Hybridoma cell strain, antibody generated by hybridoma cell strain and application of antibody

Abstract

The invention discloses a herbicide-resistant protein GAT hybridoma cell strain, an antibody generated by the herbicide-resistant protein GAT hybridoma cell strain and application of the herbicide-resistant protein GAT hybridoma cell strain and the antibody. The herbicide-resistant protein GAT hybridoma cell strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.46332. The preparation method comprises the following steps: a) purifying prokaryotic expression to obtain GAT recombinant protein; b) immunizing animals: immunizing BALB / c mice by taking the GAT recombinant protein as an antigen; c) cell fusion: splenocytes of the immune BALB / c mice are collected and fused with SP2 / 0 cells; and d) cell strain establishment: carrying out subcloning through a limited dilution method, carrying out ELISA detection after 5-7 days of subcloning until a hybridoma cell strain capable of stably secreting a positive antibody is screened out, and carrying out amplification, re-culture and storage. The monoclonal antibody secreted by the hybridoma cell strain lays a foundation for detection of herbicide-resistant protein GAT in transgenic crops, so that development, seed production and seed conservation of transgenic gat crops are supported, and stable industrial promotion of the transgenic gat crops and sustainable application of related transformants are guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a herbicide-resistant protein GAT hybridoma cell line and an antibody produced therefrom and applications thereof. Background Art

[0002] Bacillus ( Bacillus )middle gat The glyphosate acetyltransferase expressed by this gene (glyphosate-degrading gene) confers herbicide tolerance to transgenic crops. The cultivation of herbicide-tolerant crops has made herbicide spraying an effective weed control method, making herbicide tolerance one of the most advantageous traits in agricultural production. Currently, herbicide-tolerant crops account for over 80% of the global genetically modified crop population. The proportion of genetically modified crops continues to increase.

[0003] The development and advancement of transgenic technology has driven the advancement of biology. While genetically modified foods can meet consumer expectations for yield, insect resistance, and herbicide tolerance, they also pose potential threats to human health. These include the potential for certain genes to become toxic after introduction into the host; the potential for allergens and drug resistance in genetically modified foods; and altered nutritional value. During the research, development, and commercialization of genetically modified foods, comprehensive safety assessments are crucial to enable consumers to quickly distinguish genetically modified foods from natural foods. Establishing appropriate methods for identifying and detecting genetically modified ingredients in genetically modified foods can facilitate the safety management of agricultural genetically modified organisms, ensuring the safety of humans, animals, and microorganisms, while also protecting the ecological environment and promoting further research in agricultural genetically modified biotechnology. Therefore, developing and obtaining monoclonal antibodies against GAT proteins is crucial for rapidly analyzing herbicide-tolerant GAT proteins in genetically modified crops and their derivatives. Summary of the Invention

[0004] The purpose of the present invention is to provide a herbicide-resistant protein GAT hybridoma cell line and the antibodies produced and applications thereof. The secreted monoclonal antibody lays the foundation for the detection of the herbicide-resistant protein GAT in transgenic crops.

[0005] To achieve the above-mentioned purpose, the present invention provides a hybridoma cell line, which is 1C2 and has been deposited in the General Microbiology Center of China Culture Collection Administration on March 14, 2025, with the deposit number CGMCC No. 46332.

[0006] Specifically, the method for preparing the hybridoma cell line comprises the following steps: a) Purifying the GAT recombinant protein obtained by prokaryotic expression; b) Immunization of animals: BALB / c mice were immunized with GAT recombinant protein as an antigen; c) Cell fusion: spleen cells from immunized BALB / c mice were collected and fused with SP2 / 0 cells; d) Cell line establishment: Subcloning is performed by limiting dilution method. ELISA testing is performed 5-7 days after subcloning until a hybridoma cell line that stably secretes positive antibodies is screened for expansion, re-cultivation, and preservation.

[0007] Among them, the fusion ratio of mouse spleen cells and SP2 / 0 cells is 1:5-1:10.

[0008] 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 a mouse to prepare ascites, which is then purified using a Protein A-agarose affinity chromatography column to obtain the monoclonal antibody.

[0009] Specifically, the titer of the monoclonal antibody produced by the hybridoma cell line 1C2 was 1:1280000 as determined by indirect ELISA, and the type of the antibody was IgG2a. The amino acid sequence of the heavy chain variable region of the monoclonal antibody produced by hybridoma cell line 1C2 is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2 in the sequence listing. The specific sequences are: cell lines Heavy chain variable region amino acid sequence Light chain variable region amino acid sequence 1C2 EVQLQESGPSLVKPYQTLSLTCSVTGDSSTSDYWNWIRKFPGNKFEYMGYISYSGTTYYNPSLKSRISITRDTSKNQSYLQLKSVTTEDTGTYYCANVRMNYWGLGTSVTVSS DIVITQTPSSLAMSVGQKVTMSCKSSQSLLNSRDQKNYLAWYQQKPGQSPKLLIYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADFFCQQHFRLPLTFGSGTRLEIK The present invention also provides the use of the monoclonal antibody in the detection of the herbicide-resistant protein GAT.

[0010] Compared with the prior art, the present invention has the following beneficial effects: the present 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 ascites purification 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 the GAT protein in transgenic soybeans. The construction of a hybridoma cell line that secretes a mouse monoclonal antibody against the GAT herbicide-resistant protein provides material and technical support for the detection of this protein in transgenic crops.

[0011] Collection information: The hybridoma cell line 1C2 provided by the present invention was deposited with the General Microbiology Center of the China Culture Collection Administration on March 14, 2025, and is classified as GAT monoclonal antibody hybridoma cell line. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Postal Code: 100101, and the deposit number is CGMCC No. 46332. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 FIG1 is a graph showing the SDS-PAGE electrophoresis results of the monoclonal antibody purified from the hybridoma cell line 1C2 according to the present invention; Figure 2 is the titer of the monoclonal antibody produced by the hybridoma cell line 1C2 according to the present invention; Figure 3 This is a Western blot result of the monoclonal antibody produced by the hybridoma cell line 1C2 according to the present invention specifically detecting GAT in transgenic soybeans, wherein: lane 1 is a protein extract of GAT-positive material; lane 2 is a protein extract of GAT-negative material; the primary antibody for western detection is 1C2; Figure 4 1C2 antibody 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; lane 6 is 80 ng of GAT protein and 10 μg / ml of 1C2 antibody; Figure 5 The figures are the specificity detection results of the monoclonal antibodies produced by the hybridoma cell line 1C2 according to the present invention, wherein: the left figure is a staining diagram, lane 1 is GAT protein, lane 2 is Pat / pat protein, lane 3 is Pat / bar protein, and lane 4 is G2 EPSPS protein; the right figure is a Western result diagram, lane 1 is GAT protein 100 ng, 1C2 antibody 10 μg / ml, lane 2 is Pat / pat protein 100 ng, 1C2 antibody 10 μg / ml, lane 3 is Pat / bar protein 100 ng, 1C2 antibody 10 μg / ml, lane 4 is G2 EPSPS protein 100 ng, 1C2 antibody 10 μg / ml. DETAILED DESCRIPTION

[0013] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0014] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0015] Unless otherwise specified, the materials and reagents used in the following examples can be purchased from commercial sources.

[0016] Example 1: Obtaining Hybridoma Cells and Preparing Monoclonal Antibodies 1. Preparation of Immunizing Antigen 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 competent Escherichia coli BL21 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.

[0017] 2. Immunizing Animals 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 the 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充分混合至油包水状态后进行皮下多点免疫,2-3 次加强免疫,每次免疫间隔周期 2 周,之后进行效价检测,高于>1:10000 后 1 周内进行腹腔冲击,直接将免疫剂量的抗原溶于250μL的 PBS 中。具体免疫程序及免疫剂量如表1所示。

[0018] Table 1 Immunization Procedures and Doses 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.

[0019] 3. Cell Fusion Three days after the last boost, the positive control blood was collected, the spleen was taken, and a single-cell suspension was prepared. After treating the SP2 / 0 cells in the logarithmic growth phase, they were mixed with spleen cells at a certain ratio (1:5 - 1:10), and 50% PEG 1450 was allowed to act for 1 min, terminated with dilution in the basic medium DMEM, centrifuged at low speed, and then gently suspended and mixed with HAT medium containing 20% fetal bovine serum. It was plated onto a pre-prepared feeder cell plate at 2×10 7 / plate and cultured at 5% CO2 and 37°C.

[0020] 4. Cell Line Establishment 1) Detection of Fusion Plate Detection was started when the cells in the fusion plate grew to medium size, about more than 10,000 cells. After the ELISA quality control was qualified (i.e., the OD of the negative control 450 <0.2, and the OD of the positive control 450 >1.0), positive wells (generally OD 450 ≥0.5) were selected for subcloning.

[0021] 2) Subcloning methods and detection: Pick out the fusion plate with high positive value (OD 450 >2.0) for limiting dilution, and count 60% of the monoclonal wells on each plate for subcloning. Each time, the monoclonal wells with higher positive values are picked for limiting dilution. ELISA testing can be performed 5-7 days after each subcloning until a monoclonal cell line that can stably secrete positive antibodies is finally screened for expanded culture.

[0022] 3) Cell line establishment: The cell lines that stably secreted positive antibodies screened in the subcloning stage were expanded and cultured in 24-well plates. After expansion, the supernatant was collected for antigen detection. ELISA gradient dilution and western-blotting were used to verify its stability. The monoclonal antibody secreted by the GAT monoclonal antibody hybridoma cell line 1C2 could specifically detect the GAT protein in the transgenic soybean sample. The cells were collected and expanded in 10 cm culture dishes, and the supernatant was collected again and the antibody titer was detected. The cells with OD 450 The cell line with a p<0.05 was cultured in a cell flask and frozen, namely the hybridoma cell line 1C2, which was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) on March 14, 2025, with the deposit number CGMCC No. 46332.

[0023] For identification of frozen cell lines, one cell line from the same batch must be revived for identification after the cell line is frozen. The identification standards are as follows: ① The number of revived living cells is ≥ 1 million cells / strain; ② The number of viable cells in the living cells is ≥ 500,000 / strain; ③ No other microorganisms (such as bacteria, fungi, mycoplasma, etc.) other than cell line cells are present in the revived cells; ④ After the revived cells grow to a certain number, the well-grown cells are selected for monoclonal counting and plated, and the monoclonal antibody secretion ability is tested to see whether it is positive or has antibody secretion; ⑤ The cell culture supernatant is also required to be subjected to ELISA (OD 450 >2.0) to determine whether positive antibodies are secreted and to perform western-blotting identification. Figure 3 It can be seen that the monoclonal antibody secreted by the GAT monoclonal antibody hybridoma cell line 1C2 can specifically detect the GAT protein in transgenic soybeans.

[0024] 5. Preparation of Ascites First, pristane or liquid paraffin was injected into the mouse peritoneal cavity. One week later, the hybridoma cell line 1C2 was inoculated into the mouse peritoneal cavity. After the cells were established, 10% fetal bovine serum medium was used for expansion culture. When the cell density reached 1×10 6 -2×10 6When the concentration of the protein was 100 μg / mL, the solution was centrifuged at 800 rpm, the precipitate was collected, resuspended in PBS, and injected into the peritoneal cavity of mice (liquid paraffin). After 7-10 days, the ascites was collected for purification.

[0025] 6. Antibody Purification The collected ascites was pretreated and then purified using a Protein A-agarose affinity chromatography column. The specific steps are as follows: 1) Buffer: The starting buffer is 20 mM phosphate buffer, pH 7.0; the elution buffer is 0.1 mM glycine-HCl, pH 2.7.

[0026] Prepare collection tubes: Take 1.5mL centrifuge tubes and add 70μL pH 9.0 1M Tris-HCl to each centrifuge tube.

[0027] Sample preparation: The sample obtained by 50% SAS precipitation was dialyzed overnight against the starting buffer and filtered through a 0.22 μm microporous membrane.

[0028] Purification: Equilibrate a 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 (10.2-21.1mg of protein per mL) onto the column at a flow rate of 0.5mL / min. Then, wash the column sequentially with 7-8mL of starting buffer, 6-7mL of elution buffer, and 5mL of starting buffer at the same flow rate. Collect 1mL of the eluate into each tube.

[0029] Purity and activity identification: The purity of the purified monoclonal antibody (McAb) was identified by SDS-PAGE. Figure 1 The monoclonal antibody of hybridoma cell line 1C2 was purified to remove almost all foreign proteins and had two specific main bands (55kDa and 30kDa).

[0030] 7. Determination of Monoclonal Antibody Titer The recombinant His-GAT protein was used as the antigen and the titer of the purified monoclonal antibody was detected by indirect ELISA. Figure 2 It can be seen that the titer of the purified 1C2 monoclonal antibody was 1:1280000 as determined by ELISA.

[0031] Table 2 Concentrations of monoclonal antibodies produced by hybridoma cell line 1C2 Monoclonal antibody hybridoma cell number Antibody (IgG) concentration 1C2 2.5 mg / mL 8. Monoclonal Antibody Specificity Detection 8.1 Specific Detection of GAT in Samples Derived from GM Crops Endogenous proteins of GAT-transgenic soybean and its transformed parent were extracted and run on SDS-PAGE gel. The membrane was transferred using purified monoclonal antibody (1C2) as the primary antibody and Alexa FluorTM 680 goat anti-mouse IgG (H+L) (Invitrogen) as the secondary antibody. Western blot was performed using an Odyssey infrared740 imager (9120, Li-COR Biosciences, Lincolin, NE). Figure 3 It can be seen that the purified 1C2 monoclonal antibody can specifically recognize GAT in endogenous samples.

[0032] Among them, the genetically modified soybean protein extraction method: Quickly freeze the tissue in liquid nitrogen, grind it, add 1 mL (depending on the sample size, generally 1-2 mL for 0.5 g) of protein extract, mix at 4°C for 30 minutes, centrifuge at 12,000 rpm at 4°C for 15 minutes, and collect the supernatant. The protein extract formula is shown in Table 3: Table 3 Protein extract formula 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 using 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma) Add 50 μL 8.2 GAT monoclonal antibody sensitivity test GAT protein gradient was loaded (the loading amount of lanes 1-6 of 5 ng / μl GAT protein was 1, 2, 4, 8, 12, and 16 μl, respectively), and SDS-PAGE gel was run. The membrane was transferred using purified monoclonal antibody (1C2, 10 μg / ml) as the primary antibody and goat anti-mouse IgG (H+L)-HRP (1:10000) as the secondary antibody. The western blotting results were detected by chemiluminescence, and the ultrasensitive color solution was exposed for 30 s. Figure 4 It can be seen that the sensitivity of the purified 1C2 monoclonal antibody WB is 5 ng.

[0033] 8.3 GAT monoclonal antibody specificity detection In addition to GAT, other commonly used herbicide-resistant proteins in transgenic crops include PAT / pat, PAT / bar, CP4 EPSPS, G2EPSPS, and G10 EPSPS. Monoclonal antibody 1C2 (10 μg / ml) was used for Western detection of GAT, PAT / pat, PAT / bar, and G2EPSPS (all concentrations were 10 ng / μl, and the sample volume was 10 μl) (SDS-PAGE gel was run, and the membrane was transferred using purified monoclonal antibody (1C2) as the primary antibody, goat anti-mouse IgG (H+L)-HRP (1:10000) as the secondary antibody, and the western blotting results were detected by chemiluminescence, with the ultrasensitive color development solution exposed for 2 seconds). The detection results are shown in the table. 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.

[0034] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. 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 was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number of CGMCC No.46332.

2. The monoclonal antibody produced by the hybridoma cell line according to claim 1, characterized in that The hybridoma cell line is inoculated into the peritoneal cavity of mice to prepare ascites, and the collected ascites is then purified using a Protein A-agarose affinity chromatography column to obtain monoclonal antibodies.

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

4. The monoclonal antibody according to claim 2, characterized in that The type of monoclonal antibody produced by the hybridoma cell line with the deposit number CGMCC No. 46332 is 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 the deposit 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. Use of the monoclonal antibody according to any one of claims 2 to 5 in the qualitative or quantitative detection of herbicide-resistant GAT protein.

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