A hybridoma cell line, its produced antibodies, and their applications

Abstract

This invention discloses a hybridoma cell line containing the herbicide-resistant protein GAT, its produced antibody, and its applications. The hybridoma cell line is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 46332. The preparation method includes: a) purifying prokaryotic expression to obtain recombinant GAT protein; b) immunizing animals: using the recombinant GAT protein as an antigen to immunize BALB / c mice; c) cell fusion: collecting spleen cells from immunized BALB / c mice and fusing them with SP2 / 0 cells; d) cell line establishment: subcloning using the limiting dilution method, performing ELISA detection on subclones after 5-7 days, until a stable hybridoma cell line secreting positive antibodies is selected for expansion, reculturing, and preservation. The monoclonal antibody secreted by the hybridoma cell line of this invention lays the foundation for the detection of the herbicide-resistant protein GAT in transgenic crops, thus supporting the development of transgenic crops. street The development, seed production, and preservation of genetically modified crops have ensured the survival of genetically modified organisms (GMOs). street The industrialization of genetically modified crops is progressing steadily, and the sustainable application of related transformants is advancing.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to a herbicide-resistant protein GAT hybridoma cell line, the antibodies it produces, and its applications. Background Technology

[0002] Bacillus ( Bacillus )middle gat The glyphosate acetyltransferase expressed by the glyphosate degradation gene confers herbicide tolerance to crops transgenic with this gene. The cultivation of herbicide-tolerant crops has made field spraying of herbicides an effective method of weed control, thus making herbicide tolerance one of the most advantageous traits in agricultural production. Currently, herbicide-tolerant crops account for more than 80% of all genetically modified crops globally. The trend shows that the proportion of genetically modified crops continues to increase.

[0003] The development and advancement of genetically modified (GM) technology have propelled the progress of biology. While GM foods can meet people's demands for increased yield, insect resistance, and herbicide tolerance, they also pose potential threats to human life. For example, certain genes introduced into the host can cause food toxicity; GM foods can produce allergens, leading to antibiotic resistance; and the nutritional value of food can be altered. Simultaneously with the research, development, and commercialization of GM foods, it is crucial to conduct comprehensive safety assessments, enable consumers to quickly distinguish between GM and natural foods, and establish appropriate methods for identifying and detecting GM components in GM foods. This will promote the safe management of agricultural GMOs, protect the safety of humans, animals, and microorganisms, protect the ecological environment, and further advance research in agricultural GMO technology. Therefore, the rapid analysis of herbicide-resistant GAT proteins in GM crops and their derivatives, and the research and development of monoclonal antibodies against GAT proteins, are of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a hybridoma cell line of herbicide-resistant protein GAT, the antibody produced therefrom, and its application. The monoclonal antibody secreted by the cell line lays the foundation for the detection of herbicide-resistant protein GAT in transgenic crops.

[0005] To achieve the above objectives, the present invention provides a hybridoma cell line, which was deposited on March 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46332.

[0006] Specifically, the method for preparing this hybridoma cell line includes the following steps:

[0007] a) Purification and expression in prokaryotes yielded recombinant GAT protein;

[0008] b) Immunization of animals: BALB / c mice were immunized with recombinant GAT protein as an antigen;

[0009] c) Cell fusion: Spleen cells from immunized BALB / c mice were collected and fused with SP2 / 0 cells;

[0010] d) Cell line establishment: Subcloning was performed using the limiting dilution method. Subclones were tested by ELISA after 5-7 days until hybridoma cell lines that stably secrete positive antibodies were selected for expansion, reculture, and preservation.

[0011] The fusion ratio of mouse spleen cells to SP2 / 0 cells was 1:5-1:10.

[0012] The present invention also provides a monoclonal antibody produced by the aforementioned hybridoma cell line. The hybridoma cell line is inoculated into the peritoneal cavity of mice to prepare ascites, and then purified by Protein A-agarose affinity chromatography to obtain the monoclonal antibody.

[0013] Specifically, the monoclonal antibody produced by hybridoma cell line 1C2 had a titer of 1:1280000 as detected by indirect ELISA, and the antibody type was IgG2a.

[0014] The heavy chain amino acid sequence of the variable region of the monoclonal antibody produced by hybridoma cell line 1C2 is shown in SEQ ID NO: 1 of the sequence listing, and the light chain amino acid sequence of the variable region is shown in SEQ ID NO: 2 of the sequence listing. The specific sequences are as follows:

[0015] cell lines Heavy chain variable region amino acid sequence Light chain variable region amino acid sequence 1C2 EVQLQESGPSLVKPYQTLSLTCSVTGDSSTSDYWNWIRKFPGNKFEYMGYISYSGTTYYNPSLKSRISITRDTSKNQSYLQLKSVTTEDTGTYYCANVRMNYWGLGTSVTVSS DIVITQTPSSLAMSVGQKVTMSCKSSQSLLNSRDQKNYLAWYQQKPGQSPKLLIYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADFFCQQHFRLPLTFGSGTRLEIK

[0016] This invention also provides the application of the above-mentioned monoclonal antibody in the detection of herbicide-resistant protein GAT.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This application uses the recombinant herbicide-resistant protein GAT obtained by prokaryotic expression and purification as an antigen, and prepares a hybridoma cell line 1C2 that secretes a specific and sensitive monoclonal antibody against GAT through hybridoma technology. The antibody obtained after purification of the ascites fluid of the monoclonal antibody secreted by the cell line has an indirect ELISA titer of 1:1280000 and the antibody subtype is IgG2a. The monoclonal antibody can specifically recognize GAT protein in transgenic soybeans. The construction of the mouse monoclonal antibody hybridoma cell line that secretes GAT herbicide-resistant protein provides material and technical support for the detection of this protein in transgenic crops.

[0018] Preservation information:

[0019] The hybridoma cell line 1C2 provided by this invention was deposited on March 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), classified and named GAT monoclonal antibody hybridoma cell line. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 46332. Attached Figure Description

[0020] Figure 1 This is an SDS-PAGE electrophoresis result of the monoclonal antibody purified from the hybridoma cell line 1C2 according to the present invention;

[0021] Figure 2 The titer of monoclonal antibodies produced by the hybridoma cell line 1C2 according to the present invention;

[0022] Figure 3 This is a Western spectral image of the specific detection of GAT in transgenic soybeans using a monoclonal antibody produced by the hybridoma cell line 1C2 according to the present invention. Lane 1 is the protein extract of GAT-positive material; lane 2 is the protein extract of GAT-negative material; and the primary antibody for Western spectral detection is 1C2.

[0023] Figure 4 This is a graph showing the sensitivity detection results of monoclonal antibodies produced by the hybridoma cell line 1C2 according to the present invention, wherein: lane 1 is 5 ng of GAT protein and 10 μg / ml of 1C2 antibody; lane 2 is 10 ng of GAT protein and 10 μg / ml of 1C2 antibody; lane 3 is 20 ng of GAT protein and 10 μg / ml of 1C2 antibody; lane 4 is 40 ng of GAT protein and 10 μg / ml of 1C2 antibody; lane 5 is 60 ng of GAT protein and 10 μg / ml of 1C2 antibody; and lane 6 is 80 ng of GAT protein and 10 μg / ml of 1C2 antibody.

[0024] Figure 5 The above figures show the results of specific detection of monoclonal antibodies produced by the hybridoma cell line 1C2 according to the present invention. The left figure is a cocoon staining diagram, with lane 1 representing GAT protein, lane 2 representing Pat / pat protein, lane 3 representing Pat / bar protein, and lane 4 representing G2 EPSPS protein. The right figure is a Western chromatogram, with lane 1 representing 100 ng of GAT protein and 10 μg / ml of 1C2 antibody, lane 2 representing 100 ng of Pat / pat protein and 10 μg / ml of 1C2 antibody, lane 3 representing 100 ng of Pat / bar protein and 10 μg / ml of 1C2 antibody, and lane 4 representing 100 ng of G2 EPSPS protein and 10 μg / ml of 1C2 antibody. Detailed Implementation

[0025] The following will describe in detail the specific embodiments of the present invention in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0026] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0027] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be purchased from commercial channels.

[0028] Example 1: Obtaining hybridoma cells and preparing monoclonal antibodies

[0029] 1. Preparation of immunizing antigen

[0030] The gat gene was amplified from the genomic DNA of transgenic soybean Huang 6106, the expression vector pET28a-gat was constructed, and it was transformed into Escherichia coli BL21 competent cells. After seed activation, it was expanded in culture and induced to express overnight at 16 °C with IPTG. The relatively pure His-GAT protein was obtained through Ni affinity chromatography column and gel filtration purification.

[0031] 2. Immunizing animals

[0032] Eight 8-week-old SPF-grade BALB / c female mice (purchased from Hubei Experimental Animal Research Center, license number: SCXK(E)2015-0018) were immunized with His-GAT protein as an antigen. The antigen was mixed with an equal volume of complete Freund's adjuvant (primary immunization) or incomplete Freund's adjuvant (boost immunization) and emulsified. After充分混合 to the water-in-oil state, it was immunized subcutaneously at multiple points. There were 2-3 boost immunizations, and the interval between each immunization cycle was 2 weeks. Then the titer was detected. One week after the titer was higher than 1:10000, intraperitoneal shock was carried out. The immunization dose of the antigen was directly dissolved in 250 μL of PBS. The specific immunization procedure and immunization dose are shown in Table 1.

[0033] Table 1 Immunization procedure and immunization dose

[0034]

[0035] Immunization example: In the primary immunization, 50 μg of the antigen was dissolved in PBS and then mixed with the adjuvant at a volume ratio of 1:1.

[0036] 3. Cell fusion

[0037] Three days after the final shock, blood was collected from positive controls, and spleens were harvested and prepared into single-cell suspensions. SP2 / 0 cells in the logarithmic growth phase were treated and then mixed with spleen cells at a ratio of 1:5-1:10. The mixture was incubated with 50% PEG 1450 for 1 min, diluted with DMEM basal medium, centrifuged at low speed, and then gently resuspended and mixed in HAT medium containing 20% ​​fetal bovine serum. The suspension was then incubated at a rate of 2 × 10⁻⁶ cells / year. 7 The cells were plated into a pre-prepared feeder cell plate and incubated at 37°C with 5% CO2.

[0038] 4. Cell line establishment

[0039] 1) Fusion board testing:

[0040] Once the cells in the fusion plate have grown to a medium size (approximately 10,000 or more), testing can begin. The ELISA test should be performed after the cells have passed quality control (i.e., the negative control OD). 450 <0.2, positive control OD 450 After >1.0), select the positive well (generally OD). 450 Subcloning (≥0.5) is performed.

[0041] 2) Subcloning methods and detection:

[0042] High positive values ​​(OD) were selected from the fusion plates. 450 Limiting dilutions were performed on wells with a density greater than 2.0 g / L. Subcloning was performed by counting 60% of the single-clone wells per plate. Each time, single-clone wells with higher positive values ​​were selected for limiting dilution. Each subcloning was performed 5-7 days after the initial dilution, and ELISA detection was performed until a single-clone cell line that could stably secrete positive antibodies was finally selected for expansion culture.

[0043] 3) Cell line establishment:

[0044] Stable cell lines secreting positive antibodies, selected during the subcloning stage, were expanded and cultured in 24-well plates. After expansion, the supernatant was collected for antigen detection. Stability was verified using serially diluted ELISA and Western blotting. The monoclonal antibody secreted by the GAT hybridoma cell line 1C2 specifically detected GAT protein in transgenic soybean samples. Cells were collected and expanded in 10cm culture dishes, and the supernatant was collected again to detect antibody titers. OD cells were selected. 450 The cell line with a growth rate of >2.0 was cultured in cell culture flasks and cryopreserved, namely hybridoma cell line 1C2, which was deposited on March 14, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46332.

[0045] Cell line cryopreservation identification: After cryopreservation, one cell line from the same batch must be thawed for identification. Identification criteria:

[0046] ① The number of resuscitated live cells should be ≥1 million cells / vial; ② The number of viable cells among the live cells should be ≥500,000 / cell line; ③ The resuscitated cells should not contain any microorganisms other than the cell line itself (e.g., bacteria, fungi, mycoplasma, etc.); ④ After the resuscitated cells have grown to a certain number, select the well-grown cells for monoclonal counting and plate-laying, and test the antibody secretion ability of the monoclonal cells to see if they are all positive or have antibody secretion; ⑤ The cell culture supernatant should also be subjected to ELISA (OD). 450 >2.0), while performing Western blotting to determine whether positive antibodies are secreted, through... Figure 3 It can be seen that the monoclonal antibody secreted by the GAT monoclonal antibody hybridoma cell line 1C2 can specifically detect GAT protein in transgenic soybeans.

[0047] 5. Preparation of ascites

[0048] Mice were first injected intraperitoneally with phytidine or liquid paraffin. One week later, hybridoma cell line 1C2 was inoculated into the peritoneum of mice. After cell line establishment, the cells were expanded and cultured in 10% fetal bovine serum medium. When the cell density reached 1×10⁻⁶ cells / mL... 6 -2×10 6 Centrifuge at 800 rpm at a concentration of / mL, collect the precipitate, resuspend it in PBS, and inject it into mice via intraperitoneal injection (liquid paraffin). After 7-10 days, collect the ascites fluid for purification.

[0049] 6. Antibody purification

[0050] After pretreatment, the collected ascites fluid was purified using a Protein A-agarose affinity chromatography column. The specific steps are as follows:

[0051] 1) Buffer: The starting buffer is pH 7.0, 20mM phosphate buffer; the elution buffer is pH 2.7, 0.1mM glycine hydrochloride.

[0052] Prepare collection tubes: Take 1.5 mL centrifuge tubes and add 70 μL of pH 9.0 1M Tris-HCl to each centrifuge tube.

[0053] Sample preparation: The sample obtained by precipitation with 50% SAS was dialyzed overnight in the starting buffer and then filtered through a 0.22 μm microporous membrane.

[0054] Purification process: Equilibrate the Protein A-agarose affinity chromatography column (HiTrap Protein A 1mL, Pharmacia Biotech) with sufficient starting buffer (8-10mL). Load 15-25mL of the sample to be purified (containing 10.2-21.1mg of protein per mL) onto the column at a flow rate of 0.5mL / min. Then wash sequentially with 7-8mL of starting buffer, 6-7mL of elution buffer, and 5mL of starting buffer at the same flow rate, collecting 1mL of eluent from each tube.

[0055] Purity and activity determination: The purity of the purified monoclonal antibody (McAb) was determined by SDS-PAGE. See details. Figure 1 The monoclonal antibody from the hybridoma cell line 1C2 was purified to remove almost all impurities and showed two specific main bands (55kDa and 30kDa).

[0056] 7. Monoclonal antibody titer determination

[0057] Using recombinant His-GAT protein as the antigen, the titer of the purified monoclonal antibody was detected by indirect ELISA. Figure 2 It can be seen that, as determined by ELISA, the titer of the purified 1C2 monoclonal antibody is 1:1280000.

[0058] Table 2. Concentrations of monoclonal antibodies produced by hybridoma cell line 1C2

[0059] Monoclonal antibody hybridoma cell numbering Antibody (IgG) concentration 1C2 2.5 mg / mL

[0060] 8. Monoclonal antibody specificity detection

[0061] 8.1 Specific detection of GAT in genetically modified crop-derived samples

[0062] Endogenous proteins were extracted from GAT-transgenic soybeans and their transformed parents, and subjected to SDS-PAGE gel chromatography. For membrane transfer, purified monoclonal antibody (1C2) was used as the primary antibody, and Alexa Fluror™ 680 goat anti-mouse IgG (H+L) (Invitrogen) was used as the secondary antibody. Results were analyzed using an Odyssey infrared 740 imager (9120, Li-COR Biosciences, Lincolin, NE) infrared scanner. Figure 3 It can be seen that the purified 1C2 monoclonal antibody can specifically recognize GAT in endogenous samples.

[0063] The method for extracting genetically modified soybean protein is as follows:

[0064] The tissue was flash-frozen in liquid nitrogen, ground, and 1 mL (based on sample volume, generally 1-2 mL for 0.5 g) of protein extraction solution was added. The mixture was stirred at 4°C for 30 minutes, then centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected. The protein extraction solution formulation is shown in Table 3.

[0065] Table 3 Protein Extract Formulation

[0066] Element Dosage 1M Tris, pH 7.5 500 μL 1M NaCl 1.5 mL 0.5M EDTA 20 μL 50% glycerin 2 mL 10% SDS 1 mL <![CDATA[Double-distilled water (DDH2O)]]> Prepare 10mL Protease inhibitors (Roche) Add one piece when needed. 1mM PMSF (Benzyl sulfonyl fluoride, Sigma) Add 50μL when needed.

[0067] 8.2 Sensitivity Detection of GAT Monoclonal Antibody

[0068] GAT protein was gradient-loaded (5 ng / μl GAT protein, loading volumes in lanes 1-6 were 1, 2, 4, 8, 12, and 16 μl, respectively), and run on an SDS-PAGE gel. For membrane transfer, purified monoclonal antibody (1C2, 10 μg / ml) was used as the primary antibody, and goat anti-mouse IgG (H+L)-HRP (1:10000) as the secondary antibody. Western blotting results were detected using chemiluminescence immunoassay, with exposure to ultrasensitive chromogenic solution for 30 seconds. Figure 4 It can be seen that the sensitivity of the purified 1C2 monoclonal antibody WB is 5 ng.

[0069] 8.3 Specificity detection of GAT monoclonal antibodies

[0070] Besides GAT, other commonly used herbicide-resistant proteins in transgenic crops include PAT / pat, PAT / bar, CP4 EPSPS, G2 EPSPS, and G10 EPSPS. Western blotting was performed using monoclonal antibody 1C2 (10 μg / ml) to detect GAT, PAT / pat, PAT / bar, and G2 EPSPS (all concentrations were 10 ng / μl, and the loading volume was 10 μl). (SDS-PAGE gel was run; purified monoclonal antibody (1C2) was used as the primary antibody for transfer, and goat anti-mouse IgG (H+L)-HRP (1:10000) was used as the secondary antibody. Western blotting results were detected using chemiluminescence immunoassay with exposure to ultrasensitive chromogenic solution for 2 seconds). Results are shown below. Figure 5 The results showed that the monoclonal antibody produced by the purified 1C2 could specifically recognize GAT, but could not effectively recognize other herbicide-resistant proteins.

[0071] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46332.

2. The monoclonal antibody produced by the hybridoma cell line of claim 1, characterized in that, Hybridoma cell lines were inoculated into the peritoneal cavity of mice to prepare ascites fluid, and the collected ascites fluid was then purified by Protein A-agarose affinity chromatography to obtain monoclonal antibodies.

3. The monoclonal antibody according to claim 2, characterized in that, The monoclonal antibody produced by the hybridoma cell line with accession number CGMCC No. 46332 had a titer of 1:1280000 as detected by indirect ELISA.

4. The monoclonal antibody according to claim 2, characterized in that, The hybridoma cell line with accession number CGMCC No. 46332 produces monoclonal antibodies of type IgG2a.

5. The monoclonal antibody according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody produced by the hybridoma cell line with accession number CGMCC No. 46332 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

6. The use of the monoclonal antibody according to any one of claims 2-5 in the qualitative or quantitative detection of herbicide-resistant GAT protein.

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