Herbicide-tolerant protein hybridoma cell strain, antibodies produced thereby and uses thereof

Abstract

The application discloses a herbicide-resistant protein GAT hybridoma cell strain, an antibody produced by the hybridoma cell strain and application of the hybridoma cell strain, and the hybridoma cell strain is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No.46333.A preparation method of the hybridoma cell strain comprises the following steps: a) purifying a GAT recombinant protein obtained by prokaryotic expression; b) immunizing animals: using the GAT recombinant protein as an antigen to immunize BALB / c mice; c) cell fusion: collecting spleen cells of the immunized BALB / c mice and fusing the spleen cells with SP2 / 0 cells; d) cell strain establishment: performing subcloning by a limited dilution method, performing ELISA detection on the subcloning for 5-7 days, and screening the hybridoma cell strain stably secreting positive antibodies until the hybridoma cell strain is expanded, cultured and preserved again.The monoclonal antibody secreted by the hybridoma cell strain lays a foundation for realizing detection of the herbicide-resistant protein GAT in a transgenic crop, thereby supporting the development, seed production, seed preservation and guarantee of the transgenic crop, and stably promoting industrialization of the transgenic crop and sustainable application of related transformants. gat The application discloses a herbicide-resistant protein GAT hybridoma cell strain, an antibody produced by the hybridoma cell strain and application of the hybridoma cell strain, and the hybridoma cell strain is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No.46333.A preparation method of the hybridoma cell strain comprises the following steps: a) purifying a GAT recombinant protein obtained by prokaryotic expression; b) immunizing animals: using the GAT recombinant protein as an antigen to immunize BALB / c mice; c) cell fusion: collecting spleen cells of the immunized BALB / c mice and fusing the spleen cells with SP2 / 0 cells; d) cell strain establishment: performing subcloning by a limited dilution method, performing ELISA detection on the subcloning for 5-7 days, and screening the hybridoma cell strain stably secreting positive antibodies until the hybridoma cell strain is expanded, cultured and preserved again.The monoclonal antibody secreted by the hybridoma cell strain lays a foundation for realizing detection of the herbicide-resistant protein GAT in a transgenic crop, thereby supporting the development, seed production, seed preservation and guarantee of the transgenic crop, and stably promoting industrialization of the transgenic crop and sustainable application of related transformants. gat The application discloses a herbicide-resistant protein GAT hybridoma cell

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 1D5 and has been deposited in the General Microbiology Center of China Culture Collection Administration on March 14, 2025, with the deposit number CGMCC No. 46333.

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

[0007] a) Purifying the GAT recombinant protein obtained by prokaryotic expression;

[0008] b) Immunization of animals: BALB / c mice were immunized with GAT recombinant 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 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.

[0011] Among them, the fusion ratio of mouse spleen cells and SP2 / 0 cells is 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 a mouse to prepare ascites, which is then purified using a Protein A-agarose affinity chromatography column to obtain the monoclonal antibody.

[0013] Specifically, the titer of the monoclonal antibody produced by the hybridoma cell line 1D5 was 1:5118000 as determined by indirect ELISA, and the type of the antibody was IgG1.

[0014] The amino acid sequence of the variable region heavy chain of the monoclonal antibody produced by hybridoma cell line 1D5 is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the variable region light chain is shown in SEQ ID NO: 2 in the sequence listing. The specific sequences are:

[0015] cell lines Heavy chain variable region amino acid sequence Light chain variable region amino acid sequence 1D5 RIQLVQSGPELKKPGETVKISCKTSGYIFINYGMNWVKQAPGEGLKWMGWLNTNTGQPTYAEDFKGRFVFSLETSANTAYLQINNLKTEDTATYFCTRDHFVGDYFDYWGQGTLLTVSS DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYNCQQSKEDPYTFGGGTKLEIKR

[0016] The present invention also provides the use of the monoclonal antibody in the detection of the herbicide-resistant protein GAT.

[0017] 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 1D5 that secretes a specific and sensitive monoclonal antibody against GAT through hybridoma technology. The antibody secreted by the cell line and purified from ascites has an indirect ELISA titer of 1:5118000, and the antibody subtype is IgG1. 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.

[0018] Collection information:

[0019] The hybridoma cell line 1D5 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. 46333. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 FIG1 is a graph showing the SDS-PAGE electrophoresis results of the monoclonal antibody purified from the hybridoma cell line 1D5 according to the present invention;

[0021] Figure 2 is the titer of the monoclonal antibody produced by the hybridoma cell line 1D5 according to the present invention;

[0022] Figure 3 This is a Western blot result of the monoclonal antibody produced by the hybridoma cell line 1D5 according to the present invention specifically detecting GAT in transgenic soybeans, wherein: lane 1 is a protein extract of GAT-negative material; lane 2 is a protein extract of GAT-positive material, and the primary antibody for western detection is 1D5;

[0023] Figure 4 1D5 antibody 10 μg / ml; Lane 1: 5 ng of GAT protein, 10 μg / ml of 1D5 antibody; Lane 2: 10 ng of GAT protein, 10 μg / ml of 1D5 antibody; Lane 3: 20 ng of GAT protein, 10 μg / ml of 1D5 antibody; Lane 4: 40 ng of GAT protein, 10 μg / ml of 1D5 antibody; Lane 5: 60 ng of GAT protein, 10 μg / ml of 1D5 antibody; Lane 6: 80 ng of GAT protein, 10 μg / ml of 1D5 antibody;

[0024] Figure 5 The figures are the specificity detection results of the monoclonal antibodies produced by the hybridoma cell line 1D5 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, 1D5 antibody 10 μg / ml, lane 2 is Pat / pat protein 100 ng, 1D5 antibody 10 μg / ml, lane 3 is Pat / bar protein 100 ng, 1D5 antibody 10 μg / ml, lane 4 is G2 EPSPS protein 100 ng, 1D5 antibody 10 μg / ml. DETAILED DESCRIPTION

[0025] The specific embodiments of the present application are described in detail below with reference to the accompanying drawings, but the scope of protection of the present application is not limited by the specific embodiments.

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0027] The materials, reagents, etc. used in the following examples can be commercially available unless otherwise specified.

[0028] Example 1: Hybridoma cell acquisition and preparation of monoclonal antibodies

[0029] 1. Preparation of immunizing antigen

[0030] The gat gene was amplified from the transgenic soybean genome DNA 6106, and the expression vector pET28a-gat was constructed. The competent cells of Escherichia coli BL21 were transformed, activated with seeds, expanded and cultured, and induced to express at 16℃ overnight with IPTG. The relatively pure His-GAT protein was obtained by Ni affinity chromatography column and gel filtration purification.

[0031] 2. Immunization of animals

[0032] His-GAT protein was used as antigen to immunize 8 8-week-old SPF BALB / c female mice (purchased from Hubei Province Experimental Animal Research Center, license number: SCXK (E) 2015-0018). The antigen was mixed with an equal volume of complete Freund's adjuvant (first immunization) or incomplete Freund's adjuvant (boosting immunization) and emulsified, and then subcutaneously immunized in multiple points after mixing thoroughly to the water-in-oil state. 2-3 times of boosting immunization, each immunization interval 2 weeks, then detection of titer, higher than 1:10000 within 1 week intraperitoneal shock, directly dissolve the immunization dose of antigen in 250 μL of PBS. The specific immunization program and immunization dose are shown in Table 1.

[0033] Table 1 Immunization program and immunization dose

[0034]

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

[0036] 3. Cell fusion

[0037] Three days after the last shock, positive control blood was collected, spleens were taken, and single-cell suspensions were prepared. SP2 / 0 cells in the logarithmic phase were treated and mixed with spleen cells at a certain ratio (1:5-1:10), and 50% PEG 1450 was used for 1 min. The suspension was terminated by dilution with basal medium DMEM, centrifuged at low speed, and then gently suspended and mixed with HAT medium containing 20% ​​fetal bovine serum. The suspension was then incubated at 2×10 7 / Plate onto a pre-prepared feeder cell plate and culture at 37°C with 5% CO2.

[0038] 4. Cell Line Establishment

[0039] 1) Fusion board detection:

[0040] When the cells in the fusion plate have grown to a medium size of about 10,000 or more, the test should begin. If the ELISA quality control is qualified (i.e., the negative control OD 450 <0.2, positive control OD 450 >1.0) and then select the positive wells (generally OD 450 ≥0.5) for subcloning.

[0041] 2) Subcloning methods and detection:

[0042] 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.

[0043] 3) Cell line establishment:

[0044] 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 1D5 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 OD 450 The cell line with a p<0.05 was cultured in a cell flask and frozen, namely the hybridoma cell line 1D5, 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. 46333.

[0045] 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:

[0046] 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 1D5 can specifically detect the GAT protein in transgenic soybeans.

[0047] 5. Preparation of Ascites

[0048] First, pristane or liquid paraffin was injected into the mouse peritoneal cavity. One week later, the hybridoma cell line 1D5 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 6 When the concentration of the protein was 100 μg / mL, the pellet was collected by centrifugation at 800 rpm, resuspended in PBS, and injected into the peritoneal cavity of mice (liquid paraffin). After 7-10 days, the ascites was collected for purification.

[0049] 6. Antibody Purification

[0050] The collected ascites was pretreated and then purified using a Protein A-agarose affinity chromatography column. The specific steps are as follows:

[0051] 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.

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

[0053] 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.

[0054] Purification: Equilibrate the Protein A-Sepharose affinity column (HiTrap Protein A 1 mL, Pharmacia Biotech) with enough start buffer (8-10 mL). Load the sample (10.2-21.1 mg protein / mL sample) 15-25 mL on the column at a flow rate of 0.5 mL / min, then wash with start buffer 7-8 mL, elution buffer 6-7 mL, and start buffer 5 mL at the same flow rate. Collect the eluate 1 mL per tube.

[0055] Purity and activity identification: The purified McAb was identified by SDS-PAGE for its purity, as described in detail in Figure 1 , and the hybridoma cell strain 1D5 monoclonal antibody was almost all impurities removed after purification, with two specific main bands (55 kDa and 30 kDa).

[0056] 7. Determination of the titer of the monoclonal antibody

[0057] The titer of the purified monoclonal antibody was determined by indirect ELISA using recombinant His-GAT protein as antigen, and it was found that the titer of the purified 1D5 monoclonal antibody was 1:5118000 by ELISA. Figure 2

[0058] Table 2 Concentration of the monoclonal antibody produced by the hybridoma cell strain 1D5

[0059] Monoclonal antibody hybridoma cell number Antibody (IgG) concentration 1D5 5.0 mg / mL

[0060] 8. Specific detection of the monoclonal antibody

[0061] 8.1 Specific detection of GAT in samples from transgenic crops

[0062] Endogenous proteins were extracted from transgenic GAT soybeans and their transformation parents, and SDS-PAGE gels were run, and the purified monoclonal antibody (1D5) was used as the primary antibody, and Alexa FlurorTM 680 goat anti-mouse IgG (H+L) (Invitrogen) was used as the secondary antibody, and the western detection results were scanned by an Odyssey infrared 740 imager (9120, Li-COR Biosciences, Lincolin, NE), and it was found that the purified 1D5 monoclonal antibody could specifically recognize GAT in endogenous samples. Figure 3

[0063] ​​Among them, the genetically modified soybean protein extraction method:

[0064] 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:

[0065] Table 3 Protein extract formula

[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[双蒸水(DDH2O)]]> Prepare 10mL Protease inhibitors (Roche) Add one piece when using 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma) Add 50 μL when using

[0067] 8.2 GAT monoclonal antibody sensitivity test

[0068] 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 (1D5, 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 1D5 monoclonal antibody WB is 5 ng.

[0069] 8.3 GAT monoclonal antibody specificity detection

[0070] 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 1D5 (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 (1D5) 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 Figure 5 The results showed that the monoclonal antibody produced by the purified 1D5 could specifically recognize GAT, but could not effectively recognize other herbicide-resistant proteins.

[0071] 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 is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No.46333.

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. 46333 was 1:5118000 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. 46333 is IgG1.

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. 46333 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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