CN114426955B

CN114426955B - Insect-resistant protein Cry3Bb hybridoma cell strain, antibody produced by same and application thereof

Info

Publication number: CN114426955B
Application number: CN202111623970.3A
Authority: CN
Inventors: 刘卫晓; 金芜军; 王磊; 苗朝华; 宛煜嵩; 黄卫红; 董美; 高进; 孟丽霞; 赵伟玲; 王迪; 文婷婷
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2021-12-28
Filing date: 2021-12-28
Publication date: 2023-05-12
Anticipated expiration: 2041-12-28
Also published as: CN114426955A

Abstract

The invention discloses Cry3Bb hybridoma cell strains 1D71G8 and 2H111H1, antibodies produced by the same and application thereof, wherein the hybridoma cell strains 1D71G8 and 2H111H1 are sequentially preserved in China general microbiological culture Collection center (CGMCC) with the preservation numbers of CGMCC No.23865 and 23866 on the 11 month 08 of 2021, and the preparation method comprises the following steps: a) Purifying Cry3Bb proteins in transgenic corn seeds; b) Immunizing a BALB/c mouse by taking Cry3Bb protein as an antigen; c) Spleen cells from immunized BALB/c mice were collected and fused with SP2/0 cells; d) 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, in particular to insect-resistant proteins Cry3Bb hybridoma cell strains 1D71G8 and 2H111H1, antibodies 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 two hybridoma cell strains 1D71G8 and 2H111H1, antibodies produced by the same and a preparation method thereof, and the secreted monoclonal antibodies lay a foundation for realizing detection of insect-resistant protein Cry3Bb in transgenic crops.

In order to achieve the above object, the present invention provides hybridoma cell lines including hybridoma cell lines 1d7 g8 and 2h11 h1 which are deposited with the China general microbiological culture Collection center with the biological accession numbers CGMCC No.23865 and CGMCC No.23866 at the year 11 and month 08.

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

a) Purifying Cry3Bb proteins in transgenic corn seeds;

b) Immunization of animals: immunizing a BALB/c mouse by taking Cry3Bb 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 titers of monoclonal antibodies produced by hybridoma cell lines 1D71G8 and 2H111H1, which were detected by indirect ELISA, were 1:920000 and 1:640000, respectively, and the types of antibodies were IgG1.

The heavy chain amino acid sequence of the variable region of the monoclonal antibody produced by the hybridoma cell strain 1D71G8 is shown as SEQ ID NO:1, and the amino acid sequence of the light chain is shown as SEQ ID NO: 2; the heavy chain amino acid sequence of the variable region of the monoclonal antibody generated by the hybridoma cell strain 2H 11H1 is shown as SEQ ID NO:3, and the amino acid sequence of the light chain is shown as SEQ ID NO:4. the specific sequence is as follows:

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

Compared with the prior art, the invention has the following beneficial effects: according to the method, the anti-insect protein Cry3Bb obtained by purification in transgenic corn seeds is used as an antigen, 2 hybridoma cell strains 1D71G8 and 2H111H1 which secrete specific and sensitive monoclonal antibodies against the Cry3Bb are prepared by a hybridoma technology, indirect ELISA titers of antibodies obtained after ascites purification of the monoclonal antibodies secreted by the cell strains are respectively 1:920000 and 1:640000, the monoclonal antibodies can specifically identify Cry3Bb proteins in transgenic corn, and the construction of the mouse monoclonal antibody hybridoma cell strains secreting the anti-Cry 3Bb anti-insect proteins provides a material and a technical support for the detection of the proteins in transgenic crops.

Preservation information

The hybridoma cell strains 1D71G8 and 2H111H1 provided by the invention are sequentially preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the 11 th month 08 of 2021, and are classified into Cry3Bb monoclonal antibody hybridoma cell strains, and the preservation addresses are as follows: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. Post code: 100101 and the preservation numbers are CGMCC No.23865 and CGMCC No.23866 in sequence.

Drawings

FIG. 1 is a diagram showing the result of SDS-PAGE electrophoresis of purified monoclonal antibodies of hybridoma cell lines 1D71G8 and 2H111H1 according to the invention;

FIG. 2 is the monoclonal antibody titres generated by hybridoma cell lines 1D71G8 and 2H111H1 according to the invention;

FIG. 3 is a graph showing the Western results of monoclonal antibodies generated by hybridoma cell lines 1D71G8 and 2H111H1 according to the invention for specifically detecting Cry3Bb in transgenic maize;

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

About 1kg of transgenic corn seeds are fully ground by liquid nitrogen and then dissolved in protein extracting solution, and relatively pure Cry3Bb protein is obtained through a plurality of purification steps such as high-speed freezing centrifugation, denaturation solution dissolution and precipitation, centrifugal separation, ion exchange chromatography, gel filtration, renaturation and the like.

2. Immunization of animals

8 SPF-class BALB/c female mice (purchased at Experimental animal research center in Hubei province, license number: SCXK (jaw) 2015-0018) were immunized with Cry3Bb protein as antigen, mixed with equal volumes of complete Freund's adjuvant (first immunization) and incomplete Freund's adjuvant (booster immunization) and emulsified, thoroughly mixed to a water-in-oil state for subcutaneous multipoint immunization, 2-3 booster immunizations, each immunization for 2 weeks at intervals, followed by potency detection, peritoneal impact within 1 week after >1:10000, and directly dissolving the immunizing dose of antigen in 250. Mu.L PBS, the specific immunization times and immunizing doses are shown in Table 1:

TABLE 1 immunization times and immunization doses

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 Post selection of Positive wells (general OD) ₄₅₀ 0.5) was subcloned.

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:

amplifying and culturing cell strains screened in a subcloning stage and stably secreting positive antibodies in a 24-hole plate, collecting supernatant after amplification for antigen detection, adopting ELISA gradient dilution and western-blotting to verify the stability of the cell strains, wherein Cry3Bb monoclonal antibody hybridoma cell strains 1D71G8 and 2H111H1 can specifically detect Cry3Bb proteins in transgenic corn samples, collecting cells, amplifying the cells in a 10cm culture dish, collecting the supernatant again, detecting the titer of the antibodies, and selecting OD ₄₅₀ >2.0 in a cell line of 2 strainsIn the cell bottle, freezing and preserving are carried out, namely hybridoma cell strains 1D71G8 and 2H111H1 are preserved in China general microbiological culture collection center (CGMCC) in the 11 th month 08 of 2021, and the preservation numbers are CGMCC No.23865 and CGMCC No.23866 in sequence.

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 simultaneously carrying out western-blotting identification, as can be seen from fig. 3, the Cry3Bb monoclonal antibody hybridoma cell strains 1d71g8 and 2h111h1 can specifically detect the Cry3Bb protein in the transgenic corn.

5. Preparation of ascites

Injecting pristane or liquid paraffin into the abdominal cavity of mouse, inoculating hybridoma cell lines 1D71G8 and 2H111H1 into the abdominal cavity of mouse, 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 pH2.70.1 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 antibodies (McAb) the purity was determined by SDS-PAGE, see in particular FIG. 1, and hybridoma cell lines 1D71G8 and 2H111H1 were 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 titers of the purified monoclonal antibodies were detected by indirect ELISA using Cry3Bb purified from transgenic maize as antigen, and as shown in FIG. 2, the titers of the purified monoclonal antibodies were 1:920000 and 1:640000, respectively, of 1D71G8 and 2H111H1, as determined by ELISA.

TABLE 2 concentration of monoclonal antibodies produced by hybridoma cell lines 1D71G8 and 2H111H1

Monoclonal antibody hybridoma cell numbering	Antibody (IgG) concentration
		1D7 1G8	3.6mg/mL
2H11 1H1	5.0mg/mL

8. Monoclonal antibody specific detection

The transgenic corn and the parent endogenous proteins transformed by the transgenic corn are respectively extracted, SDS-PAGE gel is run, purified monoclonal antibodies (1D 71G8 and 2H111H 1) are used as primary antibody western detection results in the transfer membrane, and as can be seen from figure 3, the purified 1D71G8 and 2H111H1 monoclonal antibodies can specifically identify Cry3Bb in an endogenous sample.

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 3:

table 3 protein extract formulation

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Sequence listing

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

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Claims

1. The hybridoma cell strain is characterized by comprising a hybridoma cell strain 1D71G8 with the biological preservation number of CGMCC No.23865 and a hybridoma cell strain 2H 11H1 with the biological preservation number of CGMCC No.23866, which are preserved in the China general microbiological culture Collection center for type culture Collection of microorganisms at 11 and 08 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 antibodies produced by hybridoma cell lines 1d71g8 and 2h111h1 have titers of 1:920000 and 1:640000, respectively, as detected by indirect ELISA.

4. The monoclonal antibody of claim 2, wherein the hybridoma cell lines 1d71g8 and 2h111h1 produce monoclonal antibodies of the type IgG1.

5. The monoclonal antibody according to claim 2, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody produced by hybridoma cell line 1d71g8 is 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; the amino acid sequence of the heavy chain variable region of the monoclonal antibody produced by the hybridoma cell strain 2H 11H1 is shown as SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:4.

6. use of a monoclonal antibody according to any one of claims 2-5 in qualitative, quantitative detection of a Cry3Bb insect-resistant protein.