Insect-resistant protein cry 1f hybridoma cell strain, antibodies produced thereby and uses thereof

Abstract

The application discloses Cry 1F hybridoma cell strains 1D7 1C6 and 1F7 1D8 and antibodies and applications generated by the hybridoma cell strains 1D7 1C6 and 1F7 1D8, which have been preserved in the China General Microbiological Culture Collection Center on December 10, 2021, with preservation numbers of CGMCC No. 45008 and 45009, and a preparation method thereof comprises the following steps: a) purifying Cry 1F protein in transgenic corn seeds; b) immunizing BALB / c mice with the Cry 1F protein as an antigen; c) collecting spleen cells of the immunized BALB / c mice and fusing the spleen cells with SP2 / 0 cells; d) subcloning through a limited dilution method, and performing ELISA detection on the subcloning for 5-7 days until a hybridoma cell strain stably secreting positive antibodies is screened out for expansion and culture and preservation, and the monoclonal antibodies secreted by the hybridoma cell strain lay a foundation for realizing detection in an anti-insect protein Cry 1F transgenic crop.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to the insect-resistant protein Cry 1F hybridoma cell lines 1D7 1C6 and 1F7 1D8, the antibodies produced from them, and their applications. Background Technology

[0002] Bacillus thuringiensis (Bt) is a widespread Gram-positive bacterium. The insect-resistant crystal proteins it secretes are currently the main biological pesticides. These secreted insect-resistant proteins are divided into two categories based on amino acid sequence similarity: Cry and Cryδ-endotoxin. Cry proteins are toxic to the larvae of various harmful insects (such as Lepidoptera, Diptera, Coleoptera, nematodes, and protozoa). Cry toxins have been transferred into crops to give them insect resistance. Cry 1F protein is one type of Cry toxin. Currently, crops mainly transgenic with the Cry gene include corn, potatoes, rice, and cotton.

[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 and insect resistance, 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, lead to antibiotic resistance, and alter the nutritional value of food. 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 GM organisms, protect the safety of humans, animals, and microorganisms, protect the ecological environment, and further advance research in agricultural GM biotechnology. Furthermore, the rapid analysis of Cry 1F protein in GM crops and their derivatives, and the development of monoclonal antibodies against Cry 1F protein, are of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide two hybridoma cell lines, 1D7 1C6 and 1F7 1D8, along with the antibodies they produce and their preparation methods. The monoclonal antibodies secreted by these cells lay the foundation for the detection of the insect-resistant protein Cry 1F in transgenic crops.

[0005] To achieve the above objectives, the present invention provides hybridoma cell lines, including hybridoma cell lines 1D7 1C6 and 1F7 1D8, which were deposited on December 10, 2021, at the China General Microbiological Culture Collection Center with bioaccession numbers CGMCC No. 45008 and CGMCC No. 45009.

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

[0007] a) Purify Cry 1F protein from transgenic maize seeds;

[0008] b) Immunization of animals: BALB / c mice were immunized with Cry 1F 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 antibodies produced by hybridoma cell lines 1D7 1C6 and 1F7 1D8 had titers of 1:6140000 and 1:2050000, respectively, as determined by indirect ELISA, and the antibody types were IgG1 and IgG2b, respectively.

[0014] The heavy chain amino acid sequence of the variable region of the monoclonal antibody produced by hybridoma cell line 1D7 1C6 is shown in SEQ ID NO: 1 of the sequence listing, and the light chain amino acid sequence is shown in SEQ ID NO: 2 of the sequence listing; the heavy chain amino acid sequence of the variable region of the monoclonal antibody produced by hybridoma cell line 1F7 1D8 is shown in SEQ ID NO: 3 of the sequence listing, and the light chain amino acid sequence is shown in SEQ ID NO: 4 of the sequence listing. The specific sequences are as follows:

[0015]

[0016] This invention also provides the application of the above-mentioned monoclonal antibody in the qualitative and quantitative detection of Cry 1F insect-resistant protein.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This application uses the insect-resistant protein Cry 1F purified from transgenic maize seeds as an antigen, and prepares two hybridoma cell lines 1D7 1C6 and 1F7 1D8 that secrete specific and sensitive monoclonal antibodies against Cry 1F through hybridoma technology. The indirect ELISA titers of the antibodies obtained after purification of the ascites fluid secreted by the cell lines are 1:6140000 and 1:2050000, respectively. The monoclonal antibodies can specifically recognize Cry 1F protein in transgenic maize. The construction of mouse monoclonal antibody hybridoma cell lines that secrete Cry 1F insect-resistant protein provides material and technical support for the detection of this protein in transgenic crops.

[0018] Preservation Information

[0019] The hybridoma cell lines 1D7 1C6 and 1F7 1D8 provided by this invention were deposited on December 10, 2021, at the China General Microbiological Culture Collection Center (CGMCC), classified as Cry1Fa monoclonal antibody hybridoma cell lines. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession numbers CGMCC No. 45008 and CGMCC No. 45009, respectively. Attached Figure Description

[0020] Figure 1 This is an SDS-PAGE electrophoresis result of monoclonal antibodies purified from hybridoma cell lines 1D7 1C6 and 1F7 1D8 according to the present invention.

[0021] Figure 2 The titer of monoclonal antibodies produced by the hybridoma cell lines 1D7 1C6 and 1F7 1D8 according to the present invention;

[0022] Figure 3 The image shows the Western blotting results of the specific detection of Cry 1F in transgenic maize by monoclonal antibodies produced by the hybridoma cell lines 1D7 1C6 and 1F7 1D8 according to the present invention. Detailed Implementation

[0023] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] Example 1: Acquisition of hybridoma cells and preparation of their monoclonal antibodies

[0027] 1. Preparation of immunizing antigen

[0028] Approximately 1 kg of transgenic corn seeds were fully ground in liquid nitrogen, then dissolved in a protein extraction solution. Through multiple purification steps including high-speed refrigerated centrifugation, dissolution of the precipitate with a denaturing solution, centrifugal separation, ion exchange chromatography, gel filtration and renaturation, a relatively pure Cry 1F protein was obtained.

[0029] 2. Immunization of animals

[0030] Eight 8-week-old SPF-grade female BALB / c mice (purchased from Hubei Provincial Laboratory Animal Research Center, License No.: SCXK(E)2015-0018) were immunized with the Cry 1F protein as the antigen. The antigen was mixed with an equal volume of complete Freund's adjuvant (for primary immunization) or incomplete Freund's adjuvant (for booster immunization) and emulsified, fully mixed to form a water-in-oil state for multi-point subcutaneous immunization. 2 to 3 booster immunizations were performed at an interval of 2 weeks each. After that, titer detection was carried out, and intraperitoneal challenge was performed within 1 week after the titer was higher than 1:10000. The immunizing dose of antigen was directly dissolved in 250 μL of PBS. The specific number of immunizations and immunizing doses are shown in Table 1:

[0031] Table 1 Number of immunizations and immunizing doses

[0032]

[0033] Immunization example: In the primary immunization, 50 μg of antigen is dissolved in PBS, and then mixed with the adjuvant at a volume ratio of 1:1.

[0034] 3. Cell fusion

[0035] Three days after the final challenge, positive control blood was collected, the spleen was taken to prepare a single-cell suspension. After the SP2 / 0 cells in the logarithmic phase were treated, they were mixed with the spleen cells at a certain ratio (1:5 to 1:10), treated with 50% PEG1450 for 1 minute, then diluted with the basal medium DMEM to terminate the reaction. After low-speed centrifugation, the cells were gently resuspended and mixed well with HAT medium containing 20% fetal bovine serum, at a density of 2×10 7 / well, the cell suspension was plated onto pre-prepared feeder layer cell plates, and cultured at 37°C in 5% CO2.

[0036] 4. Cell line establishment

[0037] 1) Fusion plate detection:

[0038] Detection was started when the cells on the fusion plate after medium exchange grew to a medium size of about 10,000 cells or more. After the ELISA quality control was qualified (i.e., the negative control OD 450 <0.2, and the positive control OD 450 >1.0), positive wells (generally OD450 Subcloning (≥0.5) is performed.

[0039] 2) Subcloning methods and detection:

[0040] 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 / dL. 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.

[0041] 3) Cell line establishment:

[0042] Stable cell lines secreting positive antibodies, selected during the subcloning stage, were expanded into 24-well plates. After expansion, the supernatant was collected for antigen detection. Stability was verified using serially diluted ELISA and Western blotting. The monoclonal antibodies secreted by the Cry 1F monoclonal antibody hybridoma cell lines 1D7 1C6 and 1F7 1D8 specifically detected Cry 1F protein in transgenic maize samples. Cells were collected and expanded into 10cm culture dishes, and the supernatant was collected again to detect antibody titers. Cells with high OD were selected. 450 Two cell lines with a growth rate of >2.0 were cultured in cell culture flasks and cryopreserved. These were hybridoma cell lines 1D7 1C6 and 1F7 1D8, which were deposited on December 10, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 45008 and CGMCC No. 45009, respectively.

[0043] 4) Cell line cryopreservation identification: After the cell lines have been cryopreserved, one line from the same batch must be thawed for identification. Identification criteria:

[0044] ① The number of revived 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 revived cells should not contain any microorganisms other than the cell line itself (e.g., bacteria, fungi, mycoplasma, etc.); ④ After the revived 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), to determine whether positive antibodies are secreted while performing Western blotting identification, through Figure 3 It can be seen that the monoclonal antibodies secreted by the Cry 1F monoclonal antibody hybridoma cell lines 1D7 1C6 and 1F71D8 can specifically detect the Cry 1F protein in transgenic maize.

[0045] 5. Preparation of ascites

[0046] Mice were first injected intraperitoneally with phytidine or liquid paraffin. One week later, hybridoma cell lines 1D7 1C6 and 1F7 1D8 were inoculated into the peritoneum of mice, respectively. After cell line establishment, the cells were expanded in 10% fetal bovine serum medium. When the cell density reached 1×10⁻⁶, the cells were cultured at a concentration of 1×10⁻⁶. 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.

[0047] 6. Antibody purification

[0048] After pretreatment, the collected ascites fluid was purified using a Protein A-agarose affinity chromatography column. The specific steps are as follows:

[0049] 1) Buffer: The starting buffer is pH 7.0, 20mM phosphate buffer; the elution buffer is pH 2.7, 0.1mM glycine hydrochloride.

[0050] 2) Prepare collection tubes: Take 1.5 mL centrifuge tubes and add 70 μL of pH 9.0 1M Tris-HCl to each centrifuge tube.

[0051] 3) Sample preparation: The sample obtained by precipitation with 50% SAS was dialyzed overnight in the starting buffer and filtered through a 0.22 μm microporous membrane.

[0052] 4) Purification process: Equilibrate the Protein A-agarose affinity chromatography column (HiTrap Protein A 1 mL, Pharmacia Biotech) with sufficient starting buffer (8-10 mL). Load 15-25 mL of the sample to be purified (containing 10.2-21.1 mg of protein per mL) onto the column at a flow rate of 0.5 mL / min. Then wash sequentially with 7-8 mL of starting buffer, 6-7 mL of elution buffer, and 5 mL of starting buffer at the same flow rate, collecting 1 mL of eluent from each tube.

[0053] 5) Purity and Activity Identification: The purity of the purified monoclonal antibody (McAb) was determined by SDS-PAGE. See details... Figure 1 The monoclonal antibodies of hybridoma cell lines 1D7 1C6 and 1F7 1D8 were purified to remove almost all impurities and showed two specific main bands (55kDa and 30kDa).

[0054] 7. Monoclonal antibody titer determination

[0055] Using Cry 1F purified from transgenic maize 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 purified 1D7 1C6 and 1F7 1D8 monoclonal antibody titers were 1:6140000 and 1:2050000, respectively.

[0056] Table 2. Concentrations of monoclonal antibodies produced by hybridoma cell lines 1D7 1C6 and 1F7 1D8

[0057] Monoclonal antibody hybridoma cell numbering Antibody (IgG) concentration 1D7 1C6 6.0 mg / mL 1F7 1D8 2.0 mg / mL

[0058] 8. Monoclonal antibody specificity detection

[0059] Endogenous proteins were extracted from transgenic maize and its transformed parents, and subjected to SDS-PAGE gel chromatography. The transferred membranes were then analyzed using purified monoclonal antibodies (1D7, 1C6, and 1F7, 1D8) as primary antibodies. Western spectroscopy was used to detect the results. Figure 3 It can be seen that the purified 1D7 1C6 and 1F7 1D8 monoclonal antibodies can specifically recognize Cry 1F in endogenous samples.

[0060] The method for extracting protein from genetically modified corn is as follows:

[0061] 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, and the supernatant was collected. The protein extraction solution formulation is shown in Table 3.

[0062] Table 3 Protein Extract Formulation

[0063] Element Dosage 1M Tris, pH 7.5 500uL 1M NaCL 1.5mL 0.5M EDTA 20uL 50% glycerol 2mL 10% SDS 1mL <![CDATA[Double distilled water (DDH2O)]]> Prepare 10mL Protease inhibitors (Roche) Add one piece when needed. 1mM PMSF (Benzylsulfonyl fluoride, Sigma) Add 50uL at a time.

[0064] 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, This includes hybridoma cell line 1D7 1C6 with accession number CGMCC No. 45008 and hybridoma cell line 1F7 1D8 with accession number CGMCC No. 45009, which were deposited at the China General Microbiological Culture Collection Center on December 10, 2021.

2. The monoclonal antibody produced by the hybridoma cell line according to claim 1, characterized in that, Hybridoma cell lines were inoculated into the peritoneal cavity of mice to prepare ascites, which 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 antibodies produced by hybridoma cell lines 1D7 1C6 and 1F7 1D8 had titers of 1:6140000 and 1:2050000, respectively, as determined by indirect ELISA.

4. The monoclonal antibody according to claim 2, characterized in that, The monoclonal antibodies produced by the hybridoma cell lines 1D71C6 and 1F71D8 are of the types IgG1 and IgG2b, respectively.

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 hybridoma cell line 1D7 1C6 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 amino acid sequence of the heavy chain variable region of the monoclonal antibody produced by hybridoma cell line 1F7 1D8 is shown in SEQ ID NO: 3 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4 in the sequence listing.

6. The application of the monoclonal antibody according to any one of claims 2-5 in the qualitative and quantitative detection of Cry 1F insect-resistant protein.

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