Preparation method and application of pig LAG3 protein specific blocking agent

Abstract

The invention is applicable to the biomedical technology, and provides a preparation method and application of a porcine LAG3 protein specific blocker, and the preparation method comprises the following steps: constructing a sequence of an extracellular region of a coding porcine immune checkpoint into a plasmid vector by an enzyme digestion connection method to obtain a recombinant plasmid; transforming the recombinant plasmid into a competent cell, carrying out MSX pressurized screening to obtain a stable expression recombinant protein, and purifying to obtain a recombinant protein; after animals are immunized by using the recombinant protein, a positive hybridoma cell strain for blocking pig immune checkpoints is obtained through cell fusion and cloning screening; after the positive hybridoma cell strain is injected into an animal pretreated by liquid paraffin, ascites is collected and purified, and the pig LAG3 protein specific blocking agent is obtained; the blocker has relatively strong specificity and relatively high affinity to the porcine LAG3, blocks an inhibition signal transmitted after the LAG3 is combined with a ligand of the LAG3, and promotes activation of host cells and presentation and killing ability of the host cells to viruses.

Description

Technical Field

[0001] The present invention belongs to biomedical technology, and in particular relates to a preparation method and application of a porcine LAG3 protein specific blocker. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded positive-sense RNA virus belonging to the order Nidovirales, family Arteriviridae, and genus Arterivirus. Pigs are the only natural host of PRRSV. The main clinical symptoms are abortion in pregnant sows, or severe reproductive disorders such as stillbirth and mummified fetuses. Piglets show difficulty breathing. Some infected pigs often have blue-purple skin due to cyanosis of the ear roots and extremities, also known as "blue ear disease". Porcine reproductive and respiratory syndrome is a type of infectious disease that currently causes serious harm and huge losses to the pig farming industry worldwide. After PRRSV infects the host, it mainly invades alveolar macrophages and monocytes, and proliferates in large numbers in the cells, thereby inducing cell apoptosis and cell autophagy. As the infection progresses, the virus continues to proliferate in monocytes and macrophages and spreads into the blood circulation and systemic lymphoid tissues, inhibiting the function of the immune system, reducing the adaptive and acquired immune response capabilities, and causing severe immunosuppression in the body. At the same time, after the host is infected with PRRSV, dendritic cells (DC), as the most functional professional antigen presenting cells (APC) in the body, will process viral antigens and further present them to naive T cells. After being activated, naive T cells differentiate into effector T cells, and finally induce the body to produce antiviral immune responses. However, in the acute infection stage, PRRSV can inhibit the expression of MHC-I, MHC-II, CD14 and CD80 / 86 molecules in DC through certain pathways, thereby weakening the antigen presentation ability of APC and assisting PRRSV in fighting the host's immune response to achieve immune escape. In addition, PRRSV infection can induce the production of regulatory T cells and exert its negative immune regulatory effect, thereby helping PRRSV to escape the host's immune response, which may also be a potential reason for PRRSV to persist in the body. Regulatory T cells (Treg) are a type of T cell subset with immunosuppressive function that can regulate cellular immune responses in the body. Treg exerts its immunosuppressive function by inhibiting the activation and proliferation of T cells. There are two main ways to achieve this: one is to inhibit the activation of target cells by direct contact with them, and the other is to inhibit the body's immune response by secreting related factors such as Transforming Growth Factor-B (TGF-B) and IL-10. Studies have shown that the number of tissue-infiltrating Tregs in the lungs, trachea and bronchi of PRRSV-infected pigs is significantly higher than that of PRRSV-negative pigs, and the number of Treg cells expressing IL-10 is also significantly increased, indicating that PRRSV infection has a strong induction effect on regulatory T cells, providing a suitable microenvironment for the proliferation of PRRSV.

[0003] Lymphocyte Activation Gene 3 (LAG3), also known as CD223, is a type of co-inhibitory molecule that is mainly expressed on the surface of a variety of immune cells. Binding to its ligand transmits inhibitory signals, inhibits the activation of its host cells, and negatively regulates the proliferation and function of immune cells. MHC-Ⅱ, as the classic ligand of LAG3, is mainly expressed on the cell membrane surface of APC. After binding, they transmit inhibitory signals to APC in reverse, inhibit its antigen presentation ability, and assist PRRSV in fighting the host's immune response to achieve immune escape. In addition, the upregulation of host cell co-inhibitory molecule expression is one of the important mechanisms for the formation of persistent viral infection. The binding of co-inhibitory molecules to the corresponding ligands will lead to low T cell proliferation function, reduced or deficient secretion of cytokines such as INF-γ and TNF-α, and failure or loss of cytotoxic function, resulting in the body's inability to effectively and timely eliminate the virus, thereby forming a persistent viral infection. Studies have shown that by blocking the interaction between LAG3 and MHC-II molecules, the inhibition of transmission to T cells can be relieved, the T cell immune response can be activated and restored, and the antiviral ability of T cells can be enhanced. Therefore, the present invention uses monoclonal antibody technology to prepare a porcine LAG3 protein-specific blocker, which competitively and specifically binds to LAG3, blocks the inhibitory signal transmitted after LAG3 binds to its ligand, promotes host cell activation and its presentation and killing ability to PRRSV, restores the antiviral ability of porcine T cells, enhances the immune function of pigs, and improves the disease resistance of pigs. It provides a new idea for the effective treatment of PRRS and also provides a new method in the field of porcine LAG3 protein-specific blocking technology. Summary of the invention

[0004] The purpose of the embodiments of the present invention is to provide a method for preparing a porcine LAG3 protein-specific inhibitor, aiming to solve the problems raised in the background technology.

[0005] The embodiment of the present invention is implemented as follows: a method for preparing a porcine LAG3 protein specific blocker, characterized in that it comprises the following steps:

[0006] Using the gene sequence of the porcine immune checkpoint as a template, the extracellular region sequence encoding the porcine immune checkpoint with double restriction sites was synthesized; the gene sequence of the porcine immune checkpoint was referenced to GenBank No. MK813967.1;

[0007] The sequence encoding the extracellular region of the porcine immune checkpoint is constructed into a plasmid vector by restriction digestion and ligation to obtain a recombinant plasmid;

[0008] The recombinant plasmid was transformed into competent cells, and the stably expressed recombinant protein was obtained through MSX pressure screening, and the recombinant protein was obtained after purification;

[0009] After immunizing animals with recombinant proteins, positive hybridoma cell lines that block pig immune checkpoints were obtained through cell fusion and cloning screening;

[0010] After the positive hybridoma cell lines were injected into animals pretreated with liquid paraffin, the ascites was collected and purified to obtain a porcine LAG3 protein-specific inhibitor.

[0011] Preferably, the animal is a mouse.

[0012] Preferably, the primer set used for synthesis includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 in the sequence listing and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2 in the sequence listing.

[0013] Preferably, the double restriction enzyme cleavage sites include BamH I and Hind III restriction enzyme cleavage sites.

[0014] Preferably, the plasmid vector is pET-28a.

[0015] Preferably, the competent cells are Trans5α competent cells.

[0016] Another object of the present invention is to provide a porcine LAG3 protein-specific inhibitor prepared by the above preparation method.

[0017] Another object of the present invention is to provide an application of the above-mentioned porcine LAG3 protein specific blocker in the preparation of drugs for preventing and treating porcine reproductive and respiratory syndrome virus infection.

[0018] Another object of the present invention is to provide an application of the above-mentioned porcine LAG3 protein-specific blocker in the preparation of a Western blot detection kit or an IFA detection kit for porcine LAG3 in tissue cell samples.

[0019] The present invention provides a method for preparing a porcine LAG3 protein-specific blocker. The prepared porcine LAG3 protein-specific blocker has strong specificity and high affinity for porcine LAG3 and porcine LAG3 protein-specific blocker, can block the inhibitory signal transmitted after LAG3 binds to its ligand, promote host cell activation and its ability to present and kill viruses. Among them, the porcine LAG3 protein-specific blocker can be used for immunofluorescence staining (IFA) and Western blot of porcine LAG3, and it is verified by in vivo experiments that it is used for the treatment of porcine respiratory and reproductive syndrome and has a significant therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the result of PCR amplification of the pig LAG3 extracellular region gene fragment with the signal peptide deleted;

[0021] Figure 2 This is the SDS-PAGE identification result of the induced expressed porcine LAG3 recombinant protein;

[0022] Figure 3 The figure shows the result of detecting the induced expressed porcine LAG3 recombinant protein using anti-His mAb by Western blot method;

[0023] Figure 4 This is a diagram showing the purification results of porcine LAG3 recombinant protein;

[0024] Figure 5 This is a diagram showing the results of the blocker titer test in the serum of immune mice;

[0025] Figure 6 This is the Western blot identification result of the pig LAG3 protein specific inhibitor;

[0026] Figure 7 This is the IFA identification result of the porcine LAG3 protein specific inhibitor;

[0027] Figure 8 This is a diagram showing the subtype identification results of the porcine LAG3 protein specific inhibitor;

[0028] Fig. 9 This is a graph showing the results of the blocker titer test in ascites;

[0029] Fig.10 This is a graph showing the saturation determination result of the binding between the porcine LAG3 protein-specific blocker and the antigen;

[0030] Fig.11 This is a diagram showing the purification results of the porcine LAG3 protein specific inhibitor;

[0031] Fig.12 This is a schematic diagram of the extracellular domain segmentation of porcine LAG3 protein;

[0032] Fig.13 The figure shows the Western blot identification of the segmentally expressed pig LAG3 protein and the reactivity detection result of the blocker with the segmentally expressed pig LAG3 protein;

[0033] Fig.14 This is a graph showing the reactivity test results between the specific blocker and the porcine LAG3-3 protein expressed in HEK 293T cells;

[0034] Fig.15 qPCR was used to detect the copy number of PRRSV N gene for cell samples;

[0035] Fig.16 TCID of PRRSV in cell supernatant 50 Test result graph;

[0036] Fig.17 This is a statistical analysis result diagram of the survival rate of experimental animals;

[0037] Fig.18 This is a graph showing the viral load test results of the spleen and lung tissues of experimental animals. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0039] In one embodiment of the present invention, a method for preparing a porcine LAG3 protein-specific blocker is provided, comprising the following steps:

[0040] Using the gene sequence of porcine immune checkpoint (GenBank No.MK813967.1) as a template, the sequence encoding the extracellular region of porcine immune checkpoint with BamH I and Hind III restriction sites was synthesized and constructed into the pET-28a vector by restriction ligation. The plasmid was extracted and transformed into Trans5α competent cells (Full Gold). The recombinant protein was stably expressed by MSX pressure screening, and the porcine immune checkpoint recombinant protein was obtained after purification. The recombinant protein was used to immunize mice, and cell fusion was performed when the titer reached 1:256000 until a positive hybridoma cell line that secreted efficient and stable blocking of immune checkpoints was screened. The positive hybridoma cell line was injected into BALB / c mice pretreated with liquid paraffin (Sigma), and the ascites was collected and purified to obtain a specific stress blocker with high concentration and good purity.

[0041] The primer set used in the above synthesis includes an upstream primer (sLAG3-109-peF: 5'-CGGGATCCATGGGGGCTCC-3') with a nucleotide sequence as shown in SEQ ID NO.1 in the sequence listing and a downstream primer (sLAG3-1305-peR: 5'-CCCAAGCTTGAGGTGGCCTGTTTTC-3') with a nucleotide sequence as shown in SEQ ID NO.2 in the sequence listing.

[0042] In another embodiment of the present invention, there is also provided an application of a porcine LAG3 protein-specific blocker in the field of antigen identification technology, comprising the following steps:

[0043] (1) Western blot identification: HEK 293T cells transfected with the recombinant plasmid pcDNA3.1 / V5-HisB-LAG3 containing the full-length gene of porcine LAG3 were used for Western blot detection using a blocker as the primary antibody, anti-His mAb as the positive control, and non-immunized mouse serum as the negative control.

[0044] (2) IFA identification: The hybridoma cell culture supernatant was used as the primary antibody to perform IFA identification on HEK 293T cells transfected with the pcDNA3.1 / V5-HisB-LAG3 recombinant plasmid.

[0045] (3) Subtype identification: The hybridoma cell culture supernatant was used to perform subtype identification according to the IsoStrip instructions, and it was determined that the heavy chains of the two protein-specific blockers were both IgG1 and the light chains were both κ.

[0046] (4) Ascites preparation and titer detection: Hybridoma cells were injected intraperitoneally into BALB / c mice pretreated with liquid paraffin. Ascites were collected around 14 days later and the blocker titer in the ascites was detected by indirect ELISA.

[0047] (5) Identification of the identity of the antigenic epitopes recognized by the two blockers, including the following steps: 400 ng of purified porcine LAG3 recombinant protein per well was used to coat the ELISA plate, the mouse ascites was diluted 10 times and then subjected to ELISA detection, and the OD 450 The previous dilution at which the nm value dropped significantly was taken as the saturation of the binding between the blocker and the antigen, and then the two blockers were tested to see whether they recognized the same antigen epitope.

[0048] (6) Purification of specific blocking agents and identification of the 1C2 recognition antigen region, including the following steps: using the ascites prepared from the above hybridoma cell line, purification by affinity chromatography using Protein G, and obtaining a high-purity LAG3 protein-specific blocking agent. According to the obtained porcine LAG3 gene sequence, its possible B cell antigen epitopes were analyzed through the ABCpred website. Combined with the epitope distribution results predicted by the Proteam module of DNAStar software, the porcine LAG3 extracellular domain without the signal peptide was divided into three overlapping segments, namely LAG3-1, LAG3-2 and LAG3-3. The corresponding gene fragments were cloned and inserted into the pET-21b plasmid to construct a prokaryotic expression vector for segmented expression. Then, Western blot detection was performed using anti-His mAb and blocker as primary antibodies, respectively. The gene fragment encoding LAG3-3 was inserted into the pCMV-Flag eukaryotic expression vector to construct the recombinant plasmid pCMV-Flag-LAG3-3, which was transfected into HEK 293T cells and cultured for 48 h. The cells were collected and lysed with NP40, and Western blot analysis was performed using the purified blocker as the primary antibody. At the same time, anti-Flag mAb was used as the positive control blocker, and cells transfected with the pCMV-Flag empty vector were used as negative control samples.

[0049] In another embodiment of the present invention, there is also provided a use of a porcine LAG3 protein-specific inhibitor in porcine reproductive and respiratory syndrome, comprising the following steps:

[0050] (1) In vitro test: Since the main host cell of PRRSV is porcine alveolar macrophage, porcine alveolar macrophage was selected to construct the in vitro test model in this experiment, and three groups were designed: blank control group, PRRSV infection group and blocker treatment group. After the plated porcine alveolar macrophages were placed in a 37℃ incubator containing 5% CO2 for 3 h, the virus-challenged group was diluted with serum-free RPMI1640 medium for PRRSVSD16 strain, and serum-free RPMI 1640 medium without virus was set as control. At 24 h and 48 h after the virus-challenged group, the LAG3 protein-specific blocker with a final concentration of 0.05 mg / mL was blocked on the cell surface. After 1 h of culture in a 37℃ incubator, the cell samples and supernatant were collected respectively. The cell samples were tested for the number of copies of PRRSVN gene by qPCR, and the cell supernatant was used for PRRSV TCID50 detection.

[0051] (2) In vivo test: In order to verify the protective effect of the specific inhibitor targeting porcine LAG3 protein on PRRSV-challenged pigs, this test divided PRRSV, PCV2, and ASFV-negative pigs into a normal control group, a challenge (PRRSV virulent strain) control group, an immune challenge group, and a targeted inhibitor treatment group. It was found that the targeted inhibitor was given twice, 3 days before the challenge and at the same time as the challenge, which could provide 100% protection for PRRSV-challenged pigs. The body temperature of the pigs in the targeted inhibitor treatment group was lower than 40°C throughout the test period, the lung tissue lesions were significantly alleviated, and the viral loads of nasal swabs, serum, lung tissue, and spleen tissue were significantly reduced. This indicates that the targeted inhibitor can restore the immune function of pigs by blocking the inhibitory signals of lymphocytes, thereby promoting the clearance of PRRSV by pigs.

[0052] Example 1: This example provides a method for preparing a porcine LAG3 protein specific inhibitor, which specifically comprises the following steps:

[0053] (1) Porcine LAG3 gene cloning: Using the porcine LAG3 gene sequence (GenBank No. MK813967.1) as a template, the following primers were designed: sLAG3-109-peF: 5'-CGGGATCCATGGGGGCTCC-3' (BamH Ⅰ, as shown in SEQ ID NO. 1 in the sequence listing), sLAG3-1305-peR: 5'-CCCAAGCTTGAGGTGGCCTGTTTTC-3' (Hind Ⅲ, as shown in SEQ ID NO. 2 in the sequence listing);

[0054] Clone the gene fragment encoding porcine LAG3 37-435 aa (eg Figure 1 The PCR products were recovered by 1% agarose gel electrophoresis using a gel recovery kit.

[0055] (2) Construction of recombinant expression vector: The PCR product and pET-28a vector were double-digested by BamH I and Hind III endonucleases (NEB), respectively. The digested products were recovered from the gel and then ligated with T4 DNA ligase (NEB) at a molar ratio of 1:5 between the plasmid and the target fragment at 16°C overnight.

[0056] The next day, the competent cells were transformed with the ligation product according to the instructions of Trans5α competent cells (Full Gold), and then the routine operations were followed to plate, culture, and pick monoclonal colonies to inoculate kanamycin-resistant LB liquid culture medium. After shaking culture for 6 hours, PCR identification of the bacterial solution was performed, and the positive bacterial solution was continued to be cultured for 12-14 hours. The plasmid was then extracted and double-enzyme digestion was performed for identification using BamH I and Hind III. The positive plasmid was sequenced, and the sequencing results were compared. The recombinant plasmid with correct sequencing was named pET28a-sLAG3.

[0057] (3) Preparation and preservation of recombinant bacteria: According to the instructions, the pET28a-sLAG3 recombinant plasmid was transferred into BL21 (DE3) competent cells. After plating, culturing, picking monoclonal bacteria for shaking culture, and identification, the positive monoclonal recombinant bacteria BL21-pET28a-sLAG3 were selected for preservation.

[0058] (4) Preparation of electrophoresis samples: Take the recombinant bacteria BL21-pET28a-sLAG3 and inoculate it into kanamycin-resistant LB liquid medium at a ratio of 1:100, and activate it by shaking overnight. The next day, transfer it to new medium at a ratio of 1:20 and shake it until the OD 600 nm ≈ 0.6. Take 1 mL of bacterial solution and centrifuge to collect the cells as the uninduced control. Add 1.0 mmol / L IPTG to the remaining bacterial solution and continue shaking culture for 6 h to induce the expression of the target protein. Take 1 mL of bacterial solution and centrifuge to collect the cells as the induced sample. The remaining bacterial solution is centrifuged and collected. The cells are resuspended in PBS with 1 / 20 volume of the bacterial solution and ultrasonically lysed. Then centrifuge to collect the supernatant and precipitate respectively. The precipitate is dissolved in a buffer containing 8 mol / L urea at 4°C overnight. Treat the uninduced control, induced, supernatant and dissolved precipitate samples according to SDS-PAGE requirements.

[0059] (5) Preparation of recombinant protein: Prepare 10% separation gel, perform SDS-PAGE on the treated samples, and then stain with Coomassie Brilliant Blue to observe and confirm that the target protein is mainly expressed in the form of inclusion bodies (such as Figure 2 The uninduced bacteria, induced bacteria, and supernatant and precipitate samples after ultrasonic lysis were subjected to SDS-PAGE and then transferred to a membrane. Western blot detection was performed using anti-His mAb (full gold) as the primary antibody. It was determined that the porcine LAG3 recombinant protein with the His tag could specifically react with the anti-His mAb (as shown in Figure 2A). Figure 3 As shown in Figure 2 ). Due to the background expression of the target protein, a weak imprint band was also detected in the uninduced bacterial sample; the expression of the target protein was also detected in the supernatant after ultrasound, but the expression level was too low, so the target protein expressed in the form of inclusion bodies was selected for subsequent research. The target protein was induced to express in large quantities and purified using Ni-NTA resin (Tian Di Ren He). SDS-PAGE detection confirmed that the target protein could be successfully eluted using an eluent containing 250 mmol / L imidazole (as shown in Figure 2 ). Figure 4 As shown in the figure), after dialysis, renaturation and concentration, the purified porcine LAG3 recombinant protein with a concentration of 0.56 mg / mL was obtained using a BCA protein quantification kit (Pierce).

[0060] (6) Mouse immunization: The purified porcine LAG3 recombinant protein was mixed with Freund's adjuvant (Sigma) in equal volumes, fully emulsified on an emulsifier, and then immunized female BALB / c mice by subcutaneous injection (50 µg / mouse, Chengdu Dashuo). Freund's complete adjuvant was used for the first immunization, followed by Freund's incomplete adjuvant. After 4 immunizations, blood was collected from the tail vein, and the serum was separated and tested by indirect ELISA to detect the blocking agent titer in the serum to reach 1:256000 (e.g. Figure 5 3 days before cell fusion, purified porcine LAG3 recombinant protein (50 µg / mouse) was used for impulse immunization by intraperitoneal injection.

[0061] (7) Preparation of SP2 / 0 cells: After taking out the SP2 / 0 cells stored in liquid nitrogen, immediately place them in a 37°C water bath and shake them gently to thaw them quickly. Centrifuge at 300 g for 10 min at room temperature. Resuspend the cells in RPMI 1640 medium (Gibco) containing 10% FBS (Gibco), add them to the cell bottle, and culture them in a 37°C, 5% CO2 incubator. Adjust the cell state and expand the culture so that the cells used for fusion are in the logarithmic growth phase.

[0062] (8) Cell fusion: Take the impact immunized mice, remove the eyeballs, collect blood, separate the serum and store it. The mice were killed by cervical dislocation, and after being thoroughly disinfected by soaking in 75% ethanol, the spleen was aseptically dissected in a clean bench to prepare a spleen cell suspension. The spleen cell suspension was added to the SP2 / 0 cell suspension at a ratio of 10:1, and PEG1450 (Sigma) was added to promote cell fusion. Then, the cells were gently suspended in preheated RPMI 1640 medium containing 1×HAT (Sigma) and 10% FBS to prepare a cell suspension. 200 μL of the cell suspension was added to each well of a 96-well cell culture plate and cultured in a 37°C, 5% CO2 incubator. After 3 days of culture, half of the medium was replaced with preheated RPMI 1640 medium containing 1×HAT and 10% FBS. After 7-9 days of culture, the cell supernatant was aspirated and tested by indirect ELISA to screen the positive cell wells.

[0063] (9) Positive cell screening: Gently blow away the hybridoma cells in the wells with positive indirect ELISA test results, count and dilute them by limiting dilution, take the diluted hybridoma cells and add them to a 96-well cell culture plate, 100 µL / well; culture the subcloned cells for about 8-10 days, observe and record the cell wells marked with single cell clusters, continue to detect by indirect ELISA method, and select the wells with higher OD450nm values ​​for the next round of subcloning. Repeat subcloning 3-5 times until a 100% positive hybridoma cell line is screened, expand the culture and freeze the hybridoma cells in time. Finally, two hybridoma cell lines that can stably secrete porcine LAG3 protein-specific inhibitors were obtained, named 1C2 and 3E11 respectively.

[0064] Example 2: Identification of LAG3 protein-specific inhibitors:

[0065] (1) Western blot identification: HEK 293T cells transfected with the recombinant plasmid pcDNA3.1 / V5-HisB-sLAG3 containing the full-length gene of porcine LAG3 were used for Western blot detection with blockers 1C2 and 3E11 as primary antibodies, anti-His mAb as positive control, and non-immunized mouse serum as negative control. It was determined that both blockers could react specifically with porcine LAG3 expressed in HEK293T cells (e.g. Figure 6 as shown).

[0066] (2) IFA identification: The hybridoma cell culture supernatant was used as the primary antibody to perform IFA identification on HEK 293T cells transfected with the pcDNA3.1 / V5-HisB-sLAG3 recombinant plasmid. It was determined that both blockers 1C2 and 3E11 could specifically react with porcine LAG3 expressed in HEK 293T cells (e.g. Figure 7 as shown).

[0067] (3) Subtype identification: The hybridoma cell culture supernatant was used to perform subtype identification according to the instructions of Roche's IsoStrip (Monoclonal Antibody Isotyping Kit). The results showed that the heavy chains of both blockers were IgG1 and the light chains were κ (e.g. Figure 8 as shown).

[0068] (4) Ascites preparation and titer detection: The two hybridoma cells were intraperitoneally injected into BALB / c mice pretreated with liquid paraffin (Sigma). The ascites were collected around 14 days and the blocker titer in the ascites was detected by indirect ELISA. The results showed that the blocker titers in the ascites prepared by the 1C2 and 3E11 hybridoma cells were 1:1048576 and 1:262144, respectively (Figure 2). Fig. 9 as shown).

[0069] (5) Identification of the identity of antigen epitopes recognized by the two blocking agents: 400 ng of purified porcine LAG3 recombinant protein was coated on the ELISA plate per well and incubated overnight. The next day, 200 μL of blocking solution (PBS'T containing 5% skim milk powder (BioFroxx)) was added to each well and placed in a 37°C incubator for blocking for 1 h. The two blocking agents were diluted according to saturation and evenly mixed and added to the ELISA plate at 100 μL / well. After incubation at 37°C for 1 h, the primary antibody was discarded. The ELISA plate was washed 3 times with PBS'T for 2 min each time. HRP-labeled goat anti-mouse IgG (H+L) (CST) diluted 1:5000 with blocking solution was added at 100 μL / well. After incubation at 37°C for 1 h, the secondary antibody was discarded. The ELISA plate was washed 3 times with PBS'T for 2 min each time. 100 μL of freshly prepared TMB (Sigma) colorimetric solution was added to each well and color was developed at room temperature in the dark for 15 min. min, 50 μL of 3 mol / L H2SO4 solution was added to each well to terminate the color development reaction. 450 The previous dilution at which the nm value dropped significantly was taken as the saturation of the binding between the blocker and the antigen. The saturation of the binding between the two blockers and the antigen was determined to be about 1:1000 (e.g. Fig.10 and Table 1).

[0070]

[0071] (6) Purification of specific blocking agent and identification of 1C2 recognition antigen region: The ascites prepared by 1C2 hybridoma cell line was purified by protein G affinity chromatography and then analyzed by SDS-PAGE. As a result, a high-purity LAG3 protein specific blocking agent (such as Fig.11 as shown).

[0072] According to the obtained porcine LAG3 gene sequence (GenBank No.MK813967.1), the possible B cell antigen epitopes were analyzed on the ABCpred website (https: / / webs.iiited.edu.in / raghava / abcpred / ABC-submission.html). Combined with the epitope distribution results predicted by the Proteam module of DNAStar software, the porcine LAG3 extracellular domain without the signal peptide was divided into three overlapping segments, namely LAG3-1 (37-175aa), LAG3-2 (167-326aa) and LAG3-3 (214-435aa). The corresponding gene fragments were cloned and inserted into the pET-21b plasmid to construct a prokaryotic expression vector for segmented expression (such as Fig.12Then, Western blot was performed using anti-His mAb and blocker 1C2 as primary antibodies. The results showed that LAG3-1 was not expressed, while anti-His mAb could detect the label control protein (swine HEV 239 protein) and the expressed LAG3-2 and LAG3-3 (as shown in Figure 2A). Fig.13 ), but the blocker 1C2 only reacts with the LAG3-3 protein (as shown in A Fig.13 Then, the gene fragment encoding LAG3-3 was inserted into the pCMV-Flag eukaryotic expression vector to construct the recombinant plasmid pCMV-Flag-LAG3-3, which was transfected into HEK 293T cells and cultured for 48 h. The cells were collected and lysed with NP40, and Western blot analysis was performed using the purified blocker 1C2 as the primary antibody. At the same time, anti-Flag mAb (full gold) was used as the positive control blocker, and cells transfected with the pCMV-Flag empty vector were used as negative control samples. Results Blocker 1C2 can specifically react with the porcine LAG3-3 protein expressed by HEK 293T (as shown in Figure 2A). Fig.14 as shown).

[0073] Example 3: Cell assay

[0074] (1) In vitro experiment: Since the main host cells of PRRSV are porcine alveolar macrophages, porcine alveolar macrophages were used to construct an in vitro experimental model. Three groups were designed: blank control group, PRRSV infection group (PRRSV group), and blocker treatment group (PRRSV+LAG3 Blockader group). First, porcine alveolar macrophages were isolated and cultured. The trachea and esophagus were carefully separated, and the lungs were taken out together with the heart. After washing the lungs with PBS containing double antibodies, the lavage fluid was filtered through eight layers of gauze. The filtered lavage fluid was washed once, centrifuged at 500 r / min for 10 min, the supernatant was discarded, and the cells were resuspended with an appropriate amount of RPMI 1640 medium containing 10% FBS. After counting, the plated porcine alveolar macrophages were placed in a 5% CO2, 37°C incubator for adherent culture for 3 h. The challenge group used serum-free RPMI 1640 medium to dilute the PRRSV SD16 strain, and a serum-free RPMI 1640 medium without virus was set as a control. In the LAG3 blocker group, a final concentration of 0.05 mg / mL LAG3 protein-specific blocker was blocked on the cell surface at 24 h and 48 h after the challenge, respectively. After culturing in a 37°C incubator for 1 h, the cell samples and supernatant were collected, and the cell samples were tested for PRRSV by qPCR. N gene copy number (results as Fig.15 The cell supernatant was used for PRRSV TCID 50 Test (results such as Fig.16as shown).

[0075] (2) In vivo test: In order to verify the protective effect of the specific blocker targeting porcine LAG3 protein on PRRSV-challenged pigs, this study divided PRRSV-, PCV2-, and ASFV-negative pigs into a normal control group (Mock group), a challenge (PRRSV virulent strain) control group (PRRSV group), an immune challenge group (MLV+PRRSV group), and a 4-targeted blocker treatment group (MLV+PRRSV+LAG3Blockader group). It was found that the targeted blocker was given twice, 3 days before and at the same time as the challenge, to provide 100% protection for PRRSV-challenged pigs (e.g., Fig.17 As shown in the figure, the body temperature of the pigs in the targeted inhibitor treatment group was below 40°C throughout the trial period, the lung tissue lesions were significantly alleviated, and the viral loads in nasal swabs, serum, lung tissues and spleen tissues were significantly reduced (as shown in the figure). Fig.18 ). This indicates that targeted blockers can restore the immune function of pigs by blocking the inhibitory signals of lymphocytes, thereby promoting the elimination of PRRSV in pigs.

[0076] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a porcine LAG3 protein specific blocker, characterized in that: The following steps are involved: Using the gene sequence of the porcine immune checkpoint as a template, the extracellular region sequence encoding the porcine immune checkpoint with double restriction sites was synthesized; the gene sequence of the porcine immune checkpoint was referenced to GenBank No. MK813967.1; The sequence encoding the extracellular region of the porcine immune checkpoint is constructed into a plasmid vector by restriction digestion and ligation to obtain a recombinant plasmid; The recombinant plasmid was transformed into competent cells, and the stably expressed recombinant protein was obtained through MSX pressure screening, and the recombinant protein was obtained after purification; After immunizing animals with recombinant proteins, positive hybridoma cell lines that block pig immune checkpoints were obtained through cell fusion and cloning screening; After the positive hybridoma cell lines were injected into animals pretreated with liquid paraffin, the ascites was collected and purified to obtain a porcine LAG3 protein-specific inhibitor.

2. The method for preparing the porcine LAG3 protein specific blocker according to claim 1, characterized in that: The animal is a mouse.

3. The method for preparing the porcine LAG3 protein specific blocker according to claim 1, characterized in that: The primer set used for the synthesis includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 in the sequence listing and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2 in the sequence listing.

4. The method for preparing the porcine LAG3 protein specific blocker according to claim 1, characterized in that: The double restriction enzyme cleavage sites include BamH I and Hind III restriction enzyme cleavage sites.

5. The method for preparing the porcine LAG3 protein specific blocker according to claim 1, characterized in that: The plasmid vector is pET-28a.

6. The method for preparing the porcine LAG3 protein specific blocker according to claim 1, characterized in that: The competent cells are Trans5α competent cells.

7. A porcine LAG3 protein-specific blocker prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the porcine LAG3 protein-specific inhibitor as claimed in claim 7 in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome virus infection.

9. Use of the porcine LAG3 protein-specific blocker according to claim 7 in preparing a Western blot detection kit or an IFA detection kit for porcine LAG3 in a tissue cell sample.

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