Chicken interleukin 2 antibody and application thereof

Abstract

The application discloses a chicken interleukin 2 antibody and application thereof, the antibody comprises a light chain and a heavy chain variable region, the amino acid sequences of three complementarity determining regions in the light chain variable region are respectively shown as SEQ ID NO. 1-3, the amino acid sequences of three complementarity determining regions in the heavy chain variable region are respectively shown as SEQ ID NO. 5-7, or the amino acid sequences of three complementarity determining regions in the light chain variable region are respectively shown as SEQ ID NO. 17-19, and the amino acid sequences of three complementarity determining regions in the heavy chain variable region are respectively shown as SEQ ID NO. 21-23. The antibody provided by the application has the advantages of high effective value, good specificity, high affinity and reaction with 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 high specificity, high sensitivity, simple operation and good repeatability, and can be used for determination of the ChIL-2 content in cell culture supernatant, chicken serum and other samples.

Description

TECHNICAL FIELD

[0001] The present application relates to a chicken interleukin 2 antibody and its application, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Interleukin is a kind of bioactive protein, which can play a role between immune cells. In 1976, Morgan et al. first discovered a factor that can help promote and maintain T cell growth in the culture supernatant of human peripheral blood lymphocytes, and named it as T cell growth factor (TCGF). In 1979, TCGF was officially named as 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), and belongs to Th1 type cytokine. It can promote CD4 + , CD8 + T cell and B cell proliferation, promote DCs activation, enhance NKs killing activity, and promote 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, it can be injected together with a vaccine to help enhance the immunogenicity of the vaccine; in addition, ChIL-2 can also be used as an evaluation index for evaluating avian immune diseases, and has been one of the research hotspots of cytokines. With the development of the breeding industry, the high-density breeding environment and improper breeding process lead to the outbreak of immune deficiency diseases in poultry. ChIL-2 plays a crucial role in the prevention and treatment of avian diseases and the evaluation of immune effect, so the detection technology of ChIL-2 shows great application potential.

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

[0004] The application aims to provide a chicken interleukin 2 antibody and application thereof.

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

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

[0007] The application further provides a chicken interleukin 2 antibody, the amino acid sequence of the light chain is shown as SEQ ID NO. 4, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 8; or the amino acid sequence of the light chain is shown as SEQ ID NO. 20, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 24.

[0008] Further, the nucleotide sequence encoding the light chain is shown as SEQ ID NO. 12, 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, the nucleotide sequence encoding the heavy chain is shown as SEQ ID NO. 32.

[0009] The heavy chain and the light chain are connected through a disulfide bond.

[0010] The application further provides application of the chicken interleukin 2 antibody fragment and the chicken interleukin 2 antibody described above in detection of chicken interleukin 2.

[0011] The application further provides a kit for detecting chicken interleukin 2, and the chicken interleukin 2 antibody described above is used as a detection antibody and / or a capture antibody in the kit.

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

[0013] Further, the kit further comprises a coating standard, a negative control, a coating solution, a blocking solution, a dilution solution, a streptavidin-labeled horseradish peroxidase, a TMB developing solution, and a reaction termination solution.

[0014] The application further provides a method for using the kit, comprising the following steps:

[0015] (1) coating an enzyme-labeled plate with a capture antibody, washing, and blocking;

[0016] (2) adding a sample to be detected, a standard, and a negative control to the enzyme-labeled plate, incubating, and washing;

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

[0018] (4) adding a streptavidin-labeled horseradish peroxidase, incubating, and washing; adding a developing solution, incubating, adding a termination solution, and reading an OD value. 450

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

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

[0021] Figure 1 : Identification of expression and purification of recombinant proteins His-ChIL-2 and GST-ChIL-2. Figure 1 A is the identification of expression and purification of His-ChIL-2 protein, lane M is 180 kDa Prestained Protein Marker, lane 1 is the lysate supernatant after induction of recombinant expression bacteria BL21(DE3)(pCold-ChIL-2), and lane 2 is the purified His-ChIL-2 protein. Figure 1 B is the identification of expression and purification of GST-ChIL-2 protein, lane M is 180 kDa Prestained Protein Marker, lane 1 is the lysate supernatant after induction of 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 antibodies to prokaryotic recombinant proteins. The 4F12 group is the identification of the reactivity of monoclonal antibody 4F12 to prokaryotic recombinant proteins, lane M is 180 kDa Prestained Protein Marker, lane 1 is the purified His-ChIL-2 protein, lane 2 is the BL21(DE3)(pColdI) empty vector control, lane 3 is the purified GST-ChIL-2 protein, and lane 4 is the BL21(DE3)(pGEX-6p-1) empty vector control. The 6E12 group is the identification of the reactivity of monoclonal antibody 6E12 to prokaryotic recombinant proteins, lane M is 180 kDa Prestained Protein Marker, lane 1 is the purified His-ChIL-2 protein, lane 2 is the BL21(DE3)(pColdI) empty vector control, lane 3 is the purified GST-ChIL-2 protein, and lane 4 is the BL21(DE3)(pGEX-6p-1) empty vector control.

[0023] Figure 3 Identification of the reactivity of anti-chicken IL-2 monoclonal antibodies to eukaryotic expression protein ChIL-2.

[0024] Figure 4 Identification of the reactivity of anti-chicken IL-2 monoclonal antibodies to natural ChIL-2.

[0025] Figure 5 Affinity determination results of anti-chicken IL-2 monoclonal antibodies.

[0026] Figure 6 Specificity identification results of double antibody sandwich ELISA of chicken interleukin 2.

[0027] Figure 7 Double antibody sandwich ELISA detection of cell supernatant sample results of chicken interleukin 2.

[0028] Figure 8 Double antibody sandwich ELISA detection of chicken serum sample results of chicken interleukin 2. DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be further described below in combination with the accompanying drawings.

[0030] Example 1 Obtaining of hybridoma cell strain

[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 coding gene published in GenBank (accession number AY029588) was selected, and after optimization according to the codon bias of E. coli, XhoI and Sail enzyme cutting sites were added at both ends of the sequence for ligation with pCold I vector (Takara Co., Ltd., item number 3361); or Smal and NotI enzyme cutting sites were added at both ends of the sequence for ligation with pGEX-6p-1 vector (Solarbio Co., Ltd., item number P0300). The sequence after adding the enzyme cutting sites was synthesized by Nanjing Kingsway Biotech Co., Ltd. and cloned into the expression vector to form recombinant expression plasmids pCold-ChIL-2 and pGEX-6p-1-ChIL-2.

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

[0035] The recombinant expression plasmids pCold-ChIL-2 and pGEX-6P-1-ChIL-2 were transformed into E. 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 protein carrying His and GST tags. The expression and purification process of His-ChIL-2 recombinant protein was as follows: a single colony of BL21(DE3)(pCold-ChIL-2) was inoculated into 5 mL of liquid LB containing ampicillin and incubated at 37°C overnight; the next day, the mother liquor was inoculated into fresh LB liquid medium (containing ampicillin) at a ratio of 1:100, and the culture was incubated at 37°C until the OD600 was about 0.6, then 0.5 mM IPTG was added and the culture was induced at 15°C for 24 h; finally, the recombinant protein His-ChIL-2 was purified using a His-tag protein purification kit (Shenguo Bioengineering Co., Ltd., item number: C600332) and identified by SDS-PAGE electrophoresis. The expression and purification process of GST-ChIL-2 recombinant protein was as follows: a single colony of BL21(DE3)(pGEX-6p-1-ChIL-2) was inoculated into 5 mL of liquid LB containing ampicillin and incubated at 37°C overnight; the next day, the mother liquor was inoculated into fresh LB liquid medium (containing ampicillin) at a ratio of 1:100, and the culture was incubated at 37°C until the OD600 was about 0.6, then 0.5 mM IPTG was added and the culture was induced at 25°C for 8 h; then the recombinant protein GST-ChIL-2 was purified using a GST-tag protein purification kit (Kingsway Biotech Co., Ltd., item number: L00207) and identified by SDS-PAGE electrophoresis.

[0036] Results as shown in Fig. 1, the expected size of the target band appeared at 14KDa (A, lane 2) and 39.4KDa (B, lane 2) respectively, which were His-ChIL-2 and GST-ChIL-2 proteins respectively. Figure 1 Figure 1 A, lane 2) and 39.4KDa (B, lane 2) respectively, which were His-ChIL-2 and GST-ChIL-2 proteins respectively. Figure 1

[0037] 2. Immunization of animals

[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 (emulsion volume ratio 1:1) was injected subcutaneously in multiple sites in the abdomen; two weeks later, 50 μg of His-ChIL-2 protein emulsified with incomplete Freund's adjuvant (emulsion volume ratio 1:1) was injected subcutaneously in multiple sites in the abdomen for booster immunization; two weeks later, 50 μg of His-ChIL-2 protein emulsified with incomplete Freund's adjuvant (emulsion volume ratio 1:1) was injected subcutaneously in multiple sites in the abdomen for the third immunization; two weeks later, blood was collected to determine the serum antibody titer; the mouse with the highest titer (the titer could reach 1:25600) was selected for intraperitoneal injection of 50 μg of purified His-ChIL-2 without adjuvant for booster immunization.

[0039] 2. Cell fusion

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

[0041] 3. Establishment of indirect ELISA detection method

[0042] ​​Screening positive cell clones by indirect ELISA method. Square test determines the coating concentration of detection antigen. Detection antigen GST-IL-2 is diluted horizontally in gradient, 100 μL per well, coated ELISA plate at 4℃ overnight; washed with PBST for 3 times, 200 μL per well, added blocking solution at 4℃ overnight; immunized mouse serum is diluted vertically in gradient, 50 μL per well, normal mouse serum is also diluted in the same gradient as negative control, incubated at 37℃ for 2 h; washed with PBST for the third time, added HRP labeled goat anti-mouse IgG secondary antibody (item number: ab205719; Abeam Company) diluted 1:10000, 100 μL per well, incubated at 37℃ for 1 h, washed with PBST, developed with TMB, and the value of OD was determined by enzyme-linked detector 450 to determine the optimal coating concentration of detection antigen.

[0043] 4. Screening positive clones

[0044] The established indirect ELISA method was used to detect the antibody secreted by hybridoma cells. The specific method is as follows: the culture supernatant of hybridoma cells was added to the pre-coated ELISA plate, 50 μL per well, with SP2 / 0 cell supernatant as negative control and immune polyclonal serum as positive control, 37℃ water bath for 2 h; washed with PBST for 3 times; added HRP labeled goat anti-mouse IgG antibody diluted 1:10000, 100 μL per well, 37℃ water bath for 1 h; after washing, developed with TMB for 10 min, after color development was stopped, the OD 450 reading was determined by enzyme-labeled detector. The experimental results were determined according to the following formula: OD 450 cell well / OD 450 negative well ≥ 2.1 was determined as positive well. The two positive clones screened were named as positive cell clone 4F12 and positive clone cell 6E12.

[0045] 5. Cloning of positive hybridoma cells

[0046] The positive cell clones 4F12 and 6E12 screened were subcloned for 3 times by limited dilution method and preserved.

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

[0048] 1. Preparation of ascites

[0049] The ascites was prepared by in vivo induction method according to the conventional method. 10-12 week old healthy BALB / c mice were injected with liquid paraffin 0.5 mL per mouse, and after 7 days, the hybridoma cells 4F12 and 6E12 cultured to logarithmic growth phase were inoculated into the abdominal cavity of the mice respectively, 5×10 5The ascites were collected after 10 days, 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 titer was determined by indirect ELISA, and the antibody was stored at -70°C after being divided into aliquots.

[0050] 2. Purification marker of the antibody

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

[0052] The purified monoclonal antibody 6E12 was labeled by 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 dissolved millimoles were calculated; the biotin Sulfo-NHS-Biotin (item number: YZM018905; Biofount Company) was equilibrated 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 min; the purification column was prewashed with 30 mL of PBS, and the sample was loaded, and the same amount of buffer as the collection amount was added, and 0.5 mL was collected in a separate tube. The monoclonal antibody protein content was determined by the absorption value at 280 nm.

[0053] Example 3: Detection of monoclonal antibody properties

[0054] 1. Identification of the subclass of the monoclonal antibody

[0055] According to the instructions of the monoclonal antibody subclass kit (Beijing Boaolong BF16001), ELISA was used. 100 μL of the culture supernatant of the hybridoma cells was added to the enzyme-labeled plate coated with GST-IL-2 antigen, 37°C incubation for 2 h, PBST washing for 3 times, 5 min each time; 100 μL of 1:1000 diluted goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM subclass antibodies were added, 37°C incubation for 0.5 h; PBST washing for 3 times, 5 min each time; 100 μL of 1:5000 diluted goat anti-mouse enzyme-labeled secondary antibody was added, 37°C incubation for 15 min, PBST washing for 3 times; 100 μL of TMB color developing liquid was added, 37°C color development for 5 min in the dark, 50 μL of 0.5M H2SO4 was added to stop the reaction, and the OD450 was detected by the enzyme-labeled instrument. 2a , IgG 2b , IgG3, IgM subclass antibodies were added, 37°C incubation for 0.5 h; PBST washing for 3 times, 5 min each time; 100 μL of 1:5000 diluted goat anti-mouse enzyme-labeled secondary antibody was added, 37°C incubation for 15 min, PBST washing for 3 times; 100 μL of TMB color developing liquid was added, 37°C color development for 5 min in the dark, 50 μL of 0.5M H2SO4 was added to stop the reaction, and the OD450 was detected by the enzyme-labeled instrument. 450 , according to the OD 450 to determine the subclass of the monoclonal antibody.

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

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

[0058] SASSSVRYIH.

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

[0060] DTFKLAS.

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

[0062] QQWGTHPRT.

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

[0064] MDFQVQIFSFLLISASVIISRGQIVLTQSPAIMSAFPGEKVTMTCSASSSVRYIHWY QQKSGTSPKRWIYDTFKLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQWGTH PRTFGGGTSLEIK.

[0065] That is, the light chain variable region of the 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 as shown in SEQ ID NO. 5, specifically as follows:

[0067] NYWIE.

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

[0069] EILPGSGSTNYNEKFKD.

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

[0071] TSTVVDY.

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

[0073] MEWTWVFLFLLSVTAGVHSQVQLQQSGAEVMKPGASVKISCKATGYIFSNYWI EWVKQRPGHGLEWIGEILPGSGSTNYNEKFKDKATFTADTSSNTAYMQLSSLTSEDSA VYYCARTSTVVDYWGQGTTLTVSS.

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

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

[0076] AGTGCCAGTTCAAGTGTACGTTACATACAC.

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

[0078] GACACATTTAAACTGGCTTCT.

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

[0080] CAGCAGTGGGGTACTCATCCACGGACG.

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

[0082] ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATTTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGTTCAAGTGTACGTTACATACACTGGTACCAGCAGAAGTCTGGCACCTCC CCCAAAAGATGGATTTATGACACATTTAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGGGTACTCATCCACGGACGTTCGGTGGAGGCACCAGCCTGGAAATCAAA.

[0083] That is, the nucleotide sequence of the light chain of monoclonal antibody 4F12 contains 384 bases.

[0084] The nucleotide sequence of the heavy chain variable region complementarity 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 complementarity 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 complementarity 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] ATGGAATGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAAGTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACATATTCAGTAATTACTGGATAGAGTGGGTAAAACAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTAGTACTAATTACAATGAGAAATTCAAGGACAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACCTCTGAGGACTCTGCCGTCTATTACTGTGCAAGAACAAGTACGGTAGTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0092] In other words, the nucleotide of the heavy chain of the monoclonal antibody 4F12 contains 405 bases.

[0093] The amino acid sequence of the complementarity determining region 1 (CDR1) of the heavy chain variable region of the monoclonal antibody 4F12 is shown as SEQ ID NO. 13, specifically as:

[0094] RASQSIGTSIH.

[0095] The amino acid sequence of the complementarity determining region 2 (CDR2) of the heavy chain variable region of the monoclonal antibody 4F12 is shown as SEQ ID NO. 14, specifically as:

[0096] YIHYSGTYT DYADDS.

[0097] The amino acid sequence of the complementarity determining region 3 (CDR3) of the heavy chain variable region of the monoclonal antibody 4F12 is shown as SEQ ID NO. 15, specifically as:

[0098] DYYGSGTYT.

[0099] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4F12 is shown as SEQ ID NO. 16, specifically as:

[0100] MVSTPQFLVFLLFWIPASRGDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQ RTNGSPRLLINYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPTYTF GGGTKLEIK.

[0101] That is, the light chain variable region of the 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 as 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 as 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 as SEQ ID NO. 23, specifically:

[0107] ESYGNYFAY.

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

[0109] MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYL MHWVKQKPGQGLEWIGYIDPYNADTKYNEKFKGKTTLTSDKSSSTAYMDLSSLTS EDSAVYYCARESYGNYFAYWGQGTLVTVSA.

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

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

[0112] AGGGCCAGTCAGAGCATTGGCACAAGCATACAT.

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

[0114] TATGCTTCTGAGTCTATCTCT.

[0115] The nucleotide sequence of the complementarity determining region 3 (CDR3) of the light chain variable region 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 the monoclonal antibody 6E12 is shown in SEQ ID NO. 28, specifically:

[0118] ATGGTATCCACACCTCAGTTCCTTGTATTTTTGCTTTTCTGGATTCCAGCCTCCAGAGGTGACATCTTGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATACATTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAACTATGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCAACGTACACATTCGGGGGGGGGACCAAGCTGGAAATAAAA.

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

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

[0121] AGCTATCTTATGCAC.

[0122] The nucleotide sequence of the complementarity determining region 2 (CDR2) of the heavy chain variable region 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 complementarity determining region 3 (CDR2) of the monoclonal antibody 6E12 is shown as SEQ ID NO. 31, specifically as follows:

[0125] GAATCCTATGGTAACTACTTTGCTTAC.

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

[0127] ATGGAATGGAGTTGGATATTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCCACTCTGAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATCTTATGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTGATCCTTACAATGCTGATACTAAGTACAATGAGAAATTCAAAGGCAAGACCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGACCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGAATCCTATGGTAACTACTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

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

[0129] 2. Detection of the titer of monoclonal antibody ascites

[0130] The GST-ChIL-2 protein was coated on a 96-well ELISA plate at 0.25 μg / mL using a coating buffer, 4°C overnight; washed 3 times, 200 μL blocking solution was added to each well, 37°C for 2 h; washed 3 times with PBST, the monoclonal antibody ascites prepared in Example 2 was diluted by equal ratio, and the SP2 / 0 ascites was diluted by the same ratio as a negative control, 37°C for 2 h; washed 5 times with PBST, 100 μL / well of 1:10,000 diluted HRP-goat anti-mouse IgG was added, 37°C for 1 h; after washing with PBST, 100 μL / well of TMB color developing solution was added for color development, 37°C for 10 min; the reaction was terminated by adding 0.5 M H2SO4, and the OD value was detected by a microplate reader 450 The titer of the monoclonal antibody ascites was determined, with P / N≥2.1 as the determination standard.

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

[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 SDS-PAGE electrophoresis was performed in a constant temperature metal bath for 10 min. Then, the electrophoresis gel was transferred to nitrocellulose membrane using a rapid transfer instrument for 10 min; blocking was performed using PBST containing 5% skim milk powder, 1 h of shaking blocking at room temperature, and washing 3 times with PBST; 1:1000 diluted monoclonal antibodies 4F12 or 6E12 were added, 4°C overnight shaking incubation, and washing 5 times with PBST; 1:10,000 diluted HRP-goat anti-mouse enzyme-labeled antibody was added, 1 h of shaking at room temperature, and washing 7 times with PBST; color development was performed using color developing solution for 1 min, and imaging was performed using a super-sensitive multifunctional imager.

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

[0135] The plasmid pCMV-myc-ChIL-2 (Han S, Huang X, Meng C, Geng S, Kang X, Pan Z, Jiao X. Prokaryotic expression of chicken interleukin-2 and its activity identification [J]. China Poultry, 2024, 46(05): 56-61.) was transfected into HeLa cells using Lipofectamine 2000, and the reactivity of the monoclonal antibody with the eukaryotic expression ChIL-2 was identified by indirect immunofluorescence 24 h after transfection. Add ice methanol, fix at room temperature for 10 min, wash with PBS for 3 times; block with 5% BSA at 37°C for 2 h, wash with PBS for 3 times; add 1:1000 diluted monoclonal antibody 4F12, 6E12 or SP2 / 0 cell culture supernatant, incubate at 37°C for 2 h, wash with PBS for 5 times; add 1:2000 diluted FITC-labeled goat anti-mouse IgG (ab7685, Abeam Company), incubate at room temperature for 1 h, wash with PBST for 5 times, and take pictures under a fluorescence microscope.

[0136] The results are shown in Figure 3 The 4F12 and 6E12 groups have obvious green fluorescence, while the SP2 / 0 control group has no obvious green fluorescence, indicating that 4F12 and 6E12 can react with eukaryotic expression ChIL-2.

[0137] Chicken spleen lymphocytes were prepared, and the chicken spleen lymphocytes were inoculated into a 24-well cell culture plate at a concentration of 5×10 6 cells / well, and the cells were stimulated with concanavalin A (ConA, 20 μg / mL) while adding 1:1000 diluted protein transport inhibitor brefeldin (BFA, 420601, BioLegend Company) to block the secretion of cytokines to the extracellular. Incubate at 37°C, 5% CO2 incubator for 24 h, and identify the reactivity of the monoclonal antibody with natural ChIL-2 by flow cytometry. Collect the cells, wash with PBS for 3 times, add 1:1000 diluted phycoerythrin (PE)-labeled CD3 antibody (555275, BD Bioscience), incubate at 4°C for 30 min, wash with PBS for 5 times; use Fixation Buffer and Intracellular Staining Perm Wash Buffer for fixation and membrane rupture treatment, wash with PBS for 3 times; add 1:2000 diluted monoclonal antibodies 4F12 and 6E12, incubate at room temperature for 1 h, wash with PBS for 3 times; add 1:2000 diluted FITC-labeled goat anti-mouse IgG, incubate at room temperature for 30 min, wash with PBS for 3 times, and use FACS to detect the proportion of T lymphocytes secreting chicken IL-2.

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

[0139] 4. Monoclonal antibody affinity identification

[0140] The GST-ChIL-2 protein was coated overnight at a concentration of 0.0625, 0.125, 0.25, and 0.5 μg / mL, respectively; the purified antibodies (4F12 and 6E12) were diluted at a starting dilution of 1:1000 as the primary antibody, a total of 16 concentration gradients, and incubated at 37°C for 2 h; 1:2000 diluted HRP-goat anti-mouse IgG was added as the secondary antibody, and incubated at 37°C for 1 h; TMB was developed for 10 min; and 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] The results are shown in Table 2. Figure 5 The 4F12 and 6E12 monoclonal antibodies stably bind to chicken IL-2 protein and have high affinity, with affinity constants of 2.44 x 10 10 M -1 and 1.28 x 10 10 M -1 , respectively.

[0142] Example 4: Establishment of chicken IL-2 double antibody sandwich ELISA method

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

[0144] (1) 100 μL / well of the capture antibody 4F12 at a final concentration of 10 μg / mL was added to a 96-well enzyme-labeled plate, and the plate was coated at 4°C for 14 h;

[0145] (2) The coating solution was discarded, and the plate was washed with PBST for 3 times, each time for 3 min;

[0146] (3) 200 μL / well of PBST containing 10% FBS was added as the blocking solution, and the plate was blocked at 37°C for 2 h;

[0147] (4) The blocking solution was discarded, and the plate was washed with PBST for 3 times, each time for 3 min;

[0148] (5) Add the sample to be tested, the serially diluted standard (natural chicken IL-2), 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 seven 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 Xinan. 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 Xinan. 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), and recombinant chicken IL-4 (ChIL-4) (Dai Hua, Zheng Jiayu, Hu Maozhi, Chen Junhua, Pan Zhiming, Jiao Xinan. 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] See the results Figure 6The ChIL-2 double antibody sandwich ELISA detection method established by the application can specifically detect ChIL-2 protein, and does not have cross-reactions with recombinant proteins ChIL-4, ChIFN-gamma, mIL-2, mIL-4 and hIL-2, and has high specificity, and can be used for ChIL-2 protein detection.

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

[0160] The natural ChIL-2 was prepared according to the following method: after 3-week-old SPF chickens (Zhejiang Li Hua Agricultural Technology Co., Ltd.) were euthanized, the chicken spleens were aseptically taken, cut, ground, filtered, centrifuged at 4 DEG C and 600 rpm / min for 5 min, repeated twice, and the red blood cells were removed. The cell suspension was added to Histopaque-1083 lymphocyte separation medium (10831, Sigma), centrifuged at 20 DEG C and 1800 rpm / min for 20 min, and the white fog layer was aspirated. After the cells were washed twice with RPMI 1640 medium, the concentration was diluted to 2x10 6 cells / well, and was placed in a 24-well plate; ConA (20 μg / mL) was used as a stimulant to stimulate the culture for 24 h, and natural ChIL-2 was obtained. The content of ChIL-2 in the cell supernatant was detected using a commercial kit Chicken IL-2 ELISA Kit (ELG-IL2-1, RayBiotech), and the concentration was adjusted to 2500 pg / mL. The prepared natural ChIL-2 protein was diluted by a factor of 2 from 2500 pg / mL, and the chicken IL-2 double antibody sandwich ELISA method in Example 4 was used for detection to determine the minimum detection limit of the method.

[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 application, indicating that the minimum detection limit of the chicken IL-2 double antibody sandwich ELISA was 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] The isolated chicken spleen lymphocytes were prepared by a conventional method, and were diluted to 5x10 6The cells were inoculated into a 24-well cell culture plate at an amount of 10 μg / mL of 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] See the 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 Serum Samples by Chicken IL-2 Double Antibody Sandwich ELISA

[0169] 1×10 7 Three 3-day-old SPF chicks (Zhejiang Lihua Agricultural Technology Co., Ltd.) were infected intramuscularly with a CFU dose of Salmonella Enteritidis C50041. A second and third inoculation with the same dose was performed 14 and 28 days after the first inoculation, respectively. Seven days after each infection, venous blood was collected, serum was isolated, and serum ChIL-2 levels were measured using the chicken IL-2 double antibody sandwich ELISA method described in Example 4. A control group was also injected with 100 μL of PBS.

[0170] See the 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 description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. 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. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions 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 includes light chain and heavy chain variable regions, the amino acid sequences of the three complementary determining regions CDR1-3 in the light chain variable region are shown in SEQ ID NOs.1-3, and the amino acid sequences of the three complementary determining regions CDR1-3 in the heavy chain variable region are shown in SEQ ID NOs.5-7; or the amino acid sequences of the three complementary determining regions CDR1-3 in the light chain variable region are shown in SEQ ID NOs.17-19, and the amino acid sequences of the three complementary determining regions CDR1-3 in the heavy chain variable region are shown in SEQ ID NOs.21-23.

2. The gene encoding the chicken interleukin-2 antibody fragment according to claim 1, characterized in that The nucleotide sequences encoding the three complementarity determining regions CDR1-3 in the light chain variable region are shown in SEQ ID NOs.9-11, and the nucleotide sequences encoding the three complementarity determining regions CDR1-3 in the heavy chain variable region are shown in SEQ ID NOs.13-15, respectively; or the nucleotide sequences encoding the three complementarity determining regions CDR1-3 in the light chain variable region are shown in SEQ ID NOs.25-27, and the nucleotide sequences encoding the three complementarity determining regions CDR1-3 in the heavy chain variable region are shown in SEQ ID NOs.29-31, respectively.

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

32.

5. Use of the chicken interleukin-2 antibody fragment according to claim 1 and the chicken interleukin-2 antibody according to claim 3 in the preparation of a reagent for detecting chicken interleukin-2.

6. A kit for detecting chicken interleukin-2, characterized in that: The chicken interleukin-2 antibody according to claim 3 is used as a detection antibody or a capture antibody in the kit.

7. The kit for detecting chicken interleukin-2 according to claim 6, wherein The detection antibody is a biotin-labeled antibody.

8. The kit for detecting chicken interleukin-2 according to claim 6, wherein The kit also contains coating standards, negative controls, coating solution, blocking solution, diluent, horseradish peroxidase labeled with streptavidin, TMB color developing solution, and reaction termination solution.

9. A method for using the kit according to any one of claims 6 to 8 in non-disease diagnosis, characterized in that: The following steps are involved: (1) Coat the ELISA plate with capture antibody, wash, and block; (2) Add the sample to be tested, the standard and the negative control to the above ELISA plate, incubate and wash; (3) Add detection antibody, incubate, and wash; (4) Add streptavidin-labeled horseradish peroxidase, incubate, and wash; 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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