Puccinia puccinia antibody detection kit and application thereof

Abstract

The invention relates to the field of biological detection, in particular to a puccinia striiformis antibody detection kit and application thereof, and provides an anti-puccinia striiformis monoclonal antibody 1A6 which comprises a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain variable region as shown in SEQ ID NO: 6. By adopting the detection kit containing the monoclonal antibody 1A6, qualitative and quantitative detection of puccinia peucedanum can be realized, and the detection kit has relatively high specificity and sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a kit for detecting antibodies against Puccinia odorifera and its application. Background Art

[0002] Puccinia graminis is a pathogenic fungus that widely infects many important crops such as wheat and barley, causing serious diseases such as wheat stem rust, which has caused significant losses to global agricultural production. The pathogen has strong transmissibility and environmental adaptability, and can be spread to distant areas through the air, posing a threat to food security. Traditional disease control methods mainly rely on chemical control and breeding of disease-resistant varieties, but because Puccinia graminis is prone to resistance and mutation, the effectiveness of a single control method has gradually decreased. Therefore, it is necessary to develop effective pathogen detection technology to achieve early diagnosis and precise control.

[0003] Monoclonal antibodies have been widely used in the detection of pathogens such as bacteria, viruses and fungi due to their high specificity and consistency. By designing monoclonal antibodies against antigenic epitopes of specific pathogens, efficient identification of pathogens can be achieved. Especially in the field of agricultural disease detection, monoclonal antibodies provide a fast, sensitive and simple detection method that can be used in field detection, monitoring and laboratory analysis. However, there is still a technical gap in the application of monoclonal antibodies against Puccinia, and existing detection methods lack specific antibodies and cannot meet the needs of high-throughput and low-error pathogen detection.

[0004] Although monoclonal antibodies show high specificity in pathogen detection, they face some challenges in specificity and sensitivity in the detection of Puccinia. First, Puccinia has certain antigenic similarities with other related rust pathogens (such as stripe rust, leaf rust, etc.), which may lead to cross-reactions and affect the specificity of detection. In addition, due to the low number of pathogens and antigen amounts, existing antibodies may be insufficient in detection sensitivity and unable to detect low concentrations of pathogens in early infections. Further improving the binding efficiency and signal amplification capabilities of antibodies, and ensuring the specificity of antibodies for Puccinia antigens and the sensitivity of detection, are still technical problems that need to be solved urgently. Summary of the invention

[0005] The present invention provides a detection kit comprising the specific monoclonal antibody 1A6, which can realize accurate detection of Puccinia repens in a sample.

[0006] The antibody detection kit provided by the present invention comprises a labeled anti-rust fungus monoclonal antibody 1A6 and a color developer or a fluorescent reagent. The monoclonal antibody 1A6 is designed for the specific antigen of the stirrup fungus, has a high binding affinity, and can effectively recognize and bind to the stirrup fungus specific antigen.

[0007] In certain embodiments, the heavy chain variable region of the 1A6 antibody comprises the amino acid sequence set forth in SEQ ID NO:2 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:6.

[0008] The present invention also provides a method for detecting Puccinia, which specifically comprises the following steps: first, contacting a sample to be detected with a detection kit containing anti-Puccinia monoclonal antibody 1A6, and if the sample contains Puccinia antigen, 1A6 antibody will bind to it. Then, by detecting the signal intensity generated by the binding of the antibody and the antigen, it can be determined whether the sample contains Puccinia antigen.

[0009] In the method of the present invention, the detection signal can be read by fluorescence or enzyme-linked immunosorbent assay to achieve high-sensitivity detection. The use of fluorescence signals can improve the sensitivity and accuracy of detection and is suitable for the detection of low-concentration samples; enzyme-linked immunosorbent assay signals are suitable for quantitative analysis and can determine the specific concentration of the Puccinia antigen by reading the absorbance. This multi-mode signal detection method enables the present invention to have a wide range of application scenarios and can meet the needs of different experimental environments.

[0010] In addition, the present invention also provides a monoclonal antibody 1A6 for detecting Puccinia serrata. The antibody comprises specific heavy chain and light chain variable regions, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

[0011] Compared with the prior art, the detection kit and monoclonal antibody 1A6 of the present invention can be used to prepare products for detecting stem rust, including equipment for laboratory diagnosis and rapid field detection. At the same time, the monoclonal antibody 1A6 of the present invention has significant specificity and sensitivity, and can provide accurate detection results at different stages of stem rust infection, providing important technical support for early warning and disease control of crop diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 SDS-PAGE analysis results of recombinant protein Pg-Avr.

[0013] Figure 2 SDS-PAGE analysis results of the light and heavy chains of monoclonal antibody 1A6.

[0014] Figure 3 Identification of subtypes of monoclonal antibody 1A6 against Avr antigen of Puccinia odorifera.

[0015] Figure 4 Specificity analysis of monoclonal antibody 1A6 against Puccinia repens.

[0016] Figure 5 Sensitivity analysis of monoclonal antibody 1A6 against Puccinia repens. DETAILED DESCRIPTION

[0017] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0018] Example 1 Preparation of Puccinia graminis Avr Antigen

[0019] The immune antigen peptide was designed according to the full-length sequence of avirulence protein [Puccinia graminisf.sp.tritici] recorded in NCBI (GenBank: WOJ46332.1): MHYITPIILMSIGQFL DILLGAGGLVGAMTPHHQINCNSPYLTFPRLTAKCDSCQLHTKVTNLVSCTS CRKSSLVYEECSTKGCPANWHKSTCQEHKFKRGILHSRCENCQQHKKETQTISCKNCKNSASTFPYCSSPECHSRW (SEQ ID NO: 1).

[0020] PCR was performed using a high-fidelity DNA polymerase, and the reaction system included Puccinia template DNA, primer Avr-F1, primer Avr-R1, dNTPs, and enzyme reaction buffer. PCR cycle conditions: pre-denaturation (95°C, 5 minutes), denaturation (95°C, 30 seconds), annealing (55-60°C, 30 seconds), extension (72°C, 1 minute), 35 cycles, and final extension (72°C, 10 minutes). Primers and dNTPs were removed using a PCR product purification kit. The pET-28a vector was digested with restriction endonucleases (NdeI and XhoI), and the enzyme digestion reaction conditions were performed according to the enzyme manufacturer's instructions. The purified PCR product was mixed with the digested vector, and the ligation reaction was performed using T4 DNA ligase. The temperature and time were determined according to the ligase instructions. The cells were mixed with the ligation product, placed in a 42°C water bath for 30 seconds, and then quickly returned to ice. Finally, after 1 hour of growth recovery in LB medium, they were spread on antibiotic-containing plates. Analyze positive clones by PCR or restriction enzyme digestion to confirm the correctness of the inserted fragment. Pick positive clones for small-scale culture, use LB medium, and add an appropriate amount of ampicillin 100 μg / mL. When the culture medium OD 600When the p-value reaches 0.6-0.8, IPTG is added to induce protein expression, with a final concentration of 0.5-1.0 mM, and culture is continued at 37°C for 4-6 hours. The induced cell pellet is collected by centrifugation (6000 rpm, 10 minutes). A lysis buffer (PBS buffer with 1 mg / mL lysozyme) is selected, and the cells are treated on ice for 30 minutes, followed by cell lysis using an ultrasonic disruptor. The ultrasonic time and power are optimized according to the sample volume and cell concentration. Centrifuge at 12,000 rpm for 30 minutes, and collect the supernatant for subsequent purification. The supernatant is transferred to a binding buffer (buffer containing 20 mM sodium chloride and 10 mM phosphate) containing a nickel column, mixed thoroughly, and placed on a shaker for binding for 1-2 hours. The nickel column is washed with a buffer containing 20, 30, and 40 mM gradient imidazole to remove unbound proteins. The bound recombinant protein Pg-Avr is eluted with an elution buffer containing 20 mM sodium chloride and 250 mM imidazole, and the eluate is collected. The purity of the protein in the eluate was analyzed by SDS-PAGE. Figure 1 .

[0021] Figure 1 The results showed that the molecular weight of the recombinant protein Pg-Avr was 16.19 kDa, which was in line with expectations.

[0022] Example 2 Screening, preparation and identification of monoclonal antibodies against Puccinia

[0023] Healthy, well-developed female BALB / C mice aged 6-8 weeks were selected for immunization: 30 μg of purified Pg-Avr recombinant protein was fully emulsified with an equal volume of Freund's complete adjuvant and injected intraperitoneally into the mice. Two weeks later, the same dose of protein was emulsified with an equal volume of Freund's incomplete adjuvant for the second immunization. Two weeks later, the same dose of protein was emulsified with an equal volume of Freund's incomplete adjuvant for the third immunization. After four immunizations, tail vein blood was collected to detect serum titer using indirect ELISA. A strong immunization was performed once 3 days before fusion. The results are shown in Table 1.

[0024] Table 1 Anti-Pg-Avr antibody titers in sera of immunized mice

[0025] BALB / C mouse model Antibody titer Mouse1 1：3200 Mouse2 1：6400 Mouse3 1：12800 Mouse4 1：6400 Mouse5 1：12800 Mouse6 1：51200 Mouse7 1：6400 Mouse8 1：25600

[0026] Select 1A6 with a serum titer of 1:51200 to immunize Mouse6, and then boost immunization once. After 3-4 days, mouse spleen cells and SP2 / 0 cells can be taken for cell fusion. Around 8 days after cell fusion, positive hybridoma cells were screened by immunofluorescence, and finally a positive hybridoma cell that can stably secrete anti-Pg-Avr monoclonal antibodies was screened and named 1A6. 96-well ELISA plates were coated with 2μg·mL-1 Pg-Avr protein, and monoclonal antibody 1A6 was horizontally diluted from 1:1000. The monoclonal antibody titer was detected by indirect ELISA, and the titer was 1:2048000.

[0027] Prepare 1-2 BALB / C mice in advance, inject 1 mL of paraffin into each mouse intraperitoneally, and inject 5 × 10 1A6 hybridoma cells intraperitoneally one week later. 6 After 10 days, the ascites was collected and diluted 10 times with PBS, centrifuged at 3500r / min for 10min, filtered with a 0.45μm filter, and purified with a Protein G gel column. A small amount of purified sample was retained for SDS-PAGE to identify the purity and content of the antibody. Figure 2 .

[0028] Figure 2 The results showed that under reducing conditions, the 1A6 antibody had one band at 50 kDa and one band at 25 kDa, respectively, which was consistent with the theoretical molecular weight of the heavy chain and light chain after reduction; no obvious mixed bands were observed, indicating that the prepared antibody 1A6 was of high purity.

[0029] The purified monoclonal antibody 1A6 was taken and the subtype of the monoclonal antibody 1A6 was identified using Mouse-Monoclonal antibody Isotyping Reagents. The specific operation was carried out according to the instructions. The subtype of the anti-Pg-Avr monoclonal antibody 1A6 was identified using the ELISA method. Figure 3 .

[0030] Figure 3 The results showed that the anti-Pg-Avr monoclonal antibody 1A6 was identified as IgG2a antibody by antibody subtype identification.

[0031] Identification of variable region sequence of monoclonal antibody 1A6: Total RNA was extracted from positive hybridoma cells that stably secreted anti-Pg-Avr monoclonal antibody 1A6. High-quality total RNA was obtained using an RNA extraction kit. Ensure that the operating procedures were strictly followed and that RNase-free consumables and reagents were used. The extracted RNA was transcribed into cDNA using reverse transcription PCR (RT-PCR). Random primers or specific primers were used, and the reverse transcriptase was selected as SuperScriptIII or M-MLV. The reaction conditions included incubation at 42°C for 60 minutes and then inactivation at 70°C for 10 minutes. Specific primers for mouse antibody 1A6 were designed to amplify the variable region gene fragment. The PCR product was verified by agarose gel electrophoresis to verify the amplified band, and the target band was extracted and purified. The nucleotide sequence of the PCR product was determined by Sanger sequencing. The sequencing results were compared with the known antibody gene sequences in the NCBI database to determine the variable region sequences of the light chain and heavy chain. The sequencing results were analyzed according to the standards of the IMGT database to determine the sequence positions of CDR1, CDR2, and CDR3.

[0032] The heavy chain variable region (VH) of monoclonal antibody 1A6: QVQLRQSGAELVKPATSIRLSC TASGFTFSTYVMSWVQQAPGKGLEWVAVIFYDGPNKYYADRVKGRYTISRDNAKNTFYHQMNSLRPEDTTVYYCGKDYGGMSGYFDYFGQGTLITVAS (SEQ ID NO: 2);

[0033] H-CDR1:TASGFTFSTY(SEQ ID NO:3);

[0034] H-CDR2:AVIFYDGPNKYYADRV (SEQ ID NO:4);

[0035] H-CDR3: GKDYGGMS (SEQ ID NO: 5).

[0036] The light chain variable region (VL) of monoclonal antibody 1A6: DIVMTQSPDSLAFSLGERVTINCKVSQNVGAAVAWYQEKPGQSAKFLIYWVSTRHTGVPNRFSGSRSGTDFTLTIGYLQAEDVAVYHCQQHYTSPYTFGQGTRVEIKN (SEQ ID NO: 6);

[0037] L-CDR1: INCKVSQNVGA (SEQ ID NO:7);

[0038] ML-CDR2: FLIYWVS (SEQ ID NO:8);

[0039] L-CDR3: CQQHYTSPY (SEQ ID NO:9).

[0040] Example 3 Specificity Identification of Monoclonal Antibody 1A6 Against Puccinia Avr Antigen

[0041] Total protein was extracted from leaves of plants infected with Puccinia striiformis and ultracentrifuged to remove impurities. The extracts were measured for protein concentration using the BCA method, and the samples were stored at -80°C for subsequent experiments. Several other control pathogens related to Puccinia striiformis were selected: Puccinia striiformis, Puccinia triticina, Uromyces appendiculatus, Blumeria graminis, Fusarium graminearum, and Magnaporthe oryzae. Total protein was extracted from each pathogen and treated in the same way as the Puccinia striiformis samples.

[0042] Enzyme-linked immunosorbent assay (ELISA) identification

[0043] Coating antigen: Extracts of Puccinia odorifera and control pathogens (200 ng / well each) were coated on 96-well ELISA plates using carbonate buffer (pH 9.6) at 4°C overnight.

[0044] Blocking: Block unbound sites with 5% bovine serum albumin (BSA) and incubate for 1 hour at room temperature.

[0045] Add primary antibody: Add 1A6 antibody (1:500 dilution) and incubate at room temperature for 1 hour.

[0046] Add secondary antibody: Add HRP-labeled anti-mouse IgG secondary antibody (1:5000 dilution) and incubate for 30 minutes.

[0047] Color development: Add TMB color development solution, incubate for 15 minutes, and then terminate the reaction with 2M H2SO4.

[0048] Detection: Read the absorbance at 450 nm using an ELISA reader, see Figure 4 .

[0049] Figure 4The results showed that at a wavelength of 450nm, the Puccinia antigen showed an absorbance value significantly higher than that of the control pathogen, while the absorbance of other pathogens was close to the negative control, indicating that the 1A6 antibody has high specificity for the Puccinia antigen, which indicates that the 1A6 antibody can specifically recognize the Puccinia protein without cross-reacting with other pathogen samples.

[0050] Example 4: Sensitivity analysis of monoclonal antibody 1A6 against Puccinia odorifera

[0051] The monoclonal antibody 1A6 with a concentration of 1 μg / mL was obtained by purification and preparation, and the purified Puccinia protein was extracted and used to prepare a series of dilution concentrations to construct a standard curve. The Puccinia antigen standard was diluted into the following series of concentrations: 1000ng / mL, 500ng / mL, 250ng / mL, 100ng / mL, 50ng / mL, 10ng / mL, 5ng / mL and 1ng / mL, and each concentration was repeated three times to ensure the accuracy and reproducibility of the data. Each concentration of antigen standard was reacted with 1A6 antibody for 30 minutes, and then FITC-labeled anti-mouse IgG secondary antibody was added and incubated at room temperature for 30 minutes. Flow cytometry was used for detection, and at least 10,000 events were collected for each sample. The fluorescence intensity was recorded and a standard curve between the fluorescence signal and the antigen concentration was generated by the flow cytometer software FlowJo. The mean fluorescence intensity (MFI) of each concentration sample was analyzed using the flow cytometer software, and the MFI value of each concentration was calculated. The standard curve was drawn with the concentration of Puccinia antigen as the abscissa and the fluorescence intensity as the ordinate to determine the linear detection range of the 1A6 antibody. Figure 5 .

[0052] Figure 5 The results show that the green dotted line marks the starting point of the linear range (50 ng / mL), and the red dotted line marks the end point of the linear range (1000 ng / mL). The figure clearly shows that within the range of 50 ng / mL to 1000 ng / mL, the detection signal (MFI) of antibody 1A6 to the concentration of Puccinia pectinosa antigen has a good linear relationship (linear correlation coefficient R 2 >0.98).

[0053] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A kit for detecting antibodies against Puccinia repens, characterized in that: The kit comprises the following components: a) a labeled anti-Puccinia serrata antibody 1A6 for binding to a Puccinia serrata-specific antigen, wherein the antibody 1A6 comprises a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain variable region as shown in SEQ ID NO: 6; b) Colorimetric or fluorescent reagents used to detect the binding of antibodies to antigens.

2. A method for detecting Puccinia repens, characterized in that: The method comprises the following steps: a) contacting a sample containing a Puccinia antigen with the antibody 1A6 as claimed in claim 1; b) Detecting the binding signal between the antibody and the antigen, and judging whether the sample contains Puccinia repens according to the detection signal.

3. The method according to claim 2, characterized in that The detection signal is a fluorescence signal or an enzyme-linked immunosorbent assay signal, so as to quantitatively determine the concentration of the Puccinia antigen.

4. A monoclonal antibody for detecting Puccinia repens, characterized in that: The antibody comprises a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:

6.

5. The antibody according to claim 4, characterized in that The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:4 and HCDR3 as shown in SEQ ID NO:5; the light chain variable region comprises LCDR1 as shown in SEQ ID NO:7, LCDR2 as shown in SEQ ID NO:8 and LCDR3 as shown in SEQ ID NO:

9.

6. Use of the detection kit according to claim 1, the antibody according to any one of claims 2 to 3 or any one of claims 4 to 5 in the preparation of a product for detecting Puccinia repens.

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