A monoclonal antibody against swine major histocompatibility complex class I and its application

Abstract

The present invention discloses a hybridoma cell with a microbial preservation number of CCTCC NO: C2024193. The antibody secreted by the hybridoma cell can efficiently recognize the α3 domain of porcine major histocompatibility complex class I antigen in vivo or in vitro, and has prospects for medical applications.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products, and relates to a monoclonal antibody against swine major histocompatibility complex class I and its application. Background Art

[0002] African swine fever (ASF) is an acute, highly virulent, and highly contagious disease of pigs caused by African swine fever virus (ASFV). The highly virulent strain of ASF is highly lethal to pigs, with a lethality rate of up to 100%. ASF was first introduced into China in 2018. The prevalence of ASFV in the fields of China is complex, and highly virulent strains with recombination of genotype I and genotype II have emerged. However, at present, there is no safe and effective vaccine or drug in China that can prevent ASFV infection.

[0003] Pigs, as important agricultural and economic animals, not only play an important role in food production but also play a key role in biomedical research. The immune system of pigs has many similarities with that of humans, making them an ideal model for studying human diseases and developing new therapies. MHC-I (major histocompatibility complex class I) molecules are key components of the immune system, and they play important roles in antigen presentation, immune response, inflammatory response, and tumor formation. MHC-I molecules are produced by human B cells and T cells, and their function is to promote the response of B cells and T cells to antigens. MHC-I molecules play an important role in the signal transduction between T cells and B cells. In infection, inflammatory response, and tumor formation, an increase in the expression level of MHC-I molecules can make antigens more easily recognized and presented by host cells, thereby increasing the immunogenicity of antigens, inducing the body to produce corresponding immune responses, and inducing downstream immune-related signaling pathways, ultimately promoting the occurrence or development of diseases.

[0004] Swine leukocyte antigen I (SLA-I, SLAI) is an important member of MHC-I molecules.

[0005] Inside cells, ASFV proteins are degraded into antigenic peptides, which are then presented to the cell surface by swine major histocompatibility complex class I (SLA I) and recognized by CD8 + T cells, thereby activating the cellular immunity of the body; therefore, screening and identifying ASFV T cell epitopes that bind to SLA I molecules will provide a theoretical basis for the design of ASF vaccines, deepen the research on ASFV proteins, and have important significance for the prevention and control of ASF. Summary of the Invention

[0006] To solve the problems existing in the prior art, in the first aspect of the present invention, a hybridoma cell is provided, and the hybridoma cell is a hybridoma cell with a microorganism deposit number of CCTCC NO: C2024193; or

[0007] A subculture cell of the hybridoma cell with a microorganism deposit number of CCTCC NO: C2024193.

[0008] In the second aspect of the present invention, a biological material is provided, and the biological material is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, and P9;

[0009] P1: Monoclonal antibody

[0010] The monoclonal antibody maintains specific binding activity to porcine class I leukocyte antigen or the α3 domain of porcine class I leukocyte antigen;

[0011] The monoclonal antibody includes a monoclonal antibody heavy chain and a monoclonal antibody light chain;

[0012] The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3;

[0013] The monoclonal antibody light chain includes light chain CDR1, light chain CDR2, and light chain CDR3;

[0014] The protein sequence of the heavy chain CDR1 is as shown in SEQ ID NO.5;

[0015] The protein sequence of the heavy chain CDR2 is as shown in SEQ ID NO.7;

[0016] The protein sequence of the heavy chain CDR3 is as shown in SEQ ID NO.9;

[0017] The protein sequence of the light chain CDR1 is as shown in SEQ ID NO.11;

[0018] The protein sequence of the light chain CDR2 is as shown in SEQ ID NO.13;

[0019] The protein sequence of the light chain CDR3 is as shown in SEQ ID NO.15;

[0020] P2: A combination of a monoclonal antibody heavy chain and a monoclonal antibody light chain

[0021] The combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain maintains specific binding activity to porcine class I leukocyte antigen or the α3 domain of porcine class I leukocyte antigen;

[0022] The monoclonal antibody heavy chain comprises the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 and a functional protein fragment or an inert protein fragment;

[0023] The monoclonal antibody light chain comprises the amino acid sequences of light chain CDR1, light chain CDR2, light chain CDR3 and a functional protein fragment or an inert protein fragment;

[0024] The protein sequence of the heavy chain CDR1 is as shown in SEQ ID NO.5;

[0025] The protein sequence of the heavy chain CDR2 is as shown in SEQ ID NO.7;

[0026] The protein sequence of the heavy chain CDR3 is as shown in SEQ ID NO.9;

[0027] The protein sequence of the light chain CDR1 is as shown in SEQ ID NO.11;

[0028] The protein sequence of the light chain CDR2 is as shown in SEQ ID NO.13;

[0029] The protein sequence of the light chain CDR3 is as shown in SEQ ID NO.15;

[0030] P3: Antibody derivative

[0031] The antibody derivative retains the specific binding activity to swine class I leukocyte antigen or the α3 domain of swine class I leukocyte antigen;

[0032] The protein sequence part of the antibody derivative contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3;

[0033] The protein sequence of the heavy chain CDR1 is as shown in SEQ ID NO.5;

[0034] The protein sequence of the heavy chain CDR2 is as shown in SEQ ID NO.7;

[0035] The protein sequence of the heavy chain CDR3 is as shown in SEQ ID NO.9;

[0036] The protein sequence of the light chain CDR1 is as shown in SEQ ID NO.11;

[0037] The protein sequence of the light chain CDR2 is as shown in SEQ ID NO.13;

[0038] The protein sequence of the light chain CDR3 is as shown in SEQ ID NO.15;

[0039] The forms of the antibody derivatives are selected from: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, humanized antibodies, single-chain antibodies, chimeric monoclonal antibodies, and engineered monoclonal antibodies;

[0040] P4: RNA combination

[0041] The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA;

[0042] The monoclonal antibody heavy chain RNA can be translated to obtain the monoclonal antibody heavy chain described in P1 or P2;

[0043] The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1 or P2;

[0044] P5: Gene combination

[0045] The coding sequence of the gene combination can encode the monoclonal antibody heavy chain described in P1 or P2 and the monoclonal antibody light chain described in P1 or P2;

[0046] P6: Gene expression cassette combination

[0047] The gene expression product in the gene expression cassette combination is the RNA combination described in P4;

[0048] P7: Genetic engineering vector

[0049] The genetic engineering vector contains the gene expression cassette described in P6;

[0050] The monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA are encoded in one or two vectors;

[0051] P8: Cell

[0052] The cell contains the genetic engineering vector described in P7;

[0053] The encoded proteins in the gene expression cassette of the genetic engineering vector are constitutively expressed or artificially induced;

[0054] When the monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or different cells;

[0055] P8: Composition

[0056] The composition contains the monoclonal antibody described in P1, the combination of the heavy chain and light chain of the monoclonal antibody described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetically engineered vector described in P7 or the cell described in P8; and

[0057] P9: Kit

[0058] The kit contains the monoclonal antibody described in P1, the combination of the heavy chain and light chain of the monoclonal antibody described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetically engineered vector described in P7 or the cell described in P8.

[0059] In some embodiments, in P1, the monoclonal antibody is the monoclonal antibody secreted by the hybridoma cells described in the first aspect of the present invention.

[0060] In some embodiments, in P2, the functional protein fragment is a tag peptide and / or signal peptide for protein isolation and purification.

[0061] In some embodiments, the protein sequence of the porcine major histocompatibility complex class I antigen is as shown in SEQ ID NO.1.

[0062] In some embodiments, the protein sequence of the α3 domain of the porcine major histocompatibility complex class I antigen is as shown in SEQ ID NO.3.

[0063] The third aspect of the present invention provides the use of the hybridoma cells described in the first aspect of the present invention or the biological material described in the second aspect of the present invention in the preparation of a preparation for the following U1, U2 or U3;

[0064] U1: Recognize the porcine major histocompatibility complex class I antigen or the α3 domain of the porcine major histocompatibility complex class I antigen in vivo or in vitro;

[0065] U2: Block the porcine major histocompatibility complex class I antigen or the α3 domain of the porcine major histocompatibility complex class I antigen in vivo; and U3: Isolate or identify the T cell epitope of the virus-encoded protein.

[0066] In some embodiments, the protein sequence of the porcine major histocompatibility complex class I antigen is as shown in SEQ ID NO.1.

[0067] In some embodiments, the protein sequence of the α3 domain of the porcine major histocompatibility complex class I antigen is as shown in SEQ ID NO.3.

[0068] In some embodiments, the virus is African swine fever virus.

[0069] The progress of the present invention lies in that: there is currently no report on antibodies for detecting and recruiting SLAI molecules. The antibodies prepared in the present invention fill the above-mentioned gap, and can not only be used for the detection of SLAI molecules, but also for recruiting SLAI molecules, analyzing the sequences of polypeptides bound to SLAI molecules, and analyzing the roles of these polypeptides in porcine immune regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Shows the results of WB test for the construction of HEK-293T-SLAⅠ stable transfected cell line.

[0071] Figure 2 Shows the reactivity of SLAⅠ monoclonal antibody MAb-3F9 to PAM detected by IFA, where the scale bar is 200μm.

[0072] Figure 3 Shows the reactivity of SLAⅠ monoclonal antibody MAb-3F9 to PAM detected by flow cytometry.

[0073] Figure 4 Shows the differential reactivity of SLAⅠ monoclonal antibody MAb-3F9 to HEK-293T and HEK-293T SLAⅠ detected by IFA, where the scale bar is 200μm.

[0074] Figure 5 Shows the differential reactivity of MAb-3F9 to HEK-293T and HEK-293T SLAⅠ stable transfected cell lines detected by flow cytometry.

[0075] Figure 6 Shows the infection efficiency of ASFV-GFP, where the scale bar is 100μm.

[0076] Figure 7 Shows the effect of WB verification of MAb-3F9 in vitro immunoadsorption chromatography of SLAⅠ. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0077] To make the objectives, technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0078] The materials and instruments not described in the present invention are conventional materials and instruments in the art. The operation details not described in the present invention are conventional operations in the art. The software used in the present invention is operated by conventional methods with reference to the usage instructions provided by the software provider. The kits used in the present invention are operated by conventional methods with reference to the kit instruction manuals.

[0079] The nucleic acid sequences shown in the present invention are written from left to right in the 5' to 3' direction, and the proteins are written from left to right in the N-terminal to C-terminal direction.

[0080] Example 1: Expression and purification of recombinant proteins MBP-SLA1 and MBP-SLA 3α3

[0081] (1) Expression and purification of recombinant protein MBP-SLA1

[0082] The gene coding sequence of porcine major histocompatibility complex class I (abbreviated as SLA I protein) used in this example can be found in GenBank accession number AF014005.1, where the name of porcine major histocompatibility complex class I is Sus scrofa MHC class I antigen; the GenBank accession number of the protein is AAB69340.1, and the name of porcine major histocompatibility complex class I in it is MHC class I antigen [Susscrofa].

[0083] The amino acid sequence of porcine major histocompatibility complex class I protein is as follows (SEQ ID NO.1):

[0084] MGPGALFLLLSGTLALTGTQAGPHSLSYFYTAVSRPDRGDSRFIAVGYVDDTQFVRFDNYAPNPRMEPRVPWIQQEGQEYWDRETRNVKETAQTYGVGLNTLRGYYNQSEAGSHTLQSMYGCYLGPDGLLLHGYRQDAYDGADYIALNEDLRSWTAADMAAQITKRKWEAADEAERRRSYLQGLCVESLRRYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTL RWDPAQPPVPIVGIIVGLVLVLVAGAMVAGVVIWRKTRSGEKGGSYTQAAGSDSDQGSDVSLTKDPRV

[0085] The amplified fragment containing the SLA I gene sequence (synthesized by Shanghai Sangon Biological Engineering Co., Ltd.) was ligated to the pMAL-c5x vector (purchased from NEB). The ligation product (recombinant plasmid) was transformed into E. coli BL21(DE3) competent cells, and the plasmid was extracted. Sanger sequencing was used to identify the plasmid, and it was found that the recombination was successful. The recombinant plasmid was named pMAL-c5x-SLA 1.

[0086] Induce expression with IPTG, extract the recombinant protein, verify the recombinant protein by WB using MBP antibody. The molecular weight of the electrophoresis result is consistent with the expectation. The recombinant protein is purified using an MBPtrap HP pre-packed column through an AKTA protein purification system according to the instructions. The expressed protein is named MBP-SLA1.

[0087] (2) Expression and purification of recombinant protein MBP-SLA 3α3

[0088] The α3 domain is a conserved region of SLAⅠ. Almost all SLAⅠ alleles have the same α3 domain. The length of this domain usually contains 101 amino acid residues and is relatively small. Therefore, three α3 regions (the 101-amino acid segment of the α3 domain selected in the present invention) are first linked together with a flexible linker and fused with the MBP tag with a strong solubilizing effect to increase the soluble expression of the protein.

[0089] Synthesize the following DNA fragment (named 3α3 fragment). Among them, the three underlined sequences are each an α3, the box represents the flexible linker, and the lowercase letters in regular font on both sides are part of the vector sequence (SEQ ID NO.2).

[0090]

[0091]

[0092] The protein sequence corresponding to the α3 fragment is as follows (SEQ ID NO.3):

[0093] AEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPAQPPVP

[0094] Ligate the synthesized 3α3 fragment (commissioned to Shanghai Sangon Biological Engineering for synthesis) with the pMAL-c5x vector (purchased from NEB). Transform the ligation product (recombinant plasmid) into E.Coli BL21(DE3) competent cells, extract the plasmid, and identify the recombinant success by Sanger sequencing of the plasmid. Name the recombinant plasmid pMAL-c5x-SLA3α3.

[0095] Induce expression with IPTG, extract the recombinant protein, verify the recombinant protein by WB using MBP antibody. The molecular weight of the electrophoresis result is consistent with the expectation. The recombinant protein is purified using an MBPtrap HP pre-packed column through an AKTA protein purification system according to the instructions. The expressed protein is named MBP-SLA 3α3.

[0096] Example 2: Preparation of Monoclonal Antibody

[0097] I. Immunization of Mice with Recombinant Protein MBP-SLA 3α3

[0098] Use the purified MBP-SLA 3α3 protein as an immunogen and prepare a protein solution at 2 mg / ml (the solvent is PBS) for use. Immunize 3 female BALB / c mice aged 6-8 weeks (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) for the first time. The second immunization is carried out 21 days after the first immunization, and the third immunization is carried out 21 days after the second immunization. At the first immunization, mix the MBP-SLA 3α3 protein solution with an equal volume of Freund's complete adjuvant, fully emulsify it, and inject it subcutaneously at multiple points. Immunize the mice at a dose of 100 μg MBP-SLA 3α3 protein per mouse. At the second and third immunizations, emulsify and mix the MBP-SLA 3α3 protein solution with an equal volume of Freund's incomplete adjuvant, and inject it subcutaneously at multiple points. Immunize the mice at a dose of 100 μg MBP-SLA 3α3 protein per mouse.

[0099] One week after the third immunization, collect blood from the medial canthus vein of the orbit. Place the collected blood at 37 °C for 30 min, then overnight at 4 °C, and centrifuge at 1000 rpm for 5 min to separate the upper serum. Dilute the serum starting from a dilution factor of 1:200 with a dilution ratio of 2, and dilute it with PBS for a total of 8 gradients. Detect the antibody titer in the serum by ELISA (the method is described below). If at a dilution factor of 2 7 the OD 450 absorbance at nm is still greater than 1, and at the same time, the negative control (serum from pre-immunized mice) is around 0.1, it is considered that the immunization is successful. Select the successfully immunized mice for booster immunization, and inject the adjuvant-free protein MBP-SLA3α3 subcutaneously, with an immunization dose of 100 μL of 1 mg / ml protein solution (the solvent is PBS) per mouse.

[0100] Use the purified recombinant protein MBP-SLA 1 as the coating antigen, determine its optimal coating concentration by the checkerboard method, and establish an ELISA method for screening monoclonal antibodies as follows:

[0101] (1) Add the purified recombinant protein MBP-SLA 1 as the coating antigen to the ELISA microtiter plate, dilute it with ELISA coating buffer (carbonate buffer, pH 9.6), start from 1.6 μg / well as the total antigen amount, 50 μl / well, with a dilution factor of 2, and dilute it for a total of 12 gradients for coating, and incubate overnight at 4 °C.

[0102] (2) Discard the coating buffer, add PBST (PBS containing 0.05 v / v% Tween 20), shake and wash the microtiter plate on the plate washer for 3 min each time, and wash 5 times.

[0103] (3) Add 200 μL of 5 w / v% skim milk (solvent: PBST) to each well for blocking, 200 μL / well, incubate at 37 °C for 2 h.

[0104] (4) Discard the blocking solution, wash the ELISA plate, the steps are the same as (2).

[0105] (5) Start diluting the immune mouse serum at a dilution ratio of 1:200, with a dilution factor of 2, dilute it with 1X PBS, a total of 8 gradients are diluted, add it to the ELISA plate, 50 μL per well.

[0106] (6) Discard the liquid in the plate, wash the ELISA plate, the steps are the same as (2).

[0107] (7) Add the secondary antibody of goat anti-mouse IgG (H+L) labeled with HRP at a dilution ratio of 1:10000 (GenScript Biotech Corporation), the diluent is PBS, 50 μL / well, incubate at 37 °C for 45 min.

[0108] (8) Discard the liquid in the plate, wash the ELISA plate, the same as step (2).

[0109] (8) Add the TMB chromogenic solution at 50 μL / well for color development, incubate in the dark at room temperature for 10 min.

[0110] (9) Add the ELISA stop solution (2 M H2SO4) at 50 μL / well. Read the absorbance at 450 nm in the microplate reader. Select the antigen coating concentration with an absorbance value of about 1.

[0111] Results: At the aforementioned antigen coating concentration, the antibody titers in the sera of 3 immune mice can all reach 1:12800, confirming that the MBP-SLA3α3 has good immunogenicity and the immunization of mice is successful.

[0112] II. Cell fusion and screening

[0113] On the 2nd day after booster immunization, take an unimmunized blank control mouse and use its peritoneal macrophages as feeder cells for hybridoma cells.

[0114] On the 3rd day after booster immunization, take the spleen of the booster immunized mouse and isolate its lymphocytes.

[0115] Cell fusion and screening:

[0116] (1) For one-time hybridoma cell fusion, prepare 5×10 9 SP2 / 0 myeloma cells with a low passage number in advance. Gently tap the bottom of the cell culture flask to make them detach. At the same time, incubate PEG (polyethylene glycol) at 37 °C.

[0117] (2) Centrifuge the SP2 / 0 cells and spleen cells (the spleen cells of three boost-immunized mice are operated independently) horizontally at 1500 rpm for 5 min.

[0118] (3) Discard the supernatant respectively, resuspend the cells with 14 mL of RPMI-1640 medium respectively, and centrifuge according to the steps in (2).

[0119] (4) Discard the supernatant respectively, take 20 mL of RPMI-1640 medium to resuspend the cells, add the SP2 / 0 cells to the spleen cells, and the dosage ratio of the two kinds of cells is 1:10, and centrifuge again according to the steps in (2).

[0120] (5) Discard the supernatant, and scrape the centrifuged cells back and forth on the EP tube rack to make the cells into a homogenate.

[0121] (6) Add the fusogen PEG to the cells evenly and adherently within 1 min, with a dosage of 1 mL. Then incubate statically in warm water at 37 °C for 95 s.

[0122] (7) Add 1 mL of RPMI-1640 medium within 1 min.

[0123] (8) Then add 2 mL of RPMI-1640 medium within 1 min.

[0124] (9) Then add 3 mL of RPMI-1640 medium within 1 min.

[0125] (10) Then add 14 mL of RPMI-1640 medium.

[0126] (11) Centrifuge the fused cells at 1500 rpm for 5 min.

[0127] (12) Discard the supernatant, resuspend the cells with 40 mL of 20% FBS-1640-HAT medium and mix well, and spread them into the prepared feeder layer cells.

[0128] (13) Observe whether the cell fusion is successful. If there are transparent round cells aggregated into clusters, it proves that the cells have fused. Five days after fusion, discard half of the original medium and add fresh 20% FBS-1640-HAT medium.

[0129] (14) Seven days after fusion, discard all the medium and replace it with 20% FBS-1640-HT medium.

[0130] After 10 days of fusion, the cell supernatant was collected for ELISA detection (using the optimized coating concentration and other conditions determined by the aforementioned checkerboard method). During screening, the enzyme-linked immunosorbent assay (ELISA) plate was coated with both the recombinant protein MBP-SLA1 and the MBP-tagged protein. Hybridoma cells that were positive for MBP-SLA1 and negative for MBP were selected and subcloned by the limited dilution method. The cells were counted, and 100 cells were inoculated into 96-well plates at a density of approximately 1 cell / well. The cells were cultured at 37 °C and 5% CO2 for 9 - 10 days, and the supernatant was collected for ELISA detection. The subcloning step was repeated three times until the positive rate reached 100%. The positive hybridoma cell lines obtained by screening were expanded and cryopreserved.

[0131] Three hybridoma cell lines that could stably secrete SLA I antibodies were obtained. One of them was named 3F9, and the monoclonal antibody it secreted was named MAb-3F9 for subsequent research.

[0132] III. Identification of Monoclonal Antibody Subtypes

[0133] MAb-3F9 was typed using a mouse immunoglobulin typing kit (supplier: SouthernBiotech, catalog number 5300-05), and it was found that this monoclonal antibody was of the κ light chain and IgG2a type.

[0134] IV. Ascites Preparation and Antibody Purification

[0135] (1) Fourteen days before injecting the hybridoma cells, 1 ml of liquid paraffin was intraperitoneally injected into each 6 - 8-week-old female BALB / c mouse.

[0136] (2) The positive hybridoma cells (3F9 strain) were resuspended in DMEM medium at a density of 1x10 6 cells / ml. Each mouse was intraperitoneally injected with 1x10 6 positive hybridoma cells.

[0137] (3) On the 7th day after injecting the positive hybridoma cells, mouse ascites was collected. The ascites was centrifuged at 5000 rpm and 4 °C for 5 min, and the supernatant was aspirated to remove the white fat layer.

[0138] (4) The ascites was diluted with PBS at a ten-fold volume of the ascites and filtered through a 0.22 μm syringe filter.

[0139] (5) According to the instructions of the HiTrap Protein G HP pre-packed column (purchased from Cytiva), IgG was purified through an AKTA protein purification system.

[0140] (6) The purified antibody was added to the antibody neutralization solution (1M Tris, pH 9.0) at a volume ratio of 9:1 (ten-fold dilution), and the antibody concentration was measured by a micro-spectrophotometer and stored at -80°C.

[0141] Example 3: Reactivity experiment of monoclonal antibody

[0142] I. Construction of HEK-293T-SLAⅠ stable cell line

[0143] (1) The plasmids (pMD2.G plasmid, psPAX2 plasmid, pLenti-CMV-BSD-SLA Ⅰ plasmid) were extracted according to the instructions of the endotoxin-free plasmid large-scale extraction kit (purchased from Qiagen). The plasmid concentration was diluted to 1 μg / μL with ddH2O and stored.

[0144] (2) HEK-293T cells were seeded into 6-well plates at a density of 5x10 5 cells / well. When the cell density was greater than 80%, transfection was performed.

[0145] (3) 200 μL of Opti-MEM serum-free medium was added to an EP tube; the pMD2.G plasmid (purchased from Addgene), psPAX2 plasmid (purchased from Addgene), and pLenti-CMV-BSD-SLA Ⅰ plasmid (the SLA Ⅰ gene coding sequence shown in GenBank AAB69340.1 was transferred into the coding region of the pLenti-CMV-BSD plasmid to obtain this pLenti-CMV-BSD-SLAⅠ) were mixed at a ratio of 1:2:3 (molar ratio), with a total plasmid usage of 2 μg and a volume of 2 μL, and added to the Opti-MEM serum-free medium. Then 6 μL of Transit-Lenti transfection reagent (purchased from Mirus) was added, mixed well, and left at room temperature for 10 min.

[0146] (4) A total of 208 μL / well was added dropwise to the 6-well plates. After 48 h, the supernatant was collected and filtered through a 0.22 μm filter.

[0147] (5) HEK-293T cells were seeded into 6-well plates at a density of 5x10 5 cells / well and grown overnight; when the cell density was greater than 80%, the medium was discarded, 600 μL of virus solution (i.e., the aforementioned artificial recombinant lentivirus) was added, and the volume was made up to 2 mL with DMEM containing 10% fetal bovine serum.

[0148] (5) After 24 h, HEK-293T cells were passaged at a ratio of 1:5.

[0149] (6) After subculture for 24 h, replace with fresh medium and add BSD to the complete medium at a working concentration of 10 μg / mL.

[0150] (7) After culturing for 3 days, subculture the surviving cells, subculture continuously for 2 - 3 times, and continue to culture using the complete medium supplemented with BSD (10 μg / mL).

[0151] Name the aforementioned transgenic cells as HEK - 293T - SLA Ⅰ cells.

[0152] Run WB detection on HEK - 293T - SLA Ⅰ lysate, HEK - 293T cell lysate, PAM cell lysate, and K562 cell lysate in parallel on one gel, and the results are as Figure 1 shown. Figure 1 In it, M: protein standard mass, lanes 1 - 3: HEK - 293T - SLA Ⅰ lysate; lanes 4 - 6: HEK - 293T cell lysate; lane 7: PAM cell lysate; lane 8: K562 cell lysate. It can be seen that a specific band of 42 kDa (corresponding to SLAI protein) appears in HEK - 293T - SLA Ⅰ lysate and PAM cell lysate, while this band is not present in other lysates.

[0153] Thus, HEK - 293T cells transfected with the SLA Ⅰ gene are obtained.

[0154] II. Testing the reactivity of monoclonal antibodies by indirect immunofluorescence (IFA) method

[0155] (1) 12 h before inoculating cells, add 100 μL of polylysine with a working concentration of 0.1 mg / mL (the solvent is PBS) to each well of a 96 - well plate, incubate at 37 °C for 30 min, then aspirate the polylysine and recover it. Wash 1 - 2 times with PBS. Store in the dark at 4 °C. Inoculate PAM cells at a density of 3x10 5 cells / well, and culture at 37 °C and 5% CO2 for 12 h to allow the cells to adhere. Inoculate HEK - 293T cells and HEK - 293T - SLA Ⅰ cells at a density of 1x10 5 cells / well respectively, and culture at 37 °C and 5% CO2 until the cell density reaches over 90%.

[0156] (2) 4 (w / w)% paraformaldehyde fixative (the solvent is PBS), act at room temperature for 15 min, then discard it, and wash the plate three times with PBS.

[0157] (3) 5 w / v% skim milk (solvent: PBS) was used to block for 30 min at room temperature. Then, the plate was washed three times with PBS, 3 min each time.

[0158] (4) The supernatant of hybridoma cells (3F9) (50 μL per well) and the cells were incubated for 1 h at room temperature, and then washed with PBS.

[0159] (5) The secondary antibody, FITC-labeled goat anti-mouse IgG (H+L) (GenScript Biotech Corporation), was diluted at a ratio of 1:100 (diluent: PBS) and incubated in the dark for 45 min at room temperature. Then, the plate was washed three times with PBS, 3 min each time.

[0160] (6) For PAM cells, the Dil cell membrane staining dye was diluted with 1X PBS at a dilution factor of 1:200, and the diluted concentration was 20 μg / mL. 50 μL per well was added to the wells, and incubated in the dark for 15 min. Then, the plate was washed three times with PBS, 3 min each time.

[0161] (7) 1X PBS was added to cover the bottom of the plate, and observations and photographs were taken under an inverted fluorescence microscope.

[0162] The reactivity of MAb-3F9 monoclonal antibody against non-denatured SLA Ⅰ molecule was examined in IFA. The results were as Figure 2 shown. Under non-denaturing conditions, MAb-3F9 showed good binding ability to SLA Ⅰ expressed in PAM cells, showing specific fluorescence. The cell membrane was stained with the membrane dye reagent Dil, and co-localization was performed with the position bound by MAb-3F9 to determine that the binding position of MAb-3F9 was on the cell membrane ( Figure 2 row 1).

[0163] Using the positive serum of the mouse corresponding to 3F9 hybridoma in Example 2 after inoculation with MBP-SLA 3α3 as a control, the hybridoma supernatant in the above step (4) was diluted and replaced with PBS at a volume ratio of 1:200. The result was the same as the fluorescence pattern of the hybridoma cell (3F9) supernatant ( Figure 2 row 2). Replacing the hybridoma supernatant in the above step (4) with PBS, the result only showed the Dil staining result ( Figure 2 row 3).

[0164] III. Flow cytometry to test the reactivity of monoclonal antibodies

[0165] (1) For PAM cells, first pipette the PAM cells down, wash them 2 - 3 times with pre - cooled PBS, and centrifuge at 5000 rpm, 4°C for 3 min. For HEK - 293T and HEK - 293T - SLAⅠ, first digest them with trypsin, then wash them 2 - 3 times with pre - cooled PBS, and centrifuge at 5000 rpm, 4°C for 3 min.

[0166] (2) Block with 5 w / v% BSA (solvent is PBS) for 10 min at 4°C. Wash 2 - 3 times with PBS. Centrifuge at 5000 rpm, 4°C for 3 min and discard the supernatant.

[0167] (3) Incubate the supernatant of hybridoma cells (3F9) with PAM cells or HEK - 293T cells, HEK - 293T - SLAⅠ cells at 4°C for 30 min. Wash 2 - 3 times with PBS. Centrifuge at 5000 rpm, 4°C for 3 min and discard the supernatant.

[0168] (4) Incubate the FITC - labeled goat anti - mouse IgG (H + L) secondary antibody at a dilution ratio of 1:100 (diluent is PBS) in the dark at 4°C for 15 min. Wash 2 - 3 times with PBS. Centrifuge at 5000 rpm, 4°C for 3 min and discard the supernatant.

[0169] (5) Resuspend the washed cells in 500 μL PBS, transfer them into a flow tube, and analyze the labeled population in a Beckman FC500 flow cytometer.

[0170] Apply MAb - 3F9 to flow cytometry, using the serum of non - immunized mice as a negative control. The results are as Figure 3 shown. The left peak corresponds to the negative control, and the right peak corresponds to the cells incubated with MAb - 3F9. The population of PAM cells bound to MAb - 3F9 shows a higher fluorescence intensity, confirming the binding ability of MAb - 3F9 to PAM cells and indicating that MAb - 3F9 can be successfully applied to flow cytometry experiments.

[0171] Incubate MAb - 3F9 with HEK - 293T - SLAⅠ and HEK - 293T respectively, and perform the aforementioned IFA and flow cytometry identification. The results are as Figure 4 and 5 shown. MAb - 3F9 shows specific fluorescence only with the over - expressed cell line HEK - 293T - SLAⅠ, recognizing SLAⅠ; while it shows no fluorescence with HEK - 293T cells, indicating that MAb - 3F9 cannot cross - recognize the HLA molecules expressed by HEK - 293T cells. Figure 4 The negative control (NC) in Figure 5The negative control (N.C) was mouse negative serum.

[0172] Example 4: SLA I immunoaffinity chromatography

[0173] The treatment of African swine fever virus in this example was carried out in the National Animal Disease Control High - level Biosafety Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0174] (1) PAM cells were cultured in a cell culture dish with a diameter of 10 cm at a density of 3x10 7 cells / dish. The culture medium was 1640 medium containing 10% (v / v) FBS.

[0175] The PAM cells used in this example were from SLA I - 1*07:02 homozygous pigs and were isolated by conventional methods.

[0176] (2) When the PAM cells adhered, the culture medium was aspirated. ASFV with a GFP reporter gene (that is, the green fluorescent protein was transferred into the African swine fever virus genome and could normally express green fluorescent protein, and the strain code was ASFV - GFP) was used to infect the cells at an MOI of 5. During the infection process, the volume of 1640 medium containing 10% (v / v) FBS was about 4 mL. The culture dish was gently shaken every 15 min. After 1 h of infection, the volume was replenished to 10 mL and cultured at 37 °C.

[0177] (3) After 6 h of infection, the cells were lysed. The floating cells were collected, and the adherent PAM cells were gently rinsed twice with PBS; the rinsed PBS and the floating cells were collected and combined into the same centrifuge tube, centrifuged at 1000 rpm for 5 min; resuspended with PBS and washed twice under the same conditions. The cells in the dish were lysed with 2 mL of NP40 (+PMSF), and 400 μL of NP40 (+PMSF) was used to lyse the cells in the centrifuge tube and added to the dish. Incubated at 4 °C for 15 min. The cell lysate was collected, centrifuged at 1000 rpm for 5 min, the supernatant was collected, and 100 μL was taken as the input sample.

[0178] (4) Take 2 1.5 - mL EP tubes, add 20 μL of protein A / G agarose beads (purchased from Santa Cruz) to each tube, wash with PBS 2 - 3 times, centrifuge at 5000 rpm for 3 min each time at 4 °C. Resuspend with 500 μL of PBS for the last time and mix with 50 μL of antibody MAb - 3F9 (1 mg / ml) respectively, and incubate at 4 °C for 1 - 2 h.

[0179] (5) Centrifuge the protein A / G agarose beads conjugated with antibodies at 5000 rpm for 5 min at 4°C. Remove the supernatant and add the lysate to EP tubes at 1.2 - 1.3 mL per tube. Incubate at 4°C for 3 - 4 h.

[0180] (6) Centrifuge for 5 min at 5000 rpm at 4°C. Take 100 μL of the supernatant as the sample of the flow - through of immunoaffinity chromatography.

[0181] (7) Wash the protein A / G agarose beads obtained after centrifugation with PBS, centrifuge at 5000 rpm for 5 min at 4°C. Wash three times with PBS. Centrifuge at 5000 rpm for 5 min at 4°C. Take 100 μL of the washing solution as the PBS washing solution sample.

[0182] (8) Add 80 μL of 10% acetic acid aqueous solution prepared with ddH2O to each tube, incubate at 4°C for 1 - 2 h to elute the sample. Collect the supernatant after centrifugation for liquid chromatography separation and mass spectrometry identification.

[0183] In vitro, PAM cells were infected with ASFV - GFP. By observing under an inverted fluorescence microscope, the infection efficiency of ASFV was determined (as Figure 6 ), which indicated that the ASFV carrying the GFP reporter gene used in this example could infect PAM cells normally; then, the SLA I - ASFV antigen - peptide complex was enriched in vitro using the monoclonal antibody MAb - 3F9 specific for SLA I, and WB verification was performed with K562 cells as the negative control, as Figure 7 shown. SLA I was detected in the eluted sample, proving that SLA I was successfully immunoprecipitated by MAb - 3F9.

[0184] Figure 7 In

[0185] lane M: protein standard mass; lane 1: PAM cell lysate; lane 2: flow - through during immunoaffinity chromatography; lane 3: PBS washing solution; lane 4: eluate; lane 5: K562 cell lysate.

[0186] Example 5: Antibody Gene Sequencing

[0187] Total RNA of hybridoma 3F9 was obtained by the Trizol method, reverse transcribed into cDNA. Multiple primer pairs were designed according to the conserved sequences of mouse antibody genes, and the cDNA was amplified by PCR. The amplified products were sequenced. By using the NCBI Nucleotide BLAST, IMGT / V Quest program and NCBI IgBLAST tools, the heavy chain coding sequence and light chain coding sequence of the antibody were spliced. The key sequences are as follows:

[0188] Heavy chain CDR1 coding sequence (SEQ ID NO.4):

[0189] TCTGGGTTCTCATTATCCAGATAT

[0190] Heavy chain CDR1 protein sequence (SEQ ID NO.5):

[0191] SGFSLSRY

[0192] Heavy chain CDR2 coding sequence (SEQ ID NO.6):

[0193] CTGGAGTGGCTGGGAATGATATGGGGTGGTGGAAACACAGACTAT AAT

[0194] Heavy chain CDR2 protein sequence (SEQ ID NO.7):

[0195] LEWLGMIWGGGNTDYN

[0196] Heavy chain CDR3 coding sequence (SEQ ID NO.8):

[0197] AGTCCTTATGATGGTTACGTCTGGTTTGCT

[0198] Heavy chain CDR3 protein sequence (SEQ ID NO.9):

[0199] SPYDGYVWFA

[0200] Light chain CDR1 coding sequence (SEQ ID NO.10):

[0201] TCAAGTGTAAGT

[0202] Light chain CDR1 protein sequence (SEQ ID NO.11):

[0203] SSVS

[0204] Light chain CDR2 coding sequence (SEQ ID NO.12):

[0205] CCCAAAGTTTGGATTTATGAAACATCCAAACTGGCT

[0206] Light chain CDR2 protein sequence (SEQ ID NO.13):

[0207] PKVWIYETSKLA

[0208] Light chain CDR3 coding sequence (SEQ ID NO.14):

[0209] TGGAAATATCCTCTGCTC

[0210] Light chain CDR3 protein sequence (SEQ ID NO.15):

[0211] WKYPLL

[0212] The hybridoma cell line 3F9 prepared by the present invention was submitted to a patent procedure-approved depository institution for preservation. The depository unit is the China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microorganism deposit number is CCTCC NO: C2024193; the name of the culture is Hybridoma cell line 3F9; the Chinese classification name is: Hybridoma cell; the English classification name is: Hybridoma Cell; the preservation time is July 26, 2024; the identification survival time is July 31, 2024.

[0213] As is known by common technical knowledge, the present invention can be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the above-disclosed embodiments are illustrative in all aspects and not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are encompassed by the present invention.

Claims

1. A hybridoma cell, wherein the hybridoma cell is a hybridoma cell with a microbial deposit number of CCTCC NO: C2024193.

2. A biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10; P1: Monoclonal antibody The monoclonal antibody maintains specific binding activity to porcine class I leukocyte antigen or the α3 domain of porcine class I leukocyte antigen; the protein sequence of the α3 domain of porcine class I leukocyte antigen is shown in SEQ ID NO.3; The monoclonal antibody comprises a monoclonal antibody heavy chain and a monoclonal antibody light chain; The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The monoclonal antibody light chain includes a light chain CDR1, a light chain CDR2 and a light chain CDR3; The heavy chain CDR1 protein sequence is shown in SEQ ID NO.5; The heavy chain CDR2 protein sequence is shown in SEQ ID NO.7; The heavy chain CDR3 protein sequence is shown in SEQ ID NO.9; The light chain CDR1 protein sequence is shown in SEQ ID NO.11; The light chain CDR2 protein sequence is shown in SEQ ID NO.13; The light chain CDR3 protein sequence is shown in SEQ ID NO.15; P2: Combination of monoclonal antibody heavy chain and monoclonal antibody light chain The monoclonal antibody heavy chain and monoclonal antibody light chain combination maintains specific binding activity to porcine class I leukocyte antigen or the α3 domain of porcine class I leukocyte antigen; the protein sequence of the α3 domain of porcine class I leukocyte antigen is shown in SEQ ID NO.3; The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 and an amino acid sequence of a tag peptide used for separating and purifying the protein; The monoclonal antibody light chain includes a light chain CDR1, a light chain CDR2, a light chain CDR3 and an amino acid sequence of a tag peptide used for separating and purifying the protein; The heavy chain CDR1 protein sequence is shown in SEQ ID NO.5; The heavy chain CDR2 protein sequence is shown in SEQ ID NO.7; The heavy chain CDR3 protein sequence is shown in SEQ ID NO.9; The light chain CDR1 protein sequence is shown in SEQ ID NO.11; The light chain CDR2 protein sequence is shown in SEQ ID NO.13; The light chain CDR3 protein sequence is shown in SEQ ID NO.15; P3: Antibody derivatives The antibody derivative retains specific binding activity to porcine class I leukocyte antigen or the α3 domain of porcine class I leukocyte antigen; the protein sequence of the α3 domain of porcine class I leukocyte antigen is shown in SEQ ID NO.3; The protein sequence portion of the antibody derivative contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3; The heavy chain CDR1 protein sequence is shown in SEQ ID NO.5; The heavy chain CDR2 protein sequence is shown in SEQ ID NO.7; The heavy chain CDR3 protein sequence is shown in SEQ ID NO.9; The light chain CDR1 protein sequence is shown in SEQ ID NO.11; The light chain CDR2 protein sequence is shown in SEQ ID NO.13; The light chain CDR3 protein sequence is shown in SEQ ID NO.15; The antibody derivative is in the form selected from the group consisting of: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, porcine antibodies, single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies; P4: RNA combination The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA; The monoclonal antibody heavy chain RNA can be translated to obtain the monoclonal antibody heavy chain described in P1 or P2; The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1 or P2; P5: Genetic combination The coding sequence of the gene combination can encode the monoclonal antibody heavy chain described in P1 or P2 and the monoclonal antibody light chain described in P1 or P2; P6: Gene expression cassette combination The gene expression product in the gene expression cassette combination is the RNA combination described in P4; P7: Genetic Engineering Vector The genetic engineering vector contains the gene expression cassette described in P6; The monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA are encoded in one or two vectors; P8: Cells The cell contains the genetic engineering vector described in P7; The encoded protein in the gene expression cassette of the genetic engineering vector is expressed constitutively or artificially induced; When the monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell; P9: Composition The composition contains the monoclonal antibody described in P1, the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8; and P10: Test kit The kit contains the monoclonal antibody described in P1, the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8.

3. The biomaterial according to claim 2, characterized in that In P1, the monoclonal antibody is the monoclonal antibody secreted by the hybridoma cell of claim 1.

4. The biomaterial according to claim 2 or 3, characterized in that The protein sequence of porcine class I leukocyte antigen is shown in SEQ ID NO.

1.

5. Use of the hybridoma cell according to claim 1 or the biological material according to claim 2 or 3 in preparing a preparation for the following U1, U2 or U3; U1: recognizes porcine class I leukocyte antigen or the α3 domain of porcine class I leukocyte antigen in vivo or in vitro; U2: blocks porcine class I leukocyte antigen or the α3 domain of porcine class I leukocyte antigen in vivo; and U3: Isolate or identify T cell epitopes of virus-encoded proteins; The protein sequence of the α3 domain of porcine class I leukocyte antigen is shown in SEQ ID NO.

3.

6. The use according to claim 5, characterized in that The protein sequence of porcine class I leukocyte antigen is shown as SEQ ID NO.

1.

7. The use according to claim 5, characterized in that The virus is African swine fever virus.

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