Chicken interleukin 2 antibody and application thereof

Abstract

The invention discloses a chicken interleukin 2 antibody and application thereof, the antibody comprises a light chain variable region and a heavy chain variable region, amino acid sequences of three complementary determining regions in the light chain variable region are respectively shown as SEQ ID NO.1-3, and amino acid sequences of three complementary determining regions in the heavy chain variable region are respectively shown as SEQ ID NO.5-7; or the amino acid sequences of the three complementary determining regions in the light chain variable region are respectively as shown in SEQ ID NO.17-19, and the amino acid sequences of the three complementary determining regions in the heavy chain variable region are respectively as shown in SEQ ID NO.21-23. The antibody provided by the invention has the advantages of high titer, good specificity, high affinity and capability of reacting with a natural ChIL-2 antigen. The ChIL-2 double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) established based on the two antibodies has the advantages of strong specificity, high sensitivity, simple operation and good repeatability, and can be used for measuring the content of ChIL-2 in cell culture supernatant, chicken serum and other samples.

Description

Technical Field

[0001] The invention relates to a chicken interleukin 2 antibody and application thereof, belonging to the field of biotechnology. Background Art

[0002] Interleukin is a biologically active protein that can play a role between immune cells. In 1976, Morgan et al. first discovered a factor in the culture supernatant of human peripheral blood lymphocytes that helps promote and maintain T cell growth and named it T cell growth factor (CGF). In 1979, TCGF was officially named interleukin 2 (IL-2) at the Second Lymphokine Symposium. IL-2 can be produced by multiple different types of cells, including activated T cells, dendritic cells (DCs) and natural killer cells (NKs). It is a Th1 cytokine that can promote CD4 + 、CD8 + It promotes the proliferation of T cells and B cells, promotes the activation of DCs, enhances the killing activity of NKs, and promotes the secretion of other cytokines. Chicken IL-2 (ChIL-2) was first discovered in 1982. ChIL-2 can be used as an immunotherapeutic agent to improve the ability of specific immune response; as an immune adjuvant and vaccine injection, it helps to enhance the immunogenicity of the vaccine; in addition, ChIL-2 can also be used as an evaluation indicator for evaluating poultry immune diseases, and has always been one of the research hotspots of cytokines. With the development of the breeding industry, high-density breeding environments and inappropriate breeding processes have led to explosive immunodeficiency diseases in poultry. ChIL-2 plays a vital role in the prevention and treatment of poultry diseases and the evaluation of immune effects. Therefore, the detection technology of ChIL-2 has shown great application potential.

[0003] The ChIL-2 immunological detection method based on monoclonal antibodies is the most important experimental method for qualitative and quantitative analysis of IL-2. ELISA is a detection method based on antigen-antibody interaction. The most commonly used method for cytokine detection is the double antibody sandwich method, which is particularly suitable for detecting samples in cell culture supernatants, serum, plasma and tissue fluids. Double antibody sandwich ELISA is widely used due to its high specificity, simplicity and sensitivity. However, the monoclonal antibodies against chicken IL-2 prepared in the prior art often have poor effects when applied to the above detection methods, especially when detecting natural chicken IL-2. Therefore, obtaining a chicken IL-2 monoclonal antibody with high titer, good specificity and high antigen affinity to natural chicken IL-2, and establishing an immunological detection method capable of detecting natural chicken IL-2 are of great significance for the evaluation of chicken immune status, the diagnosis of diseases and the study of related immune responses. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a chicken interleukin-2 antibody and its application.

[0005] Technical solution: The chicken interleukin-2 antibody fragment of the present invention comprises light chain and heavy chain variable regions, the amino acid sequences of the three complementary determining regions in the light chain variable region are respectively shown as SEQ ID NO.1-3, and the amino acid sequences of the three complementary determining regions in the heavy chain variable region are respectively shown as SEQ ID NO.5-7; or the amino acid sequences of the three complementary determining regions in the light chain variable region are respectively shown as SEQ ID NO.17-19, and the amino acid sequences of the three complementary determining regions in the heavy chain variable region are respectively shown as SEQ ID NO.21-23.

[0006] Furthermore, the nucleotide sequence encoding the three complementarity determining regions in the light chain variable region is shown as SEQ ID NO.9-11, and the nucleotide sequence encoding the three complementarity determining regions in the heavy chain variable region is shown as SEQ ID NO.13-15; or the nucleotide sequence encoding the three complementarity determining regions in the light chain variable region is shown as SEQ ID NO.25-27, and the nucleotide sequence encoding the three complementarity determining regions in the heavy chain variable region is shown as SEQ ID NO.29-31.

[0007] The present invention also provides a chicken interleukin-2 antibody, wherein the amino acid sequence of the light chain is shown in SEQ ID NO.4, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.8; or the amino acid sequence of the light chain is shown in SEQ ID NO.20, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.24.

[0008] Furthermore, the nucleotide sequence encoding the light chain is shown as SEQ ID NO.12, and the nucleotide sequence encoding the heavy chain is shown as SEQ ID NO.16; or the nucleotide sequence encoding the light chain is shown as SEQ ID NO.28, and the nucleotide sequence encoding the heavy chain is shown as SEQ ID NO.32.

[0009] The heavy and light chains are linked by disulfide bonds.

[0010] The present invention also provides the use of the chicken interleukin-2 antibody fragment and the chicken interleukin-2 antibody in detecting chicken interleukin-2.

[0011] The present invention also provides a kit for detecting chicken interleukin 2, in which the chicken interleukin 2 antibody is used as a detection antibody and / or a capture antibody.

[0012] Furthermore, the detection antibody is a biotin-labeled antibody.

[0013] Furthermore, the kit also comprises a coating standard, a negative control, a coating solution, a blocking solution, a diluent, a streptavidin-labeled horseradish peroxidase, a TMB color developing solution, and a reaction termination solution.

[0014] The present invention also provides a method for using the above kit, comprising the following steps:

[0015] (1) Coating the ELISA plate with the capture antibody, washing, and blocking;

[0016] (2) adding the sample to be tested, the standard and the negative control to the above ELISA plate, incubating and washing;

[0017] (3) adding detection antibody, incubating, and washing;

[0018] (4) Add streptavidin-labeled horseradish peroxidase, incubate, wash; add colorimetric solution, incubate, add stop solution, and read OD 450 value.

[0019] Furthermore, the coating concentration of the capture antibody is 10 μg / mL, and the dilution of the detection antibody is 1:500.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: the antibodies provided by the present invention have the advantages of high titer, good specificity, high affinity and the ability to react with natural ChIL-2 antigen. The ChIL-2 double antibody sandwich enzyme-linked immunosorbent assay (ELISA) established based on two antibodies has the advantages of strong specificity, high sensitivity, simple operation and good repeatability, and can be used for the determination of ChIL-2 content in cell culture supernatant, chicken serum and other samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :Expression, purification and identification of recombinant proteins His-ChIL-2 and GST-ChIL-2. Figure 1 A is the expression, purification and identification of His-ChIL-2 protein, lane M is 180kDa Prestained Protein Marker, lane 1 is the lysis supernatant after induction of the recombinant expression bacteria BL21 (DE3) (pCold-ChIL-2), and lane 2 is the purified His-ChIL-2 protein. Figure 1 B is the expression, purification and identification of GST-ChIL-2 protein, lane M is 180kDa Prestained Protein Marker, lane 1 is the lysis supernatant after induction of the recombinant expression bacteria BL21 (DE3) (pGEX-6p-1-p30), and lane 2 is the purified GST-ChIL-2 protein.

[0022] Figure 2 :Identification of the reactivity of anti-chicken IL-2 monoclonal antibody with prokaryotic recombinant protein. 4F12 group is the identification of the reactivity of monoclonal antibody 4F12 with prokaryotic recombinant protein, lane M is 180kDa Prestained Protein Marker, lane 1 is the purified His-ChIL-2 protein, lane 2 is the BL21 (DE3) (pColdI) empty vector bacteria control, lane 3 is the purified GST-ChIL-2 protein, and lane 4 is the BL21 (DE3) (pGEX-6p-1) empty vector bacteria control. 6E12 group is the identification of the reactivity of monoclonal antibody 6E12 with prokaryotic recombinant protein, lane M is 180kDa Prestained Protein Marker, lane 1 is the purified His-ChIL-2 protein, lane 2 is the BL21 (DE3) (pColdI) empty vector bacteria control, lane 3 is the purified GST-ChIL-2 protein, and lane 4 is the BL21 (DE3) (pGEX-6p-1) empty vector bacteria control.

[0023] Figure 3 :Identification of the reactivity of anti-chicken IL-2 monoclonal antibody with the eukaryotic expressed protein ChIL-2.

[0024] Figure 4 : Characterization of anti-chicken IL-2 monoclonal antibodies reactive with native ChIL-2.

[0025] Figure 5 : Results of affinity determination of anti-chicken IL-2 monoclonal antibody.

[0026] Figure 6 : Specific identification results of chicken interleukin-2 by double antibody sandwich ELISA.

[0027] Figure 7 : Results of double antibody sandwich ELISA for chicken interleukin-2 detection of cell supernatant samples.

[0028] Figure 8 : Results of chicken serum sample detection by double antibody sandwich ELISA of chicken interleukin-2. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0030] Example 1 Obtaining hybridoma cell lines

[0031] 1. Preparation of recombinant proteins His-ChIL-2 and GST-IL-2

[0032] (1) Construction of recombinant plasmids pCold-ChIL-2 and pGEX-6P-1-ChIL-2

[0033] The sequence of the chicken IL-2 encoding gene published by GenBank (accession number AY029588) was selected. After optimization according to the codon preference of Escherichia coli, XhoI and SalI restriction sites were added at both ends of the sequence for connection with the pCold I vector (Takara, catalog number 3361); or SmaI and NotI restriction sites were added at both ends of the sequence for connection with the pGEX-6p-1 vector (Solarbio, catalog number P0300). The sequences after adding restriction sites were commissioned to Nanjing GenScript Biotechnology Co., Ltd. for synthesis and cloned into expression vectors respectively to form recombinant expression plasmids pCold-ChIL-2 and pGEX-6p-1-ChIL-2.

[0034] (2) Expression and purification of target protein

[0035] The recombinant expression plasmids pCold-ChIL-2 and pGEX-6P-1-ChIL-2 were transformed into Escherichia coli BL21 (DE3), respectively, to obtain expression bacteria BL21 (DE3) (pCold-ChIL-2) and BL21 (DE3) (pGEX-6p-1-ChIL-2) that can express recombinant ChIL-2 proteins carrying His and GST tags. The expression and purification process of His-ChIL-2 recombinant protein is as follows: a single colony of BL21 (DE3) (pCold-ChIL-2) is inoculated into 5 mL of liquid LB (containing ampicillin) and cultured at 37°C with shaking overnight; the next day, the mother liquor is inoculated into fresh LB liquid culture medium (containing ampicillin) at a ratio of 1:100, cultured at 37°C with shaking until OD600 is about 0.6, IPTG with a final concentration of 0.5 mM is added, and the culture is induced with shaking at 15°C for 24 hours; finally, the recombinant protein His-ChIL-2 is purified using a His tag protein purification kit (Sangon Biotechnology Co., Ltd., catalog number: C600332), and identified by SDS-PAGE electrophoresis. The expression and purification process of the GST-ChIL-2 recombinant protein is as follows: a single colony of BL21 (DE3) (pGEX-6p-1-ChIL-2) was inoculated into 5 mL of liquid LB (containing ampicillin) and cultured overnight at 37°C with shaking; the next day, the mother liquor was inoculated into fresh LB liquid culture medium (containing ampicillin) at a ratio of 1:100, cultured at 37°C with shaking until OD600 was about 0.6, and IPTG with a final concentration of 0.5 mM was added for induction culture at 25°C with shaking for 8 hours; then the recombinant protein GST-ChIL-2 was purified using a GST tag protein purification kit (GenScript Biotech Co., Ltd., catalog number: L00207), and identified by SDS-PAGE electrophoresis.

[0036] The results are as follows Figure 1 As shown, at 14KDa ( Figure 1 A, lane 2) and 39.4 kDa ( Figure 1 B, target bands of expected sizes appeared in lane 2), which are His-ChIL-2 and GST-ChIL-2 proteins respectively.

[0037] 2. Animal Immunization

[0038] Three 6-week-old BALB / c mice (Comparative Medicine Center of Yangzhou University) were selected for immunization. For the first immunization, 50 μg of His-ChIL-2 protein emulsified with complete Freund's adjuvant (emulsification volume ratio of 1:1) was injected subcutaneously at multiple points in the abdomen; 2 weeks later, 50 μg of His-ChIL-2 protein emulsified with incomplete Freund's adjuvant (emulsification volume ratio of 1:1) was injected subcutaneously at multiple points in the abdomen for booster immunization, and 50 μg of His-ChIL-2 protein emulsified with incomplete Freund's adjuvant (emulsification volume ratio of 1:1) was injected subcutaneously at multiple points in the abdomen for the third immunization after an interval of two weeks. After 2 weeks, blood was collected to determine the serum antibody titer, and mice with higher titers (titer up to 1:25600) were selected for booster immunization with 50 μg purified His-ChIL-2 without adjuvant injected intraperitoneally.

[0039] 2. Cell Fusion

[0040] Three days after the intraperitoneal booster immunization, a small amount of blood was collected and the serum was separated and frozen at -20°C as a positive control for screening. The spleen cells of the immunized mice were aseptically fused with myeloma cells SP2 / 0 in the logarithmic growth phase under the action of polyethylene glycol (PEG) to prepare ICR mouse peritoneal macrophages as feeder cells. The fused cells were suspended in HAT medium (concentration of 1×10 6 cells / mL) and feeder cells (concentration of 1×10 5 cells / mL), dispensed into 96-well plates at a volume of 100 μL / well, and cultured in a 37°C, 5% carbon dioxide incubator. After 5 days, add fresh HAT medium, and culture with HT medium after turning yellow. Observe regularly, and when the cells grow to 80% of the well area, take the cell supernatant for ELISA detection. The wells with positive ELISA detection are hybridoma cells.

[0041] 3. Establishment of indirect ELISA detection method

[0042] Positive cell clones were screened by indirect ELISA. The coating concentration of the detection antigen was determined by array test. The detection antigen GST-IL-2 coating buffer was diluted horizontally, 100 μL per well was coated on the ELISA plate, and incubated at 4°C overnight; washed 3 times with PBST, 200 μL of blocking solution was added to each well, and incubated at 4°C overnight; immune mouse serum was diluted vertically in multiples, 50 μL per well, and normal mouse serum was diluted in the same multiples as a negative control, and incubated at 37°C for 2h; washed for the third time with PBST, and HRP-labeled goat anti-mouse IgG secondary antibody (Cat. No.: ab205719; Abcam) diluted 1:10000 was added, 100 μL per well, and incubated at 37°C for 1h. After washing with PBST, TMB was used for color development, and OD was measured by enzyme-linked detector. 450 The optimal coating concentration of the detection antigen was determined by the value of

[0043] 4. Screening of positive clones

[0044] The established indirect ELISA method was used to detect the antibodies secreted by hybridoma cells. The specific method is as follows: add the supernatant of hybridoma cell culture to the pre-coated ELISA plate, 50 μL / well, use SP2 / 0 cell supernatant as negative control, immune polyclonal antibody serum as positive control, 37°C water bath for 2 hours; wash 3 times with PBST; add 1:10000 diluted HRP-labeled goat anti-mouse IgG antibody, 100 μL / well, 37°C water bath for 1 hour; after washing, TMB color development for 10 minutes, after color development is terminated, measure OD with an enzyme reader 450 Reading. Determine the experimental results according to the following formula: OD 450 Cell well / OD 450 Negative wells ≥ 2.1 were judged as positive wells. The two positive clones screened were named positive cell clone 4F12 and positive clone cell 6E12.

[0045] 5. Cloning of positive hybridoma cells

[0046] The positive cell clones 4F12 and 6E12 were subcloned three times by limiting dilution method and stored.

[0047] Example 2 Preparation of anti-chicken IL-2 monoclonal antibody

[0048] 1. Preparation of Ascites

[0049] The ascites was prepared by the in vivo induction method according to the conventional method. 10-12 week old healthy BALB / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin per mouse. After 7 days, hybridoma cells 4F12 and 6E12 cultured to the logarithmic growth phase diluted with PBS were intraperitoneally inoculated with 5×10 5cells / mouse; after 10 days, the ascites was collected, the precipitate was removed by centrifugation, and the supernatant was collected. The monoclonal antibodies secreted by the hybridoma cells 4F12 and 6E12 were named 4F12 and 6E12, respectively. The antibody titers were determined by indirect ELISA, and the cells were packaged and stored at -70°C.

[0050] 2. Antibody Purification and Labeling

[0051] The prepared 4F12 and 6E12 ascites were purified using Protein G affinity chromatography, and the monoclonal antibody 6E12 was biotin-labeled.

[0052] The purified monoclonal antibody 6E12 was labeled by a standard biotin labeling method to obtain biotin-labeled chicken IL-2 monoclonal antibody Biotin-6E12. Specifically, 2 mg of monoclonal antibody 6E12 protein was dissolved in 1 mL of phosphate buffer (PBS), and the number of dissolved millimoles was calculated; biotin Sulfo-NHS-Biotin (Cat. No.: YZM018905; Biofount) was balanced to room temperature, 2 mg of Sulfo-NHS-Biotin was added to 100 μL of ultrapure water, and the above-dissolved monoclonal antibody 6E12 was added; room temperature for 30 minutes; the purification column was pre-washed with 30 mL of PBS, the sample was loaded, and the same buffer as the collection volume was added, 0.5 mL was collected in a separate tube, and the monoclonal antibody protein content was determined by the absorbance value at 280 nm.

[0053] Example 3: Monoclonal Antibody Characterization Detection

[0054] 1. Identification of monoclonal antibody subclasses

[0055] According to the instructions of the monoclonal antibody subclass kit (Beijing Biolong BF16001), the ELISA method was used. 100 μL / well of hybridoma cell culture supernatant was added to the ELISA plate coated with GST-IL-2 antigen, incubated at 37°C for 2 hours, and washed with PBST three times, each time for 5 minutes; 1:1000 diluted goat anti-mouse IgG1 and IgG 2a IgG 2b 100 μL / well of IgG3 and IgM subclass antibodies, incubate at 37℃ for 0.5h; wash 3 times with PBST for 5min each time; add 100 μL / well of 1:5000 diluted goat anti-mouse enzyme-labeled secondary antibody, incubate at 37℃ for 15min, wash 3 times with PBST; add 100 μL / well of TMB colorimetric solution, color at 37℃ in the dark for 5min, add 0.5MH2SO450 μL / well to terminate the reaction, and detect OD with a microplate reader 450 , according to OD 450 Determine the subclass of the monoclonal antibody.

[0056] The results showed that the subclass of monoclonal antibody 4F12 was IgG2a, and the subclass of monoclonal antibody 6E12 was IgG2a.

[0057] After identification, the results showed that the amino acid sequence of the light chain variable region complementary determining region 1 (CDR1) of the monoclonal antibody 4F12 is as shown in SEQ ID NO.1, specifically:

[0058] SASSSVRYIH.

[0059] The amino acid sequence of the light chain variable region complementarity determining region 2 (CDR2) of the monoclonal antibody 4F12 is shown in SEQ ID NO.2, specifically:

[0060] DTFKLAS.

[0061] The amino acid sequence of the light chain variable region complementarity determining region 3 (CDR3) of the monoclonal antibody 4F12 is shown in SEQ ID NO.3, specifically:

[0062] QQWGTHPRT.

[0063] The amino acid sequence of the light chain variable region of monoclonal antibody 4F12 is shown in SEQ ID NO.4, specifically:

[0064] MDFQVQIFSFLLISASVIISRGQIVLTQSPAIMSAFPGEKVTMTCSASSSVRYIHWY QQKSGTSPKRWIYDTFKLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQWGTH PRTFGGGTSLEIK.

[0065] That is, the light chain variable region of monoclonal antibody 4F12 contains 128 amino acids.

[0066] The amino acid sequence of the heavy chain variable region complementarity determining region 1 (CDR1) of the monoclonal antibody 4F12 is shown in SEQ ID NO.5, specifically:

[0067] NYWIE.

[0068] The amino acid sequence of the heavy chain variable region complementarity determining region 2 (CDR2) of the monoclonal antibody 4F12 is shown in SEQ ID NO.6, specifically:

[0069] EILPGSGSTNYNEKFKD.

[0070] The amino acid sequence of the heavy chain variable region complementarity determining region 3 (CDR3) of the monoclonal antibody 4F12 is shown in SEQ ID NO.7, specifically:

[0071] TSTVVDY.

[0072] The amino acid sequence of the heavy chain variable region of monoclonal antibody 4F12 is shown in SEQ ID NO.8, specifically:

[0073] MEWTWVFLFLLSVTAGVHSQVQLQQSGAEVMKPGASVKISCKATGYIFSNYWI EWVKQRPGHGLEWIGEILPGSGSTNYNEKFKDKATFTADTSSNTAYMQLSSLTSEDSA VYYCARTSTVVDYWGQGTTLTVSS.

[0074] That is, the heavy chain variable region of monoclonal antibody 4F12 contains 135 amino acids.

[0075] Correspondingly, the nucleotide sequence of the light chain variable region complementary determining region 1 (CDR1) of the monoclonal antibody 4F12 is shown in SEQ ID NO.9, specifically:

[0076] AGTGCCAGTTCAAGTGTACGTTACATACAC.

[0077] The nucleotide sequence of the light chain variable region complementary determining region 2 (CDR2) of the monoclonal antibody 4F12 is shown in SEQ ID NO.10, specifically:

[0078] GACACATTTAAACTGGCTTCT.

[0079] The nucleotide sequence of the light chain variable region complementary determining region 3 (CDR3) of the monoclonal antibody 4F12 is shown in SEQ ID NO.11, specifically:

[0080] CAGCAGTGGGGTACTCATCCACGGACG.

[0081] The nucleotide sequence of the light chain variable region of monoclonal antibody 4F12 is shown in SEQ ID NO.12, specifically:

[0082] ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATTTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGTTCAAGTGTACGTTACATACACTGGTACCAGCAGAAGTCTGGCACCTCC CCCAAAAGATGGATTTATGACACATTTAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGGGTACTCATCCACGGACGTTCGGTGGAGGCACCAGCCTGGAAATCAAA.

[0083] That is, the nucleotides of the light chain of monoclonal antibody 4F12 contain 384 bases.

[0084] The nucleotide sequence of the heavy chain variable region complementary determining region 1 (CDR1) of the monoclonal antibody 4F12 is shown in SEQ ID NO.13, specifically:

[0085] AATTACTGGATAGAG.

[0086] The nucleotide sequence of the heavy chain variable region complementary determining region 2 (CDR2) of the monoclonal antibody 4F12 is shown in SEQ ID NO.14, specifically:

[0087] GAGATTTTACCTGGAAGTGGTAGTACTAATTACAATGAGAAATTCAAGGAC.

[0088] The nucleotide sequence of the heavy chain variable region complementary determining region 3 (CDR2) of the monoclonal antibody 4F12 is shown in SEQ ID NO.15, specifically:

[0089] ACAAGTACGGTAGTTGACTAC.

[0090] The nucleotide sequence of the heavy chain variable region of monoclonal antibody 4F12 is shown in SEQ ID NO.16, specifically:

[0091] ATGGAATGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAAGTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACATATTCAGTAATTACTGGATAGAGTGGGTAAAACAGAGGCCTGGACATGGCCTTGAGTGGATTGG AGAGATTTTACCTGGAAGTGGTAGTACTAATTACAATGAGAAATTCAAGGACAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACCTCTGAGGACTCTGCCGTCTATTACTGTGCAAGAACAAGTACGGTAGTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0092] That is, the nucleotides of the heavy chain of the monoclonal antibody 4F12 contain 405 bases.

[0093] The amino acid sequence of the light chain variable region complementarity determining region 1 (CDR1) of the monoclonal antibody 6E12 is shown in SEQ ID NO.17, specifically:

[0094] RASQSIGTSIH.

[0095] The amino acid sequence of the light chain variable region complementarity determining region 2 (CDR2) of the monoclonal antibody 6E12 is shown in SEQ ID NO.18, specifically:

[0096] YASESIS.

[0097] The amino acid sequence of the light chain variable region complementarity determining region 3 (CDR3) of the monoclonal antibody 6E12 is shown in SEQ ID NO.19, specifically:

[0098] QQSNSWPTYT.

[0099] The amino acid sequence of the light chain variable region of monoclonal antibody 6E12 is shown in SEQ ID NO.20, specifically:

[0100] MVSTPQFLVFLLFWIPASRGDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQ RTNGSPRLLINYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPTYTF GGGTKLEIK.

[0101] That is, the light chain variable region of monoclonal antibody 6E12 contains 128 amino acids.

[0102] The amino acid sequence of the heavy chain variable region complementarity determining region 1 (CDR1) of the monoclonal antibody 6E12 is shown in SEQ ID NO.21, specifically:

[0103] SYLMH.

[0104] The amino acid sequence of the heavy chain variable region complementarity determining region 2 (CDR2) of the monoclonal antibody 6E12 is shown in SEQ ID NO.22, specifically:

[0105] YIDPYNADTKYNEKFKG.

[0106] The amino acid sequence of the heavy chain variable region complementarity determining region 3 (CDR3) of the monoclonal antibody 6E12 is shown in SEQ ID NO.23, specifically:

[0107] ESYGNYFAY.

[0108] The amino acid sequence of the heavy chain variable region of monoclonal antibody 6E12 is shown in SEQ ID NO.24, specifically:

[0109] MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYL MHWVKQKPGQGLEWIGYIDPYNADTKYNEKFKGKTTLTSDKSSSTAYMDLSSLTS EDSAVYYCARESYGNYFAYWGQGTLVTVSA.

[0110] That is, the heavy chain of monoclonal antibody 6E12 contains 137 amino acids.

[0111] Correspondingly, the nucleotide sequence of the light chain variable region complementary determining region 1 (CDR1) of the monoclonal antibody 6E12 is shown in SEQ ID NO.25, specifically:

[0112] AGGGCCAGTCAGAGCATTGGCACAAGCATACAT.

[0113] The nucleotide sequence of the light chain variable region complementary determining region 2 (CDR2) of the monoclonal antibody 6E12 is shown in SEQ ID NO.26, specifically:

[0114] TATGCTTCTGAGTCTATCTCT.

[0115] The nucleotide sequence of the light chain variable region complementary determining region 3 (CDR3) of the monoclonal antibody 6E12 is shown in SEQ ID NO.27, specifically:

[0116] CAACAAAGTAATAGCTGGCCAACGTACACA.

[0117] The nucleotide sequence of the light chain variable region of monoclonal antibody 6E12 is shown in SEQ ID NO.28, specifically:

[0118] ATGGTATCCACATCAGTTCCTTGTATTTTTGCTTTTCTGGATTCCAGCCTCCAGAGGTGACATCTTGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATACATTGGTATCAGCAAAGAACAAATGGTTCTCCA AGGCTTCTCATAAACTATGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCAACGTACACATTCGGGGGGGGGACCAAGCTGGAAATAAAA.

[0119] That is, the nucleotides of the light chain of monoclonal antibody 6E12 contain 384 bases.

[0120] The nucleotide sequence of the heavy chain variable region complementary determining region 1 (CDR1) of the monoclonal antibody 6E12 is shown in SEQ ID NO.29, specifically:

[0121] AGCTATCTTATGCAC.

[0122] The nucleotide sequence of the heavy chain variable region complementary determining region 2 (CDR2) of the monoclonal antibody 6E12 is shown in SEQ ID NO.30, specifically:

[0123] TATATTGATCCTTACAATGCTGATACTAAGTACAATGAGAAATTCAAAGGC.

[0124] The nucleotide sequence of the heavy chain variable region complementary determining region 3 (CDR2) of the monoclonal antibody 6E12 is shown in SEQ ID NO.31, specifically:

[0125] GAATCCTATGGTAACTACTTTGCTTAC.

[0126] The nucleotide sequence of the heavy chain variable region of monoclonal antibody 6E12 is shown in SEQ ID NO.32, specifically:

[0127] ATGGAATGGAGTTGGATATTTCTTTCCTGTCAGGAACTGCAGGTGTCCACTCTGAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATCTTATGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATA TATTGATCCTTACAATGCTGATACTAAGTACAATGAGAAATTCAAAGGCAAGACCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGACCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGAATCCTATGGTAACTACTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0128] That is, the nucleotides of the heavy chain of monoclonal antibody 6E12 contain 411 bases.

[0129] 2. Detection of monoclonal antibody ascites titer

[0130] The GST-ChIL-2 protein was coated at 0.25 μg / mL on a 96-well ELISA plate using a coating buffer, overnight at 4°C; washed 3 times, 200 μL of blocking solution was added to each well, and incubated at 37°C for 2 h; washed 3 times with PBST, the monoclonal antibody ascites prepared in Example 2 was diluted in multiples, and SP2 / 0 ascites was diluted in the same multiples as a negative control, and incubated at 37°C for 2 h; washed 5 times with PBST; HRP-sheep anti-mouse IgG diluted 1:10000 was added, 100 μL / well, and incubated at 37°C for 1 h; after washing with PBST, TMB colorimetric solution was added for color development, 100 μL / well, and incubated at 37°C for 10 min; 0.5 M H2SO4 was added to terminate the reaction, and OD was detected by an enzyme marker. 450 The value was determined by taking P / N ≥ 2.1 as the criterion to determine the monoclonal antibody ascites titer.

[0131] The results showed that the titers of monoclonal antibodies 4F12 and 6E12 were both 1:4096000, which were very high.

[0132] 3. Identification of monoclonal antibody immunoreactivity

[0133] The recombinant proteins His-ChIL-2 and GST-ChIL-2 were added to the loading buffer, and the metal bath was kept at a constant temperature for 10 minutes for SDS-PAGE electrophoresis. Then, the electrophoresis gel was transferred using a fast transfer instrument for 10 minutes, and the protein was transferred to the nitrocellulose membrane; PBST containing 5% skim milk powder was used for blocking, and the membrane was shaken at room temperature for 1 hour, and washed with PBST for 3 times; monoclonal antibody 4F12 or 6E12 was added at a dilution of 1:1000, and incubated at 4°C overnight, and washed with PBST for 5 times; HRP-sheep anti-mouse enzyme-labeled antibody was added at a dilution of 1:10000, and the membrane was exposed to room temperature for 1 hour, and washed with PBST for 7 times; the color was developed with the color developing solution for 1 minute, and the membrane was photographed using an ultra-sensitive multifunctional imager.

[0134] The results are as follows Figure 2 As shown, 4F12 and 6E12 monoclonal antibodies can react with prokaryotic recombinant expressed proteins His-ChIL-2 (lane 1) and GST-ChIL-2 (lane 3), respectively, with good reactivity.

[0135] The plasmid pCMV-myc-ChIL-2 (Han Shunzi, Huang Xia, Meng Chuang, Geng Shizhong, Kang Xilong, Pan Zhiming, Jiao Xinan. Prokaryotic expression of chicken interleukin-2 and identification of its activity [J]. China Poultry, 2024, 46(05): 56-61.) was transfected into HeLa cells using Lipofectamine 2000. The reactivity of the monoclonal antibody with eukaryotic expressed ChIL-2 was identified by indirect immunofluorescence 24 h after transfection. Add ice methanol to fix at room temperature for 10 minutes, wash three times with PBS; use 5% BSA to incubate at 37°C for 2 hours for blocking, wash three times with PBS; add 1:1000 diluted monoclonal antibody 4F12, 6E12 or SP2 / 0 cell culture supernatant, incubate at 37°C for 2 hours, wash five times with PBS; add 1:2000 diluted FITC-labeled goat anti-mouse IgG (ab7685, Abcam), act at room temperature for 1 hour, wash five times with PBST, and take pictures under a fluorescence microscope.

[0136] The results are as follows Figure 3 As shown, the 4F12 and 6E12 groups had obvious green fluorescence, while the SP2 / 0 control group had no obvious green fluorescence, indicating that 4F12 and 6E12 were able to react with eukaryotically expressed ChIL-2.

[0137] Chicken spleen lymphocytes were prepared by 5×10 6 cell / well, inoculated into a 24-well cell culture plate, stimulated with concanavalin A (ConA, 20 μg / mL), and added with a 1:1000 dilution of the protein transport inhibitor bradeletin (BFA, 420601, BioLegend) to block the secretion of cytokines into the extracellular space, and cultured in a 37°C, 5% CO2 incubator for 24 h. The reactivity of the monoclonal antibody with natural ChIL-2 was identified by flow cytometry. The cells were collected, washed three times with PBS, and phycoerythrin (PE)-labeled CD3 antibody (555275, BD Bioscience) was added at a dilution of 1:1000, incubated at 4°C for 30 min, and washed five times with PBS; fixation Buffer and Intracellular Staining Perm WashBuffer were used for fixation and permeabilization, respectively, and washed three times with PBS; monoclonal antibodies 4F12 and 6E12 were added at a dilution of 1:2000, incubated at room temperature for 1 h, and washed three times with PBS; FITC-labeled goat anti-mouse IgG was added at a dilution of 1:2000, incubated at room temperature for 30 min, washed three times with PBS, and the proportion of T lymphocytes secreting chicken IL-2 was analyzed by FACS.

[0138] Results Figure 4, using 4F12 and 6E12 as detection antibodies for ChIL-2, the proportion of T lymphocytes secreting chicken IL-2 in the stimulated group was higher than that in the unstimulated group, indicating that 4F12 and 6E12 can react with natural ChIL-2.

[0139] 4. Monoclonal antibody affinity identification

[0140] The cells were coated with GST-ChIL-2 protein at concentrations of 0.0625, 0.125, 0.25, and 0.5 μg / mL overnight; the purified antibodies (4F12 and 6E12) were diluted from 1:1000 as primary antibodies, with a total of 16 concentration gradients, and incubated at 37°C for 2 hours; HRP-sheep anti-mouse IgG diluted 1:2000 was added as secondary antibody, and incubated at 37°C for 1 hour; TMB color development was performed for 10 minutes; the reading was performed after H2SO4 termination. The affinity constant (K) of each antibody was calculated according to the formula: K = ([Ag'] / [Ag]t-1) / (2[n[Ab']-[Ab]t) 100 .

[0141] Results Figure 5 , 4F12 and 6E12 monoclonal antibodies bind stably to chicken IL-2 protein with high affinity, which are 2.44×10 10 M -1 and 1.28×10 10 M -1 .

[0142] Example 4 Establishment of Chicken IL-2 Double Antibody Sandwich ELISA Method

[0143] The optimal working concentration of capture antibody 4F12 was determined to be 10 μg / mL by the chessboard method, and the optimal dilution of detection antibody Biotin-6F12 was 1:500. After optimizing the coating solution, optimal blocking solution, optimal blocking time, optimal serum incubation time, optimal serum dilution multiple, and optimal substrate action time, the final operating procedure of chicken IL-2 double antibody sandwich ELISA was determined as follows:

[0144] (1) Add capture antibody 4F12 at a final concentration of 10 μg / mL to a 96-well ELISA plate, 100 μL / well, and coat at 4°C for 14 h.

[0145] (2) Discard the coating solution and wash with PBST three times, 3 min each time;

[0146] (3) Add PBST blocking solution containing 10% FBS, 200 μL / well, and block at 37°C for 2 h;

[0147] (4) Discard the blocking solution and wash with PBST three times, 3 min each time;

[0148] (5) Add the sample to be tested, the standard (natural chicken IL-2) diluted in multiples, and the negative control (DMEM medium) to the coated ELISA plate, 100 μL / well, and incubate at 37°C for 1 h;

[0149] (6) Discard the sample and wash with PBST five times, 3 min each time;

[0150] (7) Add 1:500 diluted detection antibody Biotin-6F12, 100 μL / well, and incubate at 37°C for 1 h;

[0151] (8) Discard the detection antibody and wash with PBST 7 times, 3 min each time;

[0152] (9) Add 1:8000 diluted avidin-horseradish peroxidase (SA-HRP), 100 μL / well, and incubate at 37°C for 45 min;

[0153] (10) Discard SA-HRP and wash with PBST seven times, 3 min each time;

[0154] (11) Add TMB colorimetric solution (100 μL / well) and incubate at 37°C in the dark for 10 min.

[0155] (12) Add 0.5M H2SO4, 50 μL / well, and use a microplate reader to read the OD 450 Measure the absorbance at the wavelength.

[0156] Example 5 Specificity determination of chicken IL-2 double antibody sandwich ELISA method

[0157] The chicken IL-2 double antibody sandwich ELISA method in Example 4 was used to detect ChIL-2, recombinant mouse IL-2 (mIL-2, 402-ML, R&D Systems), IL-4 (mIL-4, 404-ML-010, R&D Systems), recombinant chicken IL-4 (ChIL-4) (Dai Hua, Zheng Jiayu, Chen Junhua, Sun Lin, Pan Zhiming, Jiao Xin'an. Cloning and prokaryotic expression of chicken interleukin 4 gene [J]. Chinese Journal of Preventive Veterinary Medicine, 2008, (06): 473-477.), IFN-γ (ChIFN-γ) (Dai Hua, Zheng Jiayu, Hu Maozhi, Chen Junhua, Pan Zhiming, Jiao Xin'an. Preparation and identification of monoclonal antibodies against chicken gamma-interferon [J]. Journal of Yangzhou University (Agriculture and Life Sciences Edition), 2007, (04): 6-9.), recombinant human IL-2 (hIL-2, BT-002-010, R&D Systems), recombinant chicken IL-4 (ChIL-4) (Dai Hua, Zheng Jiayu, Chen Junhua, Sun Lin, Pan Zhiming, Jiao Xin'an. Cloning and prokaryotic expression of chicken interleukin 4 gene [J]. Chinese Journal of Preventive Veterinary Medicine, 2008, (06): 473-477.), IFN-γ (ChIFN-γ) (Dai Hua, Zheng Jiayu, Hu Maozhi, Chen Junhua, Pan Zhiming, Jiao Xin'an. Preparation and identification of monoclonal antibodies against chicken gamma-interferon [J]. Journal of Yangzhou University (Agriculture and Life Sciences Edition), 2007, (04): 6-9.), Systems) proteins were detected to evaluate the specificity of this method.

[0158] Results Figure 6The ChIL-2 double antibody sandwich ELISA detection method established in the present invention can specifically detect ChIL-2 protein, and does not cross-react with recombinant proteins ChIL-4, ChIFN-γ, mIL-2, mIL-4, and hIL-2. It has strong specificity and can be used for ChIL-2 protein detection.

[0159] Example 6 Determination of the detection limit of chicken IL-2 double antibody sandwich ELISA method

[0160] Natural ChIL-2 was prepared as follows: 3-week-old SPF chickens (Zhejiang Lihua Agricultural Technology Co., Ltd.) were euthanized and the spleens were aseptically removed. The spleens were minced, ground, and filtered in PBS, and centrifuged at 4°C, 600 rpm / min for 5 min, and repeated twice to remove red blood cells. The cell suspension was added to Histopaque-1083 lymphocyte separation medium (10831, Sigma), centrifuged at 20°C, 1800 rpm / min for 20 min, the white fog layer was aspirated, and the cells were washed twice with RPMI 1640 medium and diluted to 2×10 6 cell / well, and plated in a 24-well plate; ConA (20 μg / mL) was used as a stimulant to stimulate the culture for 24 hours to obtain natural ChIL-2, and the commercial kit Chicken IL-2 ELISA Kit (ELG-IL2-1, RayBiotech) was used to detect the content of ChIL-2 in the cell supernatant, and the concentration was adjusted to 2500 pg / mL. The prepared natural ChIL-2 protein was diluted in multiples starting from 2500 pg / mL, and the chicken IL-2 double antibody sandwich ELISA method in Example 4 was used for detection, and the minimum detection limit of the method was determined.

[0161] The results are shown in Table 1. When the concentration of natural ChIL-2 was 78.1 pg / mL, it could still be detected by the method of the present invention, indicating that the minimum detection limit of chicken IL-2 double antibody sandwich ELISA can reach 78.1 pg / mL.

[0162] Table 1 Determination of detection limit of chicken IL-2 double antibody sandwich ELISA method

[0163]

[0164]

[0165] Example 7 Detection of cell supernatant samples by chicken IL-2 double antibody sandwich ELISA method

[0166] Chicken spleen lymphocytes were isolated by conventional methods, and 5×10 6The cells were inoculated into a 24-well cell culture plate at an amount of 10 μg / mL cell / well, and the cells were stimulated with ConA at a final concentration of 10 μg / mL for 24 h, 48 h and 72 h, respectively, and the culture supernatant was collected. At the same time, an unstimulated group was set up, and the chicken IL-2 double antibody sandwich ELISA method in Example 4 was used to detect the ChIL-2 content in the cell supernatant.

[0167] Results Figure 7 Compared with the unstimulated group, the ChIL-2 content in the cell supernatant of the ConA stimulated group was significantly increased, and the content was highest after 48 hours of stimulation, indicating that the established chicken IL-2 double antibody sandwich ELISA method can be used for the determination of ChIL-2 in cell supernatant.

[0168] Example 8 Detection of Chicken IL-2 Double Antibody Sandwich ELISA Method Chicken Serum Samples

[0169] 1×10 7 Three 3-day-old SPF chicks (Zhejiang Lihua Agricultural Technology Co., Ltd.) were infected by intramuscular injection of CFU dose of Enteritidis Salmonella C50041, and the second and third inoculations were carried out on the 14th and 28th days after the first inoculation, respectively, with the same dose as the first; venous blood was collected 7 days after each infection, serum was separated, and the content of ChIL-2 in serum was detected using the chicken IL-2 double antibody sandwich ELISA method in Example 4. At the same time, 100 μL PBS was injected as a control group.

[0170] Results Figure 8 Compared with the uninfected group, the ChIL-2 content in the serum of the Salmonella enteritidis infected group was significantly increased, indicating that the established chicken IL-2 double antibody sandwich ELISA method can effectively detect ChIL-2 in chicken serum with good specificity and sensitivity.

[0171] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A chicken interleukin-2 antibody fragment, characterized in that The antibody fragment comprises light chain and heavy chain variable regions, the amino acid sequences of the three complementarity determining regions in the light chain variable region are respectively shown as SEQ ID NOs.1-3, and the amino acid sequences of the three complementarity determining regions in the heavy chain variable region are respectively shown as SEQ ID NOs.5-7; or the amino acid sequences of the three complementarity determining regions in the light chain variable region are respectively shown as SEQ ID NOs.17-19, and the amino acid sequences of the three complementarity determining regions in the heavy chain variable region are respectively shown as SEQ ID NOs.21-23.

2. The chicken interleukin-2 antibody fragment according to claim 1, characterized in that The nucleotide sequence encoding the three complementarity determining regions in the light chain variable region is shown in SEQ ID NOs.9-11, and the nucleotide sequence encoding the three complementarity determining regions in the heavy chain variable region is shown in SEQ ID NOs.13-15; or the nucleotide sequence encoding the three complementarity determining regions in the light chain variable region is shown in SEQ ID NOs.25-27, and the nucleotide sequence encoding the three complementarity determining regions in the heavy chain variable region is shown in SEQ ID NOs.29-31.

3. A chicken interleukin-2 antibody, characterized in that The antibody comprises a light chain and a heavy chain, the amino acid sequence of the light chain is shown in SEQ ID NO.4, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.8; or the amino acid sequence of the light chain is shown in SEQ ID NO.20, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

24.

4. The chicken interleukin-2 antibody according to claim 3, characterized in that The nucleotide sequence encoding the light chain is shown as SEQ ID NO.12, and the nucleotide sequence encoding the heavy chain is shown as SEQ ID NO.16; or the nucleotide sequence encoding the light chain is shown as SEQ ID NO.28, and the nucleotide sequence encoding the heavy chain is shown as SEQ ID NO.

32.

5. Use of the chicken interleukin-2 antibody fragment according to any one of claims 1 to 2 and the chicken interleukin-2 antibody according to any one of claims 3 to 4 in detecting chicken interleukin-2.

6. A kit for detecting chicken interleukin-2, characterized in that: The chicken interleukin-2 antibody according to any one of claims 3 to 4 is used as a detection antibody and / or a capture antibody in the kit.

7. The kit for detecting African swine fever virus according to claim 6, characterized in that: The detection antibody is a biotin-labeled antibody.

8. The African swine fever virus detection kit according to claim 6, characterized in that: The kit also comprises a coating standard, a negative control, a coating solution, a blocking solution, a diluent, a streptavidin-labeled horseradish peroxidase, a TMB color developing solution, and a reaction termination solution.

9. The method for using the kit according to any one of claims 6 to 8, characterized in that: The following steps are involved: (1) Coating the ELISA plate with the capture antibody, washing, and blocking; (2) adding the sample to be tested, the standard and the negative control to the above ELISA plate, incubating and washing; (3) adding detection antibody, incubating, and washing; (4) adding streptavidin-labeled horseradish peroxidase, incubating, and washing; Add colorimetric solution, incubate, add stop solution, and read OD 450 value.

10. The method according to claim 9, characterized in that The coating concentration of the capture antibody was 10 μg / mL, and the dilution of the detection antibody was 1:500.

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