CN114426955A

CN114426955A - Insect-resistant protein Cry3Bb hybridoma cell strain, antibody produced by same and application of antibody

Info

Publication number: CN114426955A
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: 2022-05-03
Anticipated expiration: 2041-12-28
Also published as: CN114426955B

Abstract

The invention discloses Cry3Bb hybridoma cell strains 1D 71G 8 and 2H 111H 1 as well as an antibody generated by the same and application of the antibody, wherein the hybridoma cell strains 1D 71G 8 and 2H 111H 1 are sequentially preserved in China general microbiological culture Collection center on 11 months and 08 days 2021, and the preservation numbers are CGMCC No.23865 and 23866, and the preparation method comprises the following steps: a) purifying Cry3Bb protein in transgenic corn seeds; b) cry3Bb protein is used as an antigen to immunize a BALB/c mouse; c) collecting splenocytes from immunized BALB/c mice and fusing with SP2/0 cells; d) subcloning by a limiting dilution method, carrying out ELISA detection by subcloning for 5-7 days until hybridoma cell strains which stably secrete positive antibodies are screened out, and then carrying out amplification culture and preservation, wherein monoclonal antibodies secreted by the hybridoma cell strains lay a foundation for realizing detection in insect-resistant protein Cry3Bb transgenic crops.

Description

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

Technical Field

The invention relates to the field of bioengineering, and particularly relates to insect-resistant protein Cry3Bb hybridoma cell strains 1D 71G 8 and 2H 111H 1, and antibodies generated by the cell strains and application of the cell strains.

Background

Bacillus thuringiensis (Bt) is a widely existing gram-positive bacterium, and the insect-resistant crystal protein secreted by the bacterium is the main biopesticide at present; secreted insect-resistant proteins are divided 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, protists, and the like). Cry toxins have been transferred into crops to render them insect resistant. The Cry3Bb protein is one of the Cry toxins. The crops of the forward rotation Cry gene mainly comprise corn, potato, rice, cotton and the like.

The development and progress of transgenic technology has promoted the development of biology. The transgenic food can meet the requirements of people on yield, insect resistance and the like, but also brings potential threats to human life, for example, after some genes are introduced into a host, the food generates toxicity, the transgenic food generates allergens, people generate drug resistance, the nutritional value of the food is changed, and the like. When the research and the development and the commercialization of the transgenic food are carried out, in order to comprehensively evaluate the safety of the transgenic food, a consumer can rapidly distinguish the transgenic food from the 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 guaranteed, the ecological environment can be protected, and the further research of the agricultural transgenic biotechnology can be promoted. In order to rapidly analyze Cry3Bb protein in transgenic crops and derivatives thereof, the research and the acquisition of monoclonal antibodies for resisting the Cry3Bb protein have great significance.

Disclosure of Invention

The invention aims to provide two hybridoma cell strains 1D 71G 8 and 2H 111H 1, antibodies generated by the two hybridoma cell strains and a preparation method of the antibodies, and monoclonal antibodies secreted by the two hybridoma cell strains lay a foundation for realizing detection of insect-resistant protein Cry3Bb in transgenic crops.

In order to realize the purpose, the invention provides hybridoma cell strains which comprise hybridoma cell strains 1D 71G 8 and 2H 111H 1 which are preserved in China general microbiological culture Collection center (CGMCC No. 23865) and CGMCC No.23866 on 11-month 08-2021 and have biological preservation numbers of CGMCC No.23865 and CGMCC No. 23866.

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

a) purifying Cry3Bb protein in transgenic corn seeds;

b) immunizing animals: cry3Bb protein is used as an antigen to immunize a BALB/c mouse;

c) cell fusion: collecting splenocytes from immunized BALB/c mice and fusing with SP2/0 cells;

d) cell establishment: subcloning by a limiting dilution method, and carrying out ELISA detection by subcloning for 5-7 days until hybridoma cell strains which stably secrete positive antibodies are screened out for amplification, re-culture and storage.

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

The invention also provides a monoclonal antibody produced by the hybridoma cell strain, 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 the monoclonal antibodies generated by the hybridoma cell strains 1D 71G 8 and 2H 111H 1 detected by indirect ELISA are 1:920000 and 1:640000 respectively, and the types of the antibodies are IgG 1.

The amino acid sequence of the variable region heavy chain of the monoclonal antibody generated by the hybridoma cell strain 1D 71G 8 is shown as SEQ ID NO: 1, and the light chain amino acid sequence is shown as SEQ ID NO: 2; the amino acid sequence of the variable region heavy chain of the monoclonal antibody generated by the hybridoma cell strain 2H 111H 1 is shown as SEQ ID NO: 3, and the light chain amino acid sequence 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 protein.

Compared with the prior art, the invention has the following beneficial effects: according to the application, insect-resistant protein Cry3Bb obtained by purification in transgenic corn seeds is used as an antigen, 2 hybridoma cell lines 1D 71G 8 and 2H 111H 1 secreting specific and sensitive monoclonal antibodies of Cry3Bb are prepared through a hybridoma technology, the indirect ELISA titers of antibodies obtained after the monoclonal antibody ascites is secreted by the cell lines and purified are respectively 1:920000 and 1:640000, the monoclonal antibodies can specifically recognize Cry3Bb protein in the transgenic corn, and the construction of mouse monoclonal antibody hybridoma cell lines secreting anti-Cry 3Bb insect-resistant protein provides material and technical support for detection of the protein in transgenic crops.

Preservation information

The hybridoma cell strains 1D 71G 8 and 2H 111H 1 provided by the invention have been sequentially preserved in China general microbiological culture Collection center at 11 months and 08 days 2021, and are classified as Cry3Bb monoclonal antibody hybridoma cell strains, wherein the preservation addresses are as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North. And E, postcode: 100101, and the preservation numbers are CGMCC No.23865 and CGMCC No.23866 in sequence.

Drawings

FIG. 1 is a SDS-PAGE result of purified monoclonal antibodies of hybridoma cell strains 1D 71G 8 and 2H 111H 1 according to the invention;

FIG. 2 shows the titers of monoclonal antibodies produced by hybridoma cell lines 1D 71G 8 and 2H 111H 1 according to the invention;

FIG. 3 is a diagram showing the result of specific detection of Cry3Bb Western in transgenic corn by monoclonal antibodies generated by hybridoma cell lines 1D 71G 8 and 2H 111H 1 according to the invention;

Detailed Description

The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.

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

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

EXAMPLE 1 hybridoma cell acquisition and preparation of monoclonal antibodies thereto

1. Preparation of immune antigens

And (2) fully grinding about 1kg of transgenic corn seeds by using liquid nitrogen, dissolving the ground transgenic corn seeds in a protein extracting solution, and performing multiple purification steps of high-speed freezing centrifugation, denatured solution dissolution and precipitation, centrifugal separation, ion exchange chromatography, gel filtration, renaturation and the like to obtain relatively pure Cry3Bb protein.

2. Immunizing animals

Cry3Bb protein is used as antigen to immunize 8 SPF-grade BALB/c female mice (purchased from the research center of experimental animals in Hubei province, with the license number: SCXK (Ee) 2015-0018), the antigen is mixed with equal volume of complete Freund's adjuvant (prime) and incomplete Freund's adjuvant (boost) and emulsified, fully mixed to a water-in-oil state for subcutaneous multipoint immunization, 2-3 times of boost immunization are carried out, each immunization interval is 2 weeks, then titer detection is carried out, abdominal cavity impact is carried out within 1 week after being higher than >1:10000, the antigen with the immunization dose is directly dissolved in 250 mu L PBS, and the specific immunization times and immunization dose are shown in Table 1:

TABLE 1 immunization frequency and immunization dose

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

3. Cell fusion

Collecting positive control blood 3 days after the last impact, taking spleen, and preparing into single cell suspension; treating SP2/0 cells at log phase, mixing with splenocytes at a certain ratio (1:5-1:10), allowing 50% PEG1450 to act for 1min, diluting with basal medium DMEM, centrifuging at low speed, gently suspending in HAT medium containing 20% fetal calf serum, mixing, and making into suspension at 2 × 10⁷Plating into a prepared feeder cell plate, and placing in 5% CO₂Culturing at 37 ℃.

4. Cell establishment

1) Detecting a fusion plate:

detecting when the cell growth of the fusion plate is more than 1 ten thousand cells with medium size, and performing quality control on ELISA (namely negative control OD)₄₅₀<0.2, positive control OD₄₅₀>1.0) followed by selection of positive wells (general OD)₄₅₀Not less than 0.5) as subcloning.

2) Subcloning method and detection:

high detection Positive value (OD) in picked out fusion plate₄₅₀>2.0) is subjected to limited dilution, 60 percent of monoclonal holes in each plate are counted to be used as subclones, the monoclonal holes with higher positive values are selected each time for limited dilution, ELISA detection can be carried out each time for 5-7 days by subcloning, and the monoclonal cell strains capable of stably secreting positive antibodies are screened out to be subjected to expanded culture.

3) Establishing a cell strain:

expanding and culturing cell strains which are screened in a subcloning stage and stably secrete positive antibodies in a 24-well plate, collecting supernatant after expansion for antigen detection, verifying the stability by adopting ELISA gradient dilution and western-blotting, specifically detecting Cry3Bb protein in a transgenic corn sample by monoclonal antibodies secreted by Cry3Bb monoclonal antibody hybridoma cell strains 1D 71G 8 and 2H 111H 1, collecting cells in a culture dish expanded to be more than 10cm, collecting supernatant again, detecting the titer of the antibodies in the supernatant, and selecting OD (optical density)₄₅₀>2.0 strains are cultured in a cell bottle and frozen, namely hybridoma cell strains 1D 71G 8 and 2H 111H 1 are sequentially preserved in China general microbiological culture Collection center (CGMCC for short) on 11 months and 08 days 2021, and the preservation numbers are CGMCC No.23865 and CGMCC No.23866 in sequence.

4) Cell line cryopreservation identification one cell line in the same batch must be recovered for identification after cell line cryopreservation is finished, and the identification standard is as follows:

firstly, resuscitating the number of living cells to be more than or equal to 100 ten thousand cells/branch; ② viable cells in the viable cells are more than or equal to 50 ten thousand per strain; ③ the revived cells can not have other microorganisms (such as bacteria, fungi, mycoplasma, etc.) except the cells of the cell strain; fourthly, after the cells are revived to grow to a certain number, the grown cells are selected to be used as a monoclonal counting plate, and whether the monoclonal antibody secretion ability is full positive or has antibody secretion is detected; fifthly, the cell culture supernatant also needs to be used as ELISA (OD)₄₅₀>2.0) to determine whether positive antibodies are secreted and simultaneously carry out western-blotting identification, and as can be seen from fig. 3, Cry3Bb monoclonal antibody hybridoma cell lines 1D 71G 8 and monoclonal antibodies secreted by 2H 111H 1 can specifically detect Cry3Bb proteins in transgenic corn.

5. Preparation of ascites

Injecting the mice with pristane or liquid paraffin to the abdominal cavity, inoculating hybridoma cell strains 1D 71G 8 and 2H 111H 1 to the abdominal cavity of the mice after one week, performing cell strain determination, performing amplification culture, and selecting 10% fetal calf serum culture medium when the cell density reaches 1 × 10⁶-2×10⁶at/mL, the pellet was collected by centrifugation at 800rpm, resuspended in PBS, and then intraperitoneally injected into mice (liquid paraffin), and after 7 to 10 days, ascites were collected and prepared for purification.

6. Antibody purification

The collected ascites is purified by a Protein A-agarose affinity chromatography column after being pretreated, and the method comprises the following specific steps:

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: take 1.5mL centrifuge tubes, add 70. mu.L of Tris-HCl pH9.01M to each centrifuge tube. 3) Sample preparation: the resulting sample precipitated with 50% SAS was dialyzed overnight against the starting buffer and filtered through a 0.22 μm microfiltration membrane.

4) And (3) purification process: the Protein A-Sepharose affinity column (HiTrap Protein A1 mL, Pharmacia Biotech) was equilibrated with enough starting buffer (8-10 mL). Taking 15-25mL of a sample to be purified (containing 10.2-21.1mg of protein per milliliter of sample) to load on a column at a flow rate of 0.5mL/min, then sequentially washing with 7-8mL of an initial buffer solution, 6-7mL of an elution buffer solution and 5mL of the initial buffer solution at the same flow rate, and collecting eluent in 1mL of each tube.

5) Purity and Activity identification the purity of the purified monoclonal antibody (McAb) was identified by SDS-PAGE, see FIG. 1, hybridoma cell lines 1D 71G 8 and 2H 111H 1 monoclonal antibody purified to remove almost all contaminating proteins, with 2 specific main bands (55kDa and 30 kDa).

7. Potency assay for monoclonal antibodies

The titer of the purified monoclonal antibody is detected by an indirect ELISA method by using Cry3Bb purified from the transgenic corn as an antigen, and the titer of the purified 1D 71G 8 and 2H 111H 1 monoclonal antibodies is 1:920000 and 1:640000 by ELISA determination according to the graph shown in figure 2.

TABLE 2 concentration of monoclonal antibodies produced by hybridoma cell lines 1D 71G 8 and 2H 111H 1

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

8. Monoclonal antibody specificity detection

Transgenic corn and transformed parent endogenous proteins thereof are respectively extracted, SDS-PAGE gel is run, purified monoclonal antibodies (1D 71G 8 and 2H 111H 1) for membrane transfer are used as a primary-antibody western detection result, and as can be seen from figure 3, the purified 1D 71G 8 and 2H 111H 1 monoclonal antibodies can specifically recognize Cry3Bb in endogenous samples.

The transgenic corn protein extraction method comprises the following steps:

quickly freezing tissue with liquid nitrogen, grinding, adding 1mL (generally 0.5g and 1-2mL) of protein extract, mixing at 4 deg.C for 30 min, (centrifuging at 12,000rpm at 4 deg.C for 15min, collecting supernatant), and making into protein extract with formula shown in Table 3:

TABLE 3 protein extract formula

The foregoing descriptions of specific exemplary embodiments of the present invention have been 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 certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and 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 of antibody

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Claims

1. Hybridoma cell strains which are characterized by comprising hybridoma cell strains 1D 71G 8 and 2H 111H 1 which are preserved in China general microbiological culture Collection center at 11/08 th 2021 and have the biological preservation numbers of CGMCC No.23865 and CGMCC No. 23866.

2. The hybridoma cell strain of claim 1, wherein the preparation method comprises the following steps:

a) purifying Cry3Bb protein in transgenic corn seeds;

3. The hybridoma cell line of claim 2, wherein the fusion ratio of mouse spleen cells to SP2/0 cells is 1:5 to 1: 10.

4. The monoclonal antibody produced by the hybridoma cell line of any one of claims 1 to 3, wherein the hybridoma cell line is inoculated into the abdominal cavity of a mouse to prepare ascites, and then subjected to Protein A-agarose affinity chromatography column purification to obtain the monoclonal antibody.

5. The monoclonal antibody of claim 4, wherein the titers of the monoclonal antibodies produced by the hybridoma cell lines 1D 71G 8 and 2H 111H 1 detected by indirect ELISA are 1:920000 and 1:640000, respectively.

6. The monoclonal antibody of claim 4, wherein the types of monoclonal antibodies produced by hybridoma cell lines 1D 71G 8 and 2H 111H 1 are IgG 1.

7. The monoclonal antibody of claim 4, wherein the amino acid sequence of the variable region heavy chain of the monoclonal antibody produced by the hybridoma cell line 1D 71G 8 is as shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown as SEQ ID NO: 2; the amino acid sequence of the variable region heavy chain of the monoclonal antibody generated by the hybridoma cell strain 2H 111H 1 is shown as SEQ ID NO: 3, and the light chain amino acid sequence is shown as SEQ ID NO: 4.

8. use of a monoclonal antibody according to any one of claims 4-7 in the qualitative and quantitative detection of Cry3Bb insect-resistant protein.