Antibody against chicken interleukin-1β precursor protein and its application

Abstract

The present invention discloses a hybridoma cell with a microbial deposit number of CCTCC NO: C2024399, and discloses a monoclonal antibody secreted by the hybridoma cell. The antibody can recognize chicken interleukin-1β precursor protein.

Description

Technical Field

[0001] The invention belongs to the field of biological products and relates to an antibody against chicken interleukin-1β precursor protein and application thereof. Background Art

[0002] Inflammation is the body's own defense response to tissue damage or microbial infection. These exogenous stimuli cause immune cells to migrate rapidly to the damaged or infected site. Inflammation is an important innate immune process, the purpose of which is to activate the immune system, fight infection, repair damaged tissues, and restore the body to a steady state. In addition, the hallmarks of the inflammatory response are strong targeting and obvious effects.

[0003] Inflammation is a reaction formed by multiple processes. It is generally believed that it is caused by the accumulation of platelets, mast cells and leukocytes, which release various types of mediators (e.g., cytokines, reactive oxygen species, etc.) to control the affected area in the body, initiate an immune response, eliminate the triggering factors, and thus restore to a physically intact state.

[0004] The main factors that trigger inflammation are microorganisms, viral degradation products and cytokines, such as interleukin-1β (IL-1β). These substances determine the occurrence of inflammation by interacting with multiple receptors such as Toll-like receptors and IL-1. Among them, IL-1β is one of the cytokines with the highest degree of involvement in the inflammatory process. IL-1β is a proinflammatory cytokine mainly produced by monocytes, macrophages and lymphocytes. As a key regulatory medium of inflammatory response and the body's immune system, it can trigger a variety of inflammatory responses and activate the gene expression and release of inflammatory cytokines, playing a vital role in the occurrence of inflammatory diseases. Interleukin-1β precursors (pro-IL-1β) are the forms of mature IL-1β before being cleaved. As early as 1989, scholars discovered that the proinflammatory cytokine pro-IL-1β can be cleaved into mature IL-1β by the protease caspase-1. During this process, whether pro-IL-1β is cleaved can be used to determine whether an inflammatory response occurs in the body. Therefore, pro-IL-1β plays an important role in initiating early proinflammatory immune responses and is also a marker for detecting early proinflammatory immune responses. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a hybridoma cell, wherein the hybridoma cell is a hybridoma cell with a microbial preservation number of CCTCC NO: C2024399 or a subculture cell of a hybridoma cell with a microbial preservation number of CCTCC NO: C2024399; the monoclonal antibody secreted by the subculture cell of the hybridoma cell with a microbial preservation number of CCTCC NO: C2024399 maintains specific binding activity to chicken interleukin-1β precursor protein.

[0006] In some embodiments, the amino acid sequence of the chicken interleukin-1β precursor protein is as shown in SEQ ID NO.6.

[0007] A second aspect of the present invention provides a biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10;

[0008] P1: Monoclonal antibody

[0009] The monoclonal antibody is the monoclonal antibody secreted by the hybridoma cell described in the first aspect of the present invention;

[0010] P2: Genetically engineered monoclonal antibodies

[0011] The genetically engineered monoclonal antibody retains specific binding activity to chicken interleukin-1β precursor protein;

[0012] The genetically engineered monoclonal antibody comprises a heavy chain of a genetically engineered monoclonal antibody and a light chain of a genetically engineered monoclonal antibody;

[0013] The heavy chain of the genetically engineered monoclonal antibody includes a heavy chain CDR1 of the genetically engineered monoclonal antibody, a heavy chain CDR2 of the genetically engineered monoclonal antibody, and a heavy chain CDR3 of the genetically engineered monoclonal antibody;

[0014] The light chain of the genetically engineered monoclonal antibody includes a light chain CDR1 of the genetically engineered monoclonal antibody, a light chain CDR2 of the genetically engineered monoclonal antibody, and a light chain CDR3 of the genetically engineered monoclonal antibody;

[0015] The heavy chain CDR1 of the genetically engineered monoclonal antibody is identical to the heavy chain CDR1 protein sequence of the monoclonal antibody secreted by the hybridoma cell described in the first aspect of the present invention;

[0016] The heavy chain CDR2 of the genetically engineered monoclonal antibody is identical to the heavy chain CDR2 protein sequence of the monoclonal antibody secreted by the hybridoma cell described in the first aspect of the present invention;

[0017] The heavy chain CDR3 of the genetically engineered monoclonal antibody is identical in sequence to the heavy chain CDR3 protein sequence of the monoclonal antibody secreted by the hybridoma cell described in the first aspect of the present invention;

[0018] The light chain CDR1 of the genetically engineered monoclonal antibody is identical in protein sequence to the light chain CDR1 of the monoclonal antibody secreted by the hybridoma cell described in the first aspect of the present invention;

[0019] The light chain CDR2 of the genetically engineered monoclonal antibody is identical in protein sequence to the light chain CDR2 of the monoclonal antibody secreted by the hybridoma cell described in the first aspect of the present invention;

[0020] The light chain CDR3 of the genetically engineered monoclonal antibody is identical in sequence to the light chain CDR3 protein sequence of the monoclonal antibody secreted by the hybridoma cell described in the first aspect of the present invention;

[0021] P3: Antibody derivatives

[0022] The antibody derivative retains specific binding activity to chicken interleukin-1β precursor protein;

[0023] The heavy chain CDR1 of the antibody derivative is identical to the heavy chain CDR1 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to the first aspect of the present invention;

[0024] The heavy chain CDR2 of the antibody derivative is identical to the heavy chain CDR2 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to the first aspect of the present invention;

[0025] The heavy chain CDR3 of the antibody derivative is identical to the heavy chain CDR3 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to the first aspect of the present invention;

[0026] The light chain CDR1 of the antibody derivative is identical to the light chain CDR1 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to the first aspect of the present invention;

[0027] The light chain CDR2 of the antibody derivative is identical to the light chain CDR2 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to the first aspect of the present invention;

[0028] The light chain CDR3 of the antibody derivative is identical to the light chain CDR3 protein sequence of the monoclonal antibody secreted by the hybridoma cell described in the first aspect of the present invention;

[0029] The antibody derivative is in the form of: enzyme-labeled antibody, fluorescent-labeled antibody, chemically modified antibody, antibody Fab fragment, single-chain antibody, chimeric monoclonal antibody and modified monoclonal antibody;

[0030] P4: RNA combination

[0031] The RNA combination includes genetically engineered monoclonal antibody heavy chain RNA and genetically engineered monoclonal antibody light chain RNA;

[0032] The genetically engineered monoclonal antibody heavy chain RNA can be translated to obtain the heavy chain of the genetically engineered monoclonal antibody described in P2;

[0033] The genetically engineered monoclonal antibody light chain RNA can be translated to obtain the genetically engineered monoclonal antibody light chain described in P2;

[0034] P5: Genetic combination

[0035] The coding sequence of the gene combination can encode the heavy chain of the genetically engineered monoclonal antibody described in P2 and the light chain of the genetically engineered monoclonal antibody described in P2;

[0036] P6: Gene expression cassette combination

[0037] The gene expression product in the gene expression cassette combination is the RNA combination described in P4;

[0038] P7: Genetic Engineering Vector

[0039] The genetic engineering vector contains the gene expression cassette described in P6;

[0040] The genetically engineered monoclonal antibody heavy chain RNA and the genetically engineered monoclonal antibody light chain RNA are encoded in one or two vectors;

[0041] P8: Cells

[0042] The cell contains the genetic engineering vector described in P7;

[0043] The coding RNA in the gene expression cassette of the genetic engineering vector is constitutively expressed or artificially induced;

[0044] When the genetically engineered monoclonal antibody heavy chain RNA and the genetically engineered monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells;

[0045] P9: Composition

[0046] The composition contains the monoclonal antibody described in P1, the genetically engineered 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

[0047] P10: Test kit

[0048] The kit contains the monoclonal antibody described in P1, the genetically engineered 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.

[0049] In some embodiments, the amino acid sequence of the chicken interleukin-1β precursor protein is as shown in SEQ ID NO.6.

[0050] The third aspect of the present invention provides use of the hybridoma cell 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 biological agent;

[0051] The biological agents include:

[0052] A protein containing an antigenic determinant of an amino acid sequence as shown in SEQ ID NO.7, which is used for in vivo or in vitro recognition, in vivo or in vitro detection, in vivo or in vitro identification, or in vivo intervention; wherein the antigenic determinant as shown in SEQ ID NO.7 is located on the outer surface of the protein;

[0053] Preparations for evaluating the inflammation-suppressing effects of inflammation-suppressing materials;

[0054] Preparations for evaluating the inflammation-inducing effects of inflammation-promoting materials.

[0055] In some embodiments, the protein containing the antigenic determinant of the amino acid sequence as shown in SEQ ID NO.7 is chicken interleukin-1β precursor protein; and / or

[0056] The inflammation is inflammation with high expression of interleukin-1β precursor.

[0057] The fourth aspect of the present invention provides a method for detecting chicken interleukin-1β precursor protein in cells for non-diagnostic purposes, the method comprising the following steps:

[0058] S1: Fix the cells to be detected on the solid surface to obtain immobilized cells;

[0059] S2: incubating the immobilized cells with a monoclonal antibody to obtain immobilized cells of the first incubation;

[0060] The monoclonal antibody is the monoclonal antibody or genetically engineered monoclonal antibody described in the second aspect of the present invention;

[0061] S3: adding the monoclonal antibody specific binding substance connected with the label to the fixed cells of the first incubation for incubation to obtain the fixed cells of the second incubation;

[0062] S4: Characterizing the marker indirectly combined with the fixed cells in the second incubation, and determining whether chicken interleukin-1β precursor protein exists in the cells to be detected according to the presence or absence of the marker.

[0063] In some embodiments, any one of the following situations A1, A2, A3, A4, A5 and A6 is selected:

[0064] A1: In step S1, the cells to be detected are HD11 cells;

[0065] A2: In step S1, the cells to be detected are HD11 cells treated with anti-inflammatory drugs;

[0066] A3: In step S1, the cells to be detected are HD11 cells that promote inflammatory substance treatment;

[0067] A4: In step S1, the solid surface is the inner surface of the well of the microplate;

[0068] A5: In step S3, the monoclonal antibody specific binding substance connected with the label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in the second aspect of the present invention labeled with a fluorescent molecule; in step S4, the fixed cells in the second incubation are photographed with a fluorescence microscope, and the presence of chicken interleukin-1β precursor protein in the sample to be tested is determined based on the photographic results;

[0069] A6: In step S3, the monoclonal antibody specific binder connected with a label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in the second aspect of the present invention labeled with horseradish peroxidase; in step S4, the fixed cells incubated for the second time are developed with TMB colorimetric solution, and the presence of chicken interleukin-1β precursor protein in the sample to be tested is determined based on the color development result.

[0070] The fifth aspect of the present invention provides a method for detecting chicken interleukin-1β precursor protein in a sample for non-diagnostic purposes, the method comprising the following steps:

[0071] T1: Fix the sample to be tested on the solid surface to obtain an immobilized sample;

[0072] T2: incubating the immobilized sample with a monoclonal antibody to obtain an immobilized sample of the first incubation;

[0073] The monoclonal antibody is the monoclonal antibody or genetically engineered monoclonal antibody described in the second aspect of the present invention;

[0074] T4: adding the monoclonal antibody specific binding substance connected with the label to the immobilized sample of the first incubation for incubation to obtain the immobilized sample of the second incubation;

[0075] T5: Characterizing the label indirectly combined with the immobilized sample in the second incubation, and determining whether chicken interleukin-1β precursor protein exists in the sample to be detected according to the presence or absence of the label.

[0076] In some embodiments, any one of the following situations B1, B2, B3 and B4 is selected:

[0077] B1: In step T1, the sample to be detected is chicken interleukin-1β precursor protein, or the sample to be detected contains chicken interleukin-1β precursor protein;

[0078] B2: In step T1, the solid surface is the inner surface of the well of the microplate;

[0079] B3: In step T3, the monoclonal antibody specific binding substance connected with the label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in the second aspect of the present invention labeled with a fluorescent molecule; in step T4, the immobilized sample of the second incubation is photographed with a fluorescence microscope, and the presence of chicken interleukin-1β precursor protein in the sample to be tested is determined based on the photographic result;

[0080] B4: In step T3, the monoclonal antibody specific binding substance connected with the label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in the second aspect of the present invention labeled with horseradish peroxidase; in step T4, the fixed cells incubated for the second time are developed with TMB colorimetric solution, and it is determined whether chicken interleukin-1β precursor protein exists in the sample to be tested based on the color development result.

[0081] In some embodiments, the amino acid sequence of the chicken interleukin-1β precursor protein is shown in SEQ ID NO.6; and / or

[0082] The sample to be detected is in the form of cell lysate.

[0083] A sixth aspect of the present invention provides a method for screening chicken anti-inflammatory drugs, the screening method comprising the following steps:

[0084] U1: Chicken cell lines were used as control cells; the same chicken cell lines were treated with inflammatory factors to obtain activated cells;

[0085] U2: incubating the control cells with the drug to be tested to obtain drug-treated control cells; in a parallel treatment manner, incubating the activated cells with the drug to be tested to obtain drug-treated activated cells;

[0086] U3: fixing the drug-treated control cells to a solid surface to obtain fixed control cells; fixing the drug-treated activated cells to a solid surface in a parallel treatment manner to obtain fixed activated cells;

[0087] U4: incubating the immobilized control cells with a monoclonal antibody to obtain immobilized control cells of the first incubation; in a parallel treatment manner, incubating the immobilized activated cells with a monoclonal antibody to obtain immobilized activated cells of the first incubation;

[0088] The monoclonal antibody is the monoclonal antibody or genetically engineered monoclonal antibody described in the second aspect of the present invention;

[0089] U5: adding the monoclonal antibody specific binding substance connected with the label to the fixed control cells of the first incubation for incubation to obtain the fixed control cells of the second incubation; in a parallel processing manner, adding the monoclonal antibody specific binding substance connected with the label to the fixed activated cells of the first incubation for incubation to obtain the fixed activated cells of the second incubation;

[0090] U6: Compare the relative intensity of the label in the fixed control cells in the second incubation with that in the fixed activated cells in the second incubation, and determine qualitatively, quantitatively or semi-quantitatively whether the drug to be tested inhibits inflammation.

[0091] In some embodiments, any one of the following is selected from C1, C2, C3, C4, C5, C6, C7 and C8:

[0092] C1: The anti-inflammatory drug is an anti-chicken interleukin-1β precursor drug;

[0093] C2: The inflammation is inflammation with high expression of interleukin-1β precursor;

[0094] C3: The chicken-derived cells are HD11 cells;

[0095] C4: The inflammatory factor is lipopolysaccharide;

[0096] C5: The inflammatory factor is a susceptible virus of the chicken-derived cells;

[0097] C6: The solid surface is the inner surface of the well of the microplate;

[0098] C7: In step U5, the monoclonal antibody specific binding substance connected with the label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in the second aspect of the present invention labeled with a fluorescent molecule; in step U6, the fixed control cells in the second incubation and the fixed activated cells in the second incubation are photographed respectively with a fluorescence microscope, and the difference in fluorescence intensity is used to qualitatively, quantitatively or semi-quantitatively determine whether the drug to be tested inhibits inflammation;

[0099] C8: In step U5, the monoclonal antibody specific binder connected with a label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in the second aspect of the present invention labeled with horseradish peroxidase; in step U6, the fixed control cells in the second incubation and the fixed activated cells in the second incubation are respectively developed with TMB colorimetric solution, and whether the drug to be tested inhibits inflammation is qualitatively, quantitatively or semi-quantitatively determined based on the difference in OD values.

[0100] In some embodiments, the susceptible virus of the chicken-derived cells is infectious bursal disease virus; and / or

[0101] The amino acid sequence of the chicken interleukin-1β precursor protein is shown in SEQ ID NO.6. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 This is the phylogenetic tree of the pro-IL-1β gene in chicken and different species.

[0103] Figure 2 This is a statistical chart of the ELISA titer of pro-IL-1β antibodies in the serum of immunized mice.

[0104] Figure 3 This is a statistical chart of the ELISA titer determination results of chicken pro-IL-1β monoclonal antibody 1E12.

[0105] Figure 4 This is a statistical graph showing the results of determining the monoclonal antibody secretion ability of chicken pro-IL-1β monoclonal antibody hybridoma cells 1E12 of different generations.

[0106] Figure 5 This is a photograph of the Western blot detection results of chicken pro-IL-1β monoclonal antibody on prokaryotic expressed pro-IL-1β protein.

[0107] Figure 6 The following is a photo of the immunofluorescence detection of the eukaryotic pro-IL-1β protein by using the chicken pro-IL-1β monoclonal antibody. The scale bar is 400 μm.

[0108] Figure 7This is a photograph of the Western blot detection results of chicken pro-IL-1β monoclonal antibody on eukaryotic expressed pro-IL-1β protein.

[0109] Figure 8 This is a photo of the IFA detection result of chicken pro-IL-1β monoclonal antibody on endogenous pro-IL-1β protein in HD11 cells. The scale bar is 100μm.

[0110] Fig. 9 Schematic diagram of segmented expression of chicken pro-IL-1β gene and results of antigen epitope identification, wherein: A. first round truncation of pro-IL-1β; B. further truncation of P1 segment; C. further truncation of P1' segment; D. further truncation of C2 segment; E. further truncation of N4 segment; F. Western blot detection corresponding to A; G. Western blot detection corresponding to B; H. Western blot detection corresponding to C; I. Western blot detection corresponding to D; J. Western blot detection corresponding to E. DETAILED DESCRIPTION

[0111] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0112] Materials and instruments not described in the present invention are conventional materials and instruments in the art, 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 instructions of the software provider, and the kit used in the present invention is operated by conventional methods with reference to the kit instruction manual.

[0113] The nucleic acid sequences shown in the present invention are all written from left to right in the direction of 5' to 3', and the proteins are written from left to right in the direction of N-terminus to C-terminus.

[0114] Application scenarios of the method for detecting chicken interleukin-1β precursor protein in cells for non-diagnostic purposes and detecting chicken interleukin-1β precursor protein in samples for non-diagnostic purposes (not the process of identifying, studying and determining the cause or lesion status of living human or animal bodies) include but are not limited to the following situations:

[0115] (1) Conduct scientific experiments to explore the effects of drugs on inflammation control.

[0116] (2) To study the changes in interleukin-1β precursor protein caused by inflammatory factors.

[0117] Example 1: Preparation of chicken interleukin-1β precursor protein

[0118] 1. Amplification of Chicken Pro-IL-1β Gene

[0119] According to the chicken pro-IL-1β gene sequence (GenBank accession number DQ393267), the upstream and downstream primers pro-IL-1β-pCold-F (SEQ ID NO.1) and pro-IL-1β-pCold-R (SEQ ID NO.2) were designed to amplify the full length of the chicken pro-IL-1β gene. HD11 cells that were well cultured were taken, and the total RNA of HD11 cells was extracted using an RNA extraction kit (purchased from Bao Engineering (Dalian) Co., Ltd.) according to the steps in the kit instructions. The total RNA was then reverse transcribed into cDNA using a reverse transcription kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) according to the steps in the kit instructions. Using cDNA as a template, the chicken pro-IL-1β gene sequence was amplified using primers pro-IL-1β-pCold-F and pro-IL-1β-pCold-R. The PCR reaction system was as follows: 2× PrimeSTAR 25μL, upstream primer 1μL, downstream primer 1μL, ddH2O21μL, template 2μL, a total of 50μL. PCR reaction program: 98°C for 5 min; 98°C for 10 s, 55°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 10 min extension. The PCR amplification product (amplified fragment 1) was identified by 1% agarose gel electrophoresis, and the target fragment was purified and recovered using a gel recovery kit (Omega) according to the instructions. The purified target gene was stored at -20°C for future use.

[0120] pro-IL-1β-pCold-F: CATCATCATATCGAAGGTAGGCATATGGAGCTCGGTACCCTCGAGGCGTTCGTTCC

[0121] pro-IL-1β-pCold-R: TCGACAAGCTTGAATTCATGATGATGATGATGATGGCGCCCACTTAGCTTGTAGGTGGCGATGTT

[0122] 2. Construction of chicken pro-IL-1β gene recombinant expression vector

[0123] The prokaryotic expression vector pColdⅠ (TaKaRa) was double-digested with EcoRI and Xho I restriction endonucleases, and the vector pColdⅠ recovered by restriction digestion was homologously recombined with the above-purified amplified fragment 1 using a homologous recombination kit (Novozan) according to the instructions. The correct pro-IL-1β prokaryotic expression recombinant plasmid identified by Sanger sequencing was named pCold-pro-IL-1β.

[0124] The upstream and downstream primers pro-IL-1β-pCAGGS-F (SEQ ID NO. 3) and pro-IL-1β-pCAGGS-R (SEQ ID NO. 4) were designed for cloning chicken pro-IL-1β into the eukaryotic vector pCAGGS.

[0125] pro-IL-1β-pCAGGS-F:CATCATTTTGGCAAAGAATTCGGCGGGGGTAGCATGGCGTTCGTTCCCGACCTGGAC

[0126] pro-IL-1β-pCAGGS-R: CTATTTGTCATCGTCATCTTTATAATCGCTACC CCCGCCGCGCCCACTTAGCTTGTAGGTGGCGAT

[0127] The primers pro-IL-1β-pCAGGS-F and pro-IL-1β-pCAGGS-R were used to obtain the amplified fragment 2 by PCR amplification with the purified amplified fragment 1 as a template. The eukaryotic expression vector pCAGGS was double-digested with EcoRI and Xho I restriction endonucleases, and the vector pCAGGS recovered by enzyme digestion was homologously recombined with the purified amplified fragment 2 according to the instructions using a homologous recombination kit (Novozan). The correct pro-IL-1β eukaryotic expression recombinant plasmid identified by Sanger sequencing was named pCAGGS-pro-IL-1β.

[0128] 3. Sequence Analysis of Chicken Pro-IL-1β Gene

[0129] The sequencing results showed that the amplified chicken pro-IL-1β gene was 804 bp long and encoded 267 aa, which was completely consistent with the nucleotide sequence of the chicken pro-IL-1β reference gene on GenBank (GenBank accession number DQ393267), with a homology rate of 100%.

[0130] The nucleotide sequence of the chicken pro-IL-1β gene is as follows (SEQ ID NO.5):

[0131] ATGGCGTTCGTTCCCGACCTGGACGTGCTGGAGAGCAGCAGCCTCAGCGAAGAGACCTTCTACGGCCCCTCCTGCCTCTGCCTGCAGAAGAAGCCTCGCCTGGATTCTGAGCACACCACAGTGGACGTGCAGGTGACGGTGCGGAAGGGACGTGGTGCCCGGAGCTTTCGGCGGGCCGCCGTGCTGGTGGTGGCCATGACCAAACTGCTGCGGAGGCCGAGGAGCAGGGACTTTGCTGACAGCGACCTGAGCGCGCTCCTGGAGGAGGTTTTTGAGCCCGTCACCTTCCAGCGGCTGGAGAGCAGCTACGCCGGGGCGCCCGCCTTCCGCTACACCCGCTCACAGTCCTTCGACATCTTCGACATCAACCAGAAGTGCTTCGTGCTGGAGTCACCCACCCAGCTGGTGGCCCTGCACCTCCAGGGGCCCTCCTCCAGCCAGAAAGTGAGGCTCAACATTGCGCTGTACCGGCCCCGAGGCCCACGGGGCAGCGCTGGAACTGGGCAGATGCCAGTGGCACTGGGCATCAAGGGCTACAAGCTCTACATGTCGTGTGTGATGAGCGGCACCGAGCCCACACTGCAGCTGGAGGAAGCCGACGTCATGCGGGACATCGACAGCGTCGAGCTGACCCGCTTCATCTTCTACCGCCTGGACAGCCCGACTGAGGGCACCACGCGCTTCGAGTCGGCCGCCTTCCCCGGGTGGTTCATCTGCACCTCCCTGCAGCCCCGGCAGCCCGTGGGCATCACCAACCAACCCGACCAGGTCAACATCGCCACCTACAAGCTAAGTGGGCGCTGA

[0132] The amino acid sequence of the chicken pro-IL-1β gene-encoded protein is as follows (SEQ ID NO.6):

[0133] MAFVPDLDVLESSSLSEETFYGPSCLCLQKKPRLDSEHTTVDVQVTVRKGRGARSFRRAAVLVVAMTKLLRRPRSRDFADSDLSALLEEVFEPVTFQRLESSYAGAPAFRYTRSQSFDIFDINQKCFVLESPT QLVALHLQGPSSSQKVRLNIALYRPRGPRGSAGTGQMPVALGIKGYKLYMSCVMSGTEPTLQLEEADVMRDIDSVELTRFIFYRLDSPTEGTTRFESAAFPGWFICTSLQPRQPVGITNQPDQVNIATYKLSGR

[0134] An evolutionary tree of gene nucleotide sequences was constructed using MAGA6.0 software for chicken pro-IL-1β gene (GenBank accession number DQ393267), duck (pro-IL-1β gene GenBank accession number DQ393268), goose pro-IL-1β gene (GenBank accession number DQ393269), turkey pro-IL-1β gene (GenBank accession number DQ393271), pigeon pro-IL-1β gene (GenBank accession number DQ393270), mouse pro-IL-1β gene (GenBank accession number M15131), porcine pro-IL-1β gene (GenBank accession number NM214055) and human pro-IL-1β gene (GenBank accession number M15840).

[0135] The results are as follows Figure 1 Specifically, chicken pro-IL-1β is closely related to duck, goose, turkey and pigeon, with nucleotide homology of 82.2%-99.9% and amino acid sequence homology of 78.6%-100%; while chicken pro-IL-1β is more distantly related to human, mouse and pig, with nucleotide homology of 38.4%-50.7% and amino acid sequence homology of 9.1%-12.1%.

[0136] 3. Expression and Purification of Chicken Pro-IL-1β

[0137] The recombinant plasmid pCold-pro-IL-1β was transformed into competent cells BL21 (DE3), and a single colony was picked and inoculated into 4 mL of LB culture medium containing ampicillin for 12 h, and then transferred to LB culture medium containing ampicillin at a ratio of 1%, and cultured at 37 ° C and 200 r / min until OD 450nmWhen the pH value was 0.6, add 1.0mmol / LIPTG at a final concentration of 1.0mmol / LI for induction for 22h, the speed was 180r / min, and the temperature was 20℃. The induced bacterial solution was centrifuged at 4500g for 10min, and the supernatant was discarded to collect the bacteria. The bacteria were resuspended in 10mL pre-cooled PBS and washed, repeated 3 times, and finally the bacterial precipitate was resuspended in 1.5mL pre-cooled PBS. The resuspended bacteria were placed in an ice-water mixture environment for full ultrasonic disruption. The supernatant and precipitate obtained after the disrupted bacteria were centrifuged at 13000g, 4℃, and 30min were prepared separately, and SDS-PAGE and Coomassie Brilliant Blue staining were performed on 12.5% ​​separation gel to identify that the target protein was expressed in the form of inclusion bodies.

[0138] The chicken pro-IL-1β recombinant protein expressed in the form of inclusion bodies was purified by gel cutting. Take 500μL of inclusion body sample and perform SDS-PAGE according to the conventional method (S1: 80V, 20min; S2: 120V, 1h 20min). Put the protein gel after electrophoresis into a clean plate, stain it with 0.25mol / L KCl solution precooled at 4℃ for 5min, and carefully cut out the silver-white target band with a surgical blade. Wash the collected target band with PBS once, place it in a 5mL syringe, and then inject it into the glass tube of the electroelution instrument, set the voltage to 120V and the time to 1h for electroelution.

[0139] Take 10 μL of a small amount of eluted protein, dilute it 10 times with PBS, and add SDS-PAGE protein loading buffer (5×) to prepare the sample. The protein purification effect was identified by SDS-PAGE and Western blot. The primary antibody used for Western blot identification was mouse anti-His tag antibody (Sigma), and the secondary antibody was goat anti-mouse IgG labeled with IRDye800CW (Sigma). SDS-PAGE results showed that the molecular weight of the purified chicken pro-IL-1β recombinant protein was about 37 kDa, and Western blot results showed that it could be recognized by anti-His antibody.

[0140] Example 2: Preparation and identification of chicken pro-IL-1β monoclonal antibody

[0141] 1. Mouse Immunization

[0142] The prokaryotic expressed and purified chicken pro-IL-1β recombinant protein (hereinafter referred to as pro-IL-1β protein) solution obtained in Example 1 (the solvent was sterile PBS (0.01 mol / L, pH 7.4)) was mixed with Freund's complete adjuvant at a volume ratio of 1:1, and fully emulsified on a tissue grinder to obtain immunogenic composition 1. The same pro-IL-1β protein solution was mixed with Freund's incomplete adjuvant at a volume ratio of 1:1, and emulsified in the same way to obtain immunogenic composition 2.

[0143] Six-week-old BALB / c mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were selected for immunization, and a total of 5 mice were immunized. Immunogen composition 1 was injected subcutaneously at multiple points on the back to immunize mice, and the immunization dose was 0.1 mg pro-IL-1β protein / mouse. Immunogen composition 2 was used for the second immunization 14 days after the first immunization, and immunogen composition 2 was used for the third immunization 14 days after the second immunization. The immunization method and dose were the same as the first immunization. Blood was collected from the tip of the mouse tail 7 days after the third immunization, and the serum was separated for antibody titer detection.

[0144] 2. Detection of Mouse Serum Antibodies

[0145] The purified pro-IL-1β protein was coated on the ELISA plate, and the antibody titer of the sera of immune mice was detected by ELISA method. Unimmunized mice were used as negative controls. The specific operations are as follows: (1) The purified pro-IL-1β protein (prepared in Example 1) was diluted to 12 μg / mL with carbonate coating solution, 100 μL was added to each well of the ELISA plate, and coated overnight at 4°C; (2) The coated ELISA plate was taken out, the coating solution in the well was discarded, and patted dry; (3) 100 μL of 5% skim milk (solvent is PBS) was added to each well and incubated at 37°C for 1 hour, the liquid in the well was discarded, and patted dry; (4) PBST (200 μL / well) was added and washed 3 times, and patted dry; (5) The serum sample to be tested (mouse serum obtained in step 1) was diluted with serum diluent The release solution (PBS) was diluted 7 times in a 2-fold ratio starting from 1:1000 (volume ratio), and 100 μL was added to each well. The wells were incubated at 37°C for 1 hour, and the liquid in the wells was discarded and patted dry; (6) PBST (200 μL / well) was added to wash 4 times, and the wells were patted dry; (7) HRP-labeled goat anti-mouse IgG secondary antibody (purchased from Sigma) was diluted with PBS at a volume ratio of 1:3000, 100 μL / well, and incubated at 37°C for 1 hour. The liquid in the wells was discarded and patted dry; (8) PBST (200 μL / well) was added to wash 4 times, and the wells were patted dry; (9) 100 μL of TMB substrate colorimetric solution was added to each well and incubated at 37°C for 15 minutes; (10) 100 μL of stop solution (2M H2SO4) was added to each well, and the ELISA plate was immediately placed in an ELISA instrument to measure the absorbance OD 450 nm, when the ratio of the absorbance value of the sample well to the negative control well is greater than 2.1, it is determined to be positive. The ELISA titer statistics of pro-IL-1β antibodies in the serum of the immunized mouse with the highest antibody titer (mouse a) refer to Figure 2 It can be seen that the ELISA titer of pro-IL-1β serum can reach 1:32000, and the anti-pro-IL-1β antibody produced by this mouse has good efficacy, and subsequent research will be carried out on this mouse.

[0146] 3. Hybridoma Cell Preparation

[0147] (1) Cultivation of myeloma cells SP2 / 0

[0148] Cell recovery: Take out the frozen myeloma cells SP2 / 0 from liquid nitrogen, immediately put them in a 37℃ water bath for 5 minutes, and take them out immediately after the cells are thawed. Transfer the cells to a sterile 15mL centrifuge tube in a biosafety cabinet, add 5mL of preheated DMEM culture medium, gently pipette to mix, and centrifuge at 1000g for 5min. Discard the supernatant, add 2mL of DMEM culture medium, pipette to mix, transfer to a cell culture bottle, add 4mL of DMEM culture medium and mix, and culture in a 37℃, 5% CO2 incubator.

[0149] Cell passaging: Observe the cell status and density. When the cell density reaches more than 90%, perform cell passaging. Discard the original DMEM culture medium in the culture bottle, add 4mL of sterile PBS along the side wall, rinse slowly once, add appropriate amount of DMEM culture medium, and blow away the cells. Transfer the cell suspension to a new culture bottle, add appropriate amount of DMEM culture medium, mix well, and culture in a 37℃, 5% CO2 incubator.

[0150] (2) Preparation of mouse spleen cells

[0151] The serum antibody titer of mouse a meets the standard, and mouse a is boosted. The boosting immunization method is the same as the second immunization. Splenocytes are prepared 3 days after the boosting immunization. ① Take the aforementioned mouse a for boosting immunization, remove the eyeballs, bleed and collect serum; ② After the mouse is killed by dislocation of the neck, soak it in 75% alcohol for 15 minutes, then fix the mouse, cut the peritoneum, bluntly separate the spleen, take an appropriate amount of DMEM culture fluid, rinse the surface, and remove excess fat and connective tissue as much as possible; ③ Place the spleen in a clean culture dish, add 25mL DMEM culture fluid, pierce the spleen with a needle, and then use a 20mL syringe to take DMEM and inject it from one end of the spleen, and rinse repeatedly until the spleen turns white; ④ Transfer the spleen cell suspension to a 50mL centrifuge tube, mix well, and count the cells.

[0152] (3) Cell fusion

[0153] ① Resuspend SP2 / 0 cells with 25mL warm DMEM culture medium and count the cells; ② Mix spleen cells and SP2 / 0 cells at a ratio of 7:1, centrifuge at 1200g for 5min at room temperature, discard the supernatant, and gently tap the bottom to resuspend and mix the cells; ③ Place the centrifuge tube in 37℃ warm sterile water, drop 1mL PEG cell fusion agent (sigma) into the centrifuge tube within 1min, and then let it stand for 4min; ④ Slowly add 20mL warm DMEM culture medium within 4min to terminate the fusion, centrifuge at 1200g for 5min at room temperature, and discard the supernatant; ⑤ Resuspend the cells with HAT culture medium, add 10mL Hybridoma Feeder to add factors, and then add HAT culture medium to 200mL culture medium to resuspend the cells; ⑥ Dispense the cell suspension into five 96-well cell plates at a dose of 200μL / well, observe the cell growth after 5d and supplement DMEM culture medium.

[0154] After 7 days of fusion, when HAT medium was used for supplementation, hybridoma cells with initial cell clusters could be seen. After 10 days of fusion, the hybridoma cell clusters in the 96-well plate became larger, the cells were round in shape, and in good condition.

[0155] 4. Screening and subcloning of positive hybridoma cells

[0156] The indirect ELISA method (the detection method is the same as in Section 2 above) was used to detect the titer of pro-IL-1β antibody in the supernatant of hybridoma cells. Fifteen wells with high OD values ​​and only single cell clusters were selected from five 96-well cell plates. The culture medium in the wells was discarded, and the HT culture medium was added again to gently disperse the cell clusters. The wells were transferred to 15 48-well plates respectively. After full growth, the cells were expanded and cultured, and the cell supernatants were tested for antibodies again. Then, one well with the highest absorbance value and only a single cell cluster was selected and subcloned using a flow cytometry sorting system. The cells with the highest absorbance value and only a single cell cluster were blown into a suspension, and the cells were diluted with HT culture medium. The cells were divided into 1 cell / well by a flow cytometry sorting system and added to a 96-well cell culture plate (each well containing 200 μL HT culture medium). The cells were placed in a 37°C constant temperature incubator for 5-8 days, and the supernatant was tested for titer, and then a second subcloning was performed (the method is the same as above). After secondary subcloning, all wells with single cell clusters can be detected as positive and have high OD values, which means that the cells in the positive wells are monoclonal hybridoma cell lines that secrete pro-IL-1β antibodies. The obtained hybridoma cell line that can secrete pro-IL-1β monoclonal antibodies is named hybridoma cell line ch-pro-IL-1β-1E12, also called pro-IL-1β-MAb-1E12 strain (abbreviated as 1E12 strain). The monoclonal antibody secreted by the 1E12 strain is called antibody 1E12.

[0157] The hybridoma cell line 1E12 prepared by the present invention is submitted to a patent procedure approved depository for deposit. The depository is China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microbial deposit number is CCTCC NO: C2024399; the culture name is: Hybridoma cell line ch-pro-IL-1β-1E12 Hybridoma cell line ch-pro-IL-1β-1E12; the Chinese classification name is: Hybridoma cell line; the English classification name is: Hybridoma CellLine; the deposit time is December 4, 2024; the identified survival time is December 12, 2024.

[0158] 5. Expansion of hybridoma cells and preparation of monoclonal antibody ascites

[0159] The hybridoma cell line 1E12 identified as positive was gradually expanded and cultured. When cultured to a 6-well plate, the titer of the hybridoma cell supernatant was again determined by ELISA (the detection method was the same as in Section 2 above). Cells that were still positive were further expanded and cultured for cell cryopreservation and preparation of ascites monoclonal antibodies.

[0160] The process of ascites preparation is as follows: take 5 healthy BALB / c female mice aged 6 weeks, inject 0.3 mL of Freund's incomplete adjuvant per mouse intraperitoneally, and use it 5-7 days later. Prepare a suspension of positive hybridoma cells 1E12 in the logarithmic growth phase with PBS, and adjust the cell density to 6.7×10 6 Each mouse was injected intraperitoneally with approximately 2.0×10 6 One week later, the ascites was collected and centrifuged at 10,000 g for 10 min. The upper layer of light yellow clear liquid was carefully extracted, which was the monoclonal antibody ascites and stored at -80°C for later use.

[0161] 6. Characterization of Monoclonal Antibodies

[0162] (1) Determination of monoclonal antibody titer

[0163] Mouse ascites was diluted with PBS at a ratio of 1:2 11 Start by diluting the monoclonal antibody in 8 steps of 2-fold dilution. Use indirect ELISA to detect the titer of the monoclonal antibody. The indirect ELISA operation steps are the same as in Section 2. Figure 3 . It can be seen that the antibody titer in the monoclonal antibody ascites is 1:2 15 .

[0164] (2) Identification of monoclonal antibody subclasses

[0165] According to the instructions, the pro-IL-1β monoclonal antibody was identified by using the mouse monoclonal antibody Ig class / subclass identification ELISA kit (supplier: Beijing Biolong Immunotechnology Co., Ltd.). The 1E12 monoclonal antibody was identified as IgG1 by the mouse Ig class / subclass identification kit.

[0166] (3) Stability determination of hybridoma cell-secreted antibodies

[0167] The positive hybridoma cell line 1E12 was cultured in DMEM medium for 20 generations. The culture method and operation details of each generation were the same. The pro-IL-1β antibody titer in the supernatant of each generation of cells collected was detected by indirect ELISA (the detection method is the same as in Section 2 above). Statistical results are shown in Figure 4 It can be seen that each generation of cells can stably secrete pro-IL-1β monoclonal antibody.

[0168] (4) Initial application of monoclonal antibodies

[0169] In order to test the practicality of pro-IL-1β monoclonal antibody 1E12, IFA and Western blot were used to detect the reactivity of pro-IL-1β monoclonal antibody to prokaryotic and eukaryotic pro-IL-1β proteins. The reactivity of pro-IL-1β monoclonal antibody to endogenous pro-IL-1β expression induced by LPS and IBDV was then detected.

[0170] ① Detection of prokaryotic expressed proteins by monoclonal antibodies

[0171] The purified prokaryotic recombinant protein pro-IL-1β in Example 1 was subjected to SDS-PAGE and then transferred to the NC membrane. The diluted pro-IL-1β monoclonal antibody 1E12 (1:1000) was used as the primary antibody, and the goat anti-mouse IgG labeled with IRDye800CW was used as the secondary antibody (Sigma). The reactivity of the pro-IL-1β monoclonal antibody to the prokaryotic expression protein of pro-IL-1β was detected by Western blot technology. The results are shown in Figure 5 It can be seen that the prepared pro-IL-1β monoclonal antibody 1E12 can be used to detect the prokaryotic expression protein of pro-IL-1β by Western blot technology.

[0172] ② Detection of eukaryotic expressed proteins by monoclonal antibodies

[0173] The recombinant eukaryotic expression plasmid pCAGGS-pro-IL-1β and the empty plasmid pCAGGS obtained in Example 1 were transfected into DF-1 cells for 30 hours to perform eukaryotic expression of pro-IL-1β. 30 hours after transfection, IFA detection was performed using pro-IL-1β monoclonal antibody 1E12 (1:1000) as the primary antibody and FITC-labeled goat anti-mouse IgG (Sigma) as the secondary antibody. The results are shown in Figure 6 It can be seen that the prepared pro-IL-1β monoclonal antibody 1E12 can be used to detect eukaryotically expressed pro-IL-1β protein by IFA technology.

[0174] In addition, the recombinant eukaryotic expression plasmid pCAGGS-pro-IL-1β and the empty plasmid pCAGGS were transfected into HD11 cells for 30 h, respectively, and Western blot detection was performed using pro-IL-1β monoclonal antibody 1E12 (1:1000) as the primary antibody and IRDye800CW-labeled goat anti-mouse IgG (Sigma) as the secondary antibody.

[0175] Results Figure 7 It can be seen that the prepared pro-IL-1β monoclonal antibody 1E12 can be used to detect the pro-IL-1β eukaryotic expression protein by Western blot technology.

[0176] ③ Detection of endogenous pro-IL-1β by monoclonal antibody

[0177] The highly virulent strain of infectious bursal disease virus vvIBDV HLJ0504 (GenBank No. GQ451330.1) was cultured at 2.5×10 8 HD11 cells were inoculated with 1 μg / mL of LPS (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) for 6 h (IBDV group); in addition, HD11 cells were stimulated with 1 μg / mL of LPS (lipopolysaccharide, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) for 6 h (LPS group); blank cell controls without LPS or virus inoculation were set up at the same time (MOCK group). After 6 h of LPS stimulation or IBDV inoculation, the lysates of the three groups of cells were collected, and the pro-IL-1β monoclonal antibody 1E12 (1:1000) was used as the primary antibody. The reactivity of the pro-IL-1β monoclonal antibody 1E12 to endogenously expressed pro-IL-1β in cells was detected by IFA. FITC goat anti-mouse IgG (sigma) was used as the secondary antibody in IFA.

[0178] Results Figure 8 It can be seen that the prepared pro-IL-1β monoclonal antibody 1E12 can be used to detect endogenous pro-IL-1β protein in cells by Western blot technology.

[0179] 7. Identification of the antigenic epitope recognized by the chicken pro-IL-1β monoclonal antibody

[0180] The pro-IL-1β gene (GenBank accession number DQ393267) was truncated and expressed, and the antigenic epitope recognized by the pro-IL-1β monoclonal antibody 1E12 was identified by Western blot technology. Design and synthesize primers corresponding to each peptide segment, and use each peptide segment primer to obtain each peptide segment gene fragment by PCR amplification using the pCold-pro-IL-1β plasmid obtained in Example 1 as a template. The first round is to connect the amplification product of the primer corresponding to the peptide segment with the DNA fragment corresponding to the Flag protein by the fusion PCR method to form a recombinant DNA fragment (the downstream of the recombinant DNA fragment is the Flag coding sequence), and the second to fifth rounds are to connect the amplification product of the primer corresponding to the peptide segment with the DNA fragment corresponding to the EGFP protein by the fusion PCR method to form a recombinant DNA fragment (the upstream of the recombinant DNA fragment is the EGFP coding sequence), and each recombinant DNA fragment is connected to the eukaryotic expression vector pCAGGS by homologous recombination using a homologous recombination kit (Novozan) according to the operating steps in the manual to obtain a recombinant eukaryotic expression plasmid capable of expressing each peptide segment and marker. The recombinant eukaryotic expression plasmids were transfected into DF-1 cells 30 hours after transfection. For a fusion protein sample, two parts were divided, one with pro-IL-1β monoclonal antibody 1E12 as the primary antibody and goat anti-mouse IgG labeled with IRDye800CW as the secondary antibody, and the segment that the monoclonal antibody can recognize was identified by Western blot to determine the antibody recognition characteristics; the other part used the antibody (Sigma) of the fusion tag (Flag or EGFP) of each peptide segment as the primary antibody and goat anti-mouse IgG labeled with IRDye800CW as the secondary antibody to detect the expression of each peptide segment. The negative control (Mock) was DF-1 cells transfected with pCAGGS plasmid.

[0181] Schematic diagram of pro-IL-1β gene segmental expression, see Fig. 9 .

[0182] ① First round truncation (see Fig. 9 A): The pro-IL-1β gene was cut into three segments, namely P1 (aa 1-95), P2 (aa90-179), and P3 (aa 174-267), and a Flag tag sequence was fused to the C-terminus of each segment.

[0183] ② Second round of truncation (see Fig. 9 B): The segment identified as positive in the previous round was further truncated into 4 segments, namely P1' (aa 2-23), P2' (aa 18-45), P3' (aa 40-67), and P4' (aa 62-89), and the EGFP tag sequence was fused to the N-terminus of each segment.

[0184] ③ The third round of truncation (see Fig. 9 C): The segments identified as positive in the previous round were further truncated by gradually deleting the C-terminal amino acids, namely C1 (aa 2-22), C2 (aa 2-21), C3 (aa2-20), C4 (aa 2-19), and C5 (aa 2-18), and the EGFP tag sequence was fused to the N-terminus of each segment.

[0185] ④ The fourth round of truncation (see Fig. 9 D): The segments identified as positive in the previous round were further truncated by gradually deleting the N-terminal amino acids, namely N1 (aa 4-21), N2 (aa 6-21), N3 (aa8-21), and N4 (aa 10-21). The N-terminus of each segment was fused with an EGFP tag sequence.

[0186] ⑤ The fifth round is shortened (see Fig. 9 E): By further deleting the N-terminal amino acid, the segments identified as positive in the previous round were further truncated, namely N5 (aa 11-21), N6 (aa 12-21), N7 (aa 13-21), and N8 (aa 14-21), accurately locating the antigen epitope targeted by the pro-IL-1β monoclonal antibody. The N-terminus of each segment was fused with an EGFP tag sequence.

[0187] The results of Western blot-based epitope identification of chicken pro-IL-1β monoclonal antibody 1E12 can be found in Fig. 9 The results of the first round of truncated detection showed that all three short peptides were expressed, and the epitope recognized by monoclonal antibody 1E12 was located at P1 (aa 1-95) ( Fig. 9 A and F); The results of the second round of truncated detection showed that all four short peptides were expressed, and the epitope recognized by monoclonal antibody 1E12 was located at P1' (aa2-23) ( Fig. 9 B and G); The results of the third round of detection of truncations from the C-terminus further showed that all five truncations were expressed, and the epitope recognized by monoclonal antibody 1E12 was located at C2 (aa 2-21) ( Fig. 9 C and H); The results of the fourth round of detection by N-terminal stepwise truncation showed that all four truncations were expressed, and monoclonal antibody 1E12 reacted with all four truncations ( Fig. 9 D and I), and further truncation detection is required; the fifth round of detection results of further stepwise truncation through the N-terminus showed that all four truncations were expressed, and mAb 1E12 reacted with truncation T7 but not with T8, indicating that T7 is the minimum recognition and reaction unit of mAb 1E12. In summary, the epitope recognized by pro-IL-1β mAb 1E12 is 13SSLSEETFY21 (SEQ ID NO.7) of pro-IL-1β ( Fig. 9 ).

[0188] Pro-IL-1β (full length 267aa) is the form of mature IL-1β (located at aa 106-267 of pro-IL-1β) before being cleaved. It plays an important role in initiating early immune responses and is also a marker for detecting early proinflammatory immune responses. 13 SSLSEETFY 21 Located in the N segment of chicken pro-IL-1β protein, this is the first report of the identification of the antigenic epitope of chicken pro-IL-1β protein. This discovery reveals an antigenic structure of chicken pro-IL-1β protein, which not only provides a tool for the detection of chicken pro-IL-1β protein, but also has important value for the systematic understanding of the structure and function of chicken pro-IL-1β protein.

[0189] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A hybridoma cell, wherein the hybridoma cell is a hybridoma cell with a microbial deposit number of CCTCC NO: C2024399 or a subculture cell of a hybridoma cell with a microbial deposit number of CCTCC NO: C2024399; the monoclonal antibody secreted by the subculture cell of the hybridoma cell with a microbial deposit number of CCTCC NO: C2024399 maintains specific binding activity to chicken interleukin-1β precursor protein.

2. The hybridoma cell according to claim 1, wherein The amino acid sequence of the chicken interleukin-1β precursor protein is shown in SEQ ID NO.

6.

3. 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 is a monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; P2: Genetically engineered monoclonal antibodies The genetically engineered monoclonal antibody retains specific binding activity to chicken interleukin-1β precursor protein; The genetically engineered monoclonal antibody comprises a heavy chain of a genetically engineered monoclonal antibody and a light chain of a genetically engineered monoclonal antibody; The heavy chain of the genetically engineered monoclonal antibody includes a heavy chain CDR1 of the genetically engineered monoclonal antibody, a heavy chain CDR2 of the genetically engineered monoclonal antibody, and a heavy chain CDR3 of the genetically engineered monoclonal antibody; The light chain of the genetically engineered monoclonal antibody includes a light chain CDR1 of the genetically engineered monoclonal antibody, a light chain CDR2 of the genetically engineered monoclonal antibody, and a light chain CDR3 of the genetically engineered monoclonal antibody; The heavy chain CDR1 of the genetically engineered monoclonal antibody is identical to the heavy chain CDR1 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; The heavy chain CDR2 of the genetically engineered monoclonal antibody is identical to the heavy chain CDR2 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; The heavy chain CDR3 of the genetically engineered monoclonal antibody is identical to the heavy chain CDR3 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; The light chain CDR1 of the genetically engineered monoclonal antibody is identical to the light chain CDR1 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; The light chain CDR2 of the genetically engineered monoclonal antibody is identical to the light chain CDR2 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; The light chain CDR3 of the genetically engineered monoclonal antibody is identical to the light chain CDR3 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; P3: Antibody derivatives The antibody derivative retains specific binding activity to chicken interleukin-1β precursor protein; The heavy chain CDR1 of the antibody derivative is identical to the heavy chain CDR1 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; The heavy chain CDR2 of the antibody derivative is identical to the heavy chain CDR2 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; The heavy chain CDR3 of the antibody derivative is identical to the heavy chain CDR3 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; The light chain CDR1 of the antibody derivative is identical to the light chain CDR1 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; The light chain CDR2 of the antibody derivative is identical to the light chain CDR2 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; The light chain CDR3 of the antibody derivative is identical to the light chain CDR3 protein sequence of the monoclonal antibody secreted by the hybridoma cell according to claim 1 or 2; The antibody derivative is in the form of: enzyme-labeled antibody, fluorescent-labeled antibody, chemically modified antibody, antibody Fab fragment, single-chain antibody, chimeric monoclonal antibody and modified monoclonal antibody; P4: RNA combination The RNA combination includes genetically engineered monoclonal antibody heavy chain RNA and genetically engineered monoclonal antibody light chain RNA; The genetically engineered monoclonal antibody heavy chain RNA can be translated to obtain the heavy chain of the genetically engineered monoclonal antibody described in P2; The genetically engineered monoclonal antibody light chain RNA can be translated to obtain the genetically engineered monoclonal antibody light chain described in P2; P5: Genetic combination The coding sequence of the gene combination can encode the heavy chain of the genetically engineered monoclonal antibody described in P2 and the light chain of the genetically engineered monoclonal antibody described in 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 genetically engineered monoclonal antibody heavy chain RNA and the genetically engineered 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 coding RNA in the gene expression cassette of the genetic engineering vector is constitutively expressed or artificially induced; When the genetically engineered monoclonal antibody heavy chain RNA and the genetically engineered monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells; P9: Composition The composition contains the monoclonal antibody described in P1, the genetically engineered 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; as well as P10: Test kit The kit contains the monoclonal antibody described in P1, the genetically engineered 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.

4. The biomaterial according to claim 3, characterized in that The amino acid sequence of the chicken interleukin-1β precursor protein is shown in SEQ ID NO.

6.

5. Use of the hybridoma cells according to claims 1 and 2 or the biomaterials according to claims 3 and 4 in the preparation of biological preparations; The biological agents include: A protein containing an antigenic determinant of an amino acid sequence as shown in SEQ ID NO.7, which is used for in vivo or in vitro recognition, in vivo or in vitro detection, in vivo or in vitro identification, or in vivo intervention; wherein the antigenic determinant as shown in SEQ ID NO.7 is located on the outer surface of the protein; Preparations for evaluating the inflammation-suppressing effects of inflammation-suppressing materials; Preparations for evaluating the inflammation-inducing effects of inflammation-promoting materials.

6. The use according to claim 5, characterized in that The protein containing the antigenic determinant of the amino acid sequence as shown in SEQ ID NO.7 is chicken interleukin-1β precursor protein; and / or The inflammation is inflammation with high expression of interleukin-1β precursor.

7. A method for detecting chicken interleukin-1β precursor protein in cells for non-diagnostic purposes, the method comprising the following steps: S1: Fix the cells to be detected on the solid surface to obtain immobilized cells; S2: incubating the immobilized cells with a monoclonal antibody to obtain immobilized cells of the first incubation; The monoclonal antibody is the monoclonal antibody or genetically engineered monoclonal antibody described in claim 3 or 4; S3: adding the monoclonal antibody specific binding substance connected with the label to the fixed cells of the first incubation for incubation to obtain the fixed cells of the second incubation; S4: Characterizing the marker indirectly combined with the fixed cells in the second incubation, and determining whether chicken interleukin-1β precursor protein exists in the cells to be detected according to the presence or absence of the marker.

8. The method according to claim 7, characterized in that Selected from any one of the following: A1, A2, A3, A4, A5 and A6: A1: In step S1, the cells to be detected are HD11 cells; A2: In step S1, the cells to be detected are HD11 cells treated with anti-inflammatory drugs; A3: In step S1, the cells to be detected are HD11 cells that promote inflammatory substance treatment; A4: In step S1, the solid surface is the inner surface of the well of the microplate; A5: In step S3, the monoclonal antibody specific binding substance connected with the label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody in claim 3 or 4 labeled with a fluorescent molecule; in step S4, the fixed cells in the second incubation are photographed with a fluorescence microscope, and the presence of chicken interleukin-1β precursor protein in the sample to be tested is determined based on the photographic results; A6: In step S3, the monoclonal antibody specific binder connected with a label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in claim 3 or 4 labeled with horseradish peroxidase; in step S4, the fixed cells in the second incubation are developed with TMB colorimetric solution, and the presence of chicken interleukin-1β precursor protein in the sample to be tested is determined based on the color development results.

9. A method for detecting chicken interleukin-1β precursor protein in a sample for non-diagnostic purposes, the method comprising the following steps: T1: Fix the sample to be tested on the solid surface to obtain an immobilized sample; T2: incubating the immobilized sample with a monoclonal antibody to obtain an immobilized sample of the first incubation; The monoclonal antibody is the monoclonal antibody or genetically engineered monoclonal antibody described in claim 3 or 4; T4: adding the monoclonal antibody specific binding substance connected with the label to the immobilized sample of the first incubation for incubation to obtain the immobilized sample of the second incubation; T5: Characterizing the marker indirectly combined with the immobilized sample in the second incubation, and determining whether chicken interleukin-1β precursor protein exists in the sample to be detected according to the presence or absence of the marker.

10. The method according to claim 9, characterized in that Select any one of the following cases B1, B2, B3 and B4: B1: In step T1, the sample to be detected is chicken interleukin-1β precursor protein, or the sample to be detected contains chicken interleukin-1β precursor protein; B2: In step T1, the solid surface is the inner surface of the well of the microplate; B3: In step T3, the monoclonal antibody specific binding substance connected with the label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody in claim 3 or 4 labeled with a fluorescent molecule; in step T4, the fixed sample of the second incubation is photographed with a fluorescence microscope, and the presence of chicken interleukin-1β precursor protein in the sample to be tested is determined based on the photographic results; B4: In step T3, the monoclonal antibody specific binder connected with a label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in claim 3 or 4 labeled with horseradish peroxidase; in step T4, the fixed cells in the second incubation are colored with TMB colorimetric solution, and the presence of chicken interleukin-1β precursor protein in the sample to be tested is determined based on the colorimetric result.

11. The method according to claim 10, characterized in that The amino acid sequence of the chicken interleukin-1β precursor protein is shown in SEQ ID NO.6; and / or The sample to be detected is in the form of cell lysate.

12. A method for screening chicken anti-inflammatory drugs, the screening method comprising the following steps: U1: Chicken cell lines were used as control cells; the same chicken cell lines were treated with inflammatory factors to obtain activated cells; U2: incubating the control cells with the drug to be tested to obtain drug-treated control cells; incubating the activated cells with the drug to be tested in a parallel treatment manner to obtain drug-treated activated cells; U3: fixing the drug-treated control cells to a solid surface to obtain fixed control cells; fixing the drug-treated activated cells to a solid surface in a parallel treatment manner to obtain fixed activated cells; U4: incubating the immobilized control cells with a monoclonal antibody to obtain immobilized control cells of the first incubation; in a parallel treatment manner, incubating the immobilized activated cells with a monoclonal antibody to obtain immobilized activated cells of the first incubation; The monoclonal antibody is the monoclonal antibody or genetically engineered monoclonal antibody described in claim 3 or 4; U5: adding the monoclonal antibody specific binding substance connected with the label to the fixed control cells of the first incubation for incubation to obtain the fixed control cells of the second incubation; in a parallel processing manner, adding the monoclonal antibody specific binding substance connected with the label to the fixed activated cells of the first incubation for incubation to obtain the fixed activated cells of the second incubation; U6: Compare the relative intensity of the label in the fixed control cells in the second incubation with that in the fixed activated cells in the second incubation, and determine qualitatively, quantitatively or semi-quantitatively whether the drug to be tested inhibits inflammation.

13. The method according to claim 12, characterized in that Selected from any one of the following cases C1, C2, C3, C4, C5, C6, C7 and C8: C1: The anti-inflammatory drug is an anti-chicken interleukin-1β precursor drug; C2: The inflammation is inflammation with high expression of interleukin-1β precursor; C3: The chicken-derived cells are HD11 cells; C4: The inflammatory factor is lipopolysaccharide; C5: The inflammatory factor is a susceptible virus of the chicken-derived cells; C6: The solid surface is the inner surface of the well of the microplate; C7: In step U5, the monoclonal antibody specific binding substance connected with the label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in claim 3 or 4 labeled with a fluorescent molecule; in step U6, the fixed control cells in the second incubation and the fixed activated cells in the second incubation are photographed respectively with a fluorescence microscope, and the difference in fluorescence intensity is used to qualitatively, quantitatively or semi-quantitatively determine whether the drug to be tested inhibits inflammation; C8: In step U5, the monoclonal antibody specific binder connected with a label is a secondary antibody of the monoclonal antibody or genetically engineered monoclonal antibody described in claim 3 or 4 labeled with horseradish peroxidase; in step U6, the fixed control cells in the second incubation and the fixed activated cells in the second incubation are respectively developed with TMB colorimetric solution, and whether the drug to be tested inhibits inflammation is qualitatively, quantitatively or semi-quantitatively determined based on the difference in OD values.

14. The method according to claim 13, characterized in that The susceptible virus of the chicken-derived cells is infectious bursal disease virus; and / or The amino acid sequence of the chicken interleukin-1β precursor protein is shown in SEQ ID NO.6.

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