Grass carp CD3e hybridoma cell line and monoclonal antibody and their application

Abstract

The present invention provides a grass carp CD3e hybridoma cell line and monoclonal antibody and application. The hybridoma cell line has been deposited in the China Center for Type Culture Collection on August 17, 2022, and the deposit number is: CCTCC NO: C2022215; the grass carp CD3e monoclonal antibody is obtained by secretion of the hybridoma cell line, and the grass carp CD3e monoclonal antibody is used in the preparation of a reagent or a kit for detecting grass carp CD3e protein; the monoclonal antibody can be used to simply, quickly and accurately detect the expression level of grass carp CD3e protein by immunological methods such as enzyme-linked immunosorbent assay, protein blotting, immunofluorescence assay, immunohistochemistry and flow cytometry. In addition, the grass carp CD3e monoclonal antibody has good specificity and high sensitivity, can provide a tool for basic research in fish immunology, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a grass carp CD3e hybridoma cell line and a CD3e monoclonal antibody secreted by the hybridoma cell line, as well as a preparation method and application of the grass carp CD3e monoclonal antibody. Background Art

[0002] T cells are the main components of lymphocytes. They have multiple biological functions, such as directly killing target cells, assisting or inhibiting B cells to produce antibodies and producing cytokines, etc. They play an important role in resisting disease infection and tumor formation. The immune response produced by T cells is cellular immunity, which plays an important role in the body's immune response. The CD3e molecule, also known as CD3E, is a one-way type I membrane glycoprotein on the surface of T cells. The CD3e molecule contains an Ig-like (immunoglobulin-like) domain and an ITAM domain. The CD3e molecule forms a T cell receptor-CD3 complex together with CD3-γ, CD3-δ and CD3-ζ as well as T cell receptor α / β and γ / δ heterodimers. This complex plays an important role in coupling antigen recognition with multiple intracellular signal transduction pathways.

[0003] Grass carp is one of the most important freshwater aquaculture species in my country. It is popular among consumers because of its delicious meat and low price. In recent years, due to the increasing breeding density, excessive feeding and serious deterioration of water quality, the diseases of farmed grass carp have increased, resulting in a large number of deaths, which seriously hinders the sustainable development of grass carp farming. The immune system plays an important role in disease prevention and control and immune regulation of fish. Among them, CD3 is one of the most important cell surface markers of T lymphocytes and has an important signal transmission function in the process of antigen recognition. The preparation of grass carp CD3e monoclonal antibody can provide a tool for the study of fish immune system, and deepen the understanding of fish disease resistance mechanism while deepening the study of grass carp immune system. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide a grass carp CD3e hybridoma cell line and monoclonal antibody and applications.

[0005] To achieve the above object, the solution of the present invention is:

[0006] In the first aspect, the present invention provides a grass carp CD3e hybridoma cell strain, the preservation number of the grass carp CD3e hybridoma cell strain is: CCTCC NO: C2022215, the hybridoma cell strain is obtained by immunizing mice with grass carp CD3e protein as an antigen, and is named hybridoma cell strain GC9-CD3e. It was deposited in the China Center for Type Culture Collection on August 17, 2022, and the address is: Wuhan University, Wuhan, China.

[0007] In a second aspect, the present invention provides a grass carp CD3e monoclonal antibody, which is secreted by the above-mentioned grass carp CD3e hybridoma cell line.

[0008] As a preferred embodiment of the present invention, the method for preparing grass carp CD3e monoclonal antibody comprises: taking the culture supernatant of the hybridoma cell line mentioned above and / or injecting the hybridoma cell line mentioned above into the peritoneal cavity of mice, extracting ascites and centrifuging and purifying to obtain grass carp CD3e monoclonal antibody.

[0009] In a third aspect, the present invention provides an application of a grass carp CD3e monoclonal antibody, and an application of the grass carp CD3e monoclonal antibody in preparing a reagent or a kit for detecting grass carp CD3e protein.

[0010] In a fourth aspect, the present invention provides a reagent or kit for detecting grass carp CD3e protein, which comprises the above-mentioned grass carp CD3e monoclonal antibody.

[0011] As a preferred embodiment of the present invention, the kit is selected from one or more of an enzyme-linked immunosorbent assay kit, a protein blotting kit, an immunofluorescence assay, an immunohistochemistry kit and a flow cytometry kit.

[0012] In a fifth aspect, the present invention provides an application of a grass carp CD3e monoclonal antibody, and the application of the grass carp CD3e monoclonal antibody in enzyme-linked immunosorbent assay (ELISA), protein blotting (Western blot), immunofluorescence assay (IFA), immunohistochemistry (IHC) and flow cytometry (FCM), that is, the grass carp CD3e monoclonal antibody can use ELISA to specifically detect the protein concentration of CD3e in grass carp serum; it can also detect the expression of CD3e protein in grass carp tissues or cells by Western blot; through frozen tissue sectioning technology, the expression of CD3e in grass carp tissues can be detected using laser confocal microscopy; and the expression distribution of CD3e in grass carp cells can also be detected by flow cytometry.

[0013] Due to the adoption of the above scheme, the beneficial effects of the present invention are:

[0014] The present invention aims at the problem of lack of monoclonal antibody of grass carp CD3e, and provides a hybridoma cell line GC9-CD3e secreting monoclonal antibody of grass carp CD3e, which has good passage ability and can be amplified in large quantities, and the purified monoclonal antibody of grass carp CD3e has high purity (>90%), and affinity constants are 2.08E+08 and 2.98E+09 respectively. The monoclonal antibody can be used to simply, quickly and accurately detect the expression level of grass carp CD3e protein by immunological methods such as IHC, WB, ELISA, FCM, etc., without cross-reaction with other proteins of grass carp. It can provide tools for basic research of fish immunology and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a graph showing the antibody titer detection results after animal immunization in Example 2 of the present invention.

[0016] Figure 2 This is a purity diagram of grass carp CD3e monoclonal antibody detected by SDS-PAGE in Example 2 of the present invention.

[0017] Figure 3 This is a graph showing the affinity constant results of ELISA detection of grass carp CD3e monoclonal antibody in Example 2 of the present invention.

[0018] Figure 4 This is a graph showing the results of Western blot detection of the specificity of the grass carp CD3e monoclonal antibody in Example 2 of the present invention.

[0019] Figure 5 This is a cellular localization diagram of grass carp CD3e detected by immunofluorescence in Example 3 of the present invention.

[0020] The hybridoma cell line GC9-CD3e was deposited in the China Type Culture Collection of Wuhan University on August 17, 2022, and its deposit number is: CCTCC NO: C2022215. DETAILED DESCRIPTION

[0021] The invention provides a grass carp CD3e hybridoma cell strain, a monoclonal antibody and applications.

[0022] The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments of the present invention, but the protection scope of the present invention is not limited to the following description.

[0023] The reagents used in the present invention and their sources: Escherichia coli BL21 competent cells (stored in this laboratory), SPF BALB / c female mice (Mus muscμLus) (Beijing Huada Protein Research and Development Center Co., Ltd.), mouse myeloma cells (SP2 / 0Cell) (Beijing Huada Protein Research and Development Center Co., Ltd.), HEK293 cells (human embryonic kidney cells 293) (stored in this laboratory), ExTaq Mix and Prime star were purchased from TaKaRa; agarose gel recovery kit was purchased from OMEGA; peptone, yeast powder, agar powder, agarose, sodium chloride, glycerol, Triton X-100, 10×TBS rinse solution were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; restriction endonucleases were purchased from Thermo Fisher Company; isopropylthiogalactoside (IPTG), Tris-HCl, DTT, and EDTA were purchased from VWR Company; oxidized glutathione, reduced glutathione, and guanidine hydrochloride were purchased from Amresco Company; high-glucose DMEM basal culture medium, fetal bovine serum (FBS), penicillin-streptomycin, and phosphate buffered saline (PBS) were purchased from Gibco Company; LipofectamineTM3000 was purchased from ThermoFisher Company.

[0024] Example 1: Preparation of grass carp CD3e antigen

[0025] (1) Cloning of the grass carp CD3e genome and construction of the prokaryotic expression plasmid

[0026] According to the grass carp genome database (http: / / www.ncgr.ac.cn / grasscarp / ), the localblast technique was used to obtain the partial CDS sequence of grass carp CD3e by comparing it with the synonymous gene sequences of other vertebrates, and then the sequence was verified. Then the 5′ and 3′ terminal sequences of grass carp CD3e were obtained by 5′RACE and 3′RACE techniques. The full-length sequence was spliced ​​according to the sequencing results. The full-length sequence was amplified and sequenced by the high-fidelity enzyme PrimeSTAR.

[0027] Based on the selected NcoI and HindIII restriction sites, primers with protective bases and restriction sites were designed to clone the DNA sequence of CD3e.

[0028] Among them, the grass carp CD3e gene base sequence (SEQ ID NO.1):

[0029] ATGATTCTCACGGGCTTCGTGTTTCTGATGCTCACGGCGGCGCTGAGTCCAGCTGAGGGTCAAGAAATCAAAGAAATCACAGCAAATAGTGTCATTCTGACCTGCTCTGGAGGTGAATCTGACAAGTGGTATAAAAATAAAGAGACAAATGTGATGAGTAGCACCTATGAAGTTCAAGCGGAAAATGGAATAGTTGAAGGAGTGTACCACTGTGAATATACAGAAAATTTAAAAACCATCAAACACATGTTCTAC CTCAATGTAAAGGTGTGTGAGAACTGCTATGAGTTGAGCGGGTTCTTGGCGTGGGGCGTCATATTCGGTGACTTACTGGTTACAGGAGCAGTTATTCTCATCGTCTACATGTGTGTGCCGAAAAACACTGGCAGCACACAGCAGAAAGCATCAAAATCACGAACAGCAGGCGCTCCTCCACCTCCAAACCCAGATTATGAGAGACTGAATCATAACACGCGCAGCGCTGATTTGTACGCAGGCCTTCACAAA TAG (The underlined characters are the start codon and the stop codon, respectively).

[0030] The grass carp CD3e amino acid sequence (SEQ ID NO.2) is as follows:

[0031] MILTGFVFLMLTAALSPAEGQEIKEITANSVILTCSGGESDKWYKNKETNVMSSTYEVQAENGIVEGVYHCEYTENLKTIKHMFYLNVKVCENCYELSGFLAWGVIFGDLLVTGAVILIVYMCVPKNTGSTQQKASKSRTAGAPPPPNPDYERLNHNTRSADLYAGLHK.

[0032] The pEHISTEVb vector and the cloned grass carp CD3e gene fragment were double-digested with NcoI and HindIII, respectively. After digestion, the vector digestion products and the target fragment digestion products were recovered by gel recovery kit. The grass carp CD3eDNA fragment was connected to the pEHISTEVb vector under the action of T4DNA ligase. The ligation product was transformed into Escherichia coli DH5α competent cells, and the positive single clones were selected and sent to Shanghai Jinweizhi Company for sequencing confirmation.

[0033] (2) Inducible expression of recombinant grass carp CD3e prokaryotic protein

[0034] 1) Take out the E. coli BL21 competent cells, put them on ice to thaw, add the constructed CD3e prokaryotic expression plasmid to the E. coli BL21 competent cells, and place them on ice for 30 minutes;

[0035] 2) Place the competent cells that have been ice-bathed in a 42°C water bath for 90 seconds and immediately place them on ice to cool;

[0036] 3) Add 400 μL of antibiotic-free LB liquid medium, incubate on a shaker for 1 hour, then spread 100 μL on LB solid medium containing kanamycin and incubate in a 37°C incubator overnight;

[0037] 4) Pick monoclonal colonies, identify positive colonies, and send positive clones to the company for sequencing;

[0038] 5) The positive monoclonal colonies were transferred to LB liquid medium containing kanamycin for cultivation. After the culture reached the logarithmic growth phase, 1 mmol / L IPTG inducer was added to continue the culture;

[0039] 6) Collect the bacterial culture and identify the expression by SDS-PAGE and Western blot.

[0040] (3) Purification of grass carp CD3e recombinant protein

[0041] 1) Add the bacterial solution capable of expressing grass carp CD3e to 1 L of LB liquid medium containing kanamycin at a ratio of 1:100, culture the bacterial solution to the logarithmic growth phase, then add 1 mL, 1 mmol / L IPTG, and continue culturing for 12 h;

[0042] 2) Collect the cells by centrifugation, crush them under high pressure, and collect the supernatant and precipitate after high-speed centrifugation. SDS-PAGE analysis showed that the protein was expressed in inclusion bodies;

[0043] 3) Extract inclusion bodies: Resuspend the inclusion bodies in 20 mL of washing buffer containing 1‰ DTT (50 mmol / L Tris-cl pH 8.0, 300 mmol / L NaCl, 10 mmol / L EDTA, 0.5% Triton-100), centrifuge, and discard the supernatant. Repeat the above steps to wash the inclusion bodies once. Next, resuspend the inclusion bodies in 20 mL of resuspension buffer containing 1‰ DTT (50 mmol / L Tris pH 8.0, 100 mmol / L NaCl, 10 mmol / L EDTA), centrifuge, and discard the supernatant. Finally, the inclusion bodies were dissolved in DTT dissolution buffer (6 mol / L guanidine hydrochloride, 10% glycerol, 50 mmol / L Tris-cl, pH 8.0, 100 mmol / L NaCl, 10 mmol / L EDTA) to a final inclusion body concentration of 30 mg / mL and completely dissolved at 4°C. After centrifugation, the supernatant of the inclusion bodies was transferred to a new test tube and stored at -20°C for future use.

[0044] 4) Renaturation and purification of recombinant protein: This example adopts the dilution renaturation method, takes 10mL of the extracted 30mg / mL inclusion body, adds 1L of pre-cooled renaturation solution (100mmol / L Tris, pH 8.0, 400mmol / L L-ArgHCl, 0.5mmol / L GSH, 0.5mmol / L GSSG) and renatures for 36h. The renatured protein is added to the stirring concentration cup, and the renatured protein solution is filtered through a 10ku ultrafiltration membrane under a nitrogen pressure of 0.4kPa for concentration. After concentration to a small volume, the concentrated protein is replaced in a 300mmol / L protein buffer (300mmol / L NaCl, 20mmol / L Tris-HCl) and continued to concentrate. After concentration to a small volume, it is transferred to an ultrafiltration tube and continued to concentrate until the volume is no more than 5mL. Use AKTA pure protein purifier to purify the soluble protein by molecular sieve chromatography and concentrate it, and store the concentrated protein in a -80℃ refrigerator.

[0045] Example 2: Preparation of grass carp CD3e monoclonal antibody

[0046] The method mainly includes: (1) immunizing mice; (2) cell fusion; (3) selecting positive hybridoma cell lines; (4) culturing the hybridoma cell lines to secrete grass carp CD3e monoclonal antibodies; and (5) purifying to obtain monoclonal antibodies.

[0047] (1) Immunization of mice

[0048] Four SPF BALB / c female mice were immunized subcutaneously with purified grass carp CD3e prokaryotic protein for the first time, 60 μg / mouse, numbered CD3e-1, CD3e-2, CD3e-3 and CD3e-4. Four booster immunizations were performed thereafter, with 30 μg protein injected subcutaneously into each mouse each time, and the interval between each immunization was two weeks. Blood was collected from the eye sockets of mice 10 days after the last booster immunization.

[0049] (2) ELISA to measure serum titer

[0050] Use 2μg / mL grass carp CD3e protein, coat overnight at 4℃; 2% milk, block at 37℃ for 2h; serum is diluted 2 times from 200 times, blank control (blank) is PBS, negative control (negative) is negative serum diluted 200 times. Add the diluted serum to the protein-coated wells, incubate at 37℃ for 1h, wash 3 times with washing solution, add the secondary antibody diluted 20000 times with PBS, incubate at 37℃ for 1h; take out and wash 3 times with washing solution; add 100μL color development solution to each well for 5min; add 50μL stop solution to stop color development, and measure the absorbance at dual wavelengths (450, 630) with an enzyme marker. The results are as follows Figure 1 shown.

[0051] (3) Cell fusion

[0052] Mouse spleen cells and SP2 / 0 cells were taken and fused using the PEG method. After fusion, the cells were screened and cultured using a semi-solid culture medium (containing HAT). The specific steps are as follows:

[0053] 1) Gently blow the sp2 / 0 cells in good condition off the wall of the culture flask and aspirate them into a 50 mL centrifuge tube;

[0054] 2) The mice were eyeballed and blood was collected, then the mice were killed by pulling the neck and soaked in 75% alcohol for 5 min;

[0055] 3) Pour a small amount of serum-free IMDM into the dish, and place the cell sieve and syringe core into the dish. Use scissors and forceps to remove the spleen of the mouse and place it on the cell sieve. Use the syringe core to gently crush the spleen thoroughly, and suck the crushed cells into the centrifuge tube containing sp2 / 0, and centrifuge at 1500rpm for 5min;

[0056] 4) Use scissors and forceps to remove the mouse thymus and crush it. Place the crushed thymocytes in a 15 mL centrifuge tube, add 2 mL of HAT and 2 mL of HT and place in an incubator for later use;

[0057] 5) After centrifugation, discard the supernatant, gently and carefully blow the cells evenly with serum-free IMDM, and centrifuge (1500 rpm, 5 min);

[0058] 6) Pour off the supernatant of the centrifuged cells as much as possible. Tap the bottom of the centrifuge tube to fully suspend the cells, place the centrifuge tube in 37°C warm water, slowly add 1 mL of PEG within 1 minute, and let it stand in warm water for 1 minute. Then slowly add 2 mL of serum-free IMDM within 2 minutes, and then slowly add 8 mL of serum-free IMDM within 2 minutes. Centrifuge at 1000 rpm for 5 minutes;

[0059] 7) Pour off the supernatant, add 10 mL of serum, carefully blow the cells evenly, and pour in the thymocytes prepared earlier. Add 25 mL of sterilized semi-solid culture medium, mix thoroughly, and then pour evenly into 30 cell culture dishes. Place the cell culture dishes in a wet box and then incubate them in an incubator.

[0060] (4) Screening of positive monoclones

[0061] Pick 10 plates × 93 cell clones and culture them in 96-well cell culture plates (previously plated with thymocytes, 100 μL / well). Coat the plates with "CD3e" and use the ELISA method for the selected clones for the first screening: dilute the grass carp recombinant CD3e protein with coating solution to a final concentration of 2 μg / mL, 100 μL / well, 4°C, overnight; then wash 3 times with washing solution; block with 2% milk blocking solution, 200 μL / well, incubate at 37°C, wash 3 times with washing solution after 2 hours; add primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), Positive control (positive serum diluted 1000 times in PBS), all 100 μL / well, incubated at 37°C, washed 3 times with washing solution after 1h; added 20000 times diluted PBS secondary antibody, 100 μL / well, incubated at 37°C for 1h; washed 3 times with washing solution after taking out; color development, color development solution 100 μL / well, color development time is about 5min; 50 μL stop solution was added to each well and the OD value was measured with an enzyme marker after termination, and a total of 22 positive hybridoma cell lines were screened. Next, the 22 positive cell lines were coated with "CD3e" and "tag protein" respectively, and the ELISA method was used for the second screening to obtain 10 positive hybridoma cell lines. Finally, the 10 positive cell lines screened were subclassified, and 8 IgG type positive hybridoma cell lines were obtained. The results are shown in Table 1.

[0062] Table 1 Determination of antibody titer and subtype identification in CD3e positive hybridoma cell supernatant

[0063]

[0064] (5) Purification of monoclonal antibodies

[0065] Hybridoma cell lines No. 9 and No. 17 were selected and cultured in culture medium respectively. The cells were diluted to 3×10 6 After 7-10 days, the abdominal area of ​​the mouse was swollen, and the ascites was extracted and the ascites supernatant was collected. Centrifugation was carried out at 12000rpm for 10 minutes at 4°C, and the antibody was filtered through a 0.22μm filter membrane to remove fat, cell residues and small particles. Affinity purification was then performed using a HiTraprProtein A FF column, and the purity of the antibody was tested by SDS-PAGE. The results are shown in the figure. Figure 2 Finally, the affinity constant of the antibody was detected by ELISA, and the result was as follows Figure 3 shown.

[0066] (6) Western blot detection of antibody specificity

[0067] The CD3e eukaryotic expression plasmid was transfected into 293T cells. After 24 hours, the cell protein samples were collected and electrophoresed. The protein gel after electrophoresis was transferred to a PVDF membrane through a transfer membrane instrument, and then blocked for two hours with 5% skim milk powder. The purified antibody was diluted and incubated for two hours. The membrane was washed three times with TBST, and then incubated with mouse secondary antibody for 1 hour. After washing three times with TBST, the color was developed by a gel imaging instrument. The results showed that the antibodies secreted by the two hybridoma cells had good specificity. Figure 4 shown.

[0068] Case study 3: Application of grass carp CD3e monoclonal antibody

[0069] (1) Application in ELISA

[0070] Grass carp CD3e was used as the antigen to be tested for coating, and grass carp recombinant CD3e protein was used as the standard. The standard curve was prepared by gradient dilution with diluent, and 2μg / mL BSA was set as the negative control, and it was left to stand at 4°C overnight. Next, it was blocked with skim milk powder; after washing, CD3e monoclonal antibody was added and incubated for 2h; after washing, the secondary antibody diluted 20,000 times with PBS was added, 100μL / well, and incubated at 37°C for 1h; after washing three times, the color developing solution was added for color development for 5min; then the stop solution was added for termination, and the OD value at 450nm was measured with an enzyme marker.

[0071] (2) Application in Western blot

[0072] Grass carp tissue or cell protein samples were prepared and electrophoresed. The protein gel after electrophoresis was transferred to a PVDF membrane through a membrane transfer instrument, and blocked for two hours with 5% skimmed milk powder. The purified antibody was diluted and incubated for two hours. The membrane was washed three times with TBST and then incubated with mouse secondary antibody for 1 hour. After washing three times with TBST, the color was developed by a gel imaging instrument. The results showed that the antibodies secreted by the two hybridoma cells had good specificity.

[0073] (3) Application to IFA

[0074] The well-cultured 293T cells were spread on a cover glass plate, and the CD3e eukaryotic expression plasmid was transfected after the cells grew to a monolayer. The samples were prepared 18 hours later. They were first washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and transmitted with TritonX-100. Next, they were blocked with PBST for 2 hours, and then incubated with FITC-labeled CD3e monoclonal antibody for 2 hours. Finally, DAPI was used for nuclear staining, and then the images were taken using a laser confocal microscope. The results are shown in Figure 2. Figure 5 shown.

[0075] (4) Application in IHC

[0076] This monoclonal antibody can be used for IHC to detect the expression of CD3e in tissues.

[0077] (5) Application to FCM

[0078] This monoclonal antibody can be used in FCM to detect CD3e expressed in cells.

[0079] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

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Claims

1. A grass carp CD3e hybridoma cell line, characterized in that: The deposit number of the grass carp CD3e hybridoma cell strain is: CCTCC NO: C2022215.

2. A grass carp CD3e monoclonal antibody, characterized in that: The grass carp CD3e monoclonal antibody is obtained by secreting the grass carp CD3e hybridoma cell line described in claim 1.

3. Use of the grass carp CD3e monoclonal antibody according to claim 2 in the preparation of a reagent or kit for detecting grass carp CD3e protein.

4. A reagent or kit for detecting grass carp CD3e protein, characterized in that: It comprises the grass carp CD3e monoclonal antibody as described in claim 2.

5. The reagent or kit for detecting grass carp CD3e protein according to claim 4, characterized in that: The kit is selected from one or more of an enzyme-linked immunosorbent assay kit, a protein blotting kit, an immunofluorescence assay kit, an immunohistochemistry kit and a flow cytometry kit.

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