Anti- mirafractus cyprinus cd8 alpha monoclonal antibody and preparation method thereof

Abstract

The application discloses an anti-Oncorhynchus keta CD8 alpha monoclonal antibody and a preparation method thereof, the monoclonal antibody is secreted by a hybridoma cell strain CD8 alpha-5E3, a preservation unit is the China Center for Type Culture Collection, a preservation number is CCTCC NO: C2024283, and a preservation date is August 26, 2024. + The antibody is high in specificity and can be used for identifying, separating and researching the CD8 + T cells of the Oncorhynchus keta, which is helpful to reveal the immunological mechanism related to the function and cytotoxic effect of the CD8 + T cells of the Oncorhynchus keta, which is helpful to reveal the immunological mechanism related to the function and cytotoxic effect of the CD8 + T cells of the Oncorhynchus keta, which is helpful to reveal the immunological mechanism related to the function and cytotoxic effect of the CD8 + T cells of the Oncorhynchus keta, which is helpful to reveal the immunological mechanism related to the function and cytotoxic effect of the CD8 + T cells of the Oncorhynchus keta, which is helpful to reveal the immunological mechanism related to the function and cytotoxic effect of the CD8 + T cells of the Oncorhynchus keta, which is helpful to

Description

Technical Field

[0001] This invention relates to monoclonal antibodies and their preparation methods, specifically to a monoclonal antibody against CD8α in large yellow croaker and its preparation method. Background Technology

[0002] Bony fishes are a group of vertebrates with a classic adaptive immune system and primitive T cells, making them ideal models for studying the evolution of T-cell immunity. Similar to higher mammals, T-cell-mediated cytotoxicity in bony fishes plays a crucial role in the adaptive immune system and is primarily mediated by CD8+. + T cells execute, CD8 + T cells can proliferate and differentiate into cytotoxic effector T cells, which possess killing capabilities and are crucial for the body's defense against pathogen infection. However, they lack the ability to specifically recognize CD8+. + Monoclonal antibodies against T cells and CD8 + Our understanding of T cells primarily comes from mammalian models, while CD8 in early vertebrates... + The function of T cells and their immunological mechanisms, such as proliferation and differentiation, remain largely unclear.

[0003] CD8 is CD8 + T cell surface markers exist in two forms: αα homodimers and αβ heterodimers. Therefore, compared to CD8β, CD8α is a more potent marker for CD8 cells. + The main TCR co-receptor of T cells plays a more important role in T cell-mediated cytotoxicity. ([1]ColeDK, Gao GF. CD8: adhesion molecule, co-receptor and immuno-modulator. Cell Mol Immunol. 2004 Apr;1(2):81-8. [2]Moore LJ, Somamoto T, LieKK, Dijkstra JM, Hordvik I. Characterisation of salmon and trout CD8alpha and CD8beta. Mol Immunol. 2005Jun;42(10):1225-34.) With the help of high-quality anti-large yellow croaker CD8α monoclonal antibody, large yellow croaker CD8 can be identified and isolated. + T cells, which help reveal the CD8+ of bony fishes +The function and proliferation, differentiation and other immunological mechanisms of T cells provide the prerequisite for understanding the working principle of adaptive immune system of teleosts. In addition, analyzing the mechanism of T cell-mediated cytotoxicity helps the development of new fish vaccines, i.e. preventing diseases by enhancing the cellular immune response of fish (3) Yamaguchi T, Takizawa F, Furihata M, Soto-Lampe V, Dijkstra JM, Fischer U. Teleost cytotoxic T cells. Fish Shellfish Immunol. 2019 Dec;95:422-439.). Therefore, the development of anti-P. olivaceus CD8a monoclonal antibody helps to clarify the function of fish CD8 + The function of T cells and related immunological mechanisms and the development of new vaccines have important theoretical and practical significance for the improvement of the basic research of adaptive immune system of teleosts and the prevention and control of diseases. SUMMARY

[0004] Figure 1 : Immunofluorescence-labeled NIH\3T3-LcCD8a cells.

[0005] Figure 2 : Flow cytometry detection of lymphocytes labeled with anti-P. olivaceus CD8a monoclonal antibody;

[0006] Figure a: isotype control-labeled head kidney lymphocyte sample; Figure b: anti-P. olivaceus CD8a monoclonal antibody-labeled head kidney lymphocyte sample.

[0007] Figure 3 : Immunofluorescence-labeled P. olivaceus lymphocytes;

[0008] In the figure, blue indicates DAPI-labeled nuclei, and green indicates CD8 + T lymphocytes specifically labeled by anti-P. olivaceus CD8a monoclonal antibody.

[0009] Figure 4 : CD8 + T cells and CD8 - T cell markers. Among them, Actin is the internal reference gene, CD3 is the marker of T cells, CD4-1 and CD4-2 are CD4 + T cell markers, CD8a and CD8b are CD8 + T cell markers, IgT is the marker of B cells; CD8 + indicates CD8 + T cells, CD8 - indicates CD8 - T cells. DETAILED DESCRIPTION

[0010] In order to make the content of the present application more convenient to understand, the technical solutions of the present application will be further described below in combination with specific embodiments, but the present application is not limited thereto.

[0011] The methods in the following examples are all conventional methods in the art, unless otherwise specified.

[0012] The serum-free DMEM medium in the following examples was purchased from ThermoFisher, item number: 11965092.

[0013] The DMEM complete medium is a serum-free DMEM medium added with 10% FBS.

[0014] Example 1 Preparation of Pseudosciaena crocea CD8a monoclonal antibody

[0015] (1) Construction of pCDH-CMV-GFP-LcCD8a vector: According to the mRNA sequence of Pseudosciaena crocea CD8a (as described in SEQ ID NO: 1), design amplification primers (upstream primer F: 5'-GATTCTAGAGCTAGCATTGGACCAAAAGTGGATTCATATTCTGG-3'; downstream primer R: 5'-GTGACGACCAACAGGCACATTGGATCCGCGGCCGCAGGATC-3'). Using Pseudosciaena crocea head kidney cDNA as a template, 2 × Phanta Flash Master Mix (Dye Plus) (Novagen, P520-01) was used for amplification. The target fragment obtained by PCR amplification was detected by 1% agarose gel electrophoresis, and the target band was cut under ultraviolet light, weighed, and recovered according to the DNA gel recovery kit (OMEGA, D2500-01) instructions; pCDH-CMV-GFP was digested with XhoI and BamHI, 37 °C water bath for 4 h, and the enzyme digestion product was subjected to 1% agarose gel electrophoresis, and the pCDH-CMV-GFP vector linearization fragment was recovered; the pCDH-CMV-GFP linearization fragment was connected with the target fragment. ClonExpressUltra One Step Cloning Kit (Novagen, C115-01) was used for ligation, 50 °C incubation for 10 min. The ligation product was transformed into DH5a competent cells, and after sequencing, the pCDH-CMV-GFP-LcCD8a expression vector was obtained.

[0016] (2) Preparation of NIH\3T3-LcCD8α stable cell strain: 15 μg of expression vector pCDH-CMV-GFP-LcCD8α, 6 μg of packaging vector psPAX2 and 3 μg of envelope vector pMD2.G were added to 750 μl of serum-free DMEM medium, and 750 μl of serum-free DMEM medium was added to 24 μl of Lipo8000 liposome nucleic acid transfection reagent. The two liquids were mixed and blown to mix again, and then added to 293T cells cultured in a 10 cm dish with a confluence of 90%. After 6 h of transfection, the fresh DMEM complete culture medium was replaced, and the culture supernatant was collected after 48 h. According to the amount of the obtained culture supernatant, 5*PEG8000 was added at a ratio of 4:1 to the virus concentration liquid, and the mixture was shaken at 4°C overnight. Then, the mixture was centrifuged at 4°C and 3000 rpm for 30 min, and the precipitate was collected and resuspended in 1 mL of serum-free DMEM medium for standby. The NIH\3T3 cells cultured in a 10 dish with a confluence of 80% were replaced with DMEM complete culture medium, and then the resuspended slow virus was added. After 24 h, the DMEM complete culture medium was replaced, and the NIH\3T3 cells stably expressing LcCD8α were obtained after 72 h of infection and the addition of puromycin (5 μg / ml) for screening. After three generations of screening, the stable cell strain NIH\3T3-LcCD8α was obtained.

[0017] (3) Preparation of hybridoma cell strain: the NIH\3T3-LcCD8α was used to immunize mice, and the hybridoma cell strain was obtained by fusing SP2 / 0 myeloma cells and spleen cells of Balb / C mice producing anti-Pseudosciaena crocea CD8α antibody after immunization.

[0018] (4) Preparation of anti-Pseudosciaena crocea CD8α monoclonal antibody: the monoclonal antibody was produced by the hybridoma cell strain, and the monoclonal antibody had specificity for the Pseudosciaena crocea CD8α.

[0019] The specific steps of the mouse immunization step in step (3) are as follows:

[0020] Five 4-6 week old female Balb / C mice were immunized with NIH\3T3-LcCD8α, and another five mice were injected with an equal amount of serum-free DMEM medium as a negative control. Each time, 2*10 7 NIH\3T3-LcCD8α was mixed with serum-free DMEM medium to immunize the mice.

[0021] The immunization program adopted twice of basic immunization and twice of booster immunization. The basic immunization times were 0 d and 7 d, and the booster immunization was performed once again 3 days before fusion.

[0022] The specific steps of the preparation of hybridoma cell strain in step (3) are as follows:

[0023] S1. Preparation of myeloma cells (SP 2 / 0)

[0024] Select myeloma cells with good growth state, discard the supernatant, wash once with serum-free DMEM medium, and then blow them off with 10 mL of serum-free DMEM medium.

[0025] S2. Preparation of mouse spleen lymphocytes

[0026] The immune mice were sacrificed by cervical dislocation, and the spleen was removed after sterilization by cutting open the abdominal cavity. Wash with serum-free DMEM medium, then transfer to a plate with serum-free DMEM medium, grind the cells thoroughly with a 70 μm filter at the end of the syringe, and prepare a spleen cell suspension.

[0027] S3. Cell fusion

[0028] a. Prepare 4 sets of autoclaved dissecting instruments, i.e. 4 scissors and 4 fine forceps;

[0029] b. Put the prepared HAT medium, serum-free DMEM medium, 50% PEG1450, and distilled water (in a 500 mL beaker) into a 37°C water bath for preheating;

[0030] c. Collect the blood from the mice by eye ball bleeding, and centrifuge at 2500 rpm for 5 min after standing at room temperature for 2 h to collect the serum, i.e. polyclonal antibody. After washing the mouse body with water, soak it in 75% ethanol for 10 min for disinfection;

[0031] d. Fix the mouse on the foam plate in the clean bench, carefully cut the skin and peritoneum with autoclaved scissors and fine forceps to expose the spleen;

[0032] e. Replace the new scissors and fine forceps, carefully remove the spleen, and cut off the surface connective tissue, then put it into a 100 mm plate containing 20 mL of preheated serum-free DMEM medium;

[0033] f. Use a 5 mL syringe to suck 5 mL of serum-free DMEM medium from the 100 mm plate, and insert the needle part through the spleen but not penetrate it, push out the medium, at this time you will find that the spleen turns from red to white, repeat the blowing of the spleen until the spleen turns completely white, then collect the serum-free DMEM medium containing spleen cells through a 70 μm cell sieve into a 50 mL centrifuge tube, centrifuge at 1500 rpm for 7 min, resuspend with 25 mL of serum-free DMEM medium and place it in a 37°C incubator for standby;

[0034] g. Bone marrow cells were washed with 5 mL of serum-free DMEM medium, then blown down with 5 mL of serum-free DMEM medium and transferred to a 50 mL centrifuge tube. After being diluted with serum-free DMEM medium to 25 mL, the cells were centrifuged at 1500 rpm for 5 min, resuspended with 25 mL of serum-free DMEM medium, and repeated once. The cells were then placed in a 37°C incubator for standby;

[0035] h. The two types of cells were counted and mixed in a 50 mL centrifuge tube at a ratio of 1:8. If the volume of the mixture was less than 45 mL, the volume was adjusted to 45 mL with serum-free DMEM medium, and the mixture was centrifuged at 1500 rpm for 5 min.

[0036] i. The supernatant was discarded, and a small amount of liquid was left in the tube. The bottom of the centrifuge tube was tapped to loosen the cells into a porridge-like state. The 50 mL centrifuge tube with cells was immersed in water to keep the cells in a 30-37°C environment.

[0037] j. 1 mL of preheated 50% PEG1450 was added at a rate of 1 mL / 1 min. The centrifuge tube was rotated and gently shaken during the addition to ensure that the 50% PEG1450 and the cells were in good contact. Then the mixture was placed in a water bath for 90 s.

[0038] k. The reaction was terminated by adding serum-free DMEM medium. 1 mL of serum-free DMEM medium was added while gently shaking. Similarly, 4 mL of serum-free DMEM medium was added in the second minute, and the centrifuge tube was filled with serum-free DMEM medium in the third minute. Then the mixture was centrifuged at 1000 rpm for 7 min to collect the cell pellet.

[0039] l. The cells were resuspended with 100 mL of HAT medium and plated in 5 96-well plates. The hybridoma cell line CD8α-5E3 was obtained after incubation at 37°C for about seven days.

[0040] The following gives the method for establishing a hybridoma cell line:

[0041] The hybridoma cell strain CD8α-5E3 is gently blown from the culture hole, and the viable cells are counted with a blood cell counting board: the cells are diluted to 5, 10, and 50 cells / mL with serum-free DMEM medium, and 100 μL of each of the three cell suspensions is added to a 96-well culture plate, so that the corresponding wells contain 0.5, 1, and 5 cells on average, respectively. The medium is changed once on the fourth day of culture, and the growth of the cells in each well is observed carefully from the fifth to the sixth day, and recorded. When the cells grow to fill 1 / 3 to 1 / 2 of the culture hole from the seventh to the ninth day after cloning, the specificity of the antibody is detected by flow cytometry. If the cell well with specific antibody is detected, the cell with high antibody titer, single clone growth, and good growth condition is selected for further cloning. The cells in the positive well are transferred to a 24-well culture plate, and serum-free DMEM medium is added to the corresponding original well to prevent contamination or cell death. When the cells in the 24-well plate grow well, they are frozen.

[0042] The method for freezing the hybridoma cell strain CD8α-5E3 is as follows:

[0043] The growing and well-shaped hybridoma cell strain CD8α-5E3 is prepared into a cell suspension, centrifuged at 200 rpm for 5 min, the supernatant is removed, and freezing solution (9 parts of fetal bovine serum + 1 part of dimethyl sulfoxide) is added to make the final cell density 5×10 6 mL, the screw cap is tightened, then the freezing tube is placed in a small box containing cotton, and is placed in a -80℃ ultra-low temperature refrigerator overnight (8-12 h), and then is immersed in liquid nitrogen for long-term storage. The hybridoma cells obtained by the present application are full, round, transparent, and have vigorous division.

[0044] The method for preparing ascites by animal induction is as follows:

[0045] One to two weeks before inoculation of the hybridoma cells CD8α-5E3, the Balb / C mice are injected intraperitoneally with 0.5 mL of liquid paraffin or Freund's incomplete adjuvant; the successfully cloned hybridoma cells are centrifuged at 1000 rpm, the supernatant is discarded, the cells are suspended in serum-free DMEM medium, and the cell number is adjusted to 1×10 6 mL, and 0.5 mL of the prepared hybridoma cell suspension is injected intraperitoneally into each mouse. About 10 days later, the mouse abdomen is obviously swollen, the mouse abdomen skin is disinfected with iodine alcohol cotton ball, 5 mL of syringe is used to extract ascites, the ascites is centrifuged at 1000 rpm at 4℃ for 10 min, the supernatant is taken, and is stored at -20℃ after being divided.

[0046] Purification of ascites: take 3 mL of the ascites prepared above, add 6 mL of 0.06 mol / L, pH 4.8 acetic acid buffer, mix well, then add 99 μl of octanoic acid at room temperature while stirring, continue stirring for 30 min, then place at 4°C for 2 h or more. Centrifuge the solution at 4°C, 1000 rpm for 30 min, take the supernatant, adjust the pH to 7.4 with 2 mol / L NaOH, then add an equal volume of saturated sulfuric acid solution to the supernatant while stirring, 30 min later, place at 4°C for 2 h or more. Centrifuge at 10000 rpm for 30 min, take the precipitate, dissolve in 5 mL of 0.01M, pH 7.4 PBS, mix well, then place in 0.01M PBS (pH 7.4) for dialysis, dialyze at 4°C for 2 d, change the solution every 8 h, after 2 d, take out the solution in the dialysis bag, collect to obtain the purified anti-Pseudosciaena crocea CD8α monoclonal antibody, after determining the protein content by ultraviolet spectrophotometer, store at -80°C at a concentration of 5 mg / mL.

[0047] Collect NIH\3T3-LcCD8α cells with a confluence of 90%, wash twice with PBS (pH 7.4), count, take 1*10 6 cells, make cell smears with a cell smearing machine, fix with methanol for 5 min and air dry, then add 100 μL of 1% BSA, block at 37°C for 1 h. Wash twice with PBST (pH 7.4), then wash once with PBS (pH 7.4), add the anti-Pseudosciaena crocea CD8α monoclonal antibody (1 μg / mL) described above, incubate at 37°C for 1 h, after incubation, wash twice with PBST (pH 7.4), then wash once with PBS (pH 7.4). Add goat anti-mouse IgG H&L (Alexa Fluor® 647) (1 μg / mL, abcam, ab150115) fluorescent secondary antibody, incubate at 37°C for 1 h, after incubation, wash twice with PBST, then wash once with PBS. Add one drop of Hoechst 33342 dye color mounting agent, and examine under a fluorescence microscope. The results are shown in Figure 1 Fig. 3, it can be seen that NIH\3T3-LcCD8α cells can express Pseudosciaena crocea CD8α.

[0048] Example 2 Verification of anti-Pseudosciaena crocea CD8α specific antibody

[0049] (1) Flow cytometry detection:

[0050] Take the head kidney tissue of healthy Pseudosciaena crocea, place on a 70 μm mesh, add L-15 culture medium containing 25 IU / ml heparin sodium, use the tail end of a syringe to carefully grind, and the obtained is the head kidney leukocytes.

[0051] ① The crude leukocytes are about 1*10 7cells, resuspended with 500 μL L-15 medium containing 5% goat serum, blocked at 4°C for 1 h;

[0052] ②Add 1 ml anti-Megalobrama amblycephala CD8α monoclonal antibody (1 μg / ml), incubate at 4°C for 45 min;

[0053] ③Resuspend with 1 ml L-15 medium, centrifuge at 500 g for 5 min, wash twice;

[0054] ④Add 1 ml L-15 medium containing Goat anti mouse IgG Alexa Fluor 488 (1 μg / ml), resuspend, incubate at 4°C for 30 min;

[0055] ⑤Resuspend with 1 ml L-15 medium, centrifuge at 500 g for 5 min, wash twice;

[0056] ⑥Resuspend with 1×PBS, filter the cell suspension through a 70 μm mesh, and detect by flow cytometry.

[0057] The results are shown in Figure 2 , the monoclonal antibody labeled a group of cells with fluorescence, and the monoclonal antibody prepared in the application can specifically recognize CD8 + T lymphocytes in the head kidney leukocytes of M. amblycephala.

[0058] (2) Immunofluorescence experiment:

[0059] ①Take 3×10 5 cells from (1), fix with 4% paraformaldehyde for 10 min, wash twice with 1×PBS;

[0060] ②DAPI (2 μg / mL) staining for 5 min, washing twice with 1×PBS;

[0061] ③Transfer to a 35 mm confocal dish, centrifuge at 500 g for 5 min, and take pictures by laser confocal.

[0062] The results are shown in Figure 3 , the positive cells are round cells with a diameter of about 8 μm, with large nuclei and a ring of green fluorescence around the cells. The negative cells are cells without green fluorescence, including B lymphocytes similar in shape to positive cells, macrophages, and the like.

[0063] (3) RT-PCR:

[0064] ①Take the head kidney tissue of healthy M. amblycephala, place it on a 70 μm mesh, add L-15 medium containing 25 IU / mL heparin sodium, and carefully grind the tail end of the syringe to obtain the head kidney leukocytes.

[0065] ②The cells were sorted by flow cytometry after the same experimental procedure as (1), and the obtained cells were CD8 + T cells and CD8 - T cells, respectively.

[0066] ③The RNA was extracted and reversely converted into cDNA (Novozyme, R211-01) according to the description;

[0067] ④PCR amplification was performed according to the following procedure:

[0068]

[0069] ⑤1.5% nucleic acid gel was prepared and electrophoresed at 150 V;

[0070] The results show that CD8 + T cells contain marker genes of CD3, CD8 (CD8α, CD8β), but do not contain marker genes of B lymphocytes and CD4-1, CD4-2 T cells; on the contrary, CD8 - T cells do not contain marker genes of CD8 (CD8α, CD8β), which is consistent with the expected result, and the monoclonal antibody prepared by the application can specifically recognize CD8 + T lymphocytes in the head kidney white blood cells of Pseudosciaena crocea. Figure 4

[0071] The above description is only the preferred embodiment of the application, and any equivalent change and modification made according to the patent application scope of the application shall be included in the scope of the application.​

Claims

1. Anti-Pseudosciaena crocea CD8α monoclonal antibody, characterized in that: The monoclonal antibody is secreted by a hybridoma cell strain CD8a-5E3, deposited with the China Center for Type Culture Collection, and has a deposit number of CCTCC NO: C2024283 and a deposit date of August 26, 2024.

2. The anti-Pseudosciaena crocea CD8a monoclonal antibody of claim 1 for use in the preparation of a T cell reagent for detecting Pseudosciaena crocea CD8a + T cells.

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