An identifiable CD4-1 of multiple cyprinid fishes + Rat anti-grass carp CD4-1 monoclonal antibody of T cells, hybridoma cell strain and application

Abstract

The application belongs to the field of fish immunology, and discloses a kind of CD4-1 + The application discloses a rat anti-grass carp CD4-1 monoclonal antibody of T cell, a hybridoma cell strain and application. The preservation number of the hybridoma cell strain is CCTCC NO: C2024235. Compared with existing grass carp CD4-1 monoclonal antibodies, cell grouping by using the monoclonal antibody for flow cytometry staining is more obvious and specific, and the monoclonal antibody can be used for flow cytometry specific recognition of CD4-1 + T cell of six kinds of cyprinid fishes such as grass carp, blue carp, silver carp, bighead carp, crucian carp and / or amur catfish. + The application provides an important tool for subsequent research on the role and mechanism of CD4-1 + T cell in the immune response of cyprinid fishes.

Description

Technical Field

[0001] The present invention belongs to the field of fish immunology, and specifically relates to a method for identifying CD4-1 + Rat anti-grass carp CD4-1 monoclonal antibody, hybridoma cell line and application of T cells. Background Art

[0002] T cells are the main executors of adaptive immunity, among which CD4 + T cells play an important role in assisting and regulating CD4 + The functional specialization of T cells is determined by antigenic signals and the microenvironment. For example, viruses and intracellular bacteria trigger CD4 + T cells differentiate into Th1 cells, which help eliminate infection by activating macrophages and cytotoxic T cells through secretion of interferon (IFN)-γ, lymphotoxin (LT)-α, and interleukin (IL)-2. + T cells selectively differentiate into Th2 cells that produce IL-4 or Th17 cells that produce IL-17 to resist infections caused by extracellular parasites or bacteria. Regulatory T cells (Treg) are another type of specialized CD4 + T cell subsets mainly maintain immune homeostasis and prevent the occurrence of autoimmune diseases by secreting TGF-β and IL-10 to suppress excessive immune responses. + T cells differentiate into follicular helper T cells (Tfh) characterized by the secretion of IL-21, which participate in B cell activation and antibody production, especially playing a key role in the formation of germinal centers.

[0003] Tetrapods have only one CD4 molecule, whose extracellular region is composed of four Ig-like domains, while bony fish have two CD4 molecules, named CD4-1 and CD4-2. CD4-1, like the CD4 of tetrapods, has an extracellular region composed of four Ig-like domains, while the extracellular region of CD4-2 is composed of only two or three Ig-like domains. Therefore, CD4-1 of bony fish and CD4 of tetrapods are believed to have originated from CD4-2 through gene and domain duplication. Although the existence of two CD4 genes in bony fish was discovered as early as 2006, due to the lack of antibodies, the CD4-1 of bony fish was not identified until 2016. + and CD4-2 + The composition of T cells has been elucidated in rainbow trout (Oncorhynchus mykiss), and studies have shown that CD4-1 + CD4-2 + T cells are the main cell group that plays an important role in immune function, and CD4-1 - CD4-2+ T cells are more like primitive CD4 + T cells. + T cell studies have also shown that CD4-1 + CD4-2 + T cells are CD4 + The main types of T cells confirmed the universality of this result in bony fish. + T cell function is best assessed by flow cytometry sorting of CD4 T cells from lymphocyte populations using a monoclonal antibody against CD4-1. + T cells.

[0004] Therefore, preparing grass carp (Ctenopharyngodon idella) CD4-1 monoclonal antibodies and exploring the T cell-mediated immune response mechanism are of great significance for the prevention and control of fish diseases, such as the development of vaccines that can efficiently activate fish T cells. Summary of the Invention

[0005] In view of the above-mentioned defects in the prior art, the present invention provides a hybridoma cell line of rat anti-grass carp CD4-1 monoclonal antibody, and the deposit number of the hybridoma cell line is: CCTCC NO: C2024235.

[0006] Another object of the present invention is to provide a rat anti-grass carp CD4-1 monoclonal antibody, which is secreted by the above hybridoma cells.

[0007] The last object of the present invention is to provide a method for preparing and using the monoclonal antibodies secreted by the hybridoma cells.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] The applicant successfully prepared a hybridoma cell line 27E3H8E2E5 that can specifically secrete rat anti-grass carp CD4-1 monoclonal antibodies by co-immunizing rats with a stably transfected cell line expressing grass carp CD4-1 on its surface and a recombinant grass carp CD4-1 protein, and established a screening system combining flow cytometry and fluorescent quantitative PCR. The hybridoma cell line was deposited in the China Center for Type Culture Collection on July 16, 2024, and the classification name is: Hybridoma cell line (Hybridoma cellline) P4-27E3H8E2E5, the preservation number is: CCTCC NO: C2024235, and the address is: Wuhan University, Wuhan, China.

[0010] The protection scope of the present invention also includes:

[0011] The monoclonal antibody is secreted by the hybridoma cell with the deposit number: CCTCC NO: C2024235.

[0012] A polynucleotide encoding the above monoclonal antibody.

[0013] The CDR region of the heavy chain variable region of the monoclonal antibody described above is characterized by comprising the following three amino acid sequences:

[0014] TAWMH (CDRH1, SEQ ID NO. 1),

[0015] RIKDRSINSATDYVGAVKG (CDRH2, SEQ ID NO.2),

[0016] QYNGYSPGY(CDRH3, SEQ ID NO.3);

[0017] The CDR region of the light chain variable region contains the following three amino acid sequences:

[0018] QASQNIDEYVA(CDRL1,SEQ ID NO.4),

[0019] HTSTLVS(CDRL2, SEQ ID NO.5),

[0020] LQYDDLPYT (CDRL3, SEQ ID NO. 6).

[0021] The amino acid sequence of the heavy chain variable region of the monoclonal antibody described above is:

[0022] EVQLVETGGGLVQPGKSLKLTCATSGFTFSTAWMHWVRQSPDKRLEWIARIKDRSINSATDYVGAVKGRFTVSRDDSKSSVYLHMNSIKEEDTATYYCTTQYNGYSPGYWGQGVMVTVSS(SEQ ID NO.7);

[0023] The amino acid sequence of the light chain variable region is:

[0024] DIQMTQSPPSLSASLGDKVTITCQASQNIDEYVAWYQQKPGRAPRLLIHHTSTLVSGAPSRFSGGSGRDFSFSISNVKSEDIASYYCLQYDDLPYTFGPGTKLELK (SEQ ID NO. 8).

[0025] When the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.7, the nucleotide sequence of nucleic acid molecule A may be:

[0026] GAGGTGCAGCTCGTGGAGACAGGGGGAGGCTTGGTGCAGCCTGGGAAATCTCTGAAACTCACCTGTGCCACCTCAGGATTCACTTTCAGTACGGCCTGGATGCACTGGGTTCGCCAGTCTCCAGACAAGCGACTAGAGTGGATTGCTCGAATAAAAGACAGATCTATCAATTCTGCAACCGA CTATGTGGGGGCTGTGAAAGGAAGATTCACCGTCTCAAGAGATGATTCCAAAAGTAGCGTTTACCTGCACATGAACAGCATAAAAGAGGAAGACACTGCCACTTATTACTGTACTACTCAATATAACGGGTATTCCCCGGGTTATTGGGGCCAAGGAGTCATGGTCACAGTCTCCTCA(SEQ IDNO.9);

[0027] When the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 8, the nucleotide sequence of nucleic acid molecule B can be: GACATCCAGATGACACAGTCTCCTCCCTCCCTGTCTGCATCTCTGGGAGACAAAGTCACCATCACTTGTCAGGCAAGTCAAAACATTGACGAGTATGTGGCTTGGTATCAGCAAAAGCCTGGAAGAGCTCCTAGACTTCTCATACATCACACATCTACACTAGTGTCAGGCGCCCCATCGAGGTTCAGTGGCAGTGGATCTGGGAGAGATTTTTCATTCAGCATCAGCAACGTGAAGTCTGAAGATATTGCAAGTTATTATTGTCTACAATACGATGACCTTCCGTACACATTTGGACCTGGGACCAAGCTGGAACTGAAA (SEQ ID NO. 10).

[0028] A recombinant vector comprises the above polynucleotide.

[0029] A host cell comprising the above-mentioned vector or polynucleotide.

[0030] The rat anti-grass carp CD4-1 monoclonal antibody, hybridoma cell line P4-27E3H8E2E5, recombinant vector or host cell are used in the preparation of cyprinid fish CD4-1 + Application of T cell detection kit.

[0031] The rat anti-grass carp CD4-1 monoclonal antibody, hybridoma cell line P4-27E3H8E2E5, recombinant vector or host cell can specifically recognize CD4-1 of cyprinid fish. + Application in T cells.

[0032] The rat anti-grass carp CD4-1 monoclonal antibody, hybridoma cell line P4-27E3H8E2E5, recombinant vector or host cell are used in preparing reagents for studying T cell immune response of cyprinid fish.

[0033] In the above application, preferably, the carp fish include: grass carp, black carp, silver carp, bighead carp, crucian carp and / or bighead carp.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention, for the first time, uses a retroviral packaging method to insert a grass carp CD4-1 gene fragment into the rat homologous cell line NRK-52E, enabling the cell line to express grass carp CD4-1 protein on the cell surface. This protein restores the grass carp CD4-1 protein in its natural state to the greatest extent possible. Rats were co-immunized with the stably transfected cell line expressing grass carp CD4-1 on the surface and the grass carp CD4-1 recombinant protein, and a screening system combining flow cytometry and fluorescent quantitative PCR was established. A hybridoma cell line 27E3H8E2E5 that can specifically secrete rat anti-grass carp CD4-1 monoclonal antibodies was successfully prepared. The rat anti-grass carp CD4-1 monoclonal antibodies produced by this hybridoma cell line can specifically recognize CD4-1 of six species of cyprinid fish, including grass carp. + T cells, CD4-1 + It provides a powerful tool for T cell immune function research and vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the result of preparing the stable cell line in Example 1 of the present invention;

[0037] Among them: A: Construction of retroviral packaging plasmid; B: Western blot verification of retroviral packaging; C: Anti-HA antibody staining of NRK-52E cells after retrovirus infection; D: Anti-HA antibody staining of NRK-52E cells after drug screening; E: First flow cytometry sorting of HA +The cells were then stained with anti-HA antibodies; F: Second flow cytometry sorting of HA + The cells were then stained with anti-HA antibody.

[0038] Figure 2 This is the result of Coomassie Brilliant Blue staining after prokaryotic expression of grass carp CD4-1 protein in Example 2 of the present invention.

[0039] Figure 3 This is the Western blot verification result of the rat anti-grass carp CD4-1 monoclonal antibody prepared in Example 4 of the present invention.

[0040] Figure 4 This is the flow cytometry verification result of the rat anti-grass carp CD4-1 monoclonal antibody prepared in Example 4 of the present invention;

[0041] Among them: AB: Rat anti-grass carp CD4-1 monoclonal antibody was used to sort leukocytes from grass carp head kidney (A) and spleen (B), and lymphocytes were divided into CD4-1 positive and negative populations; CD: Quantitative analysis of CD4-1 expression in CD4-1 positive and negative cells in the sorted grass carp head kidney (C) and spleen (D), using β-actin as the internal reference. The values ​​shown are the mean ± standard error of three fish, and "***" indicates extremely significant difference (p < 0.001).

[0042] Figure 5 The results of subtype identification of rat anti-grass carp CD4-1 monoclonal antibody in Example 4 of the present invention;

[0043] Among them: A: detection results of rat anti-grass carp CD4-1 monoclonal antibody heavy chain subtype; B: detection results of rat anti-grass carp CD4-1 monoclonal antibody light chain subtype.

[0044] Figure 6 This is the result of Western blot verification of the recognition of grass carp CD4-1 recombinant protein by the supernatants of the 0th, 5th, 10th, 15th and 20th passages of hybridoma cell line 27E3H8E2E5 in Example 6 of the present invention.

[0045] Figure 7 The flow cytometry was used to verify the effect of the supernatants of the 0th, 5th, 10th, 15th and 20th generations of hybridoma cell line 27E3H8E2E5 on the CD4-1 + T cell recognition results.

[0046] Figure 8 Flow cytometry was used to detect IgM in the head kidney (A), spleen (B), peripheral blood (C), gill (D), intestine (E) and liver (F) of grass carp in Example 7 of the present invention.+ B cell and CD4-1 + T cell results.

[0047] Wherein: G is IgM in grass carp head kidney (A), spleen (B), peripheral blood (C), gill (D), intestinal tract (E) and liver (F) + B cell and CD4-1 + Statistical analysis of the proportion of T cells in total lymphocytes, and the values shown are the average values of three fish ± standard errors.

[0048] Figure 9 CD4-1 + T cell results.

[0049] Wherein: F is CD4-1 in the head kidney of grass carp (A), silver carp (B), bighead carp (C), crucian carp (D) and mandarin fish (E) + Statistical analysis of the proportion of T cells in total lymphocytes, and the values shown are the average values of three fish ± standard errors. DETAILED DESCRIPTION

[0050] The technical solutions of the present application will be described below in conjunction with the embodiments in the present application, so as to make the technical solutions of the present application clear and complete. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0051] The technical solutions of the present application, if not specifically stated, are the conventional solutions in the art, and the reagents or materials, if not specifically stated, are from commercial channels.

[0052] Example 1:

[0053] Preparation of a stable cell line

[0054] The present embodiment provides a stable cell line expressing grass carp CD4-1 on the cell surface, and the preparation method is as follows:

[0055] 1. Construction of grass carp pDisplay-CD4-1 gene expression vector:

[0056] (1) Extract total RNA from grass carp spleen, and synthesize cDNA template by reverse transcription for standby use;

[0057] (2) Through NCBI search grass carp CD4-1 gene sequence (GenBank accession number: GQ355588.1), the primer pair for PCR amplification is designed, which includes forward primer F1 and reverse primer R1, and Bgl II and Sma I restriction enzyme sites are added at the 5' end of the forward and reverse primers, wherein the sequence of forward primer F1 is GGA AGATCT GAAAGCCCCATAGTAATTTAC, and the sequence of reverse primer R1 is TCC CCCGGG TGGTCTTGTAGAATCCCTTTGG, and the above-mentioned spleen cDNA is used as a template to amplify the gene sequence (excluding the signal peptide part) encoding grass carp CD4-1 by PCR.

[0058] (3) The amplification product is gel recovered, and the pDisplay plasmid and the PCR product are double-digested with Bgl II and Sma I at 37℃ for 1h. The digested products are recovered, and the digested vector and the gene sequence are ligated at 22℃ for 2h. The ligation product is transformed into DH5α competent cells, and single colonies are picked and expanded for culture and bacterial liquid PCR verification. The bacterial liquid with a positive result is sent to GenScript (Wuhan) Biotechnology Co., Ltd. for sequencing. The bacterial liquid with correct sequencing is further expanded for culture, and a plasmid is extracted using a small amount of endotoxin-free plasmid extraction kit (purchased from Meibio Technology Co., Ltd.), to obtain pDisplay-CD4-1.

[0059] 2. Construction of grass carp pMXs-HA-CD4-1 gene expression vector:

[0060] (1) The primer pair for PCR amplification is designed, which includes forward primer F2 and reverse primer R2, and Not I and SnaB I restriction enzyme sites are added at the 5' end of the forward and reverse primers, wherein the sequence of forward primer F2 is AAGGAAAAAA GCGGCCGC TAATACGACTCACTATAGGGA, and the sequence of reverse primer R2 is GCG TACGTA CTAACGTGGCTTCTTCTGCCA, and the above-mentioned pDisplay-CD4-1 plasmid is used as a template to amplify the gene sequence (including the signal peptide, HA tag and transmembrane region in the pDisplay vector) from the T7 promoter to the stop codon part by PCR.

[0061] (2) The amplified product was recovered by gel extraction. The pMXS-IRES-Puro plasmid and PCR product were double-digested with Not I and SnaB I at 37°C for 1 hour. The digested product was recovered and the digested vector was ligated with the gene sequence at 22°C for 2 hours. The ligated product was transformed into DH5α competent cells, and a single colony was picked for expansion and verification by bacterial liquid PCR. The bacterial liquid PCR test results were as follows: Figure 1 As shown in Figure A, the results for numbers 1, 2, 5, 6, 7, and 9 were positive, and the bacterial cultures with positive results were sent to Qingke (Wuhan) Biotechnology Co., Ltd. for sequencing. The bacterial cultures that were sequenced correctly were further expanded and the plasmid was extracted using an endotoxin-free plasmid miniprep kit (purchased from Meiji Biotechnology Co., Ltd.) to obtain the retroviral packaging plasmid pMXs-HA-CD4-1.

[0062] 3. Retroviral packaging:

[0063] (1) Take out the frozen Plat-E cells from liquid nitrogen, thaw them, and perform cell recovery and cell passage for 3-5 times to obtain Plat-E passage cells.

[0064] (2) One day before transfection (20-24 hours), trypsinize the cells and count them. Plate the cells (without antibiotics) so that the density at the time of transfection is 70-95% (2-4×10 cells per well of a 12-well plate). 5 cells).

[0065] (3) Prepare according to the following system Liposomal nucleic acid transfection reagent complex:

[0066] For each well of cells, dilute 1 μg of DNA with 100 μL of serum-free medium (such as OPTI-MEM I medium), and dilute 2 to 4.5 μL of Lipofectamine nucleic acid transfection reagent (purchased from Yisheng Biotechnology Co., Ltd.) was mixed with diluted DNA and diluted lipofectamine nucleic acid transfection reagent (total volume 200 μL), gently mixed, and incubated at room temperature (15-25°C) for 20 minutes to allow DNA-liposome complexes to form.

[0067] (4) Directly transfer 200 μL DNA-Hieff The complex was added to each well of the cell culture plate and the culture plate was shaken to mix gently.

[0068] (5) After culturing in a 37°C, 5% CO2 incubator for 24 hours, replace the growth medium and continue culturing.

[0069] (6) After 48 hours of medium replacement, aspirate the cell supernatant into a 15 mL centrifuge tube, centrifuge at 4°C and 500 g for 10 min, and filter with a 0.45 μm filter membrane. This virus solution can be stored at 2-8°C for one week.

[0070] (7) Western blot verification of retroviral packaging success

[0071] 1) Prepare protein samples: Collect cell pellets and lyse with RIPA buffer. Add 5× reducing SDS-PAGE loading buffer, vortex to mix, incubate at 100°C in a metal bath for 10 min, and place on ice until ready for use or freeze at -80°C.

[0072] 2) SDS-PAGE: Prepare a 12% Tris-glycine polyacrylamide lower separating gel and a 5% upper stacking gel. Load the sample and perform electrophoresis at 80 V for 0.5 h and 120 V for 1 h.

[0073] 3) Transfer: Remove the SDS-PAGE gel from the gel plate and soak it in pre-prepared transfer buffer (25 mM Tris, 192 mM glycine, 15% methanol). Cut the NC membrane to an appropriate size and arrange it from bottom to top in the order of filter paper-NC membrane-gel-filter paper, ensuring that there are no bubbles in each layer. Perform semi-dry transfer at 9V for 50 min to transfer the proteins on the SDS-PAGE gel to the NC membrane.

[0074] 4) Blocking: After the transfer is completed, the NC membrane is transferred into a TBST solution (25 mM Tris, 150 mM NaCl, 0.1% Tween-20) containing 5% skim milk powder and incubated at room temperature for 2 h.

[0075] 5) Primary antibody incubation: Discard the blocking solution and dilute mouse anti-HA monoclonal antibody (purchased from ABclonal) at 1:5000 in TBST solution containing 5% skim milk powder and incubate at 4°C overnight.

[0076] 6) Secondary Antibody Incubation: Remove the primary antibody solution, wash the membrane three times with TBST for 10 min each time, dilute HRP-labeled goat anti-mouse IgG (H+L) antibody (purchased from ABclonal) at a ratio of 1:5000 with TBST solution containing 5% skim milk powder, and incubate at room temperature for 1 h.

[0077] 7) ECL color development: Remove the secondary antibody solution, wash the membrane three times with TBST, each time for 10 min, prepare an appropriate amount of ECL color development solution (purchased from Biosharp) at a ratio of 1:1, add the color development solution dropwise to the membrane, and after sufficient reaction, take a picture using a chemiluminescence imager.

[0078] Western blot test results Figure 1As shown in middle B, mouse anti-HA monoclonal antibody only recognized cells transfected with pMXs-HA-CD4-1 (lane 3), but did not recognize cells not transfected with the plasmid (lane 1) and cells transfected with pMXS-IRES-Puro empty vector (lane 2), indicating that the retrovirus was successfully packaged.

[0079] 4. Retroviral Infection of NRK-52E Cells

[0080] (1) The frozen NRK-52E cells were taken out from liquid nitrogen, thawed, and then the cells were revived and passaged 3 to 5 times to obtain NRK-52E passaged cells.

[0081] (2) When the confluence of NRK-52E cells cultured on the culture plate reached about 50%, the culture supernatant was discarded and 12 mL of DMEM medium (1% double antibody, without serum) containing 5 μM polybrene (purchased from Yisheng Biotechnology Co., Ltd.) was slowly added.

[0082] (3) Add 500 μL of retrovirus suspension to each culture plate, mix gently, and place in a 37°C, 5% CO2 incubator to complete the infection of NRK-52E cells.

[0083] (4) Observe the cells for lesions 4 hours after retroviral infection. If so, replace the cells with growth medium and continue culturing.

[0084] (5) 24 h after infection, the culture medium was replaced with growth medium.

[0085] 5. Drug Screening and Flow Cytometry Sorting of Retrovirally Infected Cells

[0086] (1) 48 h after infection, drug screening can be performed. Cells are trypsinized and plated with growth medium containing 4 μg / mL puromycin. Culture is continued in a 37°C, 5% CO2 incubator for 14 days.

[0087] (2) Flow cytometry detection and sorting of HA + cell

[0088] 1) Resuspend the trypsin-digested cells in growth medium and gently shake them on a shaker at room temperature for 2 hours to reduce the damage of trypsin to the cells.

[0089] 2) Primary antibody incubation: Wash twice with PBS containing 2% FBS, resuspend the cells and count them. The cell concentration is adjusted to 1×10 7 cells / mL, take 2 mL of cells, add mouse anti-HA monoclonal antibody (purchased from ABclonal) with a final antibody concentration of 2 μg / mL, incubate on ice for 1 hour, and gently vortex the cells once every 15 minutes.

[0090] 3) Secondary antibody incubation: After washing twice with PBS containing 2% FBS, PE-labeled goat anti-mouse IgG (purchased from Biolegend) was added to a final antibody concentration of 1 μg / mL. The cells were incubated on ice for 45 minutes, and the cells were gently vortexed every 15 minutes.

[0091] 4) Flow cytometry: Wash the cells twice with PBS containing 2% FBS, resuspend the cells and filter them, and then detect HA using a flow cytometer (BD). + The proportion of cells and sorting HA + Cell culture and drug screening continued.

[0092] (3) After two rounds of drug screening and flow cytometry sorting, a stable cell line expressing grass carp CD4-1 on its surface was finally obtained.

[0093] The flow cytometry results before and after drug screening are as follows: Figure 1 Middle C and Figure 1 As shown in D, the flow cytometry results of the cultured cells after the first flow cytometry sorting and the second flow cytometry sorting are shown in Figure 2. Figure 1 Zhong E and Figure 1 As shown in F, HA + The proportion of cells gradually increased and eventually maintained at 99% to 100%, indicating that the stable cell line was successfully constructed.

[0094] Example 2:

[0095] Prokaryotic expression and purification of grass carp CD4-1 recombinant protein

[0096] 1. Construction of grass carp pET28a-CD4-1 gene expression vector

[0097] (1) We designed a primer pair for PCR amplification of the first two domains of CD4-1. The primer pair includes forward primer F3 and reverse primer R3. EcoR I and Xho I restriction enzyme sites were added to the 5' end of the forward and reverse primers, respectively. The forward primer F3 sequence is CCG GAATTC GAAAGCCCCATAGTAATTTACGC, reverse primer R3 sequence is CCG CTCGA G TTAGAGGTCTATTACAAAAACAGTTGT, using the above-mentioned grass carp spleen cDNA as a template, the gene sequence of the first two domains of grass carp CD4-1 was amplified by PCR.

[0098] (2) The amplified product was recovered by gel extraction, and the pET28a plasmid and PCR product were double-digested with EcoR I and Xho I at 37°C for 1 hour. The digested product was recovered and the digested vector was ligated to the gene sequence at 22°C for 2 hours. The ligated product was transformed into DH5α competent cells, and a single colony was picked for expansion and culture and verified by PCR. The bacterial solution with positive results was sent to Qingke (Wuhan) Biotechnology Co., Ltd. for sequencing. The bacterial solution with correct sequencing was further expanded and the plasmid was extracted using an endotoxin-free plasmid mini-extraction kit (purchased from Meiji Biotechnology Co., Ltd.) to obtain the pET28a-CD4-1 plasmid.

[0099] 2. Prokaryotic expression and purification of grass carp pET28a-CD4-1

[0100] (1) The extracted plasmid was transformed into the expression strain BL21 (DE3), and the positive clone was inoculated into 500 mL of LB liquid medium containing Amp (100 μg / mL), and cultured in a constant temperature shaker at 37°C and 180 rpm until the bacterial solution OD 600 =0.6-0.8, IPTG was added to a final concentration of 1 mM to induce protein expression, and the cells were cultured on a shaker at 37°C for 4-6 h. The cells were collected and resuspended in native lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10 mM β-Me) at pH = 8.0, and placed in a high-pressure crusher for crushing. After crushing, the cells were resuspended in denature lysis buffer (8 M urea, 50 mM NaH2PO4, 10 mM Tris-HCl, 10 mM β-Me) at pH = 8.0, centrifuged at 12000g, 4°C for 30 min, and the supernatant was collected. The supernatant was incubated with Ni filler (purchased from Huiyan Biotechnology Co., Ltd.) at room temperature for 1 h, washed with denature lysis buffer at pH = 6.3, and eluted with denature lysis buffer at pH = 4.5. A small amount of protein was taken for SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining to detect the induced expression and protein purity.

[0101] (2) Refolding of denatured proteins: The collected proteins can be dialyzed in steps to remove urea and refold the proteins. The proteins are dialyzed in refolding buffer (50 mM Tris-HCl, 150 mM NaCl, 400 mM L-Arginine-HCl, 2 mM reduced glutathione, 0.2 mM oxidized glutathione, 0.005% Tween-20) containing 4 M, 3 M, 2 M, 1 M, and 0.5 M urea, respectively, and finally dialyzed against PBS.

[0102] (3) The purified protein solution was concentrated using an ultrafiltration tube, and the protein concentration was detected using a BCA protein concentration assay kit. The protein purity was detected by Coomassie brilliant blue staining. The Coomassie brilliant blue staining results are shown in FIG. 3, and the protein size was about 23 kDa, and the purity was high. Figure 2

[0103] Example 3

[0104] Preparation of hybridoma cell lines

[0105] 1. Rat immunization

[0106] Five 8-10-week-old Sprague Dawley (SD) rats were immunized with NRK-52E cells displaying CD4-1 (prepared in Example 1) and grass carp CD4-1 recombinant protein (prepared in Example 2) as immunogens. The immunization route and procedure were as follows: 1 x 10 7 NRK-52E cells displaying CD4-1 were injected into the tail vein, and 100 μg of grass carp CD4-1 recombinant protein was mixed with an equal volume of Freund's complete adjuvant to emulsify and injected subcutaneously at multiple points on the back of the rat. Booster immunization was performed three times at two-week intervals, and 1 x 10 7 NRK-52E cells displaying CD4-1 were injected into the tail vein, and 100 μg of grass carp CD4-1 recombinant protein was mixed with an equal volume of Freund's complete adjuvant to emulsify and injected subcutaneously at multiple points on the back of the rat. Booster immunization was performed three times at two-week intervals, and 1 x 10

[0107] 2. Cell fusion and culture

[0108] (1) Cell fusion: Rat spleen cells were mixed with mouse myeloma cells SP2 / 0 at a cell ratio of 5:1, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. 1 mL of PEG-4000 (purchased from Sigma Company) was slowly added to the test tube while shaking; then 9 mL of complete medium was slowly added multiple times. The whole process was carried out in a 37°C water bath, followed by centrifugation at 1000 rpm for 10 min to remove the supernatant, and cell fusion was completed.

[0109] (2) Cell culture: The cell clumps after centrifugation were gently dispersed and resuspended with HAT medium, and plated in a prepared 96-well plate containing feeder cells at 200 μL per well. It was placed in a 37°C, 5% CO2 incubator for culture. After one week, the growth of clones was observed under a microscope and the number of clones per well was recorded. When the cells grew to about 1 / 3 of the area of the culture well, the culture supernatant was removed for flow detection.

[0110] 3. Screening and cloning of fused cells

[0111] ​(1) The fusion cells are detected and screened by flow cytometry, which comprises the following steps:

[0112] 1) The white blood cells of grass carp kidney are separated by Percoll density gradient centrifugation, and the cells are resuspended after being washed twice with PBS containing 2% FBS, and the cell concentration is adjusted to 3x10 6 cells / mL.

[0113] 2) The cell suspension is divided into 1.5 mL EP tubes, 160 μL per tube, 40 μL of hybridoma cell culture supernatant is added, and incubated on ice for 1 h, and vortexed once every 15 min.

[0114] 3) Resuspend to 200 μL after washing twice with PBS containing 2% FBS, add PE-labeled goat anti-rat IgG antibody (purchased from BioLegend company), and the final concentration of the antibody is 1 μg / mL, avoid light, incubate on ice for 45 min, and vortex once every 15 min.

[0115] 4) Resuspend to 200 μL after washing twice with PBS containing 2% FBS, pass through the cell filter, and detect by flow cytometry (BD).

[0116] (2) The positive fusion cells in the well are cloned, which comprises the following steps:

[0117] The hybridoma cells positive in flow detection are gently eluted from the culture plate and counted, and the cell suspension is serially diluted with culture medium and inoculated in a 96-well plate at 100 μL per well. After 10 days of culture, when the hybridoma cell colonies grow to 1 / 3 area of the well bottom, the antibody activity in the supernatant is determined. The positive clones with antibody activity are subcloned for a total of 3 times.

[0118] After repeated screening by flow cytometry, three hybridoma cell strains (18D1C8A4G2, 27G4A3A5C3 and 27E3H8E2E5) stably expressing rat anti-grass carp CD4-1 monoclonal antibody and having a higher positive proportion are finally selected, and the monoclonal antibodies produced by the three hybridoma cell strains are detected on the CD4-1 + T cells of other cyprinid fishes (including Mylopharyngodon piceus, Hypophthalmichthys molitrix, Aristichthys nobilis, Carassius auratus and Megalobrama amblycephala), and a hybridoma cell strain capable of producing monoclonal antibodies capable of recognizing CD4-1 +The hybridoma cell (27E3H8E2E5) producing a monoclonal antibody against T cells was deposited in the China Center for Type Culture Collection on July 16, 2024, at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2024235 and the classification name: Hybridoma cell line P4-27E3H8E2E5.

[0119] Table 1 Flow cytometry results of the third subcloning cell supernatant

[0120]

[0121]

[0122] Example 4:

[0123] Preparation and identification of rat monoclonal antibody against grass carp CD4-1

[0124] 1. Ascites Preparation:

[0125] 10-week-old BALB / c mice were intraperitoneally injected with 500 μL of sterile paraffin oil. Ten days later, the hybridoma cell line 27E3H8E2E5 with good growth status was obtained, the culture medium was washed with PBS, the cells were resuspended in 300 μL of sterile PBS, and 5×10 cells were intraperitoneally injected into each mouse. 6 hybridoma cells; 10 to 14 days later, when the abdomen of the mouse is extremely swollen, ascites is extracted, and the extracted ascites is centrifuged at 4°C and 10,000 rpm for 10 minutes. The supernatant is aliquoted and stored at -80°C for later use.

[0126] 2. Antibody purification:

[0127] Take 200 μL of Protein G Agarose in a 15 mL centrifuge tube, wash three times with PBS, then add 500 μL of the stored aliquoted ascites and dilute to 8 mL with PBS, and incubate overnight at 4°C; after washing 6 times with PBS, elute with 500 μL of 0.1 M glycine-HCl (pH 2.8) to obtain the rat anti-grass carp CD4-1 monoclonal antibody, and an average of 1.16 mg of the rat anti-grass carp CD4-1 monoclonal antibody can be obtained per mL of ascites.

[0128] 3. Antibody identification:

[0129] (1) Identify the antibody by Western blot, which specifically includes the following steps:

[0130] 1) Sample preparation: Dilute CD4-1 recombinant protein to 0.5 mg / mL, add 5× reducing SDS-PAGE loading buffer, vortex to mix, incubate at 100°C in a metal bath for 10 min, and place on ice until ready for use or freeze at -80°C.

[0131] 2) SDS-PAGE: Prepare a 12% Tris-glycine polyacrylamide lower separating gel and a 5% upper stacking gel. Load the sample and perform electrophoresis at 80 V for 0.5 h and 120 V for 1 h.

[0132] 3) Transfer: Remove the SDS-PAGE gel from the gel plate and soak it in pre-prepared transfer buffer. Cut the NC membrane to an appropriate size and arrange it from bottom to top in the order of filter paper-NC membrane-gel-filter paper, ensuring that there are no bubbles in each layer. Perform semi-dry transfer at 9V for 30 minutes to transfer the proteins on the SDS-PAGE gel to the NC membrane.

[0133] 4) Blocking: After transfer, transfer the NC membrane into TBST solution containing 5% skim milk powder and incubate at room temperature for 2 h.

[0134] 5) Primary antibody incubation: Discard the blocking solution and dilute the rat anti-grass carp CD4-1 monoclonal antibody to 1 μg / mL in TBST solution containing 5% skim milk powder. Incubate at 4°C overnight.

[0135] 6) Secondary Antibody Incubation: Remove the primary antibody solution, wash the membrane three times with TBST for 10 min each time, dilute HRP-labeled goat anti-rat IgG (H+L) antibody (purchased from ABclonal) at a ratio of 1:5000 with TBST solution containing 5% skim milk powder, and incubate at room temperature for 1 h.

[0136] 7) ECL color development: Remove the secondary antibody solution, wash the membrane three times with TBST, each time for 10 min, prepare an appropriate amount of ECL color development solution (purchased from Biosharp) at a ratio of 1:1, add the color development solution dropwise to the membrane, and after sufficient reaction, take a picture using a chemiluminescence imager.

[0137] (2) Identifying the antibody by flow cytometry, specifically including the following steps:

[0138] 1) Isolate leukocytes from grass carp head kidney by Percoll density gradient centrifugation, wash twice with PBS containing 2% FBS, and resuspend the cells to adjust the cell concentration to 2×10 7 cells / mL.

[0139] 2) Take 2 mL of cells and add rat anti-grass carp CD4-1 monoclonal antibody to a final concentration of 2 μg / mL. Incubate on ice for 1 hour and vortex to mix every 15 minutes.

[0140] 3) After washing twice with PBS containing 2% FBS, resuspend to 2 mL, add PE-labeled goat anti-rat IgG antibody (purchased from BioLegend company), the final concentration of antibody is 1 μg / mL, avoid light, incubate on ice for 45 min, vortex every 15 min.

[0141] 4) After washing twice with PBS containing 2% FBS, resuspend to 2 mL, pass through the cell filter, and then sort CD4-1 + T cells and CD4-1 - lymphocytes in head kidney and spleen by flow cytometry (BD) sorting head.

[0142] 5) Centrifugal collection of sorted cells, total RNA of sorted cells was extracted using cell RNA extraction kit (purchased from Meibio company), and reverse transcription kit (purchased from Takara company) was used to reverse transcribe it into cDNA, and the expression of CD4-1 + T cells and CD4-1 - lymphocytes was detected by fluorescent quantitative PCR, wherein the forward primer F3 sequence of CD4-1 is AGATGTGTCCAGGTGTCATAGT, the reverse primer R3 sequence is TGGAATTTTGACTGTATAGGATGA, the forward primer sequence of the internal reference gene β-actin is AGCCATCCTTCTTGGGTATG, and the reverse primer sequence is GGTGGGGCGATGATCTTGAT.

[0143] The results of Western blot are shown in Figure 3 , and the rat anti-grass carp CD4-1 monoclonal antibody can specifically recognize the grass carp CD4-1 recombinant protein. The results of flow cytometry are shown in Figure 4 A-B, from which it can be seen that the rat anti-grass carp CD4-1 monoclonal antibody can divide the lymphocytes of grass carp head kidney and spleen into CD4-1 + T cells and CD4-1 - lymphocytes, and the cell grouping is obvious. The results of fluorescent quantitative PCR are shown in Figure 4 C-D, and the expression amount of CD4-1 + T cells in grass carp head kidney and spleen is about 15000 times and 8500 times of that in CD4-1 - lymphocytes, indicating that the monoclonal antibody has high specificity.

[0144] 4. Subtype identification:

[0145] The rat antibody subtype rapid test card (purchased from Taihejian Biopharmaceutical R&D Co., Ltd.) was restored to room temperature for 15 minutes, and then the rat anti-grass carp CD4-1 monoclonal antibody was diluted to 2μg / ml with PBS. 80μL was pipetted and added dropwise to the sample slots of the rat antibody heavy chain and light chain test card. After 15 minutes, the test line and quality control line of the test strip were observed, and the heavy chain and light chain subtypes of the rat antibody were determined according to the position of the test line. The identification results are as follows: Figure 5 As shown, the heavy chain subtype of the rat anti-grass carp CD4-1 monoclonal antibody prepared by the present invention is IgG1, and the light chain subtype is Igκ.

[0146] Example 5:

[0147] Determination of nucleic acid sequences of heavy and light chain variable regions of monoclonal antibodies

[0148] Total RNA was extracted from hybridoma cells 27E3H8E2E5 in the logarithmic growth phase using the Trizol method (purchased from TAKARA). Reverse transcription was performed using oligo(dT)20 (purchased from Invitrogen) as a primer to generate cDNA. The heavy and light chain variable region genes were then amplified using specific PCR primers using the cDNA as a template. The PCR products were purified and recovered by agarose gel electrophoresis and then ligated into the pMD18-T vector via TA cloning. Sequencing and sequence analysis revealed the gene encoding the heavy chain variable region as shown in SEQ ID NO.9, encoding the protein as shown in SEQ ID NO.7. The gene encoding the light chain variable region as shown in SEQ ID NO.10, encoding the protein as shown in SEQ ID NO.8.

[0149] Example 6:

[0150] Analysis of the stability of hybridoma cell lines through subculture and the stability of secreted antibodies after subculture

[0151] The hybridoma cell line 27E3H8E2E5 was serially subcultured to the 20th generation. Cells from the 0th, 5th, 10th, 15th and 20th generations were selected for amplification and culture, and the cell supernatants were collected. The grass carp CD4-1 recombinant protein was verified by Western blot, and the total leukocytes from the grass carp head kidney tissue were verified by flow cytometry. Figure 6 and Figure 7 As shown, the cell supernatants of passages 0, 5, 10, 15 and 20 had no significant effect on the expression of grass carp CD4-1 recombinant protein and CD4-1 + There was no difference in T cell recognition, indicating that the hybridoma cell line has stability in passage and stability in secreting antibodies after passage.

[0152] Example 7:

[0153] Analysis of CD4-1 in various tissues of grass carp using rat anti-grass carp CD4-1 monoclonal antibody + The proportion of T cells

[0154] Leukocytes from grass carp head kidney, spleen, peripheral blood, gills, intestine and liver were separated by Percoll density gradient centrifugation and flow cytometry was performed using mouse anti-grass carp IgM monoclonal antibody and rat anti-grass carp CD4-1 monoclonal antibody. Figure 8 As shown, IgM in the head kidney + B cells and CD4-1 + T cells accounted for approximately 44% and 17% of total lymphocytes, respectively, and IgM + B cells and CD4-1 + T cells account for approximately 39% and 7% of total lymphocytes, respectively, and IgM + B cells and CD4-1 + T cells accounted for approximately 59% and 2% of total lymphocytes, respectively, and IgM + B cells and CD4-1 + T cells account for approximately 7% and 11% of total lymphocytes, respectively, and IgM + B cells and CD4-1 + T cells account for approximately 13% and 6% of total lymphocytes, respectively, and IgM + B cells and CD4-1 + T cells account for approximately 13% and 5% of total lymphocytes, respectively. IgM is absent in any tissue. + CD4-1 + cells, indicating that the rat anti-grass carp CD4-1 monoclonal antibody does not recognize IgM + B cells, further demonstrating that the monoclonal antibody is highly specific.

[0155] Example 8:

[0156] Rat anti-grass carp CD4-1 monoclonal antibody against CD4-1 in other cyprinid fish + T cell recognition

[0157] The head kidney leukocytes of black carp, silver carp, bighead carp, crucian carp and bighead bream were separated by Percoll density gradient centrifugation and detected by flow cytometry using rat anti-grass carp CD4-1 monoclonal antibody. Figure 9 As shown, the rat anti-grass carp CD4-1 monoclonal antibody can specifically recognize the head kidney CD4-1 of black carp, silver carp, bighead carp, crucian carp and bighead carp. + T cells, CD4-1 in the head kidney of different fish + The proportion of T cells in total lymphocytes is different, among which the CD4-1 +T cells account for 6% of total lymphocytes, CD4-1 + T cells account for 8% of the total lymphocytes, CD4-1 + T cells accounted for 7% of the total lymphocytes, CD4-1 + T cells account for 19% of the total lymphocytes, CD4-1 + T cells make up 10% of total lymphocytes.

[0158] In summary, the present invention successfully prepared rat anti-grass carp CD4-1 monoclonal antibody by hybridoma cell technology, which can specifically recognize CD4-1 of six species of cyprinid fish, including grass carp, black carp, silver carp, bighead carp, crucian carp and / or bighead bream. + T cells, CD4-1 + It provides a powerful tool for T cell immune function research and vaccine development.

[0159] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A screened hybridoma cell line, P4-27E3H8E2E5, with a deposit number of CCTCC NO: C2024235.

2. The monoclonal antibody secreted by the hybridoma cell line P4-27E3H8E2E5 according to claim 1.

3. A polynucleotide encoding the monoclonal antibody according to claim 2.

4. The monoclonal antibody according to claim 2, wherein the sequences of CDRH1, CDRH2, and CDRH3 of the heavy chain variable region are shown in SEQ ID NOs. 1 to 3, respectively, and the sequences of CDRL1, CDRL2, and CDRL3 of the light chain variable region are shown in SEQ ID NOs. 4 to 6, respectively.

5. The monoclonal antibody according to claim 2, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8. A recombinant vector comprising the polynucleotide according to claim 3.

7. A host cell comprising the polynucleotide according to claim 3 or the recombinant vector according to claim 6.

8. The monoclonal antibody according to claim 2, the hybridoma cell line P4-27E3H8E2E5 according to claim 1, the polynucleotide according to claim 3, the recombinant vector according to claim 6 or the host cell according to claim 7 in the preparation of CD4-1 + Application of T cell detection kit.

9. The monoclonal antibody according to claim 2, the hybridoma cell line P4-27E3H8E2E5 according to claim 1, the polynucleotide according to claim 3, the recombinant vector according to claim 6 or the host cell according to claim 7 in non-diagnostic specific recognition of CD4-1 in Cyprinidae fish + Application in T cells.

Images