Xanthomonas oryzae antibody detection kit and application thereof

Abstract

The invention relates to the field of biological detection, in particular to a xanthomonas oryzae antibody detection kit and application thereof, and provides a monoclonal antibody 2A5 of a xanthomonas oryzae XadA antigen, 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 2A5, the qualitative detection of the xanthomonas oryzae and the quantitative detection of the xanthomonas oryzae extracellular membrane protein XadA antigen can be realized, and the specificity and the sensitivity are relatively high.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a Xanthomonas oryzae antibody detection kit and application thereof. Background Art

[0002] Xanthomonas oryzae is one of the important pathogens of rice, which can cause bacterial leaf streak and bacterial leaf blight, posing a serious threat to rice growth and yield. The pathogen destroys the cell structure of plants by secreting specific effector proteins and affects metabolic processes, leading to leaf dryness and early death. Xanthomonas oryzae is highly contagious and has a wide range of prevalence. It is one of the main diseases in rice farming systems worldwide. Rapid detection and accurate diagnosis of the pathogen have become an important part of rice disease prevention and control, while traditional detection methods such as bacterial culture or molecular biology techniques have the problems of long time and complex operation, which limits the convenience and efficiency of field application.

[0003] Monoclonal antibodies have important application value in pathogen detection due to their high specificity and good sensitivity. However, the specificity and sensitivity of monoclonal antibodies are limited by antigen selection and antibody preparation process, especially the development of antibodies against complex outer membrane proteins, which faces the problems of structural diversity and low immunogenicity. The surface protein of Xanthomonas oryzae (such as OuterMembrane Protein, OMP) is an ideal antigen target, but the development of efficient antibodies against Xanthomonas oryzae OMP is still technically challenging. Therefore, optimizing the variable region sequence design and preparation process of antibodies is the key path to improve antibody detection performance.

[0004] Xanthomonas adhesin-like protein A (XadA) is an important outer membrane protein that participates in the adhesion and infection process of pathogens and plays a key role in the pathogenic process. As a surface antigen with good immunogenicity, XadA is a potential target for the development and detection of Xanthomonas oryzae. However, the expression and purification of XadA face the difficulties of complex protein structure and low solubility. Therefore, it is necessary to optimize its antigen preparation process through advanced biotechnology means, and combine high-throughput screening technology to obtain specific monoclonal antibodies against XadA for qualitative and quantitative detection of Xanthomonas oryzae, thereby improving the accuracy and efficiency of detection and facilitating early diagnosis and prevention of diseases. Summary of the invention

[0005] The present invention provides a highly specific and highly sensitive anti-Xanthomonas oryzae monoclonal antibody 2A5 and application thereof in a detection kit, aiming to achieve rapid and accurate detection of Xanthomonas oryzae.

[0006] In an embodiment of the present invention, the monoclonal antibody 2A5 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, wherein 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.

[0007] The invention designs a Xanthomonas oryzae antibody detection kit, which comprises the monoclonal antibody 2A5.

[0008] The present invention also provides use of the monoclonal antibody 2A5 or a kit comprising the monoclonal antibody 2A5 in preparing a product for detecting Xanthomonas oryzae.

[0009] The kit of the present invention realizes qualitative and quantitative detection of target bacteria through high-affinity binding of monoclonal antibody 2A5 with specific antigen of Xanthomonas oryzae. Meanwhile, the minimum detection limit of monoclonal antibody 2A5 for Xanthomonas oryzae XadA antigen is about 0.5 ng / mL, and has good sensitivity and specificity, thus laying a foundation for its application in detection and functional research. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Molecular sieve column map and SDS-PAGE analysis results of XadA antigen fragment.

[0011] Figure 2 The titer curve of monoclonal antibody 2A5 detected by indirect ELISA.

[0012] Figure 3 Sensitivity analysis of monoclonal antibody 2A5 to XadA antigen fragment. DETAILED DESCRIPTION

[0013] 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.

[0014] Example 1 Preparation of surface XadA antigen fragments of Xanthomonas oryzae

[0015] According to the protein information of outer membrane protein XadA ([Xanthomonas oryzaepv.oryzae], GenBank: AAG01335.1) recorded by NCBI, IEDB Analysis Resource was used to predict sequences with high immunogenicity, low hydrophobicity and no secondary structure interference:

[0016] EGANAVGTATTALGTGANAVAENATAVGTNALASGQNSAAFGHNAQANGPASVAVGGAAVNEDGEPLITNGGVPVTTGATSAGVGGTAVGASAKADGFAASSFGVGAYAAGAQASAFGAVANAAGDYATAVGTQTRASGTSST (SEQ ID NO: 1).

[0017] Template DNA was extracted from the genome of Xanthomonas oryzae, and primers were designed according to the XadA protein information recorded in NCBI, and the restriction sites (NdeI / XhoI) were included; PCR was used to amplify the target fragment and recover the PCR product, and the expression vector of pET28a-HIS tag was used to cut the PCR product and the vector with restriction endonuclease (NdeI / XhoI), and incubated at 16°C for 2 hours with T4 DNA ligase. The ligation product was transformed into competent E.coli DH5α, and antibiotic screening with ampicillin was selected, and positive clones were verified by PCR or enzyme digestion. The recombinant plasmid of the positive clone was transformed into the E.coli BL21 (DE3) expression strain. Among them, the antibiotic screening was consistent with the plasmid resistance; a single colony was inoculated in 5mL LB liquid medium, cultured at 37°C to OD600 = 0.6-0.8, and IPTG (final concentration 0.1-1mM) was added to induce the expression of the target protein. The expression temperature and time were induced at 30°C for 4 hours or 16°C overnight, the bacteria were collected, ultrasonically disrupted or lysed, and the expression level and distribution of the target protein were verified by SDS-PAGE. The lysis buffer was configured as follows: 50mM Tris-HCl, pH 8.0, 300mM NaCl, 10mM imidazole, and protease inhibitors (PMSF) were added. Ultrasonic disruption was performed at 4°C (10 seconds of ultrasound + 10 seconds of interval, a total of 15 minutes), then centrifuged at 12000rpm, 4°C for 15 minutes, and the supernatant was collected. The Ni-NTA column was equilibrated with lysis buffer. The supernatant containing the target protein XadA was loaded and incubated at 4°C for 30 minutes to enhance binding. Nonspecific proteins were washed with a gradient of 20mM and 50mM imidazole, and the target protein was eluted with 250mM imidazole. The fractions were collected and the purity of the target protein in the fractions was detected by SDS-PAGE. A molecular sieve column was used to improve the purity, see. Figure 1 .

[0018] Figure 1 The results showed that the molecular weight of the XadA antigen fragment was 12.81 kDa, with high purity, which was in line with expectations and could be used in subsequent immune experiments.

[0019] Example 2 Screening, preparation and identification of monoclonal antibodies against Xanthomonas oryzae

[0020] BALB / c mice aged 6-8 weeks were selected, both male and female, with 5 mice in each group, one group for antigen immunization and one group as control, 50 μg of the purified protein XadA antigen fragment prepared in Example 1 was mixed with an equal volume of complete Freund's adjuvant and injected intraperitoneally for primary immunization, 2 weeks after the primary immunization, 50 μg of the purified protein XadA antigen fragment prepared in Example 1 was mixed with an equal volume of complete Freund's adjuvant and injected intraperitoneally once a week for booster immunization, 100 μg of XadA antigen fragment prepared in PBS was injected, and the final immunization was performed by tail vein injection, and the mice were killed 3 days after the final immunization, and the spleen was removed. Under sterile conditions, the spleen was ground with PBS, and the cell suspension was filtered to remove impurities. Red blood cell lysis: treated with red blood cell lysis solution, centrifuged to collect mononuclear cells, and SP2 / 0 myeloma cell line was used and cultured to the logarithmic growth phase. Centrifuged, collected cells, washed with PBS twice, and spleen cells and SP2 / 0 cells were mixed in a ratio of 5:1. Add 50% PEG-1500 (preheated to 37°C) and mix quickly. Slowly add RPMI 1640 medium to wash the cells. Transfer the cells to a 96-well plate containing HAT medium (RPMI 1640+HAT), with approximately 2×10 cells per well. 5 Cells. Culture for 5-7 days and observe the clone growth. Use indirect ELISA to detect positive clones. Coating plate: Use XadA antigen protein to coat the plate. Primary antibody: culture supernatant. Secondary antibody: HRP-labeled anti-mouse IgG. Substrate: TMB color development, 450nm detection, see Figure 2 .

[0021] Figure 2 The results showed that the titer of monoclonal antibody 2A5 detected by indirect ELISA was 1:25600.

[0022] Variable region sequence identification of monoclonal antibody 2A5: Total RNA was extracted from the hybridoma cell line producing the 2A5 antibody. RNA was extracted using a commercial RNA extraction kit, and the RNA concentration and purity were measured. The extracted mRNA was reverse transcribed into cDNA using a reverse transcription reaction (RT-PCR). Appropriate primers, random primers or specific primers (for the framework region sequences of the antibody heavy chain VH and light chain VL) were selected. cDNA was synthesized using a high-efficiency reverse transcriptase and an RT-PCR kit. Specific primers were designed and synthesized for the conserved regions of the variable regions of the antibody heavy chain (VH) and light chain (VL). VH and VL region gene fragments were amplified by PCR reaction. Appropriate PCR conditions (95°C denaturation, 55°C annealing, 72°C extension) were used for 30-35 cycles. The specificity and product length of PCR amplification were confirmed by agarose gel electrophoresis. The amplified VH and VL fragments were purified by gel purification or using a PCR product purification kit. The concentration of the purified product was further determined to ensure that it met the requirements for subsequent sequencing. Send the purified PCR product to a sequencing company or use a sequencer for bidirectional Sanger sequencing. Prepare samples according to the requirements of the sequencing platform, and select appropriate primers for complete sequencing of the VH and VL regions. Obtain the original sequencing data, perform base sequence alignment and correction. Use antibody sequence analysis software (Kabat) to align the VH and VL sequences, identify the specific locations of the CDR and framework region (FR), and determine the VH and VL variable region amino acid sequences of the 2A5 antibody.

[0023] Heavy chain variable region (VH) of monoclonal antibody 2A5:

[0024] MDFGLSFIFLALILKGVQCQVQLQESPFTSRLSINKDNSKSQVFKGRFTISRDDSKSSVYTTAPSVYPLVPGCSDTSGSLQSNDTAIYYCARALTYYDYEFAYWGQGTTVTVSSLTNYGVHWVRQSPMKPDHDASTVSG(SEQID NO:2);

[0025] H-CDR1: SRLSI (SEQ ID NO:3);

[0026] H-CDR2: TISRDDSKSSVYTTAP (SEQ ID NO: 4);

[0027] H-CDR3: CARALTYY (SEQ ID NO: 5).

[0028] The light chain variable region (VL) of monoclonal antibody 2A5: DIVLTQSQKFMSTSGDQASISCRSGQSLTLESNAGCYQQKPGGVPDRFSGSGSGTDFGSGSGTDFILTISNVFCSQSTHVPWTFLKFGGGTKLEIKLRCSGLPDLEIKSGTD (SEQ ID NO: 6);

[0029] L-CDR1: KFMSTSGDQAS (SEQ ID NO:7);

[0030] L-CDR2: QKPGGVP (SEQ ID NO:8);

[0031] L-CDR3: QSTHVPWTF (SEQ ID NO:9).

[0032] Example 3 Specificity Identification of Monoclonal Antibody 2A5 Against Xanthomonas oryzae

[0033] The standard samples of Xanthomonas albilineans, Xanthomonasampelina, Xanthomonas oryzae, Xanthomonas citri, Magnaporthe oryzae, Burkholderia glumae, and Rhizoctonia solani were subjected to a glass plate agglutination test with monoclonal antibody 2A5, and the above standard samples were inoculated in LB agar medium and cultured at 37°C for 24 hours. Single colonies were picked into 5 mL of physiological saline to prepare bacterial suspension, and the concentration of the bacterial suspension was adjusted to McFarland 1.0 standard (~10 8 CFU / mL). On a glass microscope slide, mark different strains in different areas, add an equal amount (20 μL) of bacterial suspension to each area, then add 10 μL of antibody 2A5 working solution, and add PBS to the control group instead of antibody, gently mix with a micro glass rod, use a separate glass rod for each slide, stand at room temperature for 2 minutes, observe the reaction, and record the agglutination strength comparison between Xanthomonas oryzae and other similar strains.

[0034] Table 1 Specificity identification of monoclonal antibody 2A5

[0035]

[0036] Note: Agglutination intensity grading: +++ indicates obvious agglutination, ++ indicates moderate agglutination, + indicates slight agglutination, and - indicates no agglutination.

[0037] The results in Table 1 show that antibody 2A5 exhibited an obvious agglutination reaction of +++ in Xanthomonas oryzae, while no agglutination (-) was observed in other strains; this indicates that monoclonal antibody 2A5 can specifically recognize Xanthomonas oryzae and exhibit a strong agglutination reaction in the glass plate agglutination test; it has no cross-reaction to similar strains, demonstrating its potential for application in the detection and diagnosis of Xanthomonas oryzae.

[0038] Example 4: Analysis of the sensitivity of monoclonal antibody 2A5 to XadA antigen fragment

[0039] The XadA antigen fragment prepared in Example 1 was gradiently diluted to prepare the following concentration series: 0.5, 1, 2, 5, 10, 20 (ng / mL), and diluted to a final volume of 100 μL using coating buffer. 100 μL of antigen solution of different concentrations was added to each well and incubated overnight at 4°C. Washed 3 times with PBS-T (PBS + 0.05% Tween-20) to remove unbound antigens. 200 μL of blocking solution (1% BSA) was added and incubated at 37°C for 1 hour. Monoclonal antibody 2A5 was diluted to 1:1000 with a final concentration of 1 μg / mL. 100 μL of diluted antibody was added to each well and incubated at 37°C for 1 hour. Washed 3 times with PBS-T. 100 μL of HRP-labeled anti-mouse IgG was added, diluted 1:5000. Incubated at 37°C for 1 hour and washed 3 times with PBS-T. 100 μL of TMB colorimetric solution was added and incubated at room temperature for 10 minutes. Add 50 μL of stop solution to stop the color development reaction. Read the OD value of each well at 450 nm on the microplate reader. The horizontal axis is the antigen concentration (ng / mL), and the vertical axis is the OD value. Draw a standard curve. Use the four-parameter logistic model (4-PL) to fit the curve, see Figure 3 ,formula:

[0040]

[0041] y: OD value.

[0042] x: antigen concentration.

[0043] A, B, C, D: fitting parameters.

[0044] The parameters obtained by fitting are:

[0045] A=0.062: lower limit of the curve.

[0046] B=1.350: slope.

[0047] C=6.700: Antigen concentration corresponding to half-maximal response (EC50).

[0048] D=3.000: upper limit of the curve.

[0049] After substituting the parameters, the fitting equation is:

[0050]

[0051] Correlation (R 2 ):

[0052] R 2 =0.998, indicating that the fitting effect is excellent and the data are highly consistent.

[0053] Figure 3 The results showed that according to the fitting equation, the minimum detection limit was about 0.5 ng / mL. At this concentration, the optical density began to be significantly higher than the background value. The optical density (OD) was in the range of 0.3-2.5. The linear range of antigen concentration was about 0.5-10 ng / mL. The antigen concentration of half-maximum response (EC50) was C = 6.700 ng / mL.

[0054] 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 Xanthomonas oryzae antibody detection kit, characterized in that: The kit comprises a monoclonal antibody 2A5 against Xanthomonas oryzae XadA antigen, wherein the monoclonal antibody 2A5 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.

2. A monoclonal antibody for detecting Xanthomonas oryzae, 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.

3. The antibody according to claim 2, 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.

4. Use of the detection kit according to claim 1 and the antibody according to any one of claims 2 to 3 in the preparation of a product for detecting Xanthomonas oryzae.

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