Rat anti-grass carp CD4-1 monoclonal antibody capable of recognizing CD4-1 + T cells of various cyprinid fishes, hybridoma cell strain and application

Abstract

The invention belongs to the field of fish immunology, and discloses a rat anti-grass carp CD4-1 monoclonal antibody capable of recognizing CD4-1 + T cells of various cyprinid fishes, a hybridoma cell strain and application. The preservation number of the hybridoma cell strain is CCTCC (China Center For Type Culture Collection) NO: C2024235. Compared with the existing grass carp CD4-1 monoclonal antibody, the monoclonal antibody has the advantages that the cell grouping of flow cytometry staining by using the monoclonal antibody is more obvious, the specificity is higher, and the monoclonal antibody can be used for specifically identifying CD4-1 + T cells of six cyprinid fishes such as grass carp, black carp, silver carp, bighead carp, crucian carp and / or megalobrama amblycephala by using the flow cytometry; an important tool is provided for the subsequent research on the effect and mechanism of the CD4-1 + T cell in the immune response of the cyprinid fish.

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 auxiliary and regulatory role. + 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 IL-4-producing Th2 cells or IL-17-producing Th17 cells to resist infections caused by extracellular parasites or bacteria. Regulatory T cells (Tregs) 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 inhibit excessive immune responses. + T cells differentiate into follicular helper T cells (Tfh) characterized by the secretion of IL-21, which participate in the activation of B cells 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, 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 type that exerts immune functions, 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 from lymphocyte populations using a monoclonal antibody to 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 defects in the prior art, the present invention provides a hybridoma cell strain of rat anti-grass carp CD4-1 monoclonal antibody, and the deposit number of the hybridoma cell strain 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 antibody 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 was classified and named: Hybridoma cell line (Hybridoma cellline) P4-27E3H8E2E5, with a deposit number of: CCTCC NO: C2024235, and the address is: Wuhan University, Wuhan, China.

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

[0011] A monoclonal antibody 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 carp 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 carp 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 carp 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 adopts the method of retrovirus packaging for the first time to insert the grass carp CD4-1 gene fragment into the rat homologous cell line NRK-52E, so that the cell line can express grass carp CD4-1 protein on the cell surface, and the protein restores the grass carp CD4-1 protein in the natural state to the greatest extent. The stable cell line expressing grass carp CD4-1 on the surface and the grass carp CD4-1 recombinant protein were used to co-immunize rats, and a screening system combining flow cytometry and fluorescent quantitative PCR was established, and a hybridoma cell line 27E3H8E2E5 that can specifically secrete rat anti-grass carp CD4-1 monoclonal antibody was successfully prepared. The rat anti-grass carp CD4-1 monoclonal antibody produced by the hybridoma cell line can specifically recognize the CD4-1 of six species of carp fish such as grass carp. + T cells, CD4-1 from Cyprinidae + 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: the results of constructing retroviral packaging plasmids; B: the results of Western blot verification of retroviral packaging; C: the results of staining with anti-HA antibody after retrovirus infection of NRK-52E cells; D: the results of staining with anti-HA antibody after drug screening of NRK-52E cells; E: the first flow cytometry sorting of HA +The cells were then stained with anti-HA antibody; 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 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 the leukocytes in the head kidney (A) and spleen (B) of grass carp, and the lymphocytes were divided into two groups, CD4-1 positive and negative; CD: Quantitative analysis of the expression of CD4-1 in the CD4-1 positive and negative cells in the sorted grass carp head kidney (C) and spleen (D), with β-actin as the internal reference. The values ​​shown are the mean ± standard error of the three fish, and "***" indicates a very 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: the detection result of the heavy chain subtype of rat anti-grass carp CD4-1 monoclonal antibody; B: the detection result of the light chain subtype of rat anti-grass carp CD4-1 monoclonal antibody.

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

[0045] Figure 7 The flow cytometry method was used to verify the effects of the supernatants of the 0th, 5th, 10th, 15th and 20th generations of the 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 cells and CD4-1 + T cell results;

[0047] Where: G is IgM in grass carp head kidney (A), spleen (B), peripheral blood (C), gill (D), intestine (E) and liver (F) + B cells and CD4-1 + Statistical analysis of the proportion of T cells to total lymphocytes, values ​​shown are the mean ± SD of three fish.

[0048] Fig. 9 The flow cytometry analysis of CD4-1 in the head kidney of black carp (A), silver carp (B), bighead carp (C), crucian carp (D) and bighead bream (E) in Example 8 of the present invention is shown in FIG. + T cell results;

[0049] Among them: F is the CD4-1 in the head kidney of black carp (A), silver carp (B), bighead carp (C), crucian carp (D) and bighead carp (E) + Statistical analysis of the proportion of T cells to total lymphocytes, values ​​shown are the mean ± SD of three fish. DETAILED DESCRIPTION

[0050] The technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or materials, unless otherwise specified, are all from commercial channels.

[0052] Embodiment 1:

[0053] Preparation of stable cell lines

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

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

[0056] (1) Extracting total RNA from grass carp spleen and reversely transcribing it to synthesize cDNA template for later use;

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

[0058] (3) The amplified product was recovered by gel extraction, and the pDisplay plasmid and PCR product were double-digested with Bgl II and Sma I at 37°C for 1 hour. The digested product was recovered, and the digested vector was connected 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 culture and PCR verification of the bacterial solution. 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 the endotoxin-free plasmid mini-extraction kit (purchased from Meiji Biotechnology Co., Ltd.) to obtain pDisplay-CD4-1.

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

[0060] (1) Designing a primer pair for PCR amplification, the primer pair comprising: a forward primer F2 and a reverse primer R2, with Not I and SnaB I restriction enzyme sites added to the 5' ends of the forward and reverse primers, respectively, wherein the sequence of the forward primer F2 is AAGGAAAAAA GCGGCCGC TAATACGACTCACTATAGGGA, reverse primer R2 sequence is GCG TACGTA CTAACGTGGCTTCTTCTGCCA, using the above-mentioned pDisplay-CD4-1 plasmid as a template, the gene sequence from the T7 promoter to the stop codon (including the signal peptide, HA tag and transmembrane region in the pDisplay vector) was amplified by PCR.

[0061] (2) The amplified product was recovered by gel extraction, and 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 connected to the gene sequence at 22°C for 2 hours. The ligated product was transformed into DH5α competent cells, and a single colony was selected for expansion culture and verification by bacterial liquid PCR. The bacterial liquid PCR test results were as follows: Figure 1 As shown in A, the results numbered 1, 2, 5, 6, 7 and 9 were positive, and 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 cultured, and the endotoxin-free plasmid mini-extraction kit (purchased from Meiji Biotechnology Co., Ltd.) was used to extract the plasmid 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 subculture for 3-5 times to obtain Plat-E subculture 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 / well in each well of a 12-well plate). 5 cells).

[0065] (3) Prepare according to the following system Liposome 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-MEMⅠ medium), and dilute 2 to 4.5 μL of Liposome nucleic acid transfection reagent (purchased from Yisheng Biotechnology Co., Ltd.) Diluted DNA and diluted liposome nucleic acid transfection reagent (total volume 200 μL) were mixed, gently mixed, and incubated at room temperature (15-25° C.) for 20 min 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 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 to verify whether retroviral packaging is successful

[0071] 1) Preparation of protein samples: Collect cell pellets and lyse with RIPA lysis buffer, add 5× reduced SDS-PAGE loading buffer, vortex mix, incubate at 100°C for 10 min, place on ice for later use or freeze at -80°C.

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

[0073] 3) Transfer: Remove the SDS-PAGE gel from the gel plate and soak it in the prepared transfer buffer (25 mM Tris, 192 mM glycine, 15% methanol). Cut the NC membrane to a suitable size and arrange it from bottom to top in the order of filter paper-NC membrane-gel-filter paper. Make sure 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, dilute the mouse anti-HA monoclonal antibody (purchased from ABclonal) at 1:5000 with 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 with TBST for 3 times, 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 with TBST for 3 times, 10 min each time, 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 take pictures using a chemiluminescence imager after sufficient reaction.

[0078] Western blot results Figure 1As shown in B, mouse anti-HA monoclonal antibody only recognizes cells transfected with pMXs-HA-CD4-1 (lane 3), but not cells not transfected with plasmid (lane 1) and cells transfected with pMXS-IRES-Puro empty vector (lane 2), indicating that retrovirus packaging is successful.

[0079] 4. Retroviral infection of NRK-52E cells

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

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

[0082] (3) Add 500 μL of retrovirus suspension to each culture plate, mix gently, and place in a 37° C., 5% CO 2 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, the cells can be screened with drugs. The cells are trypsinized and plated with growth medium containing 4 μg / mL puromycin. The cells are cultured in a 37°C, 5% CO2 incubator and screened for 14 days.

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

[0088] 1) Resuspend the cells digested with trypsin in growth medium and place 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. Adjust the cell concentration to 1×10 7 cells / mL, take 2 mL of cells, add mouse anti-HA monoclonal antibody (purchased from ABclonal), the final concentration of the antibody is 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, with a final antibody concentration of 1 μg / mL. The cells were incubated on ice for 45 minutes, and the cells were gently vortexed once 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 + Cells continued to be cultured and screened for drugs.

[0092] (3) After two rounds of drug screening and flow cytometry, 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 shown in Figure 2. Figure 1 Middle C and Figure 1 As shown in D, the flow cytometry results of the cultured cells after the first flow sorting and the second flow sorting are shown in Figure 2. Figure 1 ZhongE and Figure 1 As shown in F, HA + The proportion of cells gradually increased and finally maintained at 99% to 100%, indicating that the stable cell line was successfully constructed.

[0094] Embodiment 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 a forward primer F3 and a reverse primer R3. EcoR I and Xho I restriction enzyme sites were added to the 5' ends of the forward and reverse primers, respectively. The sequence of the forward primer F3 is CCG GAATTC GAAAGCCCCATAGTAATTTACGC, reverse primer R3 sequence is CCG CTCGA G TTAGAGGTCTATTACAAAAACAGTTGT, using the above 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 connected 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 culture and PCR verification of the bacterial solution. 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 the 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 transferred 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 reached 0. 600 =0.6-0.8, add IPTG to a final concentration of 1 mM to induce protein expression, culture at 37°C in a shaking incubator for 4-6 h, collect the cells and resuspend them in native lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10 mM β-Me) at pH = 8.0, place them in a high-pressure crusher for crushing, and resuspend the cells in denature lysis buffer (8 M urea, 50 mM NaH2PO4, 10 mM Tris-HCl, 10 mM β-Me) at pH = 8.0, centrifuge at 12000g, 4°C for 30 min, and collect the supernatant. 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, eluted with denature lysis buffer at pH = 4.5, and 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 sequence using 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, and finally dialyzed using PBS.

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

[0103] Embodiment 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 (prepared in Example 1) displaying CD4-1 and grass carp CD4-1 recombinant protein (prepared in Example 2) as immunogens. The immunization route and procedure were as follows: 1×10 7 NRK-52E cells displaying CD4-1 on their surface were used. At the same time, 100 μg of grass carp CD4-1 recombinant protein was fully mixed with an equal volume of Freund's complete adjuvant and emulsified, and then injected subcutaneously at multiple points on the back of the rats. Three booster immunizations were performed, each with an interval of two weeks. During the booster immunization, 1×10 7 NRK-52E cells displaying CD4-1 on their surface were cultured and 100 μg of grass carp CD4-1 recombinant protein was fully mixed with an equal volume of Freund's incomplete adjuvant and emulsified, and then injected subcutaneously at multiple points on the back of rats.

[0107] 2. Cell Fusion and Culture

[0108] (1) Cell fusion: Rat spleen cells and mouse myeloma cells SP2 / 0 were mixed 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) was aspirated and slowly added to the test tube while shaking; then a total of 9 mL of complete culture medium was slowly added multiple times. The entire process must be carried out in a 37°C water bath, followed by centrifugation at 1000 rpm for 10 min, and the supernatant was removed to complete the cell fusion.

[0109] (2) Cell culture: Gently flick the cell clumps after centrifugation, resuspend them in HAT medium, and plate them in the prepared 96-well plate containing feeder cells, 200 μL per well. Culture them in an incubator at 37°C and 5% CO2. After one week, observe the clone growth under a microscope and record the number of clones in each well. When the cells grow to occupy about 1 / 3 of the bottom area of ​​the culture well, aspirate the culture supernatant for flow cytometry.

[0110] 3. Screening and cloning of fused cells

[0111] (1) Detecting and screening the fusion cells by flow cytometry, specifically comprising the following steps:

[0112] 1) Separate the leukocytes from the head kidney of grass carp by Percoll density gradient centrifugation, wash twice with PBS containing 2% FBS and resuspend the cells to adjust the cell concentration to 3×10 6 cells / mL.

[0113] 2) The cell suspension was dispensed into 1.5 mL EP tubes, 160 μL per tube, and 40 μL of hybridoma cell culture supernatant was added. The cells were incubated on ice for 1 hour and vortexed every 15 minutes to mix well.

[0114] 3) Wash twice with PBS containing 2% FBS and resuspend to 200 μL, add PE-labeled goat anti-rat IgG antibody (purchased from BioLegend) to a final antibody concentration of 1 μg / mL, incubate on ice for 45 min in the dark, and vortex mix every 15 min.

[0115] 4) The cells were washed twice with PBS containing 2% FBS and resuspended in 200 μL. The cells were passed through a cell strainer and detected using a flow cytometer (BD).

[0116] (2) Cloning the fused cells in the positive wells, specifically comprising the following steps:

[0117] Gently wash and count the hybridoma cells that are positive in flow cytometry from the culture plate, and dilute the cell suspension with culture medium continuously and inoculate 100 μL per well in a 96-well plate. After culturing for about 10 days, when the hybridoma cell colonies grow to 1 / 3 of the area at the bottom of the well, begin to measure the antibody activity in the supernatant. Subclone the positive clones with antibody activity again, and perform it 3 times in total.

[0118] After repeated screening by flow cytometry, three hybridoma cell lines (18D1C8A4G2, 27G4A3A5C3 and 27E3H8E2E5) that stably expressed rat anti-grass carp CD4-1 monoclonal antibodies with a high positive rate were finally selected. The monoclonal antibodies produced by these three hybridoma cell lines were used to detect CD4-1 in the head kidney and spleen of other carp fish (including black carp (Mylopharyngodonpiceus), silver carp (Hypophthalmichthys molitrix), bighead carp (Aristichthys nobilis), crucian carp (Carassius auratus) and bighead bream (Megalobrama amblycephala)). + T cells were tested and a strain was selected that could produce CD4-1 +The hybridoma cell (27E3H8E2E5) of the monoclonal antibody of T cells was deposited in the China Center for Type Culture Collection on July 16, 2024, address: Wuhan University, Wuhan, China, with the deposit number of CCTCC NO: C2024235, and the classification name: hybridoma cell line (Hybridoma cell line) P4-27E3H8E2E5.

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

[0120]

[0121]

[0122] Embodiment 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. After 10 days, the hybridoma cell line 27E3H8E2E5 with good growth status was taken, the culture medium was washed off with PBS, the cells were resuspended in 300 μL of sterile PBS, and 5×10 6 hybridoma cells; 10 to 14 days later, when the abdomen of the mouse was extremely swollen, the ascites was extracted, and the extracted ascites was centrifuged at 4°C, 10000rpm for 10min, and the supernatant was 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 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× reduced SDS-PAGE loading buffer, vortex mix, incubate at 100°C for 10 min, place on ice for later use or freeze at -80°C;

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

[0132] 3) Transfer: Remove the SDS-PAGE gel from the gel plate and soak it in the prepared transfer buffer. Cut the NC membrane to a suitable size and arrange it from bottom to top in the order of filter paper-NC membrane-gel-filter paper. Make sure 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 the transfer is completed, the NC membrane is transferred into a TBST solution containing 5% skimmed milk powder and incubated at room temperature for 2 h.

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

[0135] 6) Secondary antibody incubation: remove the primary antibody solution, wash the membrane with TBST for 3 times, 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 with TBST for 3 times, 10 min each time, 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 take pictures using a chemiluminescence imager after sufficient reaction.

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

[0138] 1) Separate the leukocytes from the head kidney of grass carp 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, add rat anti-grass carp CD4-1 monoclonal antibody, the final concentration of the antibody is 2 μg / mL, incubate on ice for 1 hour, and vortex mix every 15 minutes.

[0140] 3) Wash twice with PBS containing 2% FBS and resuspend to 2 mL, add PE-labeled goat anti-rat IgG antibody (purchased from BioLegend) to a final antibody concentration of 1 μg / mL, incubate on ice for 45 min in the dark, and vortex mix every 15 min.

[0141] 4) Wash twice with PBS containing 2% FBS and resuspend to 2 mL. After passing through a cell filter, use flow cytometry (BD) to sort CD4-1 in the head kidney and spleen. + T cells and CD4-1 - Lymphocytes.

[0142] 5) The sorted cells were collected by centrifugation, and the total RNA of the sorted cells was extracted using a cell RNA extraction kit (purchased from Meiji Biotechnology Co., Ltd.), and reverse transcribed into cDNA using a reverse transcription kit (purchased from Takara Company). CD4-1 + T cells and CD4-1 - Expression of CD4-1 in lymphocytes, where 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] Western blot results Figure 3 As shown in Figure 2, rat anti-grass carp CD4-1 monoclonal antibody can specifically recognize grass carp CD4-1 recombinant protein. Figure 4 As shown in AB, it can be seen from the figure that the rat anti-grass carp CD4-1 monoclonal antibody can divide the lymphocytes of the grass carp head kidney and spleen into CD4-1 + T cells and CD4-1 - Lymphocytes, cell groups are obvious. The results of fluorescence quantitative PCR are as follows Figure 4 CD4-1 in the head kidney and spleen of grass carp + The expression levels of CD4-1 in T cells are CD4-1 - The expression of the monoclonal antibody in lymphocytes was about 15,000 times and 8,500 times, respectively, indicating that the monoclonal antibody had 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, and 80μL was dripped into the sample slots of the rat antibody heavy chain and light chain test cards in turn. 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] Embodiment 5:

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

[0148] The hybridoma cells 27E3H8E2E5 in the logarithmic growth phase were taken, and the total RNA was extracted by the Trizol (purchased from TAKARA) method, and oligo (dT) 20 (purchased from Invitrogen) was used as a primer for reverse transcription to generate cDNA; then, the heavy chain variable region and light chain variable region genes were amplified respectively using specific PCR primers using the cDNA as a template; the PCR products were subjected to agarose gel electrophoresis and recovered and purified, and then connected to the pMD18-T vector by TA cloning, followed by sequencing and sequence analysis, and the heavy chain variable region encoding gene was obtained as shown in SEQ ID NO.9, encoding the protein shown in SEQ ID NO.7; the light chain variable region encoding gene was shown in SEQ ID NO.10, encoding the protein shown in SEQ ID NO.8.

[0149] Embodiment 6:

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

[0151] The hybridoma cell line 27E3H8E2E5 was continuously subcultured to the 20th generation, and the cells of the 0th, 5th, 10th, 15th and 20th generations were selected for amplification and culture, and the cell supernatant was collected. The grass carp CD4-1 recombinant protein was verified by Western blot, and the total leukocytes of the grass carp head kidney tissue were verified by flow cytometry. Figure 6 and Figure 7 As shown, the supernatants of cells from the 0th, 5th, 10th, 15th and 20th generations were expressed on the 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 secreted antibodies after passage.

[0152] Embodiment 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 account for approximately 44% and 17% of total lymphocytes, respectively, and IgM + B cells and CD4-1 + T cells account for about 39% and 7% of total lymphocytes, respectively, and IgM + B cells and CD4-1 + T cells account for about 59% and 2% of total lymphocytes, respectively, and IgM + B cells and CD4-1 + T cells account for about 7% and 11% of total lymphocytes, respectively, and IgM + B cells and CD4-1 + T cells account for about 13% and 6% of total lymphocytes, respectively, and IgM in the liver + 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 indicating that the monoclonal antibody is highly specific.

[0155] Embodiment 8:

[0156] Rat anti-grass carp CD4-1 monoclonal antibody against other carp CD4-1 + 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. The results are shown in Fig. 9 As shown in Figure 2, rat anti-grass carp CD4-1 monoclonal antibody can specifically recognize CD4-1 in the head kidney of black carp, silver carp, bighead carp, crucian carp and bighead bream. + T cells, CD4-1 in head kidney of different fish + The proportion of T cells to total lymphocytes is different, among which CD4-1 +T cells account for 6% of total lymphocytes. + T cells accounted for 8% of the total lymphocytes. + T cells accounted for 7% of total lymphocytes, CD4-1 + T cells account for 19% of total lymphocytes. + 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, and the monoclonal antibody can specifically recognize CD4-1 of six species of carp, including grass carp, black carp, silver carp, bighead carp, crucian carp and / or bighead bream. + T cells, CD4-1 from Cyprinidae + 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 protection scope of the present invention.

Claims

1. A screened hybridoma cell line P4-27E3H8E2E5, the deposit number of the hybridoma cell line is: 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 in the heavy chain variable region are shown as SEQ ID NO.1 to SEQ ID NO.3, respectively, and the sequences of CDRL1, CDRL2 and CDRL3 in the light chain variable region are shown as SEQ ID NO.4 to SEQ ID NO.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 of claim 2, the hybridoma cell line P4-27E3H8E2E5 of claim 1, the polynucleotide of claim 3, the recombinant vector of claim 6 or the host cell of claim 7 in preparing CD4-1 + Application of T cell detection kit.

9. The monoclonal antibody of claim 2, the hybridoma cell line P4-27E3H8E2E5 of claim 1, the polynucleotide of claim 3, the recombinant vector of claim 6 or the host cell of claim 7 in specifically recognizing CD4-1 + Application in T cells.

10. Use of 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 preparing a reagent for studying T cell immune response of Cyprinidae fish.

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