CN114292820B - Insect-resistant protein Cry3Bb hybridoma cell strain, antibody produced by same and application thereof - Google Patents

Abstract

The invention discloses a hybridoma cell strain 1G42D1, an antibody produced by the hybridoma cell strain 1G42D1 and a preparation method thereof, wherein the hybridoma cell strain 1G42D1 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.22343 on the day of 5-13 of 2021, and the preparation method of the hybridoma cell strain comprises the following steps: a) Obtaining Cry3Bb recombinant protein through prokaryotic expression; b) Immunization of animals: immunizing a BALB/c mouse by taking the Cry3Bb 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 establishment: subcloning is carried out by a limiting dilution method, ELISA detection is carried out after 5-7 days of subcloning, until hybridoma cell strains which stably secrete positive antibodies are screened out and are subjected to expansion and culture and preservation, and monoclonal antibodies secreted by the hybridoma cell strains lay a foundation for detection in insect-resistant protein Cry3Bb transgenic crops.

Description

Insect-resistant protein Cry3Bb hybridoma cell strain, antibody produced by same and application thereof

Technical Field

The invention relates to the field of bioengineering, and relates to an insect-resistant protein Cry3Bb hybridoma cell strain 1G42D1, an antibody produced by the same and application thereof.

Background

Bacillus thuringiensis (Bacillus thuringiensis, bt) is a widely occurring gram-positive bacterium, whose secreted anti-insect crystal protein is the current major biopesticide; secreted insect-resistant proteins are classified into two classes according to amino acid sequence similarity: cry and Cry delta-endotoxins. Wherein the Cry proteins are toxic to larvae of a variety of insect pests (e.g., lepidoptera, diptera, coleoptera, nematodes, protozoa, etc.). Cry toxins have been transformed into crops to render them insect resistant. The Cry3Bb protein is one of the Cry toxins. At present, crops transformed with Cry genes mainly comprise corn, potato, rice, cotton and the like.

Advances and developments in transgenic technology have driven the development of biology. Although the transgenic food can meet the requirements of people on yield, insect resistance and the like, the transgenic food brings potential threats to the life of people, for example, certain genes can cause toxicity to the food after being introduced into a host, the transgenic food generates allergen, the people generate drug resistance, the nutritional value of the food is changed and the like. In order to comprehensively evaluate the safety of the transgenic food while researching, developing and commercializing the transgenic food, consumers can rapidly distinguish the transgenic food from natural food, and a proper method is established for identifying and detecting the transgenic ingredients in the transgenic food, so that the safety management of agricultural transgenic organisms can be promoted, the safety of people, animals and microorganisms can be ensured, the ecological environment can be protected, and the further research of agricultural transgenic biotechnology can be promoted. In order to rapidly analyze Cry3Bb proteins in transgenic crops and derivatives thereof, research and obtaining monoclonal antibodies against the Cry3Bb proteins are of great significance. .

Disclosure of Invention

The invention aims to provide a hybridoma cell strain 1G42D1, an antibody produced by the hybridoma cell strain and a preparation method thereof, and the secreted monoclonal antibody lays a foundation for realizing detection of an insect-resistant protein Cry3Bb in transgenic crops.

In order to achieve the above purpose, the invention provides a hybridoma cell strain, which is preserved in China general microbiological culture Collection center with a biological preservation number of CGMCC No.22343.

Specifically, the preparation method of the hybridoma cell strain comprises the following steps:

a) Obtaining Cry3Bb recombinant protein through prokaryotic expression;

b) Immunization of animals: immunizing a BALB/c mouse by taking the Cry3Bb 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 establishment: subcloning is carried out by limiting dilution method, ELISA detection is carried out on subclones for 5-7 days until hybridoma cell strains which stably secrete positive antibodies are screened out, and then the hybridoma cell strains are subjected to expansion and culture and preservation.

Wherein, the fusion ratio of spleen cells and SP2/0 cells of the mice is 1:5-1:10.

The invention also provides a monoclonal antibody produced by the hybridoma cell strain, wherein the hybridoma cell strain is inoculated into the abdominal cavity of a mouse to prepare ascites, and then Protein A-agarose affinity chromatography column purification is carried out to obtain the monoclonal antibody.

Specifically, the monoclonal antibody produced by hybridoma cell line 1G42D1 has a titer of 1:2800000 as measured by indirect ELISA.

The heavy chain amino acid sequence of the variable region of the monoclonal antibody produced by the hybridoma cell strain 1G42D1 is shown as SEQ ID NO:1, and the amino acid sequence of the light chain is shown as SEQ ID NO:2, the sequence shown in the figure is:

the invention also provides application of the monoclonal antibody in qualitative and quantitative Western detection of Cry3Bb insect-resistant proteins.

Compared with the prior art, the invention has the following beneficial effects: compared with the prior art, the invention has the following beneficial effects: the method uses high-purity insect-resistant protein Cry3Bb obtained by engineering strain expression and purification as antigen, and prepares 1 hybridoma cell strain 1G 4D 1 which secretes specific and sensitive monoclonal antibody of the Cry3Bb by hybridoma technology, wherein the indirect ELISA titer of the antibody obtained after ascites secretion and purification of the cell strain is respectively 1:2800000, the monoclonal antibody can specifically recognize prokaryotic expression recombinant Cry3Bb protein and endogenous Cry3Bb protein in transgenic corn, and the construction of the mouse monoclonal antibody cell strain secreting the Cry3Bb insect-resistant protein provides material and technical support for the detection of the protein in transgenic crops.

Preservation information

The hybridoma cell strain 1G42D1 provided by the invention is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, and the preservation address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. Post code: 100101 and the preservation number is CGMCC No.22343.

Drawings

FIG. 1 is a diagram showing the result of SDS-PAGE electrophoresis of a monoclonal antibody purified from hybridoma cell line 1G42D1 according to the invention;

FIG. 2 is the monoclonal antibody titer produced by hybridoma cell line 1G42D1 according to the invention;

FIG. 3 is a graph showing the Western results of monoclonal antibody specific detection of Cry3Bb in transgenic maize produced by hybridoma cell line 1G42D1 according to the invention.

Detailed Description

The following detailed description of embodiments of the invention is, therefore, to be taken in conjunction with the accompanying drawings, and it is to be understood that the scope of the invention is not limited to the specific embodiments.

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

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 hybridoma cell acquisition and preparation of monoclonal antibody thereof

1. Preparation of immune antigens

1.1 construction of recombinant protein Cry3Bb fragment expression Strain, thallus culture and cleavage

Amplification of Cry3Bb fragment encoding genes: PCR amplification was performed using high fidelity Fastpfu using transgenic maize genomic DNA as template, with the following primers:

and (3) carrying out agarose gel electrophoresis identification on the amplified PCR product, then carrying out enzyme digestion, connecting to pET28a plasmid, converting Trans10 competent cells (Beijing full gold), picking clone, carrying out colony PCR identification, and sequencing positive clone by Beijing Liuhua large gene company.

Competent cells of E.coli BL21 (DE 3) were transformed with the recombinant expression vector pET28a-Cry3Bb identified correctly, and the bacterial solution was spread on LB plates containing kanamycin (50. Mu.g/mL) and cultured overnight at 37 ℃. The following day, the colonies were picked up on plates and inoculated in liquid LB medium containing kanamycin at the same concentration and incubated overnight at 37 ℃. The obtained strain liquid is uniformly mixed with 50% glycerol solution in equal volume, and frozen at-80 ℃.

The preserved recombinant protein Cry3Bb expression strain is subjected to resuscitating culture, bacterial liquid is coated on an LB plate containing kanamycin, and the culture is carried out at 37 ℃ overnight. The monoclonal inoculated liquid LB (containing kanamycin) medium was picked up and cultured overnight at 37 ℃. The next day, 1% of the inoculation amount is used for inoculation, and the culture is carried out at 37 ℃ until OD ₆₀₀ About 0.8, IPTG was added at 16deg.C overnightInducing expression of the protein. After induction, the bacterial liquid is subjected to ice bath, bacterial bodies are centrifugally collected at the temperature of 4 ℃ and at the speed of 4000rpm for 10min, the supernatant is discarded, and the bacterial bodies are suspended by lysate after being washed by PBS; ultrasonication (2 s on/4s off, amp:45%, time:3 min); centrifuge at 15000rpm at 4℃for 1hr, and collect the supernatant.

1.2 purification of recombinant proteins

Mixing the cell lysate supernatant with Ni Sepharose 6FF Beads (GE Healthcare) equilibrated with lysate, and centrifuging at 3000rpm for 3min at 4deg.C in combination with 2-3hrs to discard supernatant; washing the protein-bound Ni Sepharose 6FF beads with a washing solution for 2-3 times; eluting with eluent. Collecting the eluent to obtain the crude and pure protein solution.

The protein sample obtained by affinity purification is dialyzed into a protein storage solution, and further purified by Superdex 200Increase 10/30GL (GE Healthcare) balanced by the same solution, protein peak components are collected, the purity of the protein sample is detected by SDS-PAGE, and finally the electrophoresis pure Cry3Bb protein fragment with the molecular weight of about 43kDa is obtained by gel filtration.

2. Immunization of animals

8 SPF grade BALB/c female mice (purchased in Hubei province laboratory animal research center, license number: SCXK (jaw) 2015-0018) with the Cry3Bb recombinant protein fragments qualified in the quality control of step 1.2 as antigens, mixing the antigens with equal volumes of complete Freund's adjuvant (first immunization) and incomplete Freund's adjuvant (booster immunization) and emulsifying, fully mixing to a water-in-oil state for subcutaneous multipoint immunization, carrying out 2-3 booster immunizations, carrying out a valence test after each immunization interval period of 2 weeks, carrying out intraperitoneal impact within 1 week after more than 1:10000, directly dissolving the antigens of immune doses in 250 mu L of PBS, wherein the specific immunization times and immune doses are shown in Table 1:

TABLE 1 immunization times and immunization doses

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Immunization examples: in one immunization, 50ug of antigen was dissolved in PBS and then mixed with adjuvant at a volume of 1:1.

3. Cell fusion

After 3 days of final impact, positive control blood is collected, spleen is taken, and single cell suspension is prepared; after SP2/0 cells in logarithmic phase are treated, they are mixed with spleen cells in a certain ratio (1:5-1:10), 50% PEG1450 is acted for 1min, diluted with basic culture medium DMEM, and after low-speed centrifugation, they are gently suspended and mixed with HAT medium containing 20% fetal bovine serum according to 2×10 ⁷ Plating onto a pre-prepared feeder cell plate, and placing into 5% CO ₂ Culturing at 37 ℃.

4. Cell strain

1) Fusion plate detection:

when the cell to be fused is grown to medium size of more than about 1 ten thousand cells, detection is started, and the ELISA quality control is qualified (namely negative control OD ₄₅₀ <0.2, positive control OD ₄₅₀ >1.0 Positive wells (with OD 450. Gtoreq.0.5) were selected as subclones.

2) Subcloning method and detection:

the melting plate was picked up to have a high positive value (OD ₄₅₀ >2.0 Limiting dilution is carried out on the holes, the number of the monoclonal holes with the positive value higher than that of each plate is counted to be used as subclones, limiting dilution is carried out on the monoclonal holes with the positive value higher than that of each plate, ELISA detection can be carried out on each subclone for 5-7 days, and finally, monoclonal cell strains capable of stably secreting positive antibodies are screened out for expansion culture.

3) Cell strain establishment:

the cell strain which is screened in the subcloning stage and is used for stably secreting positive antibodies is expanded and cultured in a 24-hole plate, the supernatant is collected for antigen detection, ELISA gradient dilution and western-blotting are adopted for verifying the stability, the monoclonal antibody secreted by the Cry3Bb monoclonal antibody hybridoma cell strain 1G42D1 can specifically detect an endogenous sample and recombinant protein Cry3Bb, the collected cells are expanded in a 10cm culture dish, the supernatant is collected again and the titer of the antibodies is detected, 1-strain cell strain with OD450 of >2.0 is selected and cultured in a cell bottle, freezing is carried out, namely the hybridoma cell strain 1G42D1 is preserved in the China general microbiological culture collection center (CGMCC) for 5 months 13 days, and the preservation number is CGMCC No.22343.

4) Cell strain cryopreservation identification one of the same batch must be recovered for identification after cell strain cryopreservation is completed, identification standard:

(1) the number of the resurrection cells is more than or equal to 100 ten thousand cells/branch; (2) the viable cells in the viable cells are more than or equal to 50 ten thousand per plant; (3) no other microorganisms (such as bacteria, fungi, mycoplasma, etc.) except the cells of the cell lines can be present in the resuscitated cells; (4) resuscitating the cells to grow to a certain number, selecting the grown cells as monoclonal counting plates, and detecting whether the monoclonal antibody secretion capacity is holyang or antibody secretion exists; (5) cell culture supernatants were also subjected to ELISA (OD ₄₅₀ >2.0 To determine whether positive antibodies are secreted and to perform western-blotting identification, it can be seen from fig. 3 that the monoclonal antibody secreted by the Cry3Bb monoclonal antibody hybridoma cell line 1g42d1 can specifically detect the endogenous sample and the recombinant protein Cry 3Bb.

5 preparation of ascites

Injecting pristane or liquid paraffin into the abdominal cavity of mouse, inoculating hybridoma cell strain 1G42D1 into the abdominal cavity of mouse after one week, expanding culture with 10% fetal bovine serum culture medium, and culturing until cell density reaches 1×10 ⁶ -2×10 ⁶ at/mL, the pellet was collected by centrifugation at 800rpm, resuspended in PBS, and then injected into the body of a mouse (liquid paraffin) from the abdominal cavity, and after 7-10 days, the ascites was collected and prepared for purification.

6 antibody purification

The collected ascites is purified by Protein A-agarose affinity chromatography column after pretreatment, and the specific steps are as follows:

1) Buffer solution: the starting buffer was pH7.0, 20mM phosphate buffer; the elution buffer was glycine hydrochloride at pH 2.7.0.1 mM.

2) Preparing a collecting pipe: a1.5 mL centrifuge tube was used, and 70. Mu.L of Tris-HCl, pH 9.0M, was added to each centrifuge tube.

3) Sample preparation: samples obtained by 50% sas precipitation were dialyzed overnight against starting buffer and filtered through 0.22 μm microporous filter membrane.

4) The purification process comprises the following steps: protein A-Sepharose affinity chromatography column (HiTrap Protein A1mL,Pharmacia Biotech) was equilibrated with sufficient starting buffer (8-10 mL). 15-25mL of the sample to be purified (containing 10.2-21.1mg of protein per milliliter of sample) is taken and put on a column, the flow rate is 0.5mL/min, and then the sample is washed with 7-8mL of starting buffer, 6-7mL of eluting buffer and 5mL of starting buffer in turn at the same flow rate, and the eluting solution is collected per 1mL of the column.

5) Purity and Activity characterization of purified monoclonal antibody (McAb) the purity was determined by SDS-PAGE, see in particular FIG. 1, and hybridoma cell line 1G42D1 monoclonal antibody was purified to remove almost all the contaminating proteins, with 2 major specific bands (55 kDa and 30 kDa).

7. Determination of the titers of monoclonal antibodies

The recombinant protein Cry3Bb is taken as an antigen, and the titer of the purified monoclonal antibody is detected by an indirect ELISA method, and the titer of the purified 1G42D1 monoclonal antibody is 1:2800000 as shown in the figure 2.

TABLE 2 concentration of monoclonal antibodies produced by hybridoma cell line 1G42D1

Monoclonal antibody hybridoma cell numbering	Antibody (IgG) concentration
		1G4 2D1	5.5mg/mL

8. Monoclonal antibody specific detection

The transgenic corn and its transformed parent endogenous protein were extracted separately, SDS-PAGE gel was run, purified monoclonal antibody (1G 42D 1) was used as primary antibody for the transfer, alexa FlourrTM 680 coat anti-mouse IgG (H+L) (Invitrogen) was used as secondary antibody, and the purified 1G42D1 monoclonal antibody was used to specifically recognize Cry3Bb in the endogenous sample as a result of western detection by an Odyssey input 740 imager (9120, li-COR Biosciences, lincolin, NE).

The method for extracting the transgenic zein comprises the following steps:

quick freezing tissue with liquid nitrogen, grinding, adding 1mL (1-2 mL is added according to sample amount, generally 0.5 g) of protein extract, mixing at 4deg.C for 30 min, (centrifuging at 12,000rpm at 4deg.C for 15min, collecting supernatant), and the formula of protein extract is shown in Table 4:

table 4 protein extract formulation

Composition of the components	Dosage of
		1M Tris,pH 7.5	500uL
1M NaCL	1.5mL
		0.5M EDTA	20uL
50％glycerol	2mL
		10％SDS	1mL
Double distilled water (DDH) 2 O)	Is prepared into 10mL
		Protease inhibitor (Roche)	When in use, one piece is added
1mM PMSF (phenylmethylsulfonyl fluoride, sigma)	When in use, 50uL is added

Sequence listing

<110> institute of biotechnology of national academy of agricultural sciences

<120> insect-resistant protein Cry3Bb hybridoma cell strain, antibody produced by same and application thereof

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Claims (6)

1. A hybridoma cell strain, which is preserved in China general microbiological culture collection center (CGMCC) under the biological preservation number of CGMCC No.22343 in the year 5 and the month 13 of 2021.

2. The monoclonal antibody produced by the hybridoma cell line according to claim 1, wherein the hybridoma cell line is inoculated into the abdominal cavity of a mouse to prepare ascites, and then the monoclonal antibody is obtained by purifying with a Protein a-sepharose affinity column.

3. The monoclonal antibody according to claim 2, wherein the monoclonal antibody produced by the hybridoma cell line has a titer of 1:2800000 as detected by indirect ELISA.

4. The monoclonal antibody of claim 2, wherein the type of monoclonal antibody produced by the hybridoma cell line is IgG2 b.

5. The monoclonal antibody according to claim 2, wherein the heavy chain variable region of the monoclonal antibody produced by the hybridoma cell line has an amino acid sequence as set forth in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 2.

6. Use of a monoclonal antibody according to any one of claims 2-5 in a Cry3Bb insect-resistant protein Western assay.

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