Anti-porcine SLA-DRA protein rabbit monoclonal antibody and application thereof

Abstract

The invention provides a rabbit monoclonal antibody for resisting porcine SLA-DRA protein and application of the rabbit monoclonal antibody, and relates to the technical field of biology. A heavy chain amino acid sequence of the antibody is shown as SEQ ID NO.2, and a light chain amino acid sequence of the antibody is shown as SEQ ID NO.3; the amino acid sequences of the complementary determining regions CDR1-3 on the heavy chain variable region are respectively as shown in SEQ ID NO.4-6, and the amino acid sequences of the complementary determining regions CDR1-3 on the light chain variable region are respectively as shown in SEQ ID NO.7-9. According to the present invention, the SLA-DRA full-length protein is intercepted and optimized, and is adopted as the immunogen, such that the prepared rabbit anti-pig SLA-DRA monoclonal antibody can identify the SLA-DRA protein with high specificity and high sensitivity, and can effectively identify the SLA-DR compound molecule. The kit is suitable for tests such as indirect ELISA, immunoblotting, surface plasmon resonance and the like for detecting expression of SLA-DR protein on cells and comparing different SLA-DR binding affinity differences.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a rabbit monoclonal antibody against porcine SLA-DRA and its application. Background Art

[0002] Antigen presentation is the cornerstone of the immune system, driving the adaptive immune response by activating T cells. Classical antigen-presenting cells, such as dendritic cells, macrophages, and B cells, etc., are the core of this process. It has been found that one of the characteristics of antigen-presenting cells is that they can present the recognized antigen peptides to CD4+ T cells in a major histocompatibility complex class II (MHC-II) molecule (including DR, DQ, DP)-dependent manner, thereby further activating T cells and playing a crucial role in regulating T cell activation.

[0003] As an important economic animal and biomedical research model, the study of the immune system of pigs is of great significance. In pigs, the MHC-II molecules, namely swine leukocyte antigen II (SLA-II), have two loci, namely SLA-DR and SLA-DQ, which are involved in presenting antigen peptides for CD4+ T cell recognition. Among them, the SLA-DR molecule is a heterodimer composed of an α chain (DRA) and a β chain (DRB), and these two chains have high polymorphism, especially the gene diversity of DRB. As of March 2025, according to the IPD-MHC database (http: / / www.ebi.ac.uk / ipd / mhc / sla / ), 115 SLA-DR alleles have been identified, including 16 SLA-DRA and 99 SLA-DRB1. The high polymorphism of the SLA-DR molecule makes it difficult to determine the peptide presentation characteristics of different SLA-DR molecules and their specific mechanisms involved in regulating the immune response. One of the main rate-limiting steps is the lack of antibody tools on the market that can bind to SLA-DR molecules with high affinity and are used to detect different DR combination molecules. Problems with existing commercial antibodies for detecting SLA-DR molecules: (1) Most of them are prepared for humans and mice, and there are few antibodies against porcine SLA-DR, and most of them are murine, which have problems not suitable for WB detection; (2) Most of them are directed against specific SLA-DR alleles and lack broad cross-reactivity, making it difficult to cover the polymorphism among different pig breeds and individuals; (3) Using antibodies against human DR to detect porcine SLA-DR molecules has problems such as species differences.

[0004] To address the above problems, this study focused on the conserved sites of the SLA-DRA molecule. Combining structural analysis, a rabbit monoclonal antibody against porcine SLA-DRA protein was designed and prepared. Compared with the DRB chain with higher polymorphism in the SLA-DR molecule, the DRA sequence is relatively conserved with lower polymorphism. Therefore, the antibody against SLA-DR has a wider applicability. The anti-SLA-DRA monoclonal antibody prepared in this study has a higher affinity compared to murine antibodies and can effectively recognize SLA-DR molecules in different pig breeds and individuals, overcoming the limitations of existing commercial antibodies. In addition, this antibody can detect the expression of SLA-DR in cells, which is of great significance for in-depth study of the mechanism of action of SLA-DR protein in immune responses, prevention and control of swine diseases, principles of swine herd health, and improvement of swine production performance. Summary of the Invention

[0005] In view of the shortcomings and deficiencies of the existing detection of SLA-DR protein, the object of the present invention is to provide a rabbit monoclonal antibody with wide application, capable of highly sensitively recognizing SLA-DRA protein and effectively recognizing SLA-DR complex molecules. This antibody is applicable to assays such as indirect ELISA, indirect immunofluorescence (IFA), immunoblotting (Western Blot), surface plasmon resonance (SPR), etc. for detecting the expression of SLA-DR protein on cells and comparing the differences in binding affinities of different SLA-DRs. To achieve the above object, the main technical solutions adopted by the present invention include:

[0006] In the first aspect, the present invention provides a rabbit monoclonal antibody against porcine SLA-DRA. The amino acid sequences of the complementarity-determining regions CDR1-VH, CDR2-VH, and CDR3-VH on the heavy chain of the rabbit monoclonal antibody against porcine SLA-DRA protein are shown as SEQ ID NO.4-6 respectively; the amino acid sequences of the complementarity-determining regions CDR1-VL, CDR2-VL, and CDR3-VL on the light chain of the rabbit monoclonal antibody against porcine SLA-DRA protein are shown as SEQ ID NO.7-9 respectively.

[0007] As a further technical solution, the amino acid sequence of the heavy chain variable region VH of the anti-SLA-DRA rabbit monoclonal antibody is shown as SEQ ID NO.10, and the amino acid sequence of the light chain variable region VL of the monoclonal antibody is shown as SEQ ID NO.11.

[0008] As a further technical solution, the amino acid sequence of the heavy chain of the anti-SLA-DRA rabbit monoclonal antibody is shown as SEQ IDNO.2, and the amino acid sequence of the light chain of the monoclonal antibody is shown as SEQ ID NO.3.

[0009] In a second aspect, the present invention provides a biomaterial selected from any one of a - c:

[0010] a. A nucleotide molecule, which nucleotide comprises a nucleotide sequence encoding the monoclonal antibody against porcine SLA - DRA protein according to any one of claims 1 - 3;

[0011] b. A vector that carries the nucleotide molecule in a;

[0012] c. A modified cell or recombinant strain for expressing the rabbit monoclonal antibody against porcine SLA - DRA protein according to any one of claims 1 - 3.

[0013] In a third aspect, the present invention provides a method for preparing a recombinant rabbit monoclonal antibody against SLA - DRA. The monoclonal antibody against porcine SLA - DRA is recombinantly expressed by mammalian cells. Specifically, the recombinant rabbit monoclonal antibody against porcine SLA - DRA provided by the present invention is produced by eukaryotic expression in 293T cells. Using the above - mentioned expression vector or recombinant plasmid to transform or transfect host cells, culturing the cells continuously, collecting the cell supernatant and purifying it through a Protein A affinity chromatography column to obtain the recombinant rabbit monoclonal antibody against SLA - DRA.

[0014] In a fourth aspect, when preparing the monoclonal antibody against porcine SLA - DRA, the antigen used for immunizing New Zealand white rabbits is the SLA - DRA protein, which is obtained by prokaryotic expression and has an amino acid sequence as shown in SEQ ID NO:1.

[0015] In a fifth aspect, the present invention provides a recombinant rabbit monoclonal antibody against SLA - DRA, which has high specificity and high sensitivity in binding to the SLA - DRA protein molecule, can specifically recognize and detect the expression of SLA - DR protein on cells, and can be applied to fields such as indirect immunofluorescence (IFA), indirect ELISA, immunoblotting (Western blotting, WB), surface plasmon resonance (SPR) and other detections and screenings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 It is the molecular sieve purification result of the immunogen of the rabbit monoclonal antibody against SLA - DRA prepared by the present invention;

[0018] Figure 2 Indirect ELISA test results of the anti-SLA-DRA monoclonal antibody prepared according to the present invention;

[0019] Figure 3 WB test results of the anti-SLA-DRA monoclonal antibody prepared according to the present invention;

[0020] Figure 4 IFA test results of the anti-SLA-DRA monoclonal antibody prepared according to the present invention;

[0021] Figure 5 SPR affinity test results of the anti-SLA-DRA monoclonal antibody prepared according to the present invention. Detailed implementation manners

[0022] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0023] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0024] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0025] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0026] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0027] The present invention provides a rabbit monoclonal antibody against porcine SLA-DRA protein. The amino acid sequence of the heavy chain of the rabbit monoclonal antibody is shown as SEQ ID NO.2, and the amino acid sequence of the light chain is shown as SEQ ID NO.3. The variable region VH of the heavy chain of the rabbit monoclonal antibody includes complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH having amino acid sequences shown as SEQ ID NOs. 4-6. The variable region VL of the light chain of the rabbit monoclonal antibody includes complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL having amino acid sequences shown as SEQ ID NOs. 7-9.

[0028] In some alternative embodiments, the amino acid sequence of the heavy chain is shown as SEQ ID NO.2:

[0029] MYRMQLLSCIALSLALVTNSQSVEESGGRLVTPGTPLTLTCTVSGVDLSAYAMNWVRQAPGKGLEWIGVIGRSGSISYASWTKGRFTISKTSTTVDLELTSLTTEDTATYFCGTNDLWGRGTLITVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSDTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKDYKDDDDK

[0030] In some alternative embodiments, the amino acid sequence of the light chain is shown as SEQ ID NO.3:

[0031] MYRMQLLSCIALSLALVTNSDGVMTQTPSSVSEPVGGTVTIKCQASQSISSYLAWLQQKPGQPPKRLIYRTSTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQAYYYSISDTYTNTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0032] In some alternative embodiments, the amino acid sequences of the heavy and light chain complementarity determining regions SEQ ID NO. 4-9 are shown in Table 1 as follows:

[0033] Table 1

[0034] Complementary determining region Number Amino acid sequence CDR1-VH SEQ ID NO.4 AYAMN CDR2-VH SEQ ID NO.5 VIGRSGSISYASWTKG CDR3-VH SEQ ID NO.6 NDL CDR1-VL SEQ ID NO.7 QASQSISSYLA CDR2-VL SEQ ID NO.8 RTSTLES CDR3-VL SEQ ID NO.9 QAYYYSISDTYTNT

[0035] The amino acid sequence of the heavy chain variable region VH of the antibody, as shown in SEQ NO. 10: QSVEESGGRLVTPGTPLTLT CTVSGVDLSAYAMNWVRQAPGKGLEWIGVIGRSGSISYASWTKGRFTISKTSTTVDLELTSLTTEDTATYFCGTNDLWGRGTLITVSS

[0036] The amino acid sequence of the light chain variable region VL of the antibody, as shown in SEQ NO. 11: DGVMTQTPSSVSEPVGGTV TIKCQASQSISSYLAWLQQKPGQPPKRLIYRTSTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQAYYYSISDTYTNTFGGGTEVVVK

[0037] Preparation and Purification of the Antigen in Example 1

[0038] Considering the low polymorphism of different SLA-DRA molecules, combined with the structure of the SLA-DRA amino acid sequence predicted by AlphaFold3, relatively conserved regions were selected where there are no long loop rings at the amino and carboxyl termini of DRA. Primers were designed to construct the recombinant expression vector pET-21a-SLA-DRA, and the target protein was expressed prokaryotically to obtain inclusion bodies. Using the renaturation conditions in the laboratory (i.e., the renaturation buffer formulation contains 100 mM Tris pH 8.0, 400 mM L-arginine, 2 mM EDTA, 1.5306 g / L reduced glutathione, and 0.3062 g / L oxidized glutathione), renaturation, concentration, molecular sieve purification, and SDS-PAGE were performed to identify the renaturation effect as Figure 1 shown. The specific amino acid sequence of the SLA-DRA antigen is as shown in SEQ ID NO.1:

[0039] SEQ ID NO.1: TNVVTVSDKVGNCDKSVVNVTWRNGSVTRGVSTVRDHRKHYMSTDVYDCVHWGDKKHWAR

[0040] Result analysis: The molecular sieve results showed that the target peak appeared at 95 - 105 mL, with a peak value of about 100 mAU. The protein size was about 11.9 kDa as identified by gel electrophoresis, which was consistent with the expected band size, and the purity was higher than 95%. The concentration measured by the BCA protein assay was 0.877 mg / mL.

[0041] Example 2 Preparation of Monoclonal Antibody Against Porcine SLA-DRA

[0042] 1. Animal Immunization

[0043] The recombinant SLA-DRA protein obtained by the above renaturation was fully mixed with complete Freund's adjuvant at a volume ratio of 1:1 to make an emulsifier (the emulsification standard is: the emulsified immunogen dropped into water at 37°C does not disperse, which is qualified). Female New Zealand white rabbits weighing about 2 kg were immunized by multiple subcutaneous injections in the abdomen and back, with an immunization dose of 400 μg / rabbit. The second immunization was carried out 14 days after the first immunization, using incomplete Freund's adjuvant at a volume ratio of 1:1 to make an emulsifier, and the immunization dose was halved. The third immunization was carried out 7 days after the second immunization. After the third immunization, blood was collected from the marginal ear vein of the rabbit, serially diluted, and the serum titer was detected by the indirect ELISA method. Rabbits with a positive determination result (OD450 nm value greater than 2.0) were boosted by subcutaneous multiple injections with 200 μg of immunogen once, and the spleen was collected four days later.

[0044] 2. Isolation and Culture of B Lymphocytes in the Spleen

[0045] Take the spleen collected above and perform aseptic operation. The specific steps are as follows:

[0046] 1) Inside the biosafety cabinet, cut the spleen tissue into pieces, remove as much excess fat and connective tissue as possible, thoroughly grind it and transfer it into a cell sieve. Centrifuge at 450×g for 5 min at room temperature, carefully aspirate and discard the supernatant, retain the cell pellet, add red blood cell lysis buffer, gently pipette the cells, centrifuge at 450×g for 5 min at room temperature, aspirate and discard the supernatant, add 20 mL of B cell medium, resuspend the cells, filter the cells, and perform cell counting;

[0047] 2) For the sorting and culture of lymphocytes, refer to the patents "Method for Efficiently Isolating Single Antigen-Specific B Lymphocytes from Spleen Cells (Publication No.: CN110016462A)" and "An In Vitro Culture System for B Lymphocytes and Its Application (Publication No. CN111518765A)".

[0048] 3. Cloning of the gene encoding the rabbit monoclonal antibody

[0049] Use antigen-coated ELISA to detect positive B lymphocyte clones that can recognize and bind to porcine SLA-DRA protein. Collect the above cells, extract cell RNA, reverse transcribe to obtain cDNA, and after sequencing, construct the selected rabbit monoclonal antibody heavy chain variable region (VH) and light chain variable region (VL) into an expression vector.

[0050] 4. Production and purification of rabbit monoclonal antibodies

[0051] 1) To obtain a positive rabbit monoclonal antibody that recognizes SLA-DRA protein, insert the light chain and heavy chain genes of the selected rabbit monoclonal antibody (23-9) into the pcDNA3.1(+) eukaryotic expression vector by seamless cloning technology to construct the recombinant vector pcDNA3.1-IgGV H 、pcDNA3.1-IgGV L 。

[0052] 2) Seed 293T cells at 1.0×10 6 cells / well in a 6-well plate, place it in a 37℃ 5% CO2 incubator and culture until the cell confluence is about 85%, then change to serum-free DMEM medium. Perform transfection operations. Mix the plasmid and PEI transfection reagent at a ratio of 1:2 per well (heavy chain of antibody: light chain = 1:2). Collect the supernatant 120 h after transfection, and observe the cell status during this period. Dilute the supernatants of the plasmid groups transfected with pcDNA3.1-IgGVH, pcDNA3.1-IgGVL, and pcDNA3.1-eGFP respectively, coat ELISA plates, and perform antibody titer detection and purification by Protein A column.

[0053] Example 3 Immunological Application Identification of Rabbit Monoclonal Antibody 23-9 Against Porcine SLA-DRA Protein

[0054] 1. Indirect ELISA analysis

[0055] To detect the titer and recognition specificity of the anti - porcine SLA - DRA protein rabbit monoclonal antibody (23 - 9) prepared by the present invention, samples of the SLA - DRA protein and SLA - DR complex (the complex names in this experiment are P30 - 48 - DR01 and P30 - 142 - DR01 complexes) purified by prokaryotic expression were selected for detection. The specific operation steps are as follows:

[0056] 1) Coating: The SLA - DRA protein, P30 - 48 - DR01 and P30 - 142 - DR01 complexes were diluted to 1 μg / mL with coating buffer, 100 μL per well. The control was set as the camel - sourced prokaryotic expression protein, and coated overnight at 4°C. The coating buffer formula: 0.795 g of Na2CO3, 1.464 g of NaHCO3, made up to 500 mL with water, and the pH of the solution was adjusted to 9.6 with NaOH.

[0057] 2) Blocking: Freshly prepared blocking solution (5% skim milk dissolved in PBST) was added, 200 μL per well, incubated at 37°C for 2 h. The liquid in the wells was flicked dry, 200 μL of PBST was added per well, and washed 3 times, 5 min each time;

[0058] 3) Primary antibody incubation: The 23 - 9 antibody was diluted to 1 μg / mL, 0.33 μg / mL, 0.11 μg / mL, 0.04 μg / mL, 0.01 μg / mL, 0.002 μg / mL with blocking solution, 100 μL per well, incubated at 37°C for 1 h. At the same time, a control was set and washed;

[0059] 4) Secondary antibody incubation: HRP - labeled goat anti - rabbit antibody was added, diluted 1:2000 with blocking solution, 100 μL per well, incubated at 37°C for 1 h, and washed;

[0060] 5) TMB color development: TMB color - developing solution was added, 90 μL per well, incubated at 37°C in the dark for 15 min;

[0061] 6) Termination of color development: 2M H2SO4 solution was added, 50 μL per well, and the absorbance was read at OD450 nm with an enzyme - linked immunosorbent assay reader within 5 min. The results are as Figure 2 shown.

[0062] Result analysis: The results showed that the prepared anti - porcine SLA - DRA protein rabbit monoclonal antibody could specifically recognize SLA - DRA and SLA - DR complex molecules, and the antibody dilution reached 0.002 μg / mL.

[0063] 2. Immunoblot (Western Blot) analysis

[0064] To detect the recognition specificity of the anti-pig SLA-DRA protein rabbit monoclonal antibody (23-9) of the present invention, samples of prokaryotically expressed SLA-DRA protein and different peptide-SLA-DR complexes (the complex names in this experiment are P30-48-DR01 and P30-142-DR01 complexes) were selected for detection. The specific operation steps are as follows:

[0065] 1) Transfer: The above-mentioned purified protein was subjected to 4-20% polyacrylamide (SDS-PAGE) gel electrophoresis. The program was set to 120V for 55 minutes. After the electrophoresis was completed, the protein in the gel was transferred to a PVDF membrane activated with methanol. The program was set to: 250mAU for 1 hour of transfer.

[0066] 2) Blocking: The PVDF membrane was placed in freshly prepared blocking solution (5% skim milk) and incubated at 37°C for 2 hours. The membrane was washed with TBST solution, washed at low speed 3 times, 5 minutes each time.

[0067] 3) Primary antibody incubation: The 23-9 antibody was diluted to 1 μg / mL with the blocking solution, mixed well, added, incubated on a low-speed shaker at room temperature for 30 minutes, then transferred to 4°C for overnight incubation, and washed 5 times with TBST solution, 5 minutes each time;

[0068] 4) Secondary antibody incubation: The HRP-Gout-rabbit IgG antibody was diluted to 1:5000 with the blocking solution, mixed well, incubated on a low-speed shaker at room temperature for 1 hour, and then washed 5 times with TBST solution, 5 minutes each time;

[0069] 5) Development: The PVDF membrane was placed face up in an exposure instrument, an appropriate amount of ECL hypersensitive color developing solution was added, and development treatment was carried out. The results are as Figure 3 shown.

[0070] Result analysis: The results show that the prepared anti-pig SLA-DRA protein rabbit monoclonal antibody can specifically recognize the SLA-DRA protein and the SLA-DR complex molecule, and cannot recognize the control group of camel-derived prokaryotically expressed protein.

[0071] 3. Indirect immunofluorescence (IFA) analysis of PAM cells

[0072] To further detect the localization of the anti-pig SLA-DRA protein rabbit monoclonal antibody (23-9) of the present invention at the cellular level, porcine alveolar macrophages (PAM cells) expressing porcine SLA-DRA protein were selected for an indirect immunofluorescence test. The specific operation is as follows:

[0073] 1) Cell fixation: Resuscitate a vial of primary porcine alveolar macrophages and culture them overnight in a 37°C, 5% CO₂ incubator until the cell confluence reaches about 75%. Aspirate the culture medium, add 250 μL of 4% paraformaldehyde fixative, and fix at 4°C for 30 min. Carefully aspirate the liquid and wash three times with PBST solution, 5 min each time.

[0074] 2) Permeabilization: Add 100 μL / well of 0.2% Triton-100 (diluted with PBS) and permeabilize at room temperature for 20 min, then wash.

[0075] 3) Blocking: Add 200 μL / well of blocking solution (5% FBS + 5% skim milk + PBST) and incubate at 37°C for 2 h.

[0076] 4) Primary antibody incubation: Add 100 μL / well of rabbit monoclonal antibody 23-9 diluted 1:100. Set the control as homologous rabbit anti-IgG and wash three times with PBST, 5 min each time.

[0077] 5) Secondary antibody incubation: Add 100 μL / well of FITC-labeled goat anti-rabbit IgG diluted 1:250 and incubate at 37°C in the dark for 1 h. Wash three times with PBST, 5 min each time.

[0078] 6) DAPI nuclear staining: Add 100 μL / well of anti-fluorescence quencher containing DAPI and incubate at room temperature in the dark for 10 min. Wash three times with PBST, 5 min each time. Add a small amount of PBS for the last time to prevent drying.

[0079] 7) Staining observation: Observe the cell staining under a fluorescence microscope and perform result interpretation. The IFA detection results are as Figure 4 shown.

[0080] Result analysis: The prepared rabbit monoclonal antibody 23-9 is mainly localized in the cytoplasm and cell membrane of PAM cells, which is consistent with the fact that SLA-DRA is mainly localized in the membrane for cytoplasmic processing and mature proteins.

[0081] 4. Surface plasmon resonance (SPR) analysis

[0082] Analyze the binding kinetics and affinity of the monoclonal antibody with SLA-DRA protein and SLA-DR complex protein by SPR (Biacore 8k). The specific operation is as follows:

[0083] 1) Antibody pre-enrichment: Set the final concentration of 50 mM NaOH and a protein-free Buffer to wash the surface of the blank CM5 chip. Couple the purified rabbit monoclonal antibody (23-9, concentration 2.68 mg / mL) as a conjugate to the CM5 chip. Dilute the antibody with 10 mM sodium acetate at pH 4.0, pH 4.5, pH 5.0, and pH 5.5 to a final concentration of 26.8 μg / 100 μL, and centrifuge at 13,000 rpm for 5 min. Pipette 75 μL into a 96-well microplate for pre-enrichment to test the electrostatic adsorption ability (principle: select the pH with the highest response value per unit time) to determine the optimal coupling pH condition;

[0084] 2) Calculate the amount of antibody: Select the above optimal coupling pH condition, use amine coupling (Amine). According to the antibody molecular weight of 140 kDa and the analytes including SLA-DRA (11.9 kDa), P30-48-DR0101 (46.02 kDa), P30-142-DR0101 (46.09 kDa), P30-142-DR0201 (46.49 kDa), calculate the amount of coupled antibody (RL):

[0085]

[0086] Where, W23-9 is the molecular weight of antibody 23-9, Rmax is the default maximum response of 100 RU, and Wanalyte represents the molecular weight of the analyte; theoretically, one antibody can bind two antigen molecules simultaneously, and here Sm is set to 2;

[0087] 3) Antibody immobilization: Activate the chip (EDC-NHS 1:1 mixture), and covalently immobilize the purified antibody to the CM5 sensor chip through the amino group in 10 mM sodium acetate (pH 5.0). Add the blocking solution ethanolamine, and the program settings are: binding for 120 s, dissociation for 60 s, and flow rate of 30 μL / min;

[0088] 4) Chip surface binding test: Dilute SLA-DRA protein (46.875 nM to 7.540 μM), P30-48-DR01 complex (3.468 μM to 5.550 μM), p30-142-DR01 complex (2.031 μM to 3.250 μM), p30-142-DR0201 complex (3.243 μM to 5.190 μM) into different gradients for binding test, and the program settings are: binding for 120 s, dissociation for 600 s, and flow rate of 30 μL / min. Fit the sensorgram to the 1:1 binding model with Biacore evaluation software, and the results are as Figure 4 shown.

[0089] Result analysis: The rabbit monoclonal antibody (23-9) against porcine SLA-DRA protein binds to SLA-DRA protein with high affinity and binds to different SLA-DR complex molecules with medium affinity. The SPR affinity detection results of the anti-SLA-DRA monoclonal antibody are as Figure 5 shown.

[0090] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A rabbit monoclonal antibody against porcine SLA-DRA protein, characterized in that, The amino acid sequences of complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH on the heavy chain of the rabbit monoclonal antibody against porcine SLA-DRA protein are shown in SEQ ID NO.4-6 respectively, or have at least 95% sequence identity with the sequences shown in SEQ ID NO.4-6; the amino acid sequences of complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL on the light chain of the rabbit monoclonal antibody against porcine SLA-DRA protein are shown in SEQ ID NO.7-9 respectively, or have at least 95% sequence identity with the sequences shown in SEQ ID NO.7-9.

2. The rabbit monoclonal antibody against porcine SLA-DRA protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region VH of the monoclonal antibody is as shown in SEQ ID NO.10, and the amino acid sequence of the light chain variable region VL of the monoclonal antibody is as shown in SEQ ID NO.

11.

3. The high-affinity rabbit monoclonal antibody against porcine SLA-DRA protein according to claim 2, wherein, The amino acid sequence of the heavy chain of the monoclonal antibody is as shown in SEQ ID NO.2, and the amino acid sequence of the light chain of the monoclonal antibody is as shown in SEQ ID NO.

3.

4. A biological material, characterized in that, The biological material is selected from any one of the following a-c: a. A nucleotide molecule, which includes a nucleotide sequence encoding the monoclonal antibody against porcine SLA-DRA protein according to any one of claims 1-3; b. A vector that carries the nucleotide molecule in a; c. A modified cell or recombinant strain that is used to express the rabbit monoclonal antibody against porcine SLA-DRA protein according to any one of claims 1-3.

5. Use of the rabbit monoclonal antibody against porcine SLA-DRA protein according to any one of claims 1-3, characterized in that, The application includes being used for preparing a product for detecting the expression of SLA-DRA antibody that is not for disease diagnosis and treatment purposes.

6. A marker for porcine SLA-DRA protein, characterized in that, It includes the rabbit monoclonal antibody against porcine SLA-DRA protein according to any one of claims 1-3 and a label.

7. The marker of the porcine SLA-DRA protein according to claim 7, characterized in that, The label includes an enzyme, fluorescence, molecular label, biotin, and streptavidin.

8. A kit for detecting porcine SLA-DRA protein, characterized in that, The kit includes the rabbit monoclonal antibody against porcine SLA-DRA protein according to any one of claims 1-3 or the label of porcine SLA-DRA protein according to claim 6 or 7.

9. The application according to claim 5, characterized in that, The product is a detection kit, including an immunochromatographic detection kit, an ELISA detection kit, or a detection chip.

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