Mouse anti-infectious bovine rhinotracheitis virus monoclonal antibody and application thereof

Abstract

The invention relates to the technical field related to livestock immunology, in particular to a mouse monoclonal antibody for resisting infectious bovine rhinotracheitis virus and application of the mouse monoclonal antibody. The amino acid sequences of complementary determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region of the monoclonal antibody or the antigen binding fragment of the monoclonal antibody are respectively AASGFTFSSYAM, WYDGSNKYYADS and MNSLRAEDTAVYYCAR, and the amino acid sequences of complementary determining regions CDR1, CDR2 and CDR3 of a light chain variable region of the monoclonal antibody or the antigen binding fragment of the monoclonal antibody are respectively RASQSISSYLH, AASSLQSGVPS and QQYNSYPWT. The monoclonal antibody can be specifically combined with gD proteins of all subtypes of the infectious bovine rhinotracheitis virus, and the infectious bovine rhinotracheitis virus detection kit prepared from the monoclonal antibody has the advantages of high sensitivity, strong specificity, wide detection range, short detection time and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of livestock antibody detection, and particularly relates to a mouse-derived anti-bovine infectious rhinotracheitis virus monoclonal antibody and application thereof. BACKGROUND

[0002] Infectious bovine rhinotracheitis (IBR) is an acute, febrile, and contagious disease of cattle caused by infectious bovine rhinotracheitis virus (IBRV). IBRV belongs to the Varicellovirus genus of the Alphaherpesvirinae subfamily of the Herpesviridae family in classification, and is also known as BoHV-1. According to the different restriction endonuclease sites, it can be further divided into three subtypes, namely BoHV-1.1, BoHV-1.2a and BoHV-1.2b. IBRV has a similar appearance to herpes viruses, and is a symmetrical structure of icosahedron, similar to a sphere, with a diameter of 150-220 nm. It is mainly composed of three parts of outer envelope, inner capsid and core. The appearance of the capsid of the virus is hexagonal, with a diameter of 80-120 nm. In the genome of bovine herpes virus 1, 73 open reading frames have been identified, and the encoded proteins have also been located. The genome can encode 33 structural proteins, of which 12 structural proteins are related to the formation of the envelope and exist on the particle envelope of the virus and the plasma membrane of the virus-infected cell. Among the encoded envelope proteins, 10 are glycosylated proteins and 2 are non-glycosylated proteins. The 10 glycosylated protein genes are located in the long unique region of the genome, with 6 of them: gL (UL1), gM (UL10), gH (UL22), gB (UL27), gC (UL44), and gK (UL53), and 4 of them in the short unique region: gE (US8), gI (US7), gG (UL4), and gD (UL6). Among them, gB, gD, gH, gL and gK are essential proteins for the growth of the virus in the cell, and gB, gC, gD and gE are the main glycosylated proteins for the expression of toxicity, which can stimulate the body to produce neutralizing antibodies. The gB protein helps the virus to adsorb and penetrate into the host cell, the gC protein is the most important adsorption protein of the virus, the gD protein can stimulate the host body to produce a persistent cellular immune response, and the gE protein mainly acts on the transmission of the virus between cells.

[0003] Among them, gD is the main glycoprotein on the surface of the IBRV envelope, and is one of the main virus immunogen genes recognized by the serum of infected cattle. Due to its high conservation characteristics, it is often used for antibody detection.

[0004] The commonly used IBRV antibody detection methods include virus neutralization test (VNT), indirect enzyme-linked immunosorbent assay (I-ELISA), indirect immunofluorescence assay (IFA), latex agglutination test (LAT) and the like. Among them: VNT operation is relatively cumbersome, cell culture is required, time-consuming is relatively long (2-5 days), and the laboratory conditions are high (biological safety facilities are required); I-ELISA has insufficient specificity, cross-reactions are prone to occur due to virus antigen homology or non-specific antibodies, false positives are prone to occur, the detection conditions are high, and the operation deviation is prone to affect the stability of the results. The sensitivity is low, and there is a risk of false negative.

[0005] The IFA result interpretation depends on subjective experience, it is difficult to accurately quantify, and the operation is relatively complex; the LAT has low sensitivity, false negatives are prone to occur, and it is usually used as a preliminary screening method, and the positive results need to be further verified. SUMMARY

[0006] To solve the above problems, the application provides a murine anti-bovine infectious rhinotracheitis virus monoclonal antibody and an application thereof. The monoclonal antibody as a detection antibody can specifically bind to the bovine infectious rhinotracheitis virus antigen, and is applied to ELISA detection test and CLIA detection test, so as to improve the detection sensitivity, improve the detection specificity, and shorten the detection time.

[0007] Specifically, the technical scheme of the application is as follows:

[0008] In a first aspect, the application provides a murine anti-bovine infectious rhinotracheitis virus monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the murine anti-bovine infectious rhinotracheitis virus monoclonal antibody or the antigen-binding fragment thereof has complementarity determining regions CDR1, CDR2 and CDR3 with amino acid sequences of positions 23-34, 52-63 and 83-98 of SEQ ID NO. 6, respectively; and the light chain variable region of the murine anti-bovine infectious rhinotracheitis virus monoclonal antibody or the antigen-binding fragment thereof has complementarity determining regions CDR1, CDR2 and CDR3 with amino acid sequences of positions 24-34, 50-60 and 89-97 of SEQ ID NO. 7, respectively.

[0009] In addition, the antibody or the antigen-binding fragment thereof can specifically bind to the bovine infectious rhinotracheitis virus.

[0010] Further, the monoclonal antibody or the antigen-binding fragment thereof comprises three heavy chain variable region framework regions: HFR1, HFR2 and HFR3, and four light chain variable region framework regions: LFR1, LFR2, LFR3 and LFR4, wherein

[0011] the amino acid sequence of the HFR1 is shown in SEQ ID NO. 6 at positions 1-22;

[0012] the amino acid sequence of the HFR2 is shown in SEQ ID NO. 6 at positions 35-51;

[0013] the amino acid sequence of the HFR3 is shown in SEQ ID NO. 6 at positions 64-82;

[0014] the amino acid sequence of the LFR1 is shown in SEQ ID NO. 7 at positions 1-23;

[0015] the amino acid sequence of the LFR2 is shown in SEQ ID NO. 7 at positions 35-49;

[0016] the amino acid sequence of the LFR3 is shown in SEQ ID NO. 7 at positions 61-88;

[0017] the amino acid sequence of the LFR4 is shown in SEQ ID NO. 7 at positions 98-107.

[0018] Further, the heavy chain variable region of the murine monoclonal antibody against bovine infectious nasal rhinotracheitis virus comprises an amino acid sequence shown in SEQ ID NO. 6; and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 7.

[0019] In a second aspect, the present application provides an isolated polynucleotide encoding the above-mentioned murine monoclonal antibody against bovine infectious nasal rhinotracheitis virus or the antigen-binding fragment thereof.

[0020] Further, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 3; and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 4.

[0021] In a third aspect, the present application provides a vector comprising the above-mentioned polynucleotide.

[0022] In a fourth aspect, the present application provides a host cell comprising the above-mentioned polynucleotide or vector.

[0023] In a fifth aspect, the present application provides a bovine infectious nasal rhinotracheitis virus gD multi-epitope fusion protein, and the amino acid sequence of the bovine infectious nasal rhinotracheitis virus gD multi-epitope fusion protein is shown in SEQ ID NO. 5.

[0024] Further, the nucleotide sequence encoding the bovine infectious nasal rhinotracheitis virus gD multi-epitope fusion protein is shown as SEQ ID NO. 2.

[0025] Further, the nucleotide sequence of SEQ ID NO. 2 encoding the bovine infectious nasal rhinotracheitis virus gD multi-epitope fusion protein is shown as SEQ ID NO. 1 before codon optimization.

[0026] In a sixth aspect, the present application provides a murine monoclonal antibody conjugate against bovine infectious nasal rhinotracheitis virus, comprising the monoclonal antibody or antigen binding fragment thereof as described above, and a label conjugated to the antibody or fragment.

[0027] Further, the label is selected from one or more of horseradish peroxidase label, biotin label, fluorescent dye label, chemiluminescent dye label.

[0028] Further, the monoclonal antibody is obtained by immunizing the bovine infectious nasal rhinotracheitis virus gD multi-epitope fusion protein.

[0029] In a seventh aspect, the present application provides a method for preparing a monoclonal antibody against bovine infectious nasal rhinotracheitis virus, comprising the following steps: immunizing an animal with the purified bovine infectious nasal rhinotracheitis virus gD multi-epitope fusion protein; isolating spleen lymphocytes from the immunized animal; fusing the cells with myeloma cells to produce hybridomas; screening the hybridomas producing the monoclonal antibody as described above, named 3D6; culturing the hybridomas and recovering the monoclonal antibody; and sequencing the variable regions of the heavy and light chains of the monoclonal antibody.

[0030] In an eighth aspect, the present application provides a method for detecting antibodies against bovine infectious nasal rhinotracheitis virus in a sample, comprising the following steps: contacting the sample with the monoclonal antibody or antigen binding fragment thereof as described above, or the bovine infectious nasal rhinotracheitis virus gD multi-epitope fusion protein; and detecting whether an antigen-antibody complex is formed.

[0031] In a ninth aspect, the present application provides the use of a monoclonal antibody or antigen binding fragment thereof, or the bovine infectious nasal rhinotracheitis virus gD multi-epitope fusion protein in the preparation of a diagnostic reagent for detecting bovine infectious nasal rhinotracheitis virus.

[0032] In a tenth aspect, the present application provides a kit for detecting antibodies against bovine infectious nasal rhinotracheitis virus, comprising the monoclonal antibody or antigen binding fragment thereof as described above, and / or the bovine infectious nasal rhinotracheitis virus gD multi-epitope fusion protein as described above.

[0033] Further, the kit is an enzyme-linked immunosorbent assay kit.

[0034] Further, the above-mentioned kit is a magnetic particle chemiluminescence kit.

[0035] Beneficial effects:

[0036] (1) The mouse-derived anti-bovine infectious rhinotracheitis virus monoclonal antibody, the conjugate or the composition thereof provided by the application can specifically bind to the gD protein of any subtype of bovine infectious rhinotracheitis virus, has no cross reaction with other similar proteins, and has high stability.

[0037] (2) Based on the monoclonal antibody, the competitive ELISA method for detecting bovine infectious rhinotracheitis virus antibody has high sensitivity, specificity and universality, and can accurately and quickly detect the level of bovine infectious rhinotracheitis virus antibody in bovine serum.

[0038] (3) Based on the monoclonal antibody, the competitive magnetic particle chemiluminescence bovine infectious rhinotracheitis virus antibody detection method has higher sensitivity, specificity and broad adaptability, and can realize accurate, high-throughput, rapid and automatic detection of the content of bovine infectious rhinotracheitis virus antibody in bovine serum, and has good application prospect in the detection of bovine infectious rhinotracheitis virus. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be described below.

[0040] Figure 1 It is the SDS-PAGE electrophoresis identification result graph of Example 1 of the application.

[0041] Among them, 1 is the supernatant of recombinant bacteria after induction; 2 is the supernatant of recombinant bacteria after induction. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the application more clear, the technical scheme of the application will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the application, not all the embodiments.

[0043] Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the application. The examples are only used to explain the application, and not to limit the scope of the application.

[0044] The above technical solutions will be described in detail below in combination with specific embodiments.

[0045] Example 1 Expression of multi-epitope fusion protein

[0046] (1) Preparation of the coding gene: 56 IBRV strains published by NCBI were analyzed, and the highly conserved gD protein of the representative strain was selected as the target protein region. The nucleotide sequence of gD protein was predicted by bioinformatics for B cell epitopes, and the linear dominant epitopes were selected as the target gene epitopes of the multi-epitope fusion protein. The nucleotide sequence encoding the dominant epitope and the nucleotide sequence encoding the flexible linker peptide were connected at the DNA level according to the correct reading frame, and the nucleotide sequence is shown as SEQ ID NO. 1. According to the codon preference of E. coli, the optimized coding gene sequence is obtained, and the nucleotide sequence is shown as SEQ ID NO. 2.

[0047] (2) Construction of recombinant plasmid: the optimized coding gene sequence is connected to the prokaryotic expression vector pET-32a (+) by DNA splicing technology to obtain the recombinant plasmid pET-32a (+)-IBRV-gD.

[0048] (3) Expression of multi-epitope fusion protein: the recombinant plasmid pET-32a (+)-IBRV-gD is transformed into BL21 (DE3) competent cells, and the transformed bacteria are inoculated on LB agar plates containing 50 μg / mL kanamycin and cultured at 37°C overnight. A single colony is picked and inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C, 220 rpm overnight. Inoculate into LB medium containing 50 μg / mL kanamycin at a volume of 1% of the total medium, and incubate at 37°C, 220 rpm for about 4 hours, until the OD 600 is 0.6. Add IPTG with a final concentration of 0.2 mmol / L, and induce at 28°C, 110 rpm for 12 hours, then collect the bacterial cells.

[0049] (4) Purification of multi-epitope fusion protein: crude purification is performed using a nickel ion affinity chromatography column, and further purification is performed using a protein G purification column. The purified multi-epitope fusion protein is the bovine infectious rhinotracheitis virus gD multi-epitope fusion protein. The amino acid sequence is shown as SEQ ID NO. 5, and the SDS-PAGE result is shown as Figure 1 . It can be seen from Figure 1 that the expression of the coding gene SEQ ID NO. 2 is about 40 kDa.

[0050] (5) The purity of the bovine infectious rhinotracheitis virus gD multi-epitope fusion protein is determined by microspectrophotometer, and the A280 / A260 ratio is 1.8-2.0, which is high in protein purity and can be used for coating, labeling, detection, etc.

[0051] Example 2 Preparation of mouse-derived anti-bovine infectious rhinotracheitis virus monoclonal antibody

[0052] (1) Immunization of animals: three BALB / c mice were immunized with purified bovine infectious nasal rhinotracheitis virus gD multi-epitope fusion protein, a total of 4 times. The immunization dose was 100 μg per mouse, and the immunization route was intraperitoneal immunization. For the first immunization, the bovine infectious nasal rhinotracheitis virus gD multi-epitope fusion protein was emulsified with an equal amount of Freund's complete adjuvant, and for the second and third immunizations, Freund's incomplete adjuvant was used for emulsification. The fourth immunization was a booster immunization 3 days before cell fusion, with an immunization dose of 50 μg per mouse, without adjuvant, and intraperitoneal injection. After immunization, the mouse spleen was ground to obtain spleen lymphocytes.

[0053] (2) Cell fusion: the spleen lymphocytes were mixed with myeloma cells (SP2 / 0) at a ratio of 1:10, and polyethylene glycol (PEG) was added as a fusion agent.

[0054] (3) HAT selective medium screening: after cell fusion, the mixed cell suspension was inoculated into a 96-well plate containing HAT medium and cultured in a 37°C, 5% CO2 incubator.

[0055] (4) Acquisition of cell strains: indirect ELISA detection was performed using recombinant gD protein as the coating antigen to screen positive hybridoma cell strains, and after subcloning to a single cell by limiting dilution method, the cell strains were expanded and cultured, and finally a stable secreting monoclonal antibody cell strain was obtained, named 3D6.

[0056] (5) Preparation of murine anti-bovine infectious nasal rhinotracheitis virus monoclonal antibody: BALB / c mice were pretreated with liquid paraffin intraperitoneally, and 1-2 weeks later, logarithmically growing hybridoma cells were inoculated. Five to ten days after inoculation, the mouse abdomen was observed to be swollen, and abdominal fluid was collected with a 16-gauge needle. Each mouse can be continuously collected 2-3 times, and a total of 5-10 ml of ascites can be obtained. The ascites was centrifuged (2000 r / min, 5 minutes) to remove cell debris, and was preliminarily purified by saturated ammonium sulfate precipitation and further purified by ion exchange chromatography to obtain monoclonal antibody with a purity of >95%.

[0057] (6) Determination of heavy chain and light chain variable region sequences: logarithmically growing hybridoma cells were collected and sent to General Biological for sequencing. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively, and the encoding gene sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0058] ​The complementarity determining region CDR1, CDR2, CDR3 amino acid sequences of the heavy chain variable region of the above monoclonal antibody or antigen binding fragment thereof are respectively 23-34, 52-63 and 83-98 of SEQ ID NO. 6; the complementarity determining region CDR1, CDR2, CDR3 amino acid sequences of the light chain variable region are respectively: 24-34, 50-60 and 89-97 of SEQ ID NO. 7.

[0059] It also includes 3 heavy chain variable region framework regions: HFR1, HFR2 and HFR3 and 4 light chain variable region framework regions: LFR1, LFR2, LFR3 and LFR4, wherein

[0060] The amino acid sequence of the HFR1 is shown in SEQ ID NO. 6: 1-22;

[0061] The amino acid sequence of the HFR2 is shown in SEQ ID NO. 6: 35-51;

[0062] The amino acid sequence of the HFR3 is shown in SEQ ID NO. 6: 64-82;

[0063] The amino acid sequence of the LFR1 is shown in SEQ ID NO. 7: 1-23;

[0064] The amino acid sequence of the LFR2 is shown in SEQ ID NO. 7: 35-49;

[0065] The amino acid sequence of the LFR3 is shown in SEQ ID NO. 7: 61-88;

[0066] The amino acid sequence of the LFR4 is shown in SEQ ID NO. 7: 98-107.

[0067] The antibody or antigen binding fragment thereof can specifically bind to bovine infectious nasal rhinotracheitis virus.

[0068] Unless otherwise specified, the CDR sequences described in the present application are defined according to the Kabat numbering system.

[0069] Example 3 Labeling of monoclonal antibody

[0070] (1) HRP labeling of monoclonal antibody

[0071] Take 2 mg HRP and dissolve it in 0.5 mL distilled water. Add 0.5 mL of freshly prepared 0.06 mol / L NaIO4 solution to the above solution, and place it in the dark at 4℃ for 30 min. Add 160 mmol / L of ethylene glycol 0.5 mL, and place it at room temperature for 30 min. Then add 2 mg of the monoclonal antibody to be labeled, and mix well.

[0072] Put the above solution into a dialysis bag, and dialyze it in 2 000 mL of 0.05 mmol / L CB Buffer (0.05 mol / L CB Buffer: Na2CO3 3.18 g + NaHCO3 5.88 g, add distilled water, and make up to 2 L) at 4℃ overnight.

[0073] Absorb the dialysate into a 15 mL centrifuge tube, add 0.2 mL of freshly prepared 5 mg / mL NaBH4 solution, mix well, and then place it at 4℃ for 2 h. Take out the labeled solution after dialysis, add an equal volume of glycerol, and store it at -20℃.

[0074] (2) Biotin labeling of the monoclonal antibody

[0075] Take 1 mg of biotin, equilibrate it to room temperature, add 180 μL of ultrapure water, and dissolve and mix well.

[0076] Add 13.5 mg of the treated IBRV monoclonal antibody, mix well, and then place it at room temperature for 60 min. Add the coupling solution to the ultrafiltration tube, add PBS to the white line at the top of the tube, and centrifuge it at 12000 rpm and 4℃ for 10 min.

[0077] Discard the waste liquid, wash it with PBS three times, and recover the coupling solution.

[0078] Transfer the coupling solution to a clean centrifuge tube, then add an equal volume of glycerol, record the final volume and concentration, and store it at -20℃ for future use.

[0079] Example 4: Kit for detecting bovine infectious nasal tracheitis virus antibody based on enzyme-linked immunosorbent assay

[0080] In this example, the main components of the kit for detecting bovine infectious nasal tracheitis virus antibody based on enzyme-linked immunosorbent assay are: coated enzyme-labeled plate, horseradish peroxidase-labeled monoclonal antibody (HRP-labeled antibody), positive control, negative control, diluent, washing solution, luminescent substrate solution A, luminescent substrate solution B, and termination solution.

[0081] The preparation method of the coated enzyme-labeled plate specifically includes the following steps:

[0082] S401, antigen coating: the bovine infectious rhinotracheitis virus gD multi-epitope fusion protein is diluted into a coating solution with a final concentration of 2 μg / mL using a CBS buffer (pH = 9.6), 100 μL / well of the coating solution is added to an enzyme-labeled plate, 4°C coating for 12-18 h, and the solution in the wells of the enzyme-labeled plate is discarded; PBST buffer (0.05% Tween-20, pH = 7.4) is added for washing, 300 μL / well, washing 5 times, 3 min / time;

[0083] S402, blocking: 200 μL / well of blocking solution is added to the enzyme-labeled plate obtained in step S301, 37°C blocking for 2 h, the solution in the wells of the enzyme-labeled plate is discarded; PBST buffer (0.05% Tween-20, pH = 7.4) is added for washing, 300 μL / well, washing 5 times, 3 min / time; to obtain a coated enzyme-labeled plate.

[0084] The blocking solution is ovalbumin (OVA) diluted with a PBS buffer (pH = 7.4), and the concentration of the OVA blocking solution is 5% (w / v).

[0085] Luminescent substrate solution A: prepared from 3,3',5,5'-tetramethylbenzidine (200 mg) and anhydrous ethanol (100 mL).

[0086] Luminescent substrate solution B: to Na2HPO4 (14.6 g) and citric acid (9.33 g), add water for injection to 900 mL, adjust the pH to 5.0-5.4, and add water for injection to 1000 mL.

[0087] In this embodiment, the working principle of the kit for detecting bovine infectious rhinotracheitis virus antibody based on enzyme-linked immunosorbent assay is as follows: the competitive method is used to determine whether the sample contains bovine infectious rhinotracheitis virus (IBRV) antibody. The IBRV antigen (i.e., the bovine infectious rhinotracheitis virus gD multi-epitope fusion protein of the present application) is used to coat the enzyme-labeled plate to form a stationary phase; the sample is added to the microwells of the coated enzyme-labeled plate, and then the HRP-labeled IBRV monoclonal antibody is added; the antibody in the sample and the HRP monoclonal antibody label compete with the IBRV antigen to form an antigen-antibody / antigen-enzyme-labeled antibody complex. After washing, the two-component 3,3',5,5'-tetramethylbenzidine (TMB) color developing solution is added; TMB is converted to blue under the catalysis of HRP and finally converted to yellow under the action of acid. The color depth is negatively correlated with the content of IBRV antibody in the sample; the absorbance (OD value) is measured at 450 nm wavelength by an enzyme-labeled instrument, and whether the sample contains IBRV antibody is calculated by the OD value.

[0088] The working process of the kit for detecting bovine infectious nasal tracheitis virus antibody based on enzyme-linked immunosorbent assay specifically comprises the following steps:

[0089] S401, preparation of a to-be-tested sample: dilute the to-be-tested serum sample 20 times with PBST buffer (0.05% Tween-20, pH=7.4); specifically, 10 μL of the to-be-tested serum sample and 180 μL of the PBST buffer;

[0090] S402, incubation: add negative quality control samples, positive quality control samples and the to-be-tested sample into the coated enzyme-labeled plate respectively, 50 μL per well, mix well; then add HRP-labeled monoclonal antibody solution, 50 μL per well, mix well; seal the enzyme-labeled plate with a sealing film, and incubate at 37°C for 30 min;

[0091] The HRP-labeled monoclonal antibody solution is prepared by diluting HRP monoclonal antibody marker solution with PBST buffer (0.05% Tween-20, pH=7.4) at a dilution ratio of 1:10000 (mass / volume ratio w / v);

[0092] S403, washing: discard the solution in the wells of the enzyme-labeled plate, add PBST buffer (0.05% Tween-20, pH=7.4) for washing, 300 μL per well, wash for 5 times, 3 min per time;

[0093] S404, color development and termination: add two-component TMB color developing liquid (luminescent substrate liquid A and luminescent substrate liquid B prepared at a volume ratio of 1:1) into the enzyme-labeled plate, 100 μL per well, mix well, develop color at 37°C for 10 min in the dark; then add 2M concentrated sulfuric acid, 50 μL per well, gently shake the enzyme-labeled plate until the color develops uniformly;

[0094] S405, reading: place the enzyme-labeled plate into an enzyme-labeled instrument, and determine and read the absorbance OD value under the condition that the test wavelength is 450 nm and the reference wavelength is 630 nm.

[0095] Result determination:

[0096] The OD value of the negative quality control sample is denoted as ODN, the OD value of the positive quality control sample is denoted as ODP, and the OD value of the to-be-tested sample is denoted as ODS; the Cut Off value (threshold value) =ODN+0.25;

[0097] The detection condition is established: ODP value≤0.3 and ODN value≥0.8, the detection result is valid, otherwise, the detection is re-performed;

[0098] Result determination: the ODS value of the to-be-tested sample≤Cut Off value, the result is positive; the ODS value of the to-be-tested sample> Cut Off value, the result is negative.

[0099] Example 5: Kit for detecting bovine infectious nasal tracheitis virus antibody based on magnetic particle chemiluminescence method

[0100] In this embodiment, the working principle of the kit for detecting bovine infectious nasal tracheitis virus antibody based on magnetic particle chemiluminescence method is as follows: using the competitive immunization principle and combining with the magnetic particle separation technology, the sample to be tested, biotin-labeled monoclonal antibody, acridinium ester-labeled bovine infectious nasal tracheitis virus gD multi-epitope fusion protein and streptavidin magnetic beads are mixed and incubated to form an immune complex. After removing the unbound impurities by washing with a solid-phase carrier magnetic particle, the luminescent substrate is added to promote luminescence, and the relative luminescence intensity (RLU) is measured. Within a certain range, RLU is inversely proportional to the titer of bovine infectious nasal tracheitis virus antibody, and the standard curve built in the instrument is used to output RLU=corresponding bovine infectious nasal tracheitis virus antibody titer value.

[0101] In this embodiment, the main components of the kit for detecting bovine infectious nasal tracheitis virus antibody based on magnetic particle chemiluminescence method are: magnetic bead working solution, antibody working solution, acridinium ester label working solution, pre-activation solution, activation solution, calibrator, positive quality control, and negative quality control.

[0102] Magnetic bead working solution: prepared from 225ul of streptavidin magnetic beads and 4275ul of PBS-BSA solution (pH=7.4).

[0103] Antibody working solution: prepared from 5.5ug of biotin-labeled monoclonal antibody and 11000ul of PBS-BSA solution (pH=7.4).

[0104] Acridinium ester label working solution: prepared from 1.1ug of acridinium ester-labeled bovine infectious nasal tracheitis virus gD multi-epitope fusion protein and 11000ul of PBS-BSA solution (pH=7.4).

[0105] Pre-activation solution: 0.1 M hydrochloric acid solution containing 0.1% hydrogen peroxide

[0106] Activation solution: 0.25 M sodium hydroxide solution containing 2% Triton X-100

[0107] The working process of the kit for detecting bovine infectious nasal tracheitis virus antibody based on magnetic particle chemiluminescence method includes the following steps:

[0108] Sample (20ul) + magnetic bead working solution (20ul) + acridinium ester label working solution (50ul) and antibody working solution (50ul), 37°C reaction for 5-30min, 0.1mol / L PBS buffer solution washing, adding luminescent substrate solution A (100ul) and luminescent substrate solution B (100ul) at 37°C for 5min, detecting the luminescence value.

[0109] Result determination:

[0110] S / N value = OD450nm value of sample to be tested / average OD450nm value of negative control

[0111] Established condition: OD450nm value of negative control 450nm Average value-OD450nm value of positive control 450nm Average value ≥ 0.3, the detection result is valid, otherwise retest.

[0112] Result determination: S / N value ≥ 0.4, judged as negative; S / N value < 0.4, judged as positive.

[0113] Example 6: Kit performance test

[0114] S601, Sensitivity test

[0115] First, the bovine infectious nasal tracheitis virus antibody positive quality control was diluted by 2 times gradient with PBS buffer (0.05% Tween-20, pH = 7.4) as diluent, and 2 times, 4 times, 8 times, 16 times, 32 times, 64 times, 128 times, 256 times, 512 times, 1024 times, 2048 times, 4096 times sensitivity quality control was obtained.

[0116] Then, the sensitivity of the sensitivity quality control was tested by the IBRV ELISA antibody detection kit of a certain product brand (Senocare), the IBRV competitive ELISA antibody detection kit of the application and the competitive CLIA antibody detection kit of the application, and the test results were compared and analyzed. Among them, the test results are shown in Table 1, the 512 times sensitivity quality control can be detected by the certain product brand, the 1024 times sensitivity quality control can be detected by the IBRV competitive ELISA antibody detection kit of the application, and the 2048 times sensitivity quality control can be detected by the competitive CLIA antibody detection kit of the application. The sensitivity of the two detection methods of the application is better than that of the certain product brand.

[0117]

[0118] S602, Specificity test

[0119] When the enzyme-linked immunosorbent assay method for detecting IBRV antibody and the magnetic particle chemiluminescence method for detecting IBRV antibody constructed by the application were used to detect IBRV, BVDV, BRSV, PRV, BCoV positive serum, the results are shown in Table 2, the method has no cross reaction with other susceptible animal viruses, and has good specificity.

[0120]

[0121] S603, repeatability test

[0122] Four known background pig serum samples (one strong positive serum sample, one positive serum sample, one weak positive serum sample and one negative serum sample of IBRV virus antibody) were selected, and 20 repeated tests were performed by the competitive ELISA antibody detection method and the competitive CLIA antibody detection method established in the present application. The coefficient of variation was calculated according to the absorbance value or luminescence value of the serum sample. The results are shown in Table 3. The coefficient of variation of the strong positive sample of the competitive ELISA antibody detection method established in the present application was 3.82%, the positive coefficient of variation was 2.72%, the weak positive coefficient of variation was 2.9%, and the negative serum coefficient of variation was 1.89%. The strong positive serum coefficient of variation of the competitive CLIA antibody detection method established in the present application was 1.42%, the positive serum coefficient of variation was 1.50%, the weak positive serum coefficient of variation was 2.06%, and the negative serum coefficient of variation was 2.59%. It is shown that the IBRV antibody detection method established in the present application has high repeatability.

[0123]

[0124] S604, clinical sample coincidence rate test

[0125] The IBRV competitive ELISA antibody detection method and the competitive CLIA antibody detection method of the present application simultaneously detected 176 clinical samples with known background, compared the detection results of the two methods, and calculated the coincidence rate of the two methods. The results are shown in Table 4. The Kappa value of the two methods is 0.98, and the overall coincidence rate is 99.32%, indicating that the coincidence rate of the two methods is high.

[0126]

[0127]

[0128]

[0129] The above provides a bovine infectious nasal tracheitis virus multi-epitope fusion protein, its encoding gene and its use in detecting bovine infectious nasal tracheitis virus antibody. The principles and implementation modes of the present application are described by specific examples. The above examples are only used to help understand the method and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A mouse anti-bovine infectious rhinotracheitis virus monoclonal antibody or an antigen-binding fragment thereof, characterized in that: The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the murine anti-bovine infectious rhinotracheitis virus monoclonal antibody or antigen-binding fragment thereof are positions 23-34, 52-63, and 83-98 of SEQ ID NO. 6, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are positions 24-34, 50-60, and 89-97 of SEQ ID NO. 7, respectively; Furthermore, the antibody or antigen-binding fragment thereof can specifically bind to bovine infectious rhinotracheitis virus.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein The monoclonal antibody or its antigen-binding fragment comprises three heavy chain variable region framework regions: HFR1, HFR2 and HFR3 and four light chain variable region framework regions: LFR1, LFR2, LFR3 and LFR4, wherein The amino acid sequence of HFR1 is shown in positions 1-22 of SEQ ID NO.6; The amino acid sequence of HFR2 is shown in positions 35-51 of SEQ ID NO.6; The amino acid sequence of HFR3 is shown in positions 64-82 of SEQ ID NO.6; The amino acid sequence of LFR1 is shown in positions 1-23 of SEQ ID NO.7; The amino acid sequence of LFR2 is shown in positions 35-49 of SEQ ID NO.7; The amino acid sequence of LFR3 is shown in positions 61-88 of SEQ ID NO.7; The amino acid sequence of LFR4 is shown in positions 98-107 of SEQ ID NO.

7.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The heavy chain variable region of the mouse-derived anti-bovine infectious rhinotracheitis virus monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO.6; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.

7.

4. An isolated polynucleotide, characterized in that It encodes the mouse-derived anti-bovine infectious rhinotracheitis virus monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2; The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.3; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.

4.

5. A carrier, characterized in that It comprises the polynucleotide according to claim 4.

6. A host cell, characterized in that It comprises the polynucleotide according to claim 4 or the vector according to claim 5.

7. A bovine infectious rhinotracheitis virus gD multi-epitope fusion protein, characterized in that: The amino acid sequence of the bovine infectious rhinotracheitis virus gD multi-epitope fusion protein is shown in SEQ ID NO.5, the nucleotide sequence encoding the bovine infectious rhinotracheitis virus gD multi-epitope fusion protein is shown in SEQ ID NO.2, and the nucleotide sequence of the nucleotide sequence SEQ ID NO.2 encoding the bovine infectious rhinotracheitis virus gD multi-epitope fusion protein before codon optimization is shown in SEQ ID NO.

1.

8. A mouse anti-bovine infectious rhinotracheitis virus monoclonal antibody conjugate, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and a label conjugated to the antibody or fragment, wherein the monoclonal antibody is obtained by immunizing with the infectious bovine rhinotracheitis virus gD multi-epitope fusion protein.

9. The mouse anti-bovine infectious rhinotracheitis virus monoclonal antibody conjugate according to claim 8, characterized in that: The marker is selected from one or more of horseradish peroxidase markers, biotin markers, fluorescent dye markers, and chemiluminescent dye markers.

10. A method for preparing monoclonal antibodies against bovine infectious rhinotracheitis virus, characterized in that: The method comprises the following steps: immunizing an animal with purified bovine infectious rhinotracheitis virus gD multi-epitope fusion protein; isolating spleen lymphocytes from the immunized animal; fusing the cells with myeloma cells to produce hybridomas; screening a hybridoma that produces the monoclonal antibody according to any one of claims 1 to 3, which is named 3D6; culturing the hybridoma and recovering the monoclonal antibody; and sequencing the heavy chain and light chain variable regions of the monoclonal antibody.

11. Use of the monoclonal antibody or antigen-binding fragment thereof according to claim 3, or the infectious bovine rhinotracheitis virus gD multi-epitope fusion protein according to claim 8, in the preparation of a diagnostic reagent for detecting infectious bovine rhinotracheitis virus.

12. A kit for detecting bovine infectious rhinotracheitis virus antibodies, characterized in that: The invention comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and / or the bovine infectious rhinotracheitis virus gD multi-epitope fusion protein according to claim 7.

13. The kit according to claim 12, wherein It is an enzyme-linked immunosorbent assay kit or a magnetic particle chemiluminescence assay kit.

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