Anti-pseudosciaena crocea CD8 alpha monoclonal antibody and preparation method thereof

Abstract

The invention discloses an anti-pseudosciaena crocea CD8 alpha monoclonal antibody and a preparation method thereof. The monoclonal antibody is secreted by a hybridoma cell strain CD8 alpha-5E3, the preservation unit is China Center for Type Culture Collection, the preservation number is CCTCC NO: C2024283, and the preservation date is August 26, 2024. The antibody is high in specificity, can be used for identifying, separating and researching the large yellow croaker CD8 + T cells, is beneficial to revealing the functions of the sclerobone fish CD8 + T cells, researching immunological mechanisms related to cytotoxic effects and the like, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to a monoclonal antibody and a preparation method thereof, and in particular to an anti-large yellow croaker CD8α monoclonal antibody and a preparation method thereof. Background Art

[0002] Teleost fish are a group of vertebrates that have both a classical adaptive immune system and primitive T cells, and therefore they are considered an ideal model for studying the evolution of T cell immunity. Similar to higher mammals, teleost T cell-mediated cytotoxicity plays a crucial role in the adaptive immune system and is mainly composed of CD8 + T cell execution, CD8 + T cells can proliferate and differentiate into cytotoxic effector T cells, which have killing ability. Their existence is particularly important for the body to resist pathogen infection. + Monoclonal antibody against CD8 T cells + Our understanding of T cells mainly comes from mammalian models, while the early vertebrate CD8 + The functions, proliferation, differentiation and other immunological-related mechanisms of T cells remain largely unclear.

[0003] CD8 is CD8 + There are two forms of T cell surface markers, αα homodimers and αβ heterodimers. Therefore, compared with CD8β, CD8α is CD8 + 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 andCD8beta. 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 revealed that teleost CD8 +The function, proliferation, differentiation and other immunological mechanisms of T cells provide the prerequisites, which are crucial for understanding the working principle of the adaptive immune system of bony fish. In addition, the analysis of the mechanism of T cell-mediated cytotoxicity will help the development of new fish vaccines, that is, to prevent 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-large yellow croaker CD8α monoclonal antibodies will help to clarify the fish CD8 + The functions of T cells, related immunological mechanisms and the development of new vaccines have important theoretical and practical significance for improving the basic research on the adaptive immune system of bony fish and for disease prevention and control. Summary of the invention

[0004] Figure 1 : NIH\3T3-LcCD8α cells labeled with immunofluorescence.

[0005] Figure 2 :Flow cytometry was used to detect lymphocytes labeled with anti-large yellow croaker CD8α monoclonal antibody; Figure a: Head kidney lymphocyte sample labeled with isotype primary antibody control; Figure b: Head kidney lymphocyte sample labeled with anti-large yellow croaker CD8α monoclonal antibody.

[0006] Figure 3 : Immunofluorescence labeling of large yellow croaker lymphocytes; In the figure, blue indicates the cell nucleus labeled with DAPI, and green indicates CD8 specifically labeled with anti-large yellow croaker CD8α monoclonal antibody. + T lymphocytes.

[0007] Figure 4 :CD8 + T cells and CD8 - The results of gene expression profiling of T cell markers. Among them, Actin is an internal reference gene, CD3 is a marker of T cells, CD4-1 and CD4-2 are CD4 + T cell markers, CD8α and CD8β are CD8 + T cell marker, IgT is a marker for B cells; CD8 + CD8 + T cells, CD8 - CD8 - T cells. DETAILED DESCRIPTION

[0008] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0009] The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0010] The serum-free DMEM medium in the following examples was purchased from ThermoFisher, catalog number: 11965092; DMEM complete medium is serum-free DMEM supplemented with 10% FBS.

[0011] Example 1 Preparation of Large Yellow Croaker CD8α Monoclonal Antibody (1) Construction of pCDH-CMV-GFP-LcCD8α vector: Based on the mRNA sequence of CD8α of large yellow croaker (described in SEQ ID NO: 1), primers were designed (upstream primer F: 5'-GATTCTAGAGCTAGCATTGGACCAAAAGTGGATTCATATTCTGG-3'; downstream primer R: 5'-GTGACGACCAACAGGCACATTGGATCCGCGGCCGCAGGATC-3'). Large yellow croaker head kidney cDNA was used as a template and amplified using 2 × Phanta Flash Master Mix (Dye Plus) (Novozymes, P520-01). The target fragment obtained by PCR amplification was detected by 1% agarose gel electrophoresis, and the target band was cut out under UV light, weighed, and gel recovered according to the instructions of the DNA gel recovery kit (OMEGA, D2500-01); pCDH-CMV-GFP was double-digested with XhoI and BamHI, and the digestion product was subjected to 1% agarose gel electrophoresis, and the pCDH-CMV-GFP vector linearized fragment was recovered by gel cutting; the pCDH-CMV-GFP linearized fragment was connected to the target fragment. The connection was carried out using ClonExpressUltra One Step Cloning Kit (Novozyme, C115-01), and incubated at 50 °C for 10 min. The ligation product was transformed into DH5α competent cells, and the pCDH-CMV-GFP-LcCD8α expression vector was obtained after correct sequencing.

[0012] (2) Preparation of NIH\3T3-LcCD8α stable transfection cell line: 15μg expression vector pCDH-CMV-GFP-LcCD8α, 6μg packaging vector psPAX2 and 3μg envelope vector pMD2.G were added to 750μl serum-free DMEM medium. Meanwhile, another 750μl serum-free DMEM medium was added to 24μl Lipo8000 liposome nucleic acid transfection reagent. The two tubes of liquid were pipetted and mixed separately and then mixed into one tube. After pipetting and mixing again, the mixture was added to 293T cells cultured in a 10 cm dish with a confluence of 90%. Fresh DMEM complete medium was replaced 6h after transfection. After 48h, the culture supernatant was collected and the virus concentrate 5*PEG8000 was added in a ratio of 4:1 according to the amount of culture supernatant obtained. The mixture was shaken at 4°C overnight. The mixture was centrifuged at 3000rpm for 30min at 4°C, the precipitate was collected, and 1mL serum-free DMEM medium was added to the precipitate to resuspend it for later use. After replacing DMEM complete medium with NIH / 3T3 cells cultured in 10 dishes with a confluence of 80%, add the above-mentioned resuspended lentivirus, replace DMEM complete medium after 24 hours, and subculture and add puromycin (5μg / ml) 72 hours after infection to select NIH\3T3 cells that stably express LcCD8α. After three generations of screening, the stable transfected cell line NIH\3T3-LcCD8α can be obtained.

[0013] (3) Preparation of hybridoma cell lines: NIH\3T3-LcCD8α mice were used to immunize mice, and SP2 / 0 myeloma cells were fused with spleen cells of Balb / C mice that produced anti-large yellow croaker CD8α antibodies after immunization to obtain hybridoma cell lines.

[0014] (4) Preparation of monoclonal antibody against large yellow croaker CD8α: The monoclonal antibody is produced by the hybridoma cell line mentioned above, and the monoclonal antibody is specific to the large yellow croaker CD8α.

[0015] The specific steps of the mouse immunization step in step (3) are as follows: Five female Balb / C mice aged 4-6 weeks were immunized with NIH\3T3-LcCD8α, and another five mice were injected with an equal amount of serum-free DMEM medium as negative control. 7 NIH\3T3-LcCD8α was mixed with serum-free DMEM medium to immunize mice.

[0016] The immunization program includes two basic immunizations and two booster immunizations. The basic immunization times are: 0d and 7d, and the booster immunization is: 14d. Three days before fusion, another booster immunization is given by intraperitoneal injection.

[0017] Step (3) Preparation of hybridoma cell lines specifically comprises the following steps: S1. Myeloma cell (SP 2 / 0) preparation Select myeloma cells with good growth status, discard the supernatant, wash them once with serum-free DMEM medium, and then gently blow them off with 10 mL serum-free DMEM medium.

[0018] S2. Preparation of mouse spleen lymphocytes The immunized mice were killed by cervical dislocation, and the spleen was removed by sterile incision of the abdominal cavity after disinfection. The spleen was washed with serum-free DMEM medium, and then transferred to a plate with serum-free DMEM medium. The cells were fully ground through the tail of a syringe and a 70 μm filter to prepare a spleen cell suspension.

[0019] S3. Cell fusion a. Prepare 4 sets of autoclaved dissection instruments, namely 4 pairs of scissors and 4 pairs of fine forceps; b. Preheat the prepared HAT medium, serum-free DMEM medium, 50% PEG1450, and distilled water (in a 500 ml beaker) in a 37°C water bath; c. Blood was collected from the eyeballs of mice until they died. The blood was allowed to stand at room temperature for 2 hours and then centrifuged at 2500 rpm for 5 minutes to collect serum, i.e., polyclonal antibodies. The mice were rinsed with clean water and then immersed in 75% ethanol for 10 minutes for disinfection; d. Fix the mouse on a foam board in a clean bench, and use autoclaved scissors and fine forceps to carefully cut the skin and peritoneum to expose the spleen; e. Use new scissors and fine forceps to carefully remove the spleen, cut off the connective tissue on the surface, and place it in a 100 mm dish containing 20 ml of pre-warmed serum-free DMEM medium; f. Use a 5mL syringe to aspirate 5mL of serum-free DMEM medium from a 100mm dish, partially penetrate the spleen with the needle but do not penetrate it, and push out the medium. At this time, you will find that the spleen turns from red to white. Repeatedly blow and beat the spleen until the spleen turns completely white. Then, pass the serum-free DMEM medium containing spleen cells through a 70μm cell sieve and collect it into a 50mL centrifuge tube. Centrifuge at 1500rpm for 7min, resuspend it with 25mL of serum-free DMEM medium, and place it in a 37℃ incubator for later use; g. Rinse the myeloma cells with 5 mL of serum-free DMEM medium, blow them off with 5 mL of serum-free DMEM medium, and transfer them to a 50 mL centrifuge tube. Make the volume up to 25 mL with serum-free DMEM medium, centrifuge at 1500 rpm for 5 min, resuspend with 25 mL of serum-free DMEM medium, repeat the above steps once, and place in a 37°C incubator for later use; h. Count the two types of cells separately and add them into the same 50mL centrifuge tube at a ratio of spleen cells to myeloma cells of 1:8. If the volume of the mixed solution is less than 45mL at this time, use serum-free DMEM medium to make up to 45mL and centrifuge at 1500rpm for 5min. i. Discard the supernatant, but leave a little liquid in the tube. Gently tap the bottom of the centrifuge tube until the cells are loosened and become porridge-like. Immerse the part of the 50ml centrifuge tube with cells in water to keep the cells in an environment of 30-37℃ at all times.

[0020] j. Pipette 1 mL of preheated 50% PEG1450 and add it dropwise at a rate of 1 mL / 1 min. Rotate the centrifuge tube and oscillate slightly during the addition to allow 50% PEG1450 to fully contact the cells. Then let it stand in a water bath for 90 seconds. k. Add serum-free DMEM medium to terminate the reaction, and add 1 mL of serum-free DMEM medium dropwise while gently shaking, and add it all over within 1 minute. Similarly, add a total of 4 mL of serum-free DMEM medium dropwise in the second minute, and fill the centrifuge tube with serum-free DMEM medium in the third minute, then centrifuge at 1000 rpm for 7 minutes to collect the cell pellet; 1. Add 100 mL of HAT medium to gently resuspend the cells, and spread them on five 96-well plates. Culture them in a 37°C incubator for about seven days to obtain the hybridoma cell line CD8α-5E3.

[0021] The following is a method for establishing hybridoma cell lines: Gently blow the hybridoma cell line CD8α-5E3 from the culture well and count the live cells with a blood cell counting plate: dilute the cells to 5, 10, and 50 cells / mL with serum-free DMEM medium, add the above three concentrations of cell suspension to a 96-well culture plate, 100 μL / well, so that the corresponding wells contain an average of 0.5, 1, and 5 cells, respectively. The culture medium can be replaced once on the 4th day of culture. From the 5th to 6th day, the growth of cells in each well is carefully observed and recorded. When the cell clones fill 1 / 3 to 1 / 2 of the culture well on the 7th to 9th day after cloning, the antibody specificity can be detected by flow cytometry. If the cell wells with positive specific antibodies are detected, cells with high antibody titer, single clone growth, and good growth conditions can be selected to continue re-cloning. Move the cells in the positive wells to a 24-well culture plate, and add serum-free DMEM medium to the corresponding original wells to prevent contamination of the two or cell death. When the cells in the 24-well plate grow well, they can be frozen.

[0022] The following is a method for freezing hybridoma cell line CD8α-5E3: The hybridoma cell line CD8α-5E3 with vigorous growth and good morphology was taken and prepared into a cell suspension. The suspension was centrifuged at 200 rpm for 5 min, the supernatant was removed, and freezing solution (9 parts of fetal bovine serum + 1 part of dimethyl sulfoxide) was added to make the final cell density 5×10 6 / mL, put 1mL of cell suspension into a 2mL cryotube, tighten the screw cap, then put the cryotube into a small box filled with cotton, put it in a -80℃ ultra-low temperature refrigerator overnight (8-12 h), and then immerse it in liquid nitrogen for long-term storage. The present invention obtains hybridoma cells with full appearance, round and translucent, and vigorous division.

[0023] The following is a method for inducing ascites in animals: One to two weeks before inoculation of hybridoma cells CD8α-5E3, 0.5 mL of liquid paraffin or Freund's incomplete adjuvant was intraperitoneally injected into Balb / C mice. The cloned hybridoma cells were centrifuged at 1000 rpm, the supernatant was discarded, the cells were suspended in serum-free DMEM medium, and the cell number was adjusted to 1×10 6 / mL, and each mouse was intraperitoneally injected with 0.5 mL of the above prepared hybridoma cell suspension for about 10 days. The mouse abdomen was obviously swollen. At this time, the mouse abdominal skin was disinfected with iodine and alcohol cotton balls, and the ascites was extracted with a 5 mL syringe. The ascites was centrifuged at 4°C, 1000 rpm for 10 min, and the supernatant was taken, aliquoted and stored at -20°C.

[0024] Purification of ascites: Take 3 mL of the ascites prepared above, add 6 mL of 0.06 mol / L, pH 4.8 acetate buffer, mix well, add 99 μl of octanoic acid while stirring at room temperature, continue stirring for 30 minutes, and then let it stand at 4°C for more than 2 hours. Centrifuge the solution at 4°C, 1000 rpm for 30 minutes, take the supernatant, adjust the pH value to 7.4 with 2 mol / L NaOH, add an equal volume of saturated sulfuric acid solution to the supernatant solution while stirring, and after 30 minutes, let it stand at 4°C for more than 2 hours. The mixture was centrifuged at 10,000 rpm for 30 min, and the precipitate was dissolved in 5 mL of 0.01 M PBS (pH 7.4). After mixing, the precipitate was dialyzed in 0.01 M PBS (pH 7.4) at 4°C for 2 d, with the solution changed every 8 h. After 2 d, the solution in the dialysis bag was taken out and collected to obtain the purified anti-large yellow croaker CD8α monoclonal antibody. The protein content was determined by ultraviolet spectrophotometer and the aliquots were frozen at a concentration of 5 mg / mL at -80°C.

[0025] NIH\3T3-LcCD8α cells with a confluence of 90% were collected, washed twice with PBS (pH 7.4), counted, and 1*10 6Cells were prepared by cell smear machine. After being fixed with methanol for 5 min and air-dried, 100 μL 1% BSA was added and blocked at 37°C for 1 h. Washed twice with PBST (pH 7.4), then washed once with PBS (pH 7.4), added with the above anti-large yellow croaker CD8α monoclonal antibody (1 μg / mL), incubated at 37°C for 1 h, washed twice with PBST (pH 7.4) after incubation, and then washed once with PBS (pH 7.4). Goat anti-mouse IgG H&L (Alexa Fluor® 647) (1 μg / mL, abcam, ab150115) fluorescent secondary antibody was added, incubated at 37°C for 1 h, washed twice with PBST after incubation, and then washed once with PBS. Add a drop of Hoechst 33342 dye-colored sealing medium and examine under a fluorescence microscope. The results are shown in Figure 1 As shown, it can be seen that NIH\3T3-LcCD8α cells can express large yellow croaker CD8α.

[0026] Example 2 Validation of anti-large yellow croaker CD8α specific antibody (1) Flow cytometry detection: Take the head kidney tissue of healthy large yellow croaker, place it on a 70 μm mesh, add L-15 culture medium containing 25 IU / ml sodium heparin, and carefully grind it with the tail end of a syringe. The result is the head kidney leukocytes.

[0027] ① The crude white blood cells are about 1×10 7 cells, resuspended in 500 μL of L-15 medium containing 5% goat serum, and blocked at 4°C for 1 h; ② Add 1 ml of anti-large yellow croaker CD8α monoclonal antibody (1 μg / ml) and incubate at 4°C for 45 min; ③ Resuspend with 1 ml L-15 medium, centrifuge at 500 g for 5 min, and wash twice; ④ Add 1 ml of L-15 medium containing Goat anti mouse IgG Alexa Fluor 488 (1 μg / ml) to resuspend and incubate at 4°C for 30 min; ⑤ Resuspend with 1 ml L-15 medium, centrifuge at 500 g for 5 min, and wash twice; ⑥ Resuspend in 1×PBS, filter the cell suspension through a 70 μm mesh, and then detect by flow cytometry.

[0028] The results are as follows Figure 2 As shown in FIG. 1 , the monoclonal antibody labels a group of fluorescent cells. The monoclonal antibody prepared by the present invention can specifically recognize CD8 in the leukocytes of the head kidney of large yellow croaker. + T lymphocytes.

[0029] (2) Immunofluorescence experiment: ① Take 3×10 from (1) cells 5 cells were fixed with 4% paraformaldehyde for 10 min and washed twice with 1× PBS; ②DAPI (2 μg / mL) staining for 5 min, and washing twice with 1× PBS; ③ Transfer to a 35 mm confocal culture dish, centrifuge at 500 g for 5 min, and take laser confocal photos.

[0030] The results are as follows Figure 3 As shown, positive cells are round cells with a diameter of about 8 μm, with a large nucleus and a circle of green fluorescence around the cells. Negative cells are cells without green fluorescence, including B lymphocytes with a morphology similar to positive cells and larger macrophages.

[0031] (3) RT-PCR: ① Take the head kidney tissue of healthy large yellow croaker, place it on a 70 μm mesh, add L-15 culture medium containing 25 IU / mL heparin sodium, and carefully grind it with the tail end of a syringe to obtain the head kidney leukocytes.

[0032] ② After the cells were treated with the same experimental procedure as (1), they were sorted using a flow cytometer. The obtained cells were CD8 + T cells and CD8 - T cells; ③ RNA was extracted and converted into cDNA according to the instructions (Novozyme, R211-01); ④Perform PCR amplification according to the following procedure:

[0033] ⑤ Prepare 1.5% nucleic acid gel and perform electrophoresis at 150 V; The results showed that CD8 + T cells contain marker genes for CD3 and CD8 (CD8α, CD8β), but not for B lymphocytes and CD4-1 and CD4-2 T cells; on the contrary, CD8 - T cells do not contain CD8 (CD8α, CD8β) marker genes, which is consistent with the expected results. The monoclonal antibody prepared by the present invention can specifically recognize CD8 in the head kidney leukocytes of large yellow croaker. + T lymphocytes ( Figure 4 ).

[0034] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Anti-large yellow croaker CD8α monoclonal antibody, characterized by: The monoclonal antibody is secreted by the hybridoma cell line CD8α-5E3, the preservation unit of which is the China Center for Type Culture Collection, the preservation number is CCTCC NO: C2024283, and the preservation date is August 26, 2024.

2. The method for preparing the anti-large yellow croaker CD8α monoclonal antibody according to claim 1, characterized in that: The following steps are involved: (1) Preparation of NIH\3T3-LcCD8α stable cell line; (2) Preparation of hybridoma cell line CD8α-5E3; (3) Preparation of anti-large yellow croaker CD8α monoclonal antibody.

3. The method for preparing the anti-large yellow croaker CD8α monoclonal antibody according to claim 2, characterized in that: The method for preparing the NIH\3T3-LcCD8α stable cell line in step (1) is as follows: the expression vector pCDH-CMV-GFP-LcCD8α, the packaging vector psPAX2 and the envelope vector pMD2.G are transfected into 293T cells together to obtain the LcCD8α lentivirus; the obtained LcCD8α lentivirus is concentrated and then infected with NIH\3T3 cells, and after puromycin selection, the cell line NIH\3T3-LcCD8α stably expressing LcCD8α is obtained.

4. The method for preparing the anti-large yellow croaker CD8α monoclonal antibody according to claim 3, characterized in that: The transfection system is as follows: 15 μg expression vector pCDH-CMV-GFP-LcCD8α, 6 μg packaging vector psPAX2 and 3 μg envelope vector pMD2.G are added to 750 μl serum-free DMEM medium, and 750 μl serum-free DMEM medium is taken to dilute 24 μl Lipo8000 liposome nucleic acid transfection reagent, and then the two are mixed.

5. The method for preparing the anti-large yellow croaker CD8α monoclonal antibody according to claim 3, characterized in that: The step of concentrating the LcCD8α lentivirus is to mix the LcCD8α lentivirus supernatant obtained by transfecting 293T cells for packaging with the virus concentrate 5*PEG8000 at a ratio of 4:1, and shake at 4°C overnight; the puromycin concentration is 5 μg / ml.

6. The method for preparing the large yellow croaker CD8α monoclonal antibody according to claim 2, characterized in that: In step (2), the specific method for preparing the hybridoma cell line CD8α-5E3 is as follows: NIH\3T3-LcCD8α cells are mixed with serum-free DMEM culture medium, used as antigen to immunize Balb / C mice, and myeloma cells are fused with mouse spleen cells producing anti-large yellow croaker CD8α monoclonal antibodies to obtain the hybridoma cell line CD8α-5E3 that secretes anti-large yellow croaker CD8α monoclonal antibodies.

7. The method for preparing the large yellow croaker CD8α monoclonal antibody according to claim 2, characterized in that: The method for preparing the anti-large yellow croaker CD8α monoclonal antibody in step (3) is to combine the limiting dilution method and flow cytometry to screen the hybridoma cell line CD8α-5E3 that secretes the anti-large yellow croaker CD8α monoclonal antibody, thereby obtaining the monoclonal antibody.

8. The anti-large yellow croaker CD8α monoclonal antibody according to claim 1 specifically recognizes large yellow croaker CD8 + Application in T cells.

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