Takifugu obscurus IFN-gamma monoclonal antibody fragment and application thereof

Abstract

The invention discloses a fugu obscurus IFN-gamma monoclonal antibody fragment and an application thereof. The fugu obscurus IFN-gamma monoclonal antibody can be used as a tool for immunological related detection and immune function evaluation to detect the expression level of the fugu obscurus IFN-gamma, so that the response of the fugu obscurus to pathogenic infection, immune stimulation and other immune challenges is studied; and an immunopotentiator or a vaccine product aiming at the takifugu obscurus can be researched, so that the healthy and sustainable breeding development of the takifugu obscurus is promoted.

Description

Technical Field

[0001] The present invention relates to a Takifugu fasciatus IFN-γ monoclonal antibody fragment and its application, belonging to the field of aquaculture. Background Art

[0002] Interferon (IFN) is a cytokine, so named because it can "interfere" with virus replication. According to its molecular structure and biological functions, it is divided into type I, type II, and type III IFN. Different types of IFN activate a series of signal transduction pathways by binding to cell surface receptors. IFN-γ is a cytokine with pro-inflammatory effects and is currently the only type II IFN. IFN-γ is also known as immune interferon and is one of the main proteins secreted by Th1 cells, which can promote the occurrence of immune responses. IFN-γ can also be produced by a variety of immune cells and is a key cytokine in the immune system, directly and indirectly participating in antiviral activities. Although the research on IFN-γ in mammals is more in-depth than that in aquatic animals, due to differences in species, immune systems, and living environments, there are significant differences in gene structure, functional performance, immune regulation, etc. between the two, that is, IFN-γ in fish and IFN-γ in mammals have different functional characteristics. Therefore, IFN-γ in fish also has certain uniqueness. At present, the research on fish IFN-γ is relatively limited. However, with the continuous development of molecular immunology and aquaculture research, IFN-γ monoclonal antibodies have been prepared in some common fish species, such as zebrafish (Danio rerio), Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and grass carp (Ctenopharyngodon idella), etc. However, currently, most fish lack IFN-γ-specific monoclonal antibodies, which limits the understanding of the fish immune system.

[0003] Takifugu fasciatus belongs to the order Tetraodontiformes, family Tetraodontidae, and genus Takifugu, and is widely distributed in the South China Sea, East China Sea, and inland China, etc. It is one of the important aquaculture economic species in China. With the development of the intensive aquaculture industry of Takifugu fasciatus, various fish diseases have continuously emerged during the breeding process, causing huge economic losses to its aquaculture industry. Many previous studies have shown that IFN-γ plays an important role in the process of coping with disease infections. However, currently, due to the lack of specific antibodies against Takifugu fasciatus IFN-γ, the specific regulatory mechanism of IFN-γ in Takifugu fasciatus in coping with disease infections is not clear. Therefore, developing antibodies applicable to Takifugu fasciatus IFN-γ is of great significance for promoting the immunological research of this species, and this research helps to reveal the immune mechanism of Takifugu fasciatus, thereby promoting disease prevention and control in the field of Takifugu fasciatus aquaculture, and has important scientific research and application values. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide a monoclonal antibody fragment of Takifugu obscurus IFN-γ and its application.

[0005] Technical Solution: The present invention provides a monoclonal antibody fragment of Takifugu obscurus IFN-γ. The antibody fragment is 1M1E3, 1M1G5 or 1M1D6. The antibody fragment comprises a light chain and a heavy chain variable region. The amino acid sequences of the three complementarity-determining regions in the light chain variable region of 1M1E3 are shown in SEQ ID NO:1 to SEQ ID NO:3 respectively, and the amino acid sequences of the three complementarity-determining regions in the heavy chain variable region are shown in SEQ ID NO:10 to SEQ ID NO:12 respectively; the amino acid sequences of the three complementarity-determining regions in the light chain variable region of 1M1G5 are shown in SEQ ID NO:4 to SEQ ID NO:6 respectively, and the amino acid sequences of the three complementarity-determining regions in the heavy chain variable region are shown in SEQ ID NO:13 to SEQ ID NO:15 respectively; the amino acid sequences of the three complementarity-determining regions in the light chain variable region of 1M1D6 are shown in SEQ ID NO:7 to SEQ ID NO:9 respectively, and the amino acid sequences of the three complementarity-determining regions in the heavy chain variable region are shown in SEQ ID NO:16 to SEQ ID NO:18 respectively.

[0006] Furthermore, the nucleotide sequence of the antibody fragment is shown in SEQ ID NO.19.

[0007] The present invention also provides a monoclonal antibody of Takifugu obscurus IFN-γ. The antibody comprises a light chain and a heavy chain. The antibody is 1M1E3, 1M1G5 or 1M1D6; the amino acid sequence of the light chain of 1M1E3 is shown in SEQ ID NO.20, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.21; the amino acid sequence of the light chain of 1M1G5 is shown in SEQ ID NO.22, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.23; the amino acid sequence of the light chain of 1M1D6 is shown in SEQ IDNO.24, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.25.

[0008] Furthermore, the preparation method of the antibody is: respectively ligating the nucleotide sequences of the light chain and the heavy chain of the antibody to a vector containing a murine IgGFc fragment to construct a dual plasmid; transfecting the paired light and heavy chain recombinant plasmids into cells, and screening out positive cells.

[0009] Furthermore, the vector containing the murine IgGFc fragment is the pcDNA3.4 vector.

[0010] The present invention also provides the application of the above-mentioned monoclonal antibody fragment of Takifugu obscurus IFN-γ and the monoclonal antibody of Takifugu obscurus IFN-γ in the preparation of IFN-γ protein detection products.

[0011] The present invention also provides a kit for detecting IFN-γ protein, and the above-mentioned Takifugu obscurus IFN-γ monoclonal antibody is used as a detection antibody in the kit.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The Takifugu obscurus IFN-γ monoclonal antibody of the present application can be used as a tool for immunological related detection and evaluation of immune function to detect the expression level of Takifugu obscurus IFN-γ, so as to study the response of Takifugu obscurus to pathogen infection, immune stimulation, and other immune challenges; and it helps to develop immune enhancers or vaccine products for Takifugu obscurus, thereby promoting the healthy and sustainable development of Takifugu obscurus farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the result of the PCR product gel diagram of the Takifugu obscurus IFN-γ gene cloning of the present invention. Among them, the length of the PCR product is 570 bp, and the band is single and bright.

[0014] Figure 2 It is the restriction endonuclease digestion effect; M: maker, 2: PET-28a plasmid, 3: PET-28a digestion product.

[0015] Figure 3 It is the result of the identification of the PCR gel diagram of the bacterial liquid.

[0016] Figure 4 It is the prokaryotic expression of the recombinant protein IFN-γ.

[0017] Figure 5 It is the purification of the recombinant protein IFN-γ.

[0018] Figure 6 It is the identification of the reactivity of the monoclonal antibody by Western Blot.

[0019] Figure 7 It is the identification of the purity of three monoclonal antibodies by SDS-PAGE.

[0020] Figure 8 It is the protein expression level of IFN-γ in various tissues of Takifugu obscurus.

[0021] Figure 9 It is the protein expression level of IFN-γ in various tissues of Takifugu obscurus under normal temperature feeding and low temperature stress. DETAILED DESCRIPTION OF THE INVENTION

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0023] Example 1: Cloning of Takifugu obscurus IFN-γ gene

[0024] (1) Extract the total RNA of Takifugu obscurus and reverse transcribe it into cDNA

[0025] ① Take about 100 mg of tissue, add 1 mL of Trizol, crush it with a tissue homogenizer, and then transfer it to a 1.5 mL centrifuge tube;

[0026] ② Incubate at room temperature for 5 min to fully lyse the tissue; add 200 μL of chloroform in the fume hood, gently pipette and mix well, and incubate at room temperature for 10 min;

[0027] ③ Centrifuge at 4 °C, 12,000×g for 15 min, transfer the supernatant to a 1.5 mL centrifuge tube, and carefully aspirate it;

[0028] ④ Add 0.5 mL of isopropanol, gently pipette and mix well, and incubate at room temperature for 10 min;

[0029] ⑤ Centrifuge at 4 °C, 12,000×g for 15 min, discard the supernatant. The RNA can be seen as a white precipitate adhering to the bottom of the tube. Add 1 mL of 75% ethanol and gently pipette the white precipitate;

[0030] ⑥ Centrifuge at 4 °C, 7500×g for 5 min, discard the supernatant, add 1 mL of 75% ethanol, gently pipette the white precipitate, and wash again;

[0031] ⑦ Centrifuge at 4 °C, 7500×g for 5 min, discard the supernatant, transfer the centrifuge tube to the laminar flow hood, and dry at room temperature for 5 - 10 min;

[0032] ⑧ Add 30 μL of DEPC water to the bottom of the tube, gently pipette to accelerate the dissolution of the RNA precipitate;

[0033] ⑨ Use a Nanodrop 2000 spectrophotometer to measure the RNA concentration and OD 260 / 280. An OD value between 1.8 and 2.2 indicates good RNA purity.

[0034] (2) cDNA synthesis

[0035] According to the instructions of Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) of Yeasen Biotech Co., Ltd., perform reverse transcription of RNA: RNase-free H2O To 15 μL, 5×gDNA digester Mix 3 μL, total RNA 500 ng. Mix the above solutions in a 0.2 mL enzyme-free and sterile tube, pipette and mix well, centrifuge, and incubate at 42 °C for 5 min in a PCR instrument; then add Add 5 μL of Ⅲ SuperMix, gently pipette and mix well, then centrifuge. Place it in a PCR instrument: react at 25 °C for 5 min, 55 °C for 15 min, 85 °C for 5 min. Finally, obtain the Takifugu obscurus cDNA product and store it in a -20 °C refrigerator for the next experiment.

[0036] (3) According to the genomic sequence of Takifugu obscurus IFN-γ (MZ 576197.1) in NCBI, use the software CEDesign V1.04 to design specific primers:

[0037] SEQ ID NO.26: 5′-atgggtcgcggatccgaattcATGGTTACCATGGTAACGGCA-3′

[0038] SEQ ID NO.27: 5′-gtggtggtggtggtgctcgagATGACCTCGAATCGACATCTGC-3′

[0039] (4) Configure the PCR amplification system to 20 μL: among which, 2 μL of cDNA template, 1 μL of each upstream and downstream primer, 10 μL of 2×Taq PCR Master Mix, and 6 μL of sterilized double-distilled water; amplification program: pre-denature at 94 °C for 5 min; denature at 94 °C for 30 s; anneal at Tm 71 °C for 30 s; extend at 72 °C for 45 s; 35 cycles; final extension at 72 °C for 10 min;

[0040] (5) The PCR product was detected by 1.2% agarose gel electrophoresis ( Figure 1 ), and it can be seen that the fragment size amplified by the primer is consistent with the expectation, the band is clear and there is no obvious trailing phenomenon.

[0041] (6) Recovery and purification of the target gene: Quickly cut the target band of the qualified agarose gel under ultraviolet light and recover it according to the instructions of the FastPure Gel DNA Extraction Mini Kit gel recovery kit of Novoprotein.

[0042] Example 2: Construction of the Takifugu obscurus expression vector

[0043] (1) Double-digest the PET-28a (Wuhan Transduction Biology Laboratory Co., Ltd.) plasmid. The reaction system is 50 μL: among which, 2 μL of EcoR I (Novoprotein), 2 μL of Xho I (Novoprotein), 5 μL of 10×QuickCut Buffer (Novoprotein), 2 ng of PET-28a plasmid, and make up to 50 μL with sterilized double-distilled water; water bath at 37 °C for 30 min.

[0044] (2) Detect by 1% agarose gel electrophoresis (Figure 2 )The digestion effect of restriction endonuclease, recover the digested sample and measure its concentration.

[0045] (3) Use the Novizan ClonExpress II One Step Cloning Kit to recombine and ligate the recovered and purified linearized vector and the target fragment by homologous recombination. The specific steps are as follows: 4 μL of 5×CE II Buffer, 2 μL of Exnase II, the linearized vector and the inserted fragment are converted according to the measured concentration, and sterilized double-distilled water is added to make up to 20 μL. Incubate and ligate at 37 °C for 30 min, and quickly transfer to ice for cooling.

[0046] (4) Take 5 μL of the product ligated in step (3) and add it to 50 μL of DH5α competent cells (Novizan, C502-02). Heat shock in a 42 °C water bath for 45 - 60 s, and let it stand on ice for 2 min; add 500 μL of sterilized and antibiotic-free LB medium to the centrifuge tube, and culture it on a shaker at 37 °C and 200 rpm for 1 h; then evenly spread 100 μL of the bacterial solution on an LB plate containing kanamycin (Beyotime, ST101), and invert the plate and place it in a 37 °C incubator overnight.

[0047] (5) Take out the plate cultured overnight, randomly pick about 10 single colonies into 20 μL of sterilized double-distilled water, use the reverse primer of the PET-28a plasmid universal primer (T7 ter: 5'-GCTAGTTATTGCTCAGCGG-3') and the forward primer of the target gene, and use the selected bacterial solution as a template for PCR reaction. Configure the PCR amplification system to 20 μL: among them, 2 μL of the bacterial solution template, 1 μL of each of the upstream and downstream primers, 10 μL of 2×Taq PCR Master Mix, and 6 μL of sterilized double-distilled water; amplification program: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s; annealing at Tm 71 °C for 30 s; extension at 72 °C for 45 s; 35 cycles; final extension at 72 °C for 10 min.

[0048] (6) Take the PCR product and detect it by 1.2% agarose gel electrophoresis ( Figure 3 ), and then send it to Shanghai Sangon Biotech for sequencing.

[0049] (7) Add the bacterial solution with correct sequencing to the LB liquid medium containing kanamycin, culture it overnight on a shaker at 37 °C and 200 rpm for 16 h, extract the plasmid using the Novizan FastPure Plasmid Mini Kit plasmid extraction kit to obtain the recombinant expression plasmid PET-28a-IFN-γ.

[0050] Example 3: Expression, identification and purification of TF-IFN-γ recombinant protein

[0051] (1) The correctly identified recombinant plasmid PET-28a-IFN-γ was transformed into competent host BL21(DE3) (Novoprotein, C504-02) by heat shock. Single colonies were randomly selected and the monoclonal bacterial liquid was verified using PCR technology.

[0052] (2) The verified positive clone bacterial liquid was transferred into a sterilized LB medium (containing kanamycin) for culture.

[0053] (3) The cultured bacterial liquid was inoculated into 5 mL of sterilized LB liquid medium (containing kanamycin) at a ratio of 1:100, and cultured at 37 °C and 200 rpm for 2.5 - 3 h to measure OD 600 to be 0.6 - 0.8.

[0054] (4) IPTG (Solarbio, I8070) was added to the remaining culture to a final concentration of 0.2 mM, and the mixture was shaken at 30 °C and 220 r / min for 8 - 10 h, and then shaken at 16 °C and 220 r / min for 15 - 18 h to induce the expression of the fusion protein.

[0055] (5) Centrifuge at 10000 rmp for 1 min, discard the supernatant, and repeat to collect all the bacteria into a centrifuge tube.

[0056] (6) Resuspend the collected bacteria with PBS and ultrasonically disrupt the resuspended bacterial liquid (500 W, 6 min, ultrasonic for 5 s, interval for 9 s) (note to operate on ice).

[0057] (7) Centrifuge the disrupted bacterial liquid at 10000 rpm and 4 °C for 10 min. Transfer the separated supernatant to a new centrifuge tube and resuspend the precipitate. Prepare a 12% SDS-PAGE gel according to the instructions of Novoprotein One-Step PAGE Gel Fast Preparation Kit. Mix the separated supernatant and precipitate with 5x Loading buffer (Beyotime) at a ratio of 3:1, and incubate in a 95 °C metal bath for 10 min. Slowly add the mixed protein sample into the wells of the 12% SDS-PAGE gel and perform electrophoresis at 120 V for 75 min. After electrophoresis, stain with Coomassie Brilliant Blue Fast Staining Solution (Beyotime, P0017) for detection ( Figure 4 ). The results showed that: neither the supernatant nor the precipitate of the non-induced (lanes 1 - 2) expressed the protein. Under the induction condition at 16 °C (lanes 3 - 4), more protein was expressed in the supernatant. Under the induction condition at 30 °C (lanes 5 - 6), more protein was expressed in the precipitate. Therefore, the supernatant induced at 16 °C was selected for purification. The specific steps are as follows:

[0058] ① Place the gel in a glass dish, add 50 mL of deionized water, and heat it in a microwave oven for 3 min.

[0059] ② Pour out the deionized water and repeat the above steps;

[0060] ③ Shake gently on a horizontal shaker for 5 min;

[0061] ④ Try to pour out the deionized water as completely as possible, add 20 mL of Coomassie Brilliant Blue Fast Staining Solution, and place it on a horizontal shaker for 20 - 30 min;

[0062] ⑤ Add 100 mL of deionized water, shake gently on the shaker for 5 min, pour out the liquid, and repeat once.

[0063] (8) After ultrasonic disruption of the protein, perform Ni column purification:

[0064] ① Use a gravity column for purification. Load the supernatant solution (PBS lysate supernatant containing 50 mM imidazole) onto a Ni - NTA affinity chromatography column pre - equilibrated with Ni - NTABinding - Buffer (1X PBS, 50 mM imidazole, pH 7.4);

[0065] ② Wash with 3 - 5 column volumes of Binding - Buffer to remove unbound proteins;

[0066] ③ Elute the target protein with Ni - NTA Elution - Buffer (1X PBS, 100 mM imidazole, pH 7.4) and collect the eluate;

[0067] ④ Elute the target protein with Ni - NTA Elution - Buffer (1X PBS, 500 mM imidazole, pH 7.4) and collect the eluate;

[0068] ⑤ Dialyze the collected protein overnight at a ratio of 1:50 into PBS, pH 7.4, change the solution the next day and then dialyze for another 8 hours; then concentrate using PEG 20000; perform SDS - PAGE quality inspection analysis ( Figure 5 ). The TF - IFN - γ protein is thus obtained.

[0069] Example 4: Monoclonal Antibody Preparation

[0070] (1) Animal Immunization

[0071] Two 6-8-week-old female BALB / c mice (Annuokang) weighing 20 g were selected as experimental subjects and immunized using the rapid immunization method. Approximately 50 μg of the TF-IFN-γ protein obtained in Example 3 was thoroughly mixed with Freund's adjuvant at a volume ratio of 1:1, and the mice were immunized by subcutaneous multi-point injection (the immunization dose for all 3 times was 50 μg); 5 days after the 3rd immunization, blood was collected from the mouse orbital cavity, and the serum titer of the mouse was detected by the indirect ELISA method. Mice with better serum titers were selected for booster immunization (immunization dose: 50 μg). The specific experimental information is as follows;

[0072] Table 1: Immunization protocol

[0073]

[0074] Serum ELISA detection:

[0075] ① IFN-γ coating: Dilute the antigen (TF-IFN-γ protein) to 2 μg / ml with 10 mM PBS buffer, add 100 μL per well to a 96-well ELISA plate, and incubate overnight at 4°C. Wash the coated ELISA plate 3 times with a plate washer and dry it. Add the blocking solution at 100 μL per well, incubate in a 37°C incubator for 2 h, and then wash the blocked ELISA plate 3 times with a plate washer and dry it

[0076] ② Incubate the primary antibody: Dilute the serum of the immunized mice with PBS buffer. PBS is used as the negative control. The dilution ratios of the mouse serum are 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000. Add the diluted serum to the corresponding wells at 100 μL per well, incubate at 37°C for 1 h. After incubation, place the ELISA plate in a plate washer, wash 3 times, and dry it;

[0077] ③ Incubate the secondary antibody: Add the HRP-labeled goat anti-mouse secondary antibody to the corresponding wells at 100 μL per well, incubate at 37°C for 30 min, place the ELISA plate in a plate washer, wash 3 times, and dry it;

[0078] ④ Color development: Add the mixed color development solution to the 96-well plate, 100 μL per well, and incubate at 37°C for 15 min;

[0079] ⑤ Termination and reading: Add the termination solution to the 96-well plate, 100 μL per well, then place it in an ELISA reader for reading. Set the detection wavelength to 450 nm, read the detection results, and an OD value greater than 2.1 times that of the negative control is considered positive.

[0080] Table 2: Serum titer data

[0081]

[0082]

[0083] The serum titers of both mice were 512K, both reaching the sorting standard; under the same conditions, when the first well was 1K, the OD value of mouse A799# was greater than that of A800#, so the spleen was isolated from mouse A799# to prepare a B cell suspension.

[0084] (2) Collect the spleens of mice

[0085] Six days after booster immunization, the mice were sacrificed by cervical dislocation, soaked in 75% ethanol for 5 minutes, taken out and placed on a sterile operating table with the left ventral side facing up; a small incision was made in the middle of the left ventral side of the mouse, the skin was torn open to expose the abdominal wall, and a red strip-shaped spleen could be seen. The spleen was lifted with forceps, and the connective tissue under the spleen was separated with ophthalmic scissors. The spleen was taken out and placed in a petri dish containing about 5 mL of 0.01 M PBS (pH 7.4) solution for rinsing, and the surrounding connective tissue was removed.

[0086] (3) Prepare a B cell suspension

[0087] Place a cell strainer (40um) on a clean 50 mL centrifuge tube, add 0.01 M PBS (pH 7.4) to the lower edge of the cell strainer; transfer the spleen to the cell strainer and gently press the spleen with the rubber end of the syringe piston to obtain a cell suspension; place the centrifuge tube in a centrifuge and centrifuge at 1000 rpm for 10 min at room temperature; discard the supernatant, add 3 mL of red blood cell lysate, and incubate on ice for 12 minutes; add 0.01 M PBS (pH 7.4) to 45 mL and centrifuge at 1500 rpm for 10 min at room temperature in the centrifuge; discard the supernatant and repeat the washing once; discard the supernatant, add 1 mL of FACs buffer to resuspend the cells, and store them temporarily at 4°C for sorting.

[0088] (4) Cell sorting

[0089] On a flow cytometer, single B cells affinity for the antigen were sorted into a 96-well plate by detecting the fluorescence signals of the antigen protein and the fluorescence signals after B cell staining.

[0090] (5) High-throughput expression

[0091] Cell lysis and light and heavy chain gene amplification: Using a single-cell antibody gene amplification system, the full sequence of the light chain and the Fab region fragment of the heavy chain were obtained respectively.

[0092] (6) Construct an expression plasmid and plasmid

[0093] The obtained antibody heavy chain target gene by amplification was directionally cloned into the pcDNA3.4 (Thermo Fisher) expression vector containing the murine IgG Fc fragment through the EcoR I (New England Biolabs) restriction enzyme digestion site; meanwhile, the antibody light chain gene was inserted into the blank pcDNA3.4 vector through the BamH I (New England Biolabs) enzyme digestion site to construct a dual plasmid; then plasmid extraction was performed according to the plasmid large-scale extraction kit.

[0094] (7) Transient transfection and expression

[0095] ① Synchronously transfect the paired light and heavy chain well plasmids into CHO cells (Procell) in deep well plates for cell culture;

[0096] ② After 5 days of cell growth, collect the cell supernatant, a total of 49 strains, and perform antigen-specific detection on the 49 strains of cell supernatant by ELISA method.

[0097] (8) ELISA screening of positive cells

[0098] ① IFN-γ coating: Dilute the antigen (TF-IFN-γ protein) to 2 μg / ml with 10 mM PBS buffer, add 100 μL per well to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, and incubate overnight at 4°C. Wash the coated ELISA plate 3 times with a plate washer and dry it. Add the blocking solution at 100 μL per well and incubate in a 37°C incubator for 2 h, then wash the blocked ELISA plate 3 times with a plate washer and dry it;

[0099] ② Incubate the primary antibody: Add the cell supernatant at 50 μL per well to the corresponding wells respectively, and PBS as the negative control. Incubate at 37°C for 1 h. After incubation, place the ELISA plate in a plate washer to wash 3 times and dry it;

[0100] ③ Incubate the secondary antibody: Add the HRP-labeled goat anti-mouse secondary antibody at 100 μL per well to the corresponding wells respectively, and incubate at 37°C for 30 min. Place the ELISA plate in a plate washer to wash 3 times and dry it;

[0101] ④ Color development: Add the mixed color development solution to the 96-well plate, 100 μL per well, and incubate at 37°C for 15 min;

[0102] ⑤ Termination and reading: Add the termination solution to the 96-well plate, 100 μL per well, then place it in an enzyme-linked immunosorbent assay reader for reading. Set the detection wavelength to 450 nm, read the detection results, and an OD value greater than 2.1 times that of the negative control is considered positive.

[0103] Table 3: Detection data of cell supernatant

[0104]

[0105]

[0106]

[0107] It can be seen from the results that the OD values of 14 strains, namely 1M1C1, 1M1F1, 1M1C2, 1M1D2, 1M1G2, 1M1E3, 1M1B4, 1M1G5, 1M1C6, 1M1D6, 1M1D7, 1M1B9, 1M1G9 and 1M1E10, are 2.1 times that of the negative control, that is, 14 positive clones are screened out from 49 cell supernatants.

[0108] (9) Identification of monoclonal antibody reactivity by Western Blot

[0109] Sample acquisition method: 20 days after the hatching of Takifugu obscurus fry, they are placed in a tank with normal temperature water in the laboratory and sampled after 90 days of feeding. During this period, the water volume in the tank is about 300 L, and the feed is uniformly transitioned to rotifers, hatched Artemia, frozen Artemia, and Tianma eel feed; other conditions are kept the same.

[0110] ① Extract the tissue protein of the above-mentioned Takifugu obscurus with reference to the instructions of the whole protein extraction kit (KeyGen Biotech). The specific steps are as follows:

[0111] (Ⅰ) Add 10 μL of phosphatase inhibitor, 1 μL of protease inhibitor and 10 μL of 100 mM PMSF to every 1 mL of cold Lysis Buffer, mix well and store on ice for several minutes for later use;

[0112] (Ⅱ) Put 100 mg of Takifugu obscurus tissue into 1 mL of cold Lysis Buffer and cut it into pieces, and homogenize it in a homogenizer at 4 °C for 2 min;

[0113] (Ⅲ) Transfer the tissue homogenate to a 1.5 mL pre-cooled centrifuge tube and centrifuge at 12000 g and 4 °C for 5 min;

[0114] (Ⅳ) Transfer the supernatant to a new pre-cooled centrifuge tube, which is the whole protein extract.

[0115] ② Mix the Takifugu obscurus protein and 5x Loading buffer at a ratio of 3:1 and heat in a metal bath at 95 °C for 10 min;

[0116] ③ Gel preparation: Prepare a 12% SDS-PAGE gel with reference to the instructions of the Novoprotein One-Step PAGE Gel Fast Preparation Kit;

[0117] ④ SDS-PAGE electrophoresis: Slowly add the protein sample into the SDS-PAGE gel wells and electrophorese at 120 V for 75 min;

[0118] ⑤ Transfer membrane: Cut the PVDF membrane and filter paper into the size of the separating gel. Soak the PVDF membrane in methanol for more than 30 s, soak the sponge pad and filter paper in pre-cooled transfer buffer. Place them in the order of 1 sponge pad, 1 filter paper, SDS-PAGE gel, PVDF membrane, 1 filter paper, 1 sponge pad. Clamp them in the transfer clip, put them into the transfer tank, pour in the transfer liquid, and apply ice water around the tank. Transfer at 300 mA for 50 min;

[0119] ⑥ Blocking: 30 min before the end of membrane transfer, prepare 10% blocking solution by mixing 1 g of skim milk powder + 10 mL of TBST, and place it on a horizontal shaker and shake slowly. After the membrane transfer is completed, put the PVDF membrane into the 10% blocking solution, block it at room temperature on the shaker for 2 h, then pour out the blocking solution, add 10 mL of TBST, and wash it thoroughly 3 times on the shaker;

[0120] ⑦ Incubate with primary antibody: Dilute the supernatant proteins of 14 strains of cells as primary antibodies with TBST at a ratio of 1:1000, and incubate overnight at 4 °C;

[0121] ⑧ Washing: Pour out the primary antibody dilution, add 10 mL of TBST, and wash it thoroughly 3 times on the shaker;

[0122] ⑨ Incubate with secondary antibody: Dilute goat anti-mouse IgG secondary antibody (TransGen Biotech) with TBST at a ratio of 1:5000, and incubate at room temperature for 2 h; After pouring out the secondary antibody dilution, use 10 mL of TBST and wash it thoroughly 3 times on the shaker;

[0123] ⑩ Observation: Mix according to the ratio of 1:1 of enhanced ECL chemiluminescence chromogenic solution (Vazyme), observe and record in a fluorescence WB imager ( Figure 6 ). In the effect diagrams of the supernatants of 14 strains of cells, the target bands of 1M1E3, 1M1G5, and 1M1D6 are relatively clear.

[0124] (10) Refer to step (8) of Example 3 for Ni column affinity purification of the supernatant proteins of the three selected strains of 1M1E3, 1M1G5, and 1M1D6; then refer to step (7) of Example 3 for Coomassie brilliant blue method to detect the purity of the antibodies of the three strains of 1M1E3, 1M1G5, and 1M1D6 ( Figure 7 ). It can be seen that under reducing conditions, the antibodies of the three strains of 1M1E3, 1M1G5, and 1M1D6 all have two electrophoresis bands, and are consistent with the sizes of their respective heavy and light chains, indicating that the purified monoclonal antibodies of these three strains have relatively high purity. (Note: The reducing condition is to add 100 mM DTT reducing agent to the monoclonal antibody sample, and non-reducing means without adding DTT. DTT is from Sangon Biotech Co., Ltd.)

[0125] (11) The nucleotide sequence of Takifugu obscurus IFN-γ and the amino acid sequences of the heavy and light chains of three monoclonal antibodies 1M1E3, 1M1G5, and 1M1D6 are shown in the following table:

[0126] Table 4: Nucleotide sequence of Takifugu obscurus IFN-γ

[0127]

[0128] Table 5: Variable region of light chain (VL) and complementarity-determining regions (CDRs)

[0129]

[0130] Table 6: Variable region of heavy chain (VL) and complementarity-determining regions (CDRs)

[0131]

[0132] Table 7: Variable regions of amino acids of heavy and light chains

[0133]

[0134] Example 5: Application of IFN-γ monoclonal antibody

[0135] (1) Expression level of IFN-γ protein at room temperature

[0136] Refer to step (9) of Example 4 to extract the tissue proteins of the brain, skin, gills, spleen, intestine, muscle, and liver of Takifugu obscurus, and use Western Blot technology to detect the protein expression levels of IFN-γ in each tissue of Takifugu obscurus. The primary antibody is 1M1D6. The results show ( Figure 8 ), under the condition of no external stimulation, the skin, gills, and muscle of Takifugu obscurus have relatively high expression levels, while the expression of IFN-γ protein in mucosa-related tissues such as the spleen and intestine is relatively weak.

[0137] (2) Expression level of IFN-γ protein under cold stress

[0138] The fry of Takifugu obscurus that had emerged for 20 days were respectively placed in water bodies at 24°C (room temperature group) and water bodies that had been gradually cooled to 18°C (cold stress group) and reared for 90 days (the process of gradual cooling was: the fry were placed in a water body at 24°C, and the water temperature was reduced by 1°C every 2 hours using a chiller until the water temperature dropped to 18°C). The volume of each water tank was 300 L, and the feed was uniformly fed with rotifers, hatched brine shrimp, frozen brine shrimp, and Tianma eel feed, and other environmental conditions were kept the same. Refer to step (9) of Example 4 to extract the brain, gill, and liver tissue proteins of Takifugu obscurus under the feeding conditions of 24°C and 18°C, and use Western Blot technology to detect the expression level of IFN-γ protein. The primary antibody is 1M1G5. The results show (Figure 9 ) Compared with the normal temperature group, the expression levels of IFN-γ protein in the brain, gills and liver of Takifugu obscurus under low temperature stress were all significantly increased, and the changes in the expression levels in the liver and gills were particularly significant. This result indicates that low temperature stress can significantly induce the expression of IFN-γ protein in Takifugu obscurus, especially showing a stronger response in the liver and gill tissues.

Claims

1. A fragment of a monoclonal antibody against Takifugu obscurus IFN-γ, characterized in that, The antibody fragment is 1M1E3, 1M1G5 or 1M1D6. The antibody fragment comprises a light chain and a heavy chain variable region. The amino acid sequences of the three complementarity-determining regions in the light chain variable region of 1M1E3 are shown as SEQ ID NO:1-3 respectively, and the amino acid sequences of the three complementarity-determining regions in the heavy chain variable region are shown as SEQ ID NO:10-12 respectively; the amino acid sequences of the three complementarity-determining regions in the light chain variable region of 1M1G5 are shown as SEQ ID NO:4-6 respectively, and the amino acid sequences of the three complementarity-determining regions in the heavy chain variable region are shown as SEQ ID NO:13-15 respectively; the amino acid sequences of the three complementarity-determining regions in the light chain variable region of 1M1D6 are shown as SEQ ID NO:7-9 respectively, and the amino acid sequences of the three complementarity-determining regions in the heavy chain variable region are shown as SEQ ID NO:16-18 respectively.

2. The monoclonal antibody fragment of Takifugu obscurus IFN-γ according to claim 1, wherein The nucleotide sequence of the antibody fragment is shown as SEQ ID NO.

19.

3. A Takifugu obscurus IFN-γ monoclonal antibody, characterized in that, The antibody comprises a light chain and a heavy chain, and the antibody is 1M1E3, 1M1G5 or 1M1D6; the amino acid sequence of the light chain of 1M1E3 is shown as SEQ ID NO.20, and the amino acid sequence of the heavy chain is shown as SEQ ID NO.21; the amino acid sequence of the light chain of 1M1G5 is shown as SEQ ID NO.22, and the amino acid sequence of the heavy chain is shown as SEQ ID NO.23; the amino acid sequence of the light chain of 1M1D6 is shown as SEQ ID NO.24, and the amino acid sequence of the heavy chain is shown as SEQ ID NO.

25.

4. The Takifugu obscurus IFN-γ monoclonal antibody according to claim 3, characterized in that, The preparation method of the antibody is as follows: the nucleotide sequences of the light chain and the heavy chain of the antibody are respectively ligated to a vector containing a murine IgGFc fragment to construct a dual plasmid; the paired light and heavy chain recombinant plasmids are transfected into cells, and positive cells are screened out.

5. The Takifugu obscurus IFN-γ monoclonal antibody according to claim 4, characterized in that The vector containing the murine IgGFc fragment is the pcDNA3.4 vector.

6. Use of the Takifugu obscurus IFN-γ monoclonal antibody fragment according to any one of claims 1-2 and the Takifugu obscurus IFN-γ monoclonal antibody according to claim 3 in the preparation of an IFN-γ protein product detection.

7. A kit for detecting IFN-γ protein, characterized in that, The Takifugu obscurus IFN-γ monoclonal antibody according to claim 3 is used as a detection antibody in the kit.

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