Anti-grass carp interleukin-21 monoclonal antibody and application

Abstract

The application provides an anti-grass carp interleukin-21 monoclonal antibody and application, the monoclonal antibody is obtained by hybridoma cell strain with preservation number of CCTCC NO: C2022214, and the anti-grass carp interleukin-21 monoclonal antibody specifically recognizes grass carp interleukin-21 recombinant protein, and the anti-grass carp interleukin-21 monoclonal antibody can be applied in the preparation of reagent or kit for detecting grass carp interleukin-21 protein, and can also be applied in enzyme-linked immunosorbent assay, Western blotting, immunofluorescence assay, immunohistochemistry or flow cytometry detection;It can be applied to detect the production of interleukin-21 in grass carp tissue, and can also specifically detect the concentration of interleukin-21 in grass carp serum, provides parameter index for monitoring grass carp inflammation, can monitor the health status of grass carp in real time, thereby providing technical support for the sustainable development of grass carp breeding industry.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a grass carp interleukin-21 recombinant protein, a hybridoma cell line secreting an anti-grass carp interleukin-21 monoclonal antibody, and a preparation method and application of the grass carp interleukin-21 monoclonal antibody. Background Art

[0002] Grass carp (Ctenopharyngodon idella) is a key aquaculture species in my country's freshwater fisheries, consistently ranking first in aquatic product output. In 2020, my country's total grass carp production reached approximately 5.571 million tons, accounting for 18.04% of the country's total freshwater aquaculture output. Grass carp is a popular species among farmers due to its wide range of feed sources, rapid growth rate, and high economic value. However, frequent grass carp diseases in recent years have severely hampered the sustainable development of the aquaculture industry. Strengthening fish immune defenses and monitoring fish health are urgent and crucial for the high-quality and sustainable development of aquaculture.

[0003] Studies have shown that cytokines play a crucial role in fish immune control. Interleukin-21 (IL-21) is a key novel cytokine, primarily produced by activated immune cells such as CD4+ T cells and NKT cells. It regulates the proliferation, differentiation, and function of various immune cells and plays a crucial role in inflammatory responses. Detecting IL-21 levels in grass carp tissues and serum using IL-21 monoclonal antibodies can be used to assess the inflammatory response in grass carp. Currently, mammalian IL-21 monoclonal antibodies have been developed, but due to low sequence homology and inability to recognize antigenic epitopes, they are not suitable for detection in fish. The lack of grass carp-specific antibodies limits the detection of IL-21 in grass carp. Therefore, there is an urgent need to develop highly specific and potent IL-21 monoclonal antibodies for detecting IL-21 levels in grass carp tissues and serum. This would provide a parameter for monitoring inflammatory processes in grass carp and other cyprinid fish, with potential application in the prevention and treatment of inflammatory diseases. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides an anti-grass carp IL-21 monoclonal antibody and its application.

[0005] To achieve the above object, the solution of the present invention is:

[0006] In a first aspect, the present invention provides an anti-grass carp interleukin-21 monoclonal antibody. The anti-grass carp interleukin-21 monoclonal antibody is secreted by a hybridoma cell line and labeled with FITC fluorescent dye. The hybridoma cell line is named GC9-IL21 and was deposited with the China Center for Type Culture Collection at Wuhan University, Wuhan, China on August 17, 2022, with a deposit number of CCTCC NO: C2022214. The anti-grass carp interleukin-21 monoclonal antibody specifically recognizes grass carp interleukin-21 recombinant protein.

[0007] Purified monoclonal antibodies: High-purity (>90%) anti-grass carp IL-21 monoclonal antibodies were obtained through affinity purification using a HiTrap rProtein A FF column. The antibody affinity constant was 1.14E+09 as detected by ELISA. Western blotting revealed that the antibody specifically recognized the grass carp IL-21 recombinant protein without reacting with other grass carp proteins. The anti-grass carp IL-21 monoclonal antibodies were labeled with FITC fluorescent dye for use in subsequent immunofluorescence experiments.

[0008] In a second aspect, the present invention provides an application of an anti-grass carp interleukin-21 monoclonal antibody, and an application of the anti-grass carp interleukin-21 monoclonal antibody in the preparation of a reagent or a kit for detecting grass carp interleukin-21 protein.

[0009] In a third aspect, the present invention provides a reagent or kit for detecting grass carp interleukin-21 protein, which comprises the above-mentioned grass carp interleukin-21 monoclonal antibody.

[0010] In a fourth aspect, the present invention provides an application of a grass carp interleukin-21 monoclonal antibody, the application of the grass carp interleukin-21 monoclonal antibody in enzyme-linked immunosorbent assay (ELISA), protein blotting (Western blot), immunofluorescence assay, immunohistochemistry (IHC) (i.e., by preparing frozen tissue sections and using laser confocal microscopy to observe the distribution of IL-21 in the tissue, providing parameter indicators for monitoring grass carp inflammation, and real-time monitoring of the health status of grass carp) or flow cytometry detection.

[0011] In a fifth aspect, the present invention provides a hybridoma cell line that secretes grass carp interleukin-21 monoclonal antibody, and the deposit number of the hybridoma cell line is: CCTCC NO: C2022214.

[0012] Preferably, the method for preparing the hybridoma cell line is as follows: injecting grass carp interleukin-21 recombinant protein into the peritoneal cavity of mice, taking the spleen cells of the mice and fusing them with myeloma cells, screening, and purifying to obtain the hybridoma cell line.

[0013] Specifically,

[0014] Immunization of mice: 4 SPF BALB / c female mice were selected, and a total of 4 immunizations were performed with an immunization cycle of 14 days. For the first immunization, each mouse was subcutaneously injected with 60 μg of grass carp IL-21 recombinant protein, and for the booster immunization, each mouse was injected with 30 μg. Ten days after the last immunization, the mice were anesthetized, and orbital blood was collected, and the titer of the immunized mice was determined by ELISA.

[0015] Cell fusion: Before fusion, the immunized mice were intraperitoneally injected with 50 μg of grass carp IL-21 recombinant protein, and mouse spleen cells were taken and fused with SP2 / 0 cells using the PEG method. The fused cells were transferred to semi-solid medium for culture.

[0016] Screening of positive hybridoma cells: The single clones growing on the semi-solid medium were picked into 96-well culture plates for culture. The selected clones were subjected to two rounds of screening and sub-class identification by enzyme-linked immunosorbent assay (ELISA), and finally seven positive hybridoma cell strains of IgG type were obtained. These strains were cultured in large quantities and stored in the laboratory.

[0017] Preferably, the preparation method of the grass carp interleukin-21 recombinant protein comprises: recombining the grass carp interleukin-21 gene in Pet-21d plasmid to obtain a recombinant, transforming it into competent cells DE3, purifying the grass carp interleukin-21 recombinant protein, and performing purification, activity identification and preservation. The base sequence of the grass carp interleukin-21 gene is shown in SEQ ID NO. 1.

[0018] The base sequence of the grass carp IL-21 gene is:

[0019] ATGAAGGCATCCGTGTTTGTTCTGTTCGCAGTGGCGTGTTGGTTCGTTTCGCAGGCAGAGCTGTCGCCGATGCAACTCACGCTCAGAAAAGTTATGAATGAACTCGAGAAGGTCAATAACGTAATGGACAAAAGCACAAGTTCGTTCAACTCACCCACCACCAACGATTTGAAGGACTGCTGCATCAGATCTGCTCTGGAGTGCTTTAGGTCCCAAGTGATGAAC CTGAATGTCACTCAAGAGAAACCTATAATAAAGTCACTGAAGAGCATCTCCAACGAGTCCCGAAAAAAAGTCATTGTGGAAAAGTTGCCCAGTTGCAACCTGGTAGAAGGTGAGAAAAAGGAAGCTCAGTGCAAACCCTGTGAATCGTACAGCAAGGTTAACAGCCAAATGTTCGTGCAGAACTTTCAAACTCTTCTTCAAAAGATCTATGCCAGTCAAGCA T AG (The underlined sequence indicates the promoter and stop codons).

[0020] Preferably, the amino acid sequence of the grass carp interleukin-21 recombinant protein is shown as SEQ ID NO.2.

[0021] Among them, the amino acid sequence of grass carp IL-21 recombinant protein is:

[0022] MKASVFVLFAVACWFVSQA ELSPMQLTLRKVMNELEKVNNVMDKSTSSFNSPTTNDLKDCCIRSALECFRSQVMNLNVTQEKPIIKSLKSISNESRKKVIVEKLPSCNLVEGEKKEAQCKPCESYSKVNSQMFVQNFQTLLQKIYASQA*. (The underline indicates the signal peptide, * indicates the stop codon, and the rest indicates the mature peptide.)

[0023] Preferably, the grass carp interleukin-21 recombinant protein is used in the preparation of vaccine molecular adjuvants.

[0024] Due to the adoption of the above solution, the beneficial effects of the present invention are:

[0025] The grass carp IL-21 recombinant protein prepared by the present invention is purified by gel filtration chromatography using an AKTA pure protein purification system, resulting in a single band on SDS-PAGE electrophoresis, high purity, and biological activity. Mice were immunized with the grass carp IL-21 recombinant protein as an antigen and screened to obtain a positive hybridoma cell line capable of specifically secreting an IgG-type anti-grass carp IL-21 monoclonal antibody. The anti-grass carp IL-21 monoclonal antibody (9#) prepared using the hybridoma cell line GC9-IL21 showed high purity (>90%) as determined by SDS-PAGE, ELISA, and Western blot, with an affinity constant of 1.14E+09, indicating that it can specifically recognize the grass carp IL-21 recombinant protein, demonstrating high detection sensitivity and accuracy. Therefore, grass carp IL-21 recombinant protein can be used as a vaccine molecular adjuvant to improve the immune protection effect of the vaccine; anti-grass carp IL-21 monoclonal antibody can be used for Western blot detection of grass carp IL-21 protein, and can also be used to specifically detect the IL-21 concentration in grass carp serum by ELISA, and the production of IL-21 in tissues can be detected by frozen tissue sections, providing parameter indicators for monitoring grass carp inflammation, and can monitor the health status of grass carp in real time, thereby providing technical support for the sustainable development of grass carp farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram showing the purification of grass carp IL-21 recombinant protein and SDS-PAGE protein electrophoresis verification in Example 1 of the present invention.

[0027] Figure 2 This is a diagram of the immune titer of the serum of immunized mice in Example 2 of the present invention.

[0028] Figure 3 This is a purity diagram of the anti-grass carp IL-21 monoclonal antibody in Example 3 of the present invention.

[0029] Figure 4 This is a graph showing the affinity constants of the anti-grass carp IL-21 monoclonal antibody in Example 3 of the present invention.

[0030] Figure 5 This is a diagram showing the specificity of antibodies detected by Western blot in Example 3 of the present invention.

[0031] The hybridoma cell line GC9-IL21 was deposited in the China Center for Type Culture Collection, Wuhan University on August 17, 2022, and its deposit number is: CCTCC NO: C2022214. DETAILED DESCRIPTION

[0032] The present invention provides an anti-grass carp IL-21 monoclonal antibody and its application.

[0033] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments of the present invention, but the protection scope of the present invention is not limited to the following.

[0034] Example 1: Preparation of Grass Carp IL-21 Antigen

[0035] (1) Cloning and prokaryotic expression plasmid construction of grass carp IL-21

[0036] Grass carp IL-21 primers (IL-21-F: CCATGGCTGAACTCAGCCCGATGC (SEQ ID NO. 3); IL-21-R: GGATCCTTAGGCTTG GCTGGCGTA (SEQ ID NO. 4)) were designed to amplify the mature peptide DNA sequence of IL-21 by PCR. The resulting DNA product was ligated into the pMD 19-T vector. The recombinant plasmids pMD 19-T-IL-21 and Pet-21d were then digested with DNA enzymes. IL-21 was then ligated into the Pet-21d vector using T4 DNA ligase to synthesize the recombinant plasmid Pet-21d-IL-21. Pet-21d-IL-21 was transfected into the competent DE3 expression strain, and single clones were selected for positive PCR screening by plating.

[0037] (2) Inducible expression and purification of grass carp IL-21 recombinant protein

[0038] The positive clone strain was inoculated at a ratio of 1:100 into 10 mL of LB liquid medium (containing 100 μg / mL ampicillin) and cultured in a 37°C shaker at 170 rpm until the OD value reached 0.6 (divide into two aliquots). The first 10 mL aliquot served as a negative control. In the second aliquot, 10 μL of 1 mol / mL IPTG was added and shaken for 12 hours to induce protein expression. Then, 1 mL of each aliquot was transferred to a 1.5 mL EP tube and centrifuged at 12,000 rpm for 2 minutes. The supernatant was discarded, and 40 μL of sterile PBS and 10 μL of 5× protein loading buffer were added. The mixture was then heated in a metal bath at 100°C for 10 minutes. After the sample was cooked, it was centrifuged briefly and analyzed by SDS-PAGE gel electrophoresis to confirm the expression of the target protein. If expression is required, induce a large amount of protein, centrifuge the induced bacteria, discard the supernatant, wash twice with sterile PBS, and then use an ATS micro-ultrahigh pressure homogenizer to break it. After breaking, extract the inclusion bodies, dilute and renature, concentrate under nitrogen pressure, and purify with an AKTA protein purifier. The obtained protein is measured with a BCA protein concentration kit and stored at -80°C for later use (such as Figure 1 shown).

[0039] (3) Application of grass carp IL-21 recombinant protein as a vaccine molecular adjuvant

[0040] The grass carp IL-21 recombinant protein prepared in this example has biological activity and can be used as a molecular adjuvant for inactivated grass carp hemorrhagic disease vaccines, subunit vaccines, DNA vaccines, or other vaccines to improve vaccine protection. The effectiveness of the IL-21 recombinant protein as a GCRV vaccine adjuvant was evaluated using immune-related gene expression levels, macrophage phagocytosis efficiency, peripheral blood hemocyte and leukocyte changes, serological analysis, and relative immune protection rates, with the goal of increasing the vaccine's immunogenicity and enhancing its immune protection. This, in turn, enhances the antiviral capacity of grass carp through immunization, contributing to the development and application of new vaccines.

[0041] Example 2: Process for obtaining hybridoma cell lines

[0042] (1) Experimental equipment and reagents: Sterile surgical instruments: three pairs of scissors, three pairs of forceps, a cell strainer, a syringe core, a petri dish, two 500 mL beakers, two 50 mL centrifuge tubes, three 15 mL centrifuge tubes; IMDM medium, IMDM complete medium (containing 15% serum) and newborn calf serum were purchased from Gibco, 2.2% methylcellulose (SIGMA, M0262-100G), mouse myeloma cells (SP2 / 0Cell) (Beijing Huada Protein Research and Development Center Co., Ltd.), PEG1500 (Roche, 78364), H AT (Sigma, H0262-10VL), HT (Sigma, H0137-10VL), coating solution (sodium carbonate-sodium bicarbonate buffer, pH 9.6), PBS buffer pH 7.4, blocking solution (2% milk), washing solution: PBS-T (0.05% Tween, PBS), color development solution: 1% solution A + 10% solution B (solution A: 1% TMB; solution B: 0.1% H2O2), stop solution: 2 mol / L sulfuric acid, etc. were purchased from Sangon Biotech (Shanghai) Co., Ltd. Secondary antibody: goat anti-mouse IgG / HRP was purchased from LI-COR.

[0043] (2) Immunization of mice: Four SPFBALB / c female mice (Beijing BGI Protein Research Center Co., Ltd.) were subcutaneously immunized with 60 μg of grass carp IL-21 recombinant protein per mouse. These mice were designated GIL21-1, GIL21-2, GIL21-3, and GIL21-4. Three booster immunizations were then performed at 30 μg per mouse, two weeks apart. Blood was collected from the eye sockets.

[0044] (3) ELISA detection of serum titer: 2 μg / mL grass carp IL-21 recombinant protein was coated overnight at 4°C; washed 3 times with washing solution, blocked at 37°C for 1 h; mouse serum was diluted by 2-fold gradient from 200 times, blank was PBS, negative control was negative serum diluted by 200 times, then the diluted serum was added to the protein coated wells, three replicates for each concentration gradient, 37°C, incubated for 2 h, then washed 3 times and dried; HRP labeled goat anti-mouse secondary antibody was diluted by 1:10000, mixed well and added 100 μL per well, 37°C, incubated for 1 h, then washed 3 times and dried; then 100 μL of TMB single-component color developing solution was added to each well, 37°C, color developed to the expected depth (10-30 min); finally 50 μL of stop solution was added to each well, and the OD value at 450 nm was measured by a microplate reader. The results are shown in Figure 2 Figure 2, the serum titer of GIL21-2 mice was greater than 25600, and the GIL21-2 mice were selected for impact immunization by intraperitoneal injection of 50 μg of immunogen for cell fusion experiment.

[0045] (4) Cell fusion experiment: The sp2 / 0 cells in good condition were gently blown off from the culture bottle wall and sucked into a 50 mL centrifuge tube. The mouse was anesthetized and the eyeball was taken out, then the mouse was killed by pulling the neck and soaked in 75% alcohol for 5 min. A small amount of serum-free IMDM was poured into a plate, and a cell sieve and a syringe inner core were placed in the plate. The mouse's spleen was removed with scissors and forceps and placed on the cell sieve. The spleen was gently crushed with the inner core of the syringe, and the crushed cells were sucked into the centrifuge tube containing sp2 / 0, and centrifuged at 1500 rpm for 5 min. The mouse's thymus was removed with scissors and forceps and crushed. The crushed thymus cells were placed in a 15 mL centrifuge tube, and 2 mL of HAT and 2 mL of HT were added and placed in an incubator. The centrifuged cells were poured off, and the cells were gently blown evenly with serum-free IMDM and centrifuged (1500 rpm, 5 min). The supernatant of the centrifuged cells was discarded, and the cells were suspended by tapping the bottom of the centrifuge tube. The centrifuge tube was placed in warm water at 37°C, and 1 mL of PEG was slowly added within 1 min. After adding, it was placed in warm water for 1 min. Then 2 mL of serum-free IMDM was slowly added within 2 min, followed by 8 mL of serum-free IMDM slowly added within 2 min. Centrifuge at 1000 rpm for 5 min. Discard the supernatant, add 10 mL of serum, and carefully blow the cells evenly into the thymus cells prepared earlier. Then add 25 mL of sterile semi-solid medium and mix well. Then evenly pour into 30 cell culture dishes. The cell culture dishes were placed in a humid box and cultured in an incubator.

[0046] (5) Monoclonal cell screening: First, 10 plates × 93 cell clones were selected and cultured in 96-well cell culture plates (previously plated with thymocytes, 100 μL / well). The selected clones were then screened using the ELISA method. Grass carp IL-21 recombinant protein was diluted to 2 μg / mL with coating solution, 100 μL / well, 4°C, and washed three times with washing solution overnight. Blocked with 2% milk blocking solution, 200 μL / well, incubated at 37°C for 2 hours; then washed three times with washing solution. Primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (positive serum diluted 1000 times with PBS) were added, all at 100 μL / well, incubated at 37°C for 1 hour; then washed three times with washing solution. Secondary antibody diluted 20,000 times with PBS was added, 100 μL / well, incubated at 37°C for 1 hour; removed and washed three times with washing solution. Color development was performed using 100 μL of color development solution per well for approximately 5 minutes. 50 μL of stop solution was added to each well to terminate the reaction. Absorbance was measured at dual wavelengths (450 and 630 nm) and the data was recorded and saved. A total of 16 positive hybridoma cell lines were screened. A second screening procedure was used, similar to that described above, resulting in a total of 8 positive hybridoma cell lines.

[0047] (6) Antibody subclass identification: Dilute the coated antibody to 0.5 μg / mL with 100 mmol / L PBS (pH 7.4), add 0.1 mL to each well, and incubate at 4°C overnight. Wash twice with PBS-T; add 200 μL of blocking solution to each well and incubate at 37°C for 2 h. Wash three times with PBS-T; add 100 μL of hybridoma supernatant to each well and incubate at 37°C for 1 h. Wash three times with PBS-T; add 0.1 mL / well of HRP-labeled antibody diluted 1:1000 (κ, λ) or 1:2000 (goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA) with blocking solution to the appropriate wells and incubate at 37°C for 1 h. Wash three times with PBS-T; add 50 μL of substrate solution to each well, measure the absorbance at dual wavelengths (450, 630) within 10-20 min, and record and save the data (results are shown in Table 1). Finally, seven IgG-positive hybridoma cell lines were obtained.

[0048] Table 1 Monoclonal cell line subtype identification and related information

[0049]

[0050] Example 3: Process for obtaining anti-grass carp IL-21 monoclonal antibodies

[0051] (1) The hybridoma cell lines screened above were cultured at a rate of 5×10 7 The cells were injected intraperitoneally into BALB / c mice at a dose of 10 cells. After about two weeks, when the abdomen of the mice was obviously swollen, ascites was collected to obtain anti-grass carp IL-21 monoclonal antibodies.

[0052] (2) Antibody specificity detection: The grass carp IL-21 recombinant protein was subjected to SDS-PAGE gel electrophoresis at 150V for 50min. After the electrophoresis, the protein gel was transferred to the membrane using a protein transfer instrument (25V, 2.5A, 7min). After the transfer was completed, the membrane was blocked with 5% skim milk powder blocking solution for 1h. Then, an appropriate amount of primary antibody (serum) diluted at 1:1000 was placed in the antibody incubation box and incubated at 4°C overnight. The PVDF membrane was washed three times with TBST for 5min each time, and then an appropriate amount of goat anti-mouse secondary antibody diluted at 1:10000 was added and incubated for 1h. The PVDF membrane was washed three times with TBST for 5min each time, and finally placed in the Odyssey CLx near-infrared fluorescence imaging system for scanning and imaging. The results are shown in Figure 2. Figure 3 As shown, grass carp IL-21 recombinant protein reacted specifically with monoclonal antibodies 7#, 9#, 10#, 11#, 12#, 15#, and 16#, that is, the antibodies produced by the seven monoclonal hybridoma cell lines could specifically recognize grass carp IL-21 recombinant protein.

[0053] (3) Then, the antibody 9# with good specificity was selected and affinity purified using HiTrap rProtein A FF column. The purity of the antibody was detected by SDS-PAGE. The results are as follows: Figure 4 As shown, the purity of antibody 9# produced by the GC9-IL21 hybridoma cell line was high and greater than 90%.

[0054] (4) The affinity constant of the purified antibody was detected by ELISA method, that is, 2 μg / mL grass carp IL-21 recombinant protein was used for coating at 4°C overnight; 2% milk was used for blocking at 37°C for 2h; the antibody was diluted 2-fold starting from 200 times, and the blank control (blank) was PBS, both at 100 μL / well, incubated at 37°C for 1h; washed 3 times with washing solution, and the secondary antibody diluted 20,000 times with PBS was added at 100 μL / well, incubated at 37°C for 1h; after taking out, washed 3 times with washing solution, and color was developed with 100 μL / well of color development solution for about 5min, and 50 μL of stop solution was added to each well to stop; the absorbance value was measured and the data was recorded and saved (as shown in Table 2). The affinity constant of monoclonal antibody 9# was 1.14E+09 (the results are shown in Table 2). Figure 5 ).

[0055] Table 2

[0056]

[0057] (5) In order to be used for immunofluorescence experiments, a portion of the anti-grass carp IL-21 monoclonal antibody was labeled with FITC fluorescent dye. Before labeling, the anti-grass carp IL-21 monoclonal antibody sample was dialyzed more than three times with 0.1 mol / L carbonate buffer at a pH of 9.0 at 4°C. The dialyzed antibody was then transferred to a brown bottle, and 75 μL of 1 mg / mL FITC fluorescent dye was added to 1.26 mg of the antibody under slow stirring and incubated at 4°C overnight. The reacted anti-grass carp IL-21 monoclonal antibody was dialyzed more than four times with 0.01 mol / L PBS buffer at a pH of 7.4 at 4°C until the dialyzate was colorless and stored at -20°C for later use.

[0058] The anti-grass carp IL-21 monoclonal antibody obtained by the above example has the following application prospects:

[0059] 1. Monoclonal antibodies used in Western blotting to detect IL-21 protein

[0060] This anti-grass carp IL-21 monoclonal antibody can specifically recognize the target protein IL-21 by Western blotting. Therefore, this antibody can be directly used to study the immune mechanism of fish by analyzing the localization and cellular expression of the IL-21 protein. The procedure is as follows:

[0061] (1) Sample preparation: Extract the target protein, lyse it, add 5× SDS-PAGE loading buffer, and boil it at 100°C for 10 min to prepare the sample.

[0062] (2) Electrophoresis and membrane transfer: The protein samples were subjected to SDS-PAGE gel electrophoresis (150 V, 50 min). After the electrophoresis, the protein gel was transferred to a PVDF membrane using a protein transfer apparatus (25 V, 2.5 A, 7 min).

[0063] (3) Blocking: Block with 5% skimmed milk powder blocking solution for 1 h.

[0064] (4) Primary antibody incubation: a 1:1000 diluted anti-grass carp IL-21 monoclonal antibody prepared in the present invention was placed in an antibody incubation box together with a PVDF membrane, and incubated at room temperature for 2 h or at 4°C overnight. The membrane was washed three times with TBST, each time for 5 min.

[0065] (5) Secondary antibody incubation: Add 1:10000 diluted goat anti-mouse secondary antibody and incubate at 4°C for 1 h. Wash the PVDF membrane three times with TBST, each time for 5 min.

[0066] (6) Imaging: Place in Odyssey CLx near-infrared fluorescence imaging system, scan and take pictures.

[0067] 2. Use of the monoclonal antibody in ELISA

[0068] The specific experimental operation for detecting the secretion level of grass carp IL-21 protein is as follows:

[0069] (1) Antigen coating: grass carp IL-21 is the antigen to be detected, and the grass carp IL-21 recombinant protein is the standard product diluted to different concentrations with coating solution to make a standard curve, 100 μL is added to each well of the enzyme-labeled plate, 2 μg / mL of BSA is set as a negative control, and it is placed at 4°C overnight.

[0070] (2) Washing: discard the coating solution, wash with washing solution for 3 times, 3 min each time, 500 μL / well, and pat dry.

[0071] (3) Blocking: add 200 μL of blocking solution to each well, incubate at 37°C for 2 h, then wash 3 times and pat dry.

[0072] (4) Incubation of primary antibody: dilute the prepared anti-grass carp IL-21 monoclonal antibody with antibody diluent at 1:1000, add 100 μL to each well, repeat three times for the same sample, incubate at 37°C for 2 h, then wash 3 times and pat dry.

[0073] (5) Incubation of secondary antibody: dilute the HRP-labeled goat anti-mouse secondary antibody at 1:10000, mix well, then add 100 μL to each well, incubate at 37°C for 1 h, then wash 3 times and pat dry.

[0074] (6) Color development: add 100 μL of TMB single-component color developing solution to each well, develop color at 37°C in the dark to the desired depth (10-30 min).

[0075] (7) Termination of reaction: add 50 μL of termination solution (H2SO4) to each well, and measure the OD value at 450 nm with an enzyme-labeled instrument.

[0076] 3. Use of the monoclonal antibody in immunohistochemistry (laser confocal)

[0077] (1) Fixation and embedding of grass carp tissue: (fixation) immediately place the sampled grass carp tissue into 4% PFA tissue fixing solution, fix at 4°C overnight; (dehydration) place the tissue sample into 20% sucrose solution for dehydration until it sinks, then replace with 30% sucrose solution for dehydration, replace with new 30% sucrose solution after sinking, and place at room temperature for 1 h after the tissue sinks to the bottom; (embedding) stack tin foil into small pieces, pour embedding agent OCT, place the tissue in position, and place in -80°C for solidification, then slice.

[0078] (2) Preparation of frozen sections: The frozen tissue was quickly placed in a cryostat that was precooled to -20°C. When the embedding agent was completely solidified, the tissue embedding block was fixed to the knife holder and the tissue embedding block was trimmed to a smooth and flat surface with a single-sided blade. Then the slice was trimmed (80 μm) and the thickness of the slice was adjusted to 6 μm. The tissue was adhered to a slide and marked.

[0079] (3) Immunofluorescence staining: Take out the slide from -80℃ and reheat for about 30 minutes until the slide is dry. Put the slide into 0.01mol / L PBS buffer to hydrate for 5 minutes (shaking). Replace with 0.3% Triton X-100 to permeabilize the membrane for 15 minutes (shaking). Wash with PBS 3 times, 5 minutes each time (shaking). Put the slide into sodium citrate solution and heat it in a microwave oven to about 98℃ (starting to bubble) for antigen retrieval. Stop for 5 minutes, reheat, repeat 3 times for a total of 15 minutes, take it out and cool to room temperature (about 1 hour). Replace with PBS and wash 3 times, 10 minutes each time (shaking). Use filter paper to absorb the liquid around the front and back tissues, draw a circle around the tissue with a histochemical pen, put the slide into a humidified box, add the serum derived from the primary antibody and block for 30 minutes (shaking). Shake off the serum, add about 35μL / slide of appropriately diluted FITC-labeled anti-grass carp IL-21 monoclonal antibody, and place it in a humidified box at 4℃ overnight. Remove the slide from the 4°C humidified chamber and allow to warm for approximately 30 minutes. Use a pipette to remove any excess liquid (primary antibody) from the slide. Wash three times in PBS (protected from light) for 10 minutes each time on a rocking platform. Add approximately 30 μL of DAPI (depending on tissue size) and stain for 3-5 minutes in the dark. Wash three times in PBS (protected from light) for 5 minutes each time on a rocking platform. Mount the slide with fluorescent signal-enhancing mounting media and place in a humidified chamber at 4°C for 1 hour. Observe and photograph using a laser confocal microscope.

[0080] The above description of the embodiments is intended to facilitate understanding and use of the present invention by those skilled in the art. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without resorting to creative effort. Therefore, the present invention is not limited to the above-described embodiments. Any improvements or modifications made by those skilled in the art based on the principles of the present invention that do not depart from the scope of the present invention should be considered within the scope of protection of the present invention.

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Claims

1. An anti-grass carp interleukin-21 monoclonal antibody, characterized in that: The anti-grass carp interleukin-21 monoclonal antibody is secreted by a hybridoma cell line with a deposit number of CCTCC NO: C2022214, and the anti-grass carp interleukin-21 monoclonal antibody specifically recognizes the grass carp interleukin-21 recombinant protein.

2. Use of the anti-grass carp interleukin-21 monoclonal antibody according to claim 1 in the preparation of a reagent or kit for detecting grass carp interleukin-21 protein.

3. A reagent or kit for detecting grass carp interleukin-21 protein, characterized in that: The invention comprises the grass carp interleukin-21 monoclonal antibody according to claim 1.

4. A hybridoma cell line secreting grass carp interleukin-21 monoclonal antibody, whose deposit number is: CCTCCNO: C2022214.

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