37°C for 4 h. After washing with PBST for 3 times, 3% skimmed milk was added for blocking for 30-60 min. 100 μL of the monoclonal antibody diluted by 1:5,000 was added to each well, and the plate was incubated at 37°C for 1-2 h. After washing with PBST for 3 times, 100 μL of alkaline phosphatase (AP) labeled goat anti-mouse IgG secondary antibody (Sigma) diluted by 1:8,000 was added to each well, and the plate was incubated at 37°C for 1-2 h. After washing with PBST for 4 times, PNPP substrate was added for color development for 30-60 min. After the reaction was terminated by 2 mol / L sodium hydroxide, the OD 405 value was read by an enzyme-labeled instrument. The sample was positive when the OD 405 value of the negative control was greater than 3.0. The results showed that the 9E1 monoclonal antibody had an immune response to PNRSV, but had no immune response to the crude extracts of PPV, CGMMV, ApNMV, ASGV and healthy plant tissues.
[0108] II. Establishment of a serological detection method
[0109] 2.1. Establishment of ACP-ELISA detection method
[0110] 2.1.1 Steps of ACP-ELISA detection method
[0111] 1) After the leaves were ground in a mortar, the homogenate was further homogenized with ELISA coating solution at a ratio of 1:20 (w / v, g / mL). After centrifugation at 8,000 rpm for 3 min, 100 μL of supernatant was added to each well of the ELISA plate. The leaves infected by PNRSV were used as a positive control, and the healthy leaves were used as a negative control. The plate was coated at 4°C overnight or at 37°C for 2 h.
[0112] 2) After coating, the enzyme-labeled plate was washed with PBST by a plate washer (Microplate Washers, ELX405). The number of cycles of the plate washer was set to 3, and 250 μL of blocking solution (containing 3% skimmed milk) was added to each well. The plate was blocked at 37°C for 0.5-1 h.
[0113] 3) Discard the blocking solution in ELISA plate, wash 3 times with PBST plate washer, then add 100 μL / well of the primary antibody diluted with blocking solution, incubate at 37°C for 1-2 h;
[0114] 4) Discard the primary antibody dilution in ELISA plate, wash 3 times with PBST plate washer, then add 100 μL / well of the secondary antibody (AP-labeled goat anti-mouse IgG, Sigma) diluted with blocking solution, incubate at 37°C for 1-2 h;
[0115] 5) Wash 4 times with PBST plate washer, each time for 3 min. Add PNPP substrate to develop color at 37°C for 30 min. Observe the color change of the wells to yellow with naked eyes, or stop the reaction with 2 M NaOH and measure OD at 405 nm with Bio-Rad 680 microplate reader 405 Take P / N > 3.0 as the positive judgment standard.
[0116] 2.1.2 Establishment of ACP-ELISA method for detecting PNRSV
[0117] Determine the optimal working concentration of the antibody in the ACP-ELISA method by square array test, i.e. use 1:20-fold diluted (w / v, g / mL) PNRSV diseased leaf crude extract as antigen to coat the ELISA plate; add 1:1000-1:512000-fold diluted PNRSV monoclonal antibody to each column of the ELISA plate horizontally and react for 1 h, then wash with PBST and add 1:1000-1:512000-fold diluted AP-labeled goat anti-mouse IgG secondary antibody to each row of the ELISA plate vertically, use healthy plant diluted crude extract as negative control, and determine the optimal working concentration of the antibody in the ACP-ELISA method by setting 3 replicates for each treatment. The results show that the optimal working concentration of 9E1 monoclonal antibody is 1:5000-fold dilution and that of the AP-labeled goat anti-mouse IgG secondary antibody is 1:8000-fold dilution, and the ACP-ELISA method for detecting PNRSV is established according to the optimal working concentration.
[0118] 2.1.3 Specificity and sensitivity analysis of the ACP-ELISA method for detecting PNRSV
[0119] Use the crude extracts of the diseased leaves infected with PNRSV and healthy leaves as positive and negative controls, respectively, take the crude extracts of the diseased leaves infected with PPV, CGMMV, ApNMV, and ASGV as detection samples, add them to the ELISA plate, repeat the experiment three times, and analyze the specificity of the PNRSV monoclonal antibody and the established ACP-ELISA method. Under the optimal antibody working concentration, the method detects the diseased leaves infected with PNRSV as positive reaction, while detects the crude extracts of the diseased leaves infected with PPV, CGMMV, ApNMV, ASGV, and healthy plant leaves as negative reaction, and the OD value difference between the positive and negative samples is extremely significant, indicating that the method and the monoclonal antibody have good specificity.
[0120] The infected PNRSV leaves and healthy leaves were gradiently diluted with PBS buffer from 1:20 times (w / v, g / mL) respectively, and the gradiently diluted crude extracts were added to the ELISA plate in turn to analyze the sensitivity of ACP-ELISA method for detecting the infected PNRSV leaves, and the experiment was repeated three times. The results showed that the sensitivity of ACP-ELISA for detecting the leaves reached 1:10240 times dilution (w / v, g / mL).
[0121] 2.2dot-ELISA detection method
[0122] 2.2.1dot-ELISA detection method steps
[0123] 1) Grind the plant tissue in a mortar, add 0.01M PBS buffer at a ratio of 1:20 (w / v, g / mL) for homogenization, centrifuge at 8,000 rpm for 3 min, and the supernatant is the crude extract of the plant tissue;
[0124] 2) Spotting: take 2 μL of the crude extract and spot it on the nitrocellulose (NC) membrane, and set the crude extracts of healthy and infected PNRSV leaves as negative and positive controls respectively, and dry in a 37°C incubator for 10 min;
[0125] 3) Blocking and primary antibody incubation: immerse the NC membrane in PBST (0.01M PBS containing 0.05% Tween-20) containing 5% skim milk powder blocking solution at 37°C for 30 min; then, put the NC membrane into the moderately diluted monoclonal antibody and incubate at room temperature for 1 h;
[0126] 5) Membrane washing: wash the membrane with PBST for 3 times, 3 min each time;
[0127] 6) Secondary antibody incubation: put the NC membrane into the moderately diluted AP enzyme-labeled goat anti-mouse IgG secondary antibody and incubate at room temperature for 1 h, and then wash the membrane 4 times, 3 min each time;
[0128] 7) Color development: mix 66 μL of NBT and 33 μL of BCIP substrate into 10 ml of substrate buffer (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl, pH 9.5), dry the membrane with absorbent paper, put the membrane into the substrate solution and react, develop color at room temperature in the dark for 15-20 min. Observe the results with the naked eye, when the positive control shows purple spots and the negative control has no change, wash the membrane in tap water to stop the reaction, take a photo and record the results.
[0129] 2.2dot-ELISA detection method for detecting PNRSV
[0130] The dot-ELISA method was established by using the optimal working concentrations of the monoclonal antibody and the enzyme-labeled secondary antibody. The optimal working concentrations of the monoclonal antibody and the enzyme-labeled secondary antibody were 1:5000 and 1:8000, respectively. The dot-ELISA method for detecting PNRSV in plants was established by using the optimal working concentrations of the above antibodies.
[0131] 2.3 Specificity and sensitivity analysis of the dot-ELISA method for detecting PNRSV
[0132] The specificity of the dot-ELISA method was analyzed by using the crude extracts of the diseased leaves infected with PNRSV and the healthy leaves as the positive and negative controls, respectively, and the crude extracts of the diseased leaves infected with PPV, CGMMV, ApNMV, and ASGV as the detection samples. The results showed that the dot-ELISA method detected the crude extract of the diseased leaves infected with PNRSV as a positive reaction, but detected the crude extracts of the diseased leaves infected with PPV, CGMMV, ApNMV, and ASGV and the healthy leaves as negative reactions (A), indicating that the specificity of the method and the monoclonal antibody was very good. Figure 3 A), indicating that the specificity of the method and the monoclonal antibody was very good.
[0133] The sensitivity of the dot-ELISA method for detecting the diseased leaves infected with PNRSV was analyzed by using the diseased leaves infected with PNRSV and the healthy leaves as the positive and negative controls, respectively, and the diseased leaves infected with PNRSV were ground in a mortar and diluted with 0.01M PBS from 1:10 (w / v, g / mL) to 1:1280 (w / v, g / mL) by gradient dilution. The results showed that the dot-ELISA method detected the diseased leaves infected with PNRSV as a positive reaction when the crude extract of the diseased leaves was diluted to 1:1280 (w / v, g / mL) (B), indicating that the sensitivity of the dot-ELISA method for detecting the diseased leaves was 1:1280. Figure 3 B), indicating that the sensitivity of the dot-ELISA method for detecting the diseased leaves was 1:1280.
[0134] 2.3 Tissue print-ELISA detection method
[0135] 2.3.1 Steps of the tissue print-ELISA detection method
[0136] 1) Sample preparation: the leaves of the detection plants were rolled into a cylindrical shape or the stems were cut into a flat cross-section with a knife blade;
[0137] 2) Dotting: the cross-section was pressed on the NC membrane for 3 seconds, and the healthy and PNRSV-infected plant tissues were used as the negative and positive controls, respectively, and dried in an oven at 37°C for 5 minutes;
[0138] 3) Blocking: NC membrane was blocked in 5% skim milk in PBST (0.01 M PBS containing 0.05% Tween-20) for 1 h at room temperature;
[0139] 4) Primary antibody incubation: NC membrane was incubated in the appropriate dilution of PNRSV monoclonal antibody for 1 h at room temperature;
[0140] 5) Secondary antibody incubation: After washing the membrane with PBST for 3 times, NC membrane was incubated in the appropriate dilution of AP-conjugated goat anti-mouse IgG for 1 h at room temperature;
[0141] 6) Washing: The membrane was washed with PBST for 4 times, 3 min each time;
[0142] 7) Substrate addition: 66 μl NBT and 33 μl BCIP substrate (Promega) were added into 10 ml substrate buffer (0.1 M Tris Cl, 0.1 M NaCl, 0.025 M MgCl, pH 9.5) and mixed well, then the membrane was immersed into the substrate buffer for color development until the positive color was obvious and the negative control was not colored, the membrane was rinsed in tap water to stop the reaction, and the color development result was recorded.
[0143] 2.3.2 Establishment of Tissue print-ELISA method for detecting PNRSV
[0144] The optimal working concentrations of monoclonal antibody and enzyme-labeled secondary antibody in Tissue print-ELISA were determined by square array experiment. The combination of the dilution degrees of primary antibody and enzyme-labeled secondary antibody with the highest detection sensitivity and specificity was selected as the optimal working concentration of Tissue print-ELISA. The results showed that the detection sensitivity and specificity of Tissue print-ELISA were the best when the monoclonal antibody 9E1 and the AP-labeled goat anti-mouse secondary antibody were diluted 1:5000 and 1:8000 times, respectively. The Tissue print-ELISA method was established according to the optimal working concentration of the antibody.
[0145] 2.3.3 Specificity analysis of Tissue print-ELISA method for detecting PNRSV
[0146] The diseased leaf crude extracts infected with PPV, CGMMV, ApNMV and ASGV, respectively, were used as detection samples, and the diseased leaf and healthy leaf tissue crude extracts infected with PNRSV were used as positive and negative controls, respectively. The established Tissue print-ELISA serological method was used for detection to analyze the specificity of the method for detecting PNRSV. The results showed that the method detected the diseased leaf crude extract infected with PNRSV as a positive reaction, while the detection of the crude extracts of the diseased leaves infected with PPV, CGMMV, ApNMV and ASGV and the healthy leaf tissue was all negative reactions. Figure 4). Three, Prunus necrotic ring spot virus dot-ELISA test kit
[0147] 1) Main components of the kit:
[0148]
[0149] The above reagents are stored at 4°C
[0150] Nitrocellulose membrane (NC) 10 sheets
[0151] Skimmed milk powder 30g
[0152] 10X PBST 1 bottle 100mL
[0153] Substrate buffer 1 bottle 100mL 2) Operation steps of the test sample:
[0154] a. Weigh the plant tissue, grind it in a mortar, and homogenize it at a ratio of 1:20-50 (w / v, g / mL) after adding 0.01M PBS (pH 7.4);
[0155] b. Centrifuge the homogenate at 8,000 rpm for 3 min;
[0156] c. Take 2.0 μL of the supernatant and spot it on the NC membrane, and set healthy and PNRSV-infected tissues as negative and positive controls, respectively, and dry it at room temperature for 10-20 min;
[0157] d. Immerse the NC membrane in a blocking solution containing 5% skimmed milk powder in PBST (0.01M PBS containing 0.05% Tween-20) at room temperature for 30 min;
[0158] e. Put the NC membrane in a 1:5000 dilution of the monoclonal antibody at room temperature for 60 min;
[0159] f. Wash the membrane with PBST for 3-4 times, each time for 3 min; put the NC membrane in a 1:8000 dilution of AP enzyme-labeled goat anti-mouse IgG secondary antibody at room temperature for 60 min;
[0160] g. Wash the membrane with PBST for 4 times, each time for 3 min;
[0161] h. Mix 66 μL of NBT and 33 μL of BCIP substrate with 10 mL of substrate buffer (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl2, pH 9.5), and then put the membrane in the substrate solution for color development, observe the results with the naked eye, and when the positive control shows obvious purple color and the negative control shows no color, rinse the membrane with tap water to terminate the reaction, and take a photo to record the results.
[0162] 3) Storage and shelf life:
[0163] Store at 2-8℃, avoid light, valid for 12 months.
[0164] 4) Phosphate buffer solution (0.01M PBS, pH7.4) formula:
[0165]
[0166] Dissolve in 950mL of distilled water, adjust pH to 7.4, and dilute to 1,000mL
[0167] Four, Prunus necrotic ring spot virus Tissue print-ELISA detection kit
[0168] 1) Main components of the kit:
[0169]
[0170] The above reagents are stored at 4℃
[0171] Nitrocellulose membrane (NC) 10 sheets
[0172] Skimmed milk powder 30g
[0173] 10X PBST 1 bottle 100mL
[0174] Substrate buffer 1 bottle 100mL 2) Operation steps of the sample:
[0175] a. Sample preparation: Take the diseased leaves or stems, roll the tender leaves into a cylinder, and cut a cross section with a knife blade. Cut the tender stems directly into a cross section;
[0176] b. Spotting: Press the cross section on the NC membrane for 3 seconds, and at the same time, take the healthy and infected PNRSV tissues as negative and positive controls, respectively. Dry in a 37℃ oven for 5min;
[0177] c. NC membrane is immersed in PBST (0.01M PBS containing 0.05% Tween-20) containing 5% skimmed milk powder blocking solution at room temperature for 30min;
[0178] d. NC membrane is placed in 1:5000 diluted monoclonal antibody at room temperature for 60min;
[0179] e. Wash the membrane with PBST 3 times, 3min each time; NC membrane is placed in 1:8000 diluted AP enzyme-labeled goat anti-mouse IgG secondary antibody at room temperature for 60min;
[0180] f. Wash the membrane with PBST 4 times, 3min each time;
[0181] g.66 μL NBT and 33 μL BCIP substrate were added into 10 mL substrate buffer (0.1 M Tris Cl, 0.1 M NaCl, 0.025 M MgCl, pH 9.5), after mixing, the membrane was put into the substrate solution for color development, the results were observed by naked eyes, when the positive control showed obvious purple color and the negative control showed no color, the membrane was rinsed with deionized water to terminate the reaction, and the results were recorded by taking a photo.
[0182] 3) Storage and shelf life:
[0183] Store at 2-8°C in the dark, and the shelf life is 12 months.
[0184] 4) Phosphate buffer solution (0.01 M PBS, pH 7.4) formula:
[0185]
[0186] Add 950 mL of distilled water, adjust the pH to 7.4, and dilute to 1,000 mL.
[0187] The above-described embodiments are only a preferred scheme of the present application, and are not intended to limit the present application. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present application. Therefore, any technical scheme obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present application.
Claims
1. A secreting anti-Prunus necrotic ring spot virus monoclonal hybridoma cell line 9E1, characterized in that The hybridoma cell strain 9E1 capable of secreting the anti-Prunus necrotic ring spot virus monoclonal antibody is preserved in the China General Microbiological Culture Collection Center on November 7, 2022, and the preservation number is CGMCC No. 45329.
2. The anti-Prunus necrotic ring spot virus monoclonal antibody secreted by the hybridoma cell strain 9E1 according to claim 1.
3. The anti-Prunus necrotic ring spot virus monoclonal antibody of claim 2, wherein The indirect ELISA titer of the ascites reached 10 -7 The antibody type and subclass was IgG2b, κ light chain. The monoclonal antibody had specific immunoreaction with the 30 kDa coat protein of Prunus necrotic ring spot virus. The sensitivity of the ACP-ELISA and dot-ELISA methods for detecting the crude extract of infected PNRSV leaf tissue reached 1:10240 and 1:1280 times dilution, respectively, with the unit being weight / volume, g / mL.
4. The anti-Prunus necrotic ring spot virus monoclonal antibody of claim 2, wherein The monoclonal antibody can specifically immunoreact with the Prunus necrotic ring spot virus, and does not immunoreact with Prunus pustulata virus, cucumber green mottle mosaic virus, apple stem grooving virus, apple necrotic leaf virus and healthy plant tissues.
5. Use of the anti-Prunus necrotic ring spot virus monoclonal antibody according to claim 2 for the detection of the virus, characterized in that The application is various immunological detection methods and immunological detection kits established by taking the anti-Prunus necrotic ring spot virus monoclonal antibody as a detection antibody.