CN106834234B

CN106834234B - Monoclonal antibody hybridoma cell strain for detecting Cry1Ie protein, monoclonal antibody and application thereof

Info

Publication number: CN106834234B
Application number: CN201610816083.0A
Authority: CN
Inventors: 何康来; 徐丽娜; 王振营; 王月琴; 孙丹丹
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences; Institute of Plant Protection and Agricultural Products Quality Safety of AAAS
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences; Institute of Plant Protection and Agricultural Products Quality Safety of AAAS
Priority date: 2016-09-09
Filing date: 2016-09-09
Publication date: 2020-08-07
Anticipated expiration: 2036-09-09
Also published as: CN106834234A

Abstract

The invention discloses a Cry1Ie monoclonal antibody cell strain which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.12297 in 2016, 5, 19 and 19 days. According to the invention, through the antigenicity, hydrophilicity and epitope analysis of the amino acid sequence of the Cry1Ie protein, an epitope is selected, and a specific peptide segment is artificially synthesized, so that a monoclonal hybridoma cell strain resisting the Cry1Ie protein is obtained, a foundation is laid for developing a sensitive and rapid Bt Cry1Ie crystal protein detection kit (test strip), and the monoclonal hybridoma cell strain has a wide prospect in the qualitative and quantitative detection aspects of the Cry1Ie protein. The monoclonal antibody of the invention has high affinity to Bt Cry1Ie protein expressed in plants, is used for qualitatively or quantitatively detecting Bt Cry1Ie protein in transgenic plants such as cotton, corn and the like, and has the advantages of strong specificity, rapidness and sensitivity.

Description

Monoclonal antibody hybridoma cell strain for detecting Cry1Ie protein, monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of immunology, and particularly relates to a Cry1Ie monoclonal antibody cell strain for detecting Cry1Ie protein, a monoclonal antibody and application thereof.

Background

Bacillus thuringiensis (Bacillus thuringiensis) is a world-recognized microbial insecticide with the largest production scale, the most extensive application and the most promising, and the produced insecticidal crystal protein is a main substance of the Bacillus thuringiensis for exerting insecticidal activity, and a gene for coding the insecticidal crystal protein is called Bt gene. In 1981, Schnepf et al successfully cloned the first Bt insecticidal crystal protein gene for the first time, and revealed the sequence of breeding insect-resistant plants by genetic engineering. Transgenic corn is the first transgenic crop to be grown on a large scale, and the growing area and growing countries have been increasing since the commercial planting in 1996.

The Bt Cry1Ie gene is an insecticidal protein gene with independent intellectual property rights separated and cloned from Bt strain Btc007 by plant protection research institute of Chinese agricultural academy of sciences (patent number: Z L03149867.1), which has very low similarity to the Cry1A gene widely used in transgenic plants at present, only 30 percent and no cross resistance among the genes.

At present, the detection of transgenic plants and products thereof is mostly based on nucleic acid and protein levels, the nucleic acid level detects whether genetic materials contain inserted exogenous toxin genes, such as PCR, quantitative PCR and the like, the protein level utilizes protein products expressed by the inserted exogenous toxin genes or functions thereof to detect, such as enzyme-linked immunosorbent assay, Western blot method, test strip method and the like, in the detection of the transgenic plants, the detection of Cry toxin by adopting an enzyme-linked immunosorbent assay technology (E L ISA) is a currently recognized technology with obvious advantages, and the technology has the advantages of rapidness, accuracy, high sensitivity, capability of detecting a large number of samples and the like.

The monoclonal antibody technology is that myeloma cells and specific B cells induced by certain antigen are fused by fusion agent such as PEG or electric laser to form hybridoma, and then single positive hybridoma is screened and separated. Then amplifying the antibody, and finally separating and purifying the antibody. Hybridoma cells have the dual property of both continuing to proliferate as myeloma cells and secreting specific antibodies to a single epitope as B cells do. The main difference between monoclonal antibodies and antisera, also known as polyclonal antibodies (pcabs), is that monoclonal antibodies are homogeneous immunoglobulin molecules with specificity for an epitope; compared with polyclonal antibodies, monoclonal antibodies have incomparable superiority: such as high specificity, high purity, good homogeneity, good repeatability, high titer and the like, and has wide application in medical treatment, diagnosis and the like.

Disclosure of Invention

In view of this, the technical problem to be solved by the present invention is to provide a Cry1Ie monoclonal antibody cell strain for detecting Cry1Ie protein, a monoclonal antibody and applications thereof.

In order to solve the technical problems, the invention discloses a Cry1Ie monoclonal antibody cell strain which is preserved in No. 3 Hospital No.1 Xilu, North Chen, the south facing Yang district, Beijing, on 19 days 5 and 2016, and the preservation number of the general microorganism center of the China Committee for culture Collection of microorganisms is CGMCC NO. 12297.

The invention also discloses a monoclonal antibody, wherein the monoclonal antibody is generated by the Cry1Ie monoclonal antibody cell strain with the preservation number of CGMCC NO.12297, and the monoclonal antibody is a monoclonal antibody with specificity to Cry1Ie protein.

The invention further discloses application of the Cry1Ie monoclonal antibody cell strain in detection of Cry1Ie protein.

The invention further discloses application of the monoclonal antibody in detection of Cry1Ie protein.

The invention further discloses Cry1Ie protein detection test paper which comprises a monoclonal antibody secreted by the Cry1Ie monoclonal antibody cell strain with the preservation number of CGMCC NO. 12297.

The invention further discloses a Cry1Ie protein detection kit, which comprises a monoclonal antibody secreted by the Cry1Ie monoclonal antibody cell strain with the preservation number of CGMCC NO. 12297.

Compared with the prior art, the invention can obtain the following technical effects:

1) according to the invention, through the antigenicity, hydrophilicity and epitope analysis of the amino acid sequence of the Cry1Ie protein, an epitope is selected, and a specific peptide segment is artificially synthesized, so that a monoclonal hybridoma cell strain resisting the Cry1Ie protein is obtained, a foundation is laid for developing a sensitive and rapid Bt Cry1Ie crystal protein detection kit (test strip), and the monoclonal hybridoma cell strain has a wide prospect in the qualitative and quantitative detection aspects of the Cry1Ie protein.

2) The monoclonal antibody of the invention has high affinity to Bt Cry1Ie protein expressed in plants, is used for qualitatively or quantitatively detecting Bt Cry1Ie protein in transgenic plants such as cotton, corn and the like, and has the advantages of strong specificity, rapidness and sensitivity.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a diagram of Cry1le miniexpression gel in example 1 of the present invention;

FIG. 2 is a diagram of a large amount of expression gel of Cry1le in example 1 of the present invention;

FIG. 3 is a diagram of Cry1le purified gel in example 1 of the present invention;

FIG. 4 is an SDS-PAGE pattern of the positive Cry1Ie plant material in example 5 of the present invention;

FIG. 5 is an SDS-PAGE pattern of negative Cry1Ie plant material in example 5 of the present invention;

FIG. 6 is a Westernblot diagram of Cry1Ie protein antibodies against positive and negative Cry1Ie plant materials in example 5 of the present invention.

Detailed Description

The following detailed description of the embodiments of the present invention will be provided with reference to the accompanying drawings and examples, so that how to implement the embodiments of the present invention by using technical means to solve the technical problems and achieve the technical effects can be fully understood and implemented.

Example 1 Cry1Ie (full length) antigen was prepared as follows:

1. cry1Ie (full-length) DNA sequence

The Cry1Ie (full-length) DNA sequence is shown in SEQ ID NO. 1.

2. Transformation of

(1) Competent cells (B L21) stored at-80 ℃ were removed and thawed slowly on ice.

(2) Competent cells were added to the ligation product and mixed well and left on ice for 30 min.

(3) Heat shock at 42 ℃ for 90 s.

(4) After ice-cooling for 2min, 800. mu.l of non-resistant L B medium was added.

(5) Culturing at 37 deg.C for 45 min.

(6) Centrifuging at 5000rpm for 3min, discarding most of the supernatant, leaving about 100-.

(7) Air-dried and cultured in an incubator at 37 ℃ for overnight in an inverted state.

3. Small amount of expression

(1) From the transformed plate, single colonies were picked up into 1.5ml of L B liquid medium, cultured at 37 ℃ and 200 rpm.

(2) Culturing until OD is 0.6-0.8, inducing with IPTG (0.5 mmol/L), and culturing at 37 deg.C and 200rpm for 2 h.

(3) Taking 1ml of induced bacterial liquid, carrying out centrifugation at 12000rpm for 1min, discarding the supernatant, blowing off the precipitate by 50-100 mu l of 10 mmol/L Tris-HCl (pH8.0) solution (the amount of the added buffer solution depends on the amount of the bacteria), adding 2 × loading buffer with the same volume as the buffer solution, boiling for 5min at 100 ℃, and carrying out electrophoresis detection, wherein the result is shown in figure 1, and each strip in the figure is respectively marked by M, a cell which is not induced by IPTG (isopropyl thiogalactoside), and a cell which is induced by IPTG (isopropyl thiogalactoside) 2-5, which indicates that Cry1Ie is successfully expressed in the cell.

4. High-volume expression

(1) Mu.l of the activated bacterial suspension was inoculated into 10ml of L B liquid medium and cultured at 37 ℃ and 200 rpm.

(2) The cultured bacterial suspension was transferred to 500ml of L B liquid medium and cultured at 37 ℃ and 200rpm until OD becomes 0.6-0.8, and IPTG (0.5 mmol/L) was induced at 37 ℃ for 4 hours.

(3) Collecting bacteria: centrifuge at 6000rpm for 5min, and discard the supernatant.

(4) Ultrasonic disruption, the cells were blown off with 20-30ml 10 mmol/L Tris-HCl (pH8.0) solution, and disrupted by ultrasonication (500W, 60 times, 10s each time, 15s apart).

(5) And determining the expression form by electrophoresis, namely taking 100 mu l of the bacterial suspension after ultrasonic treatment, carrying out 12000rpm centrifugation for 10min, taking 50 mu l of supernatant to another EP tube, removing the supernatant, blowing off the precipitate by 50 mu l of 10 mmol/L Tris-HCl (pH8.0), carrying out electrophoresis detection, and obtaining the result shown in figure 2, wherein the bands in the figure are respectively M which is a Marker, 1 which is a cell which is not induced by IPTG, 2 which is a cell which is induced by IPTG, 3 which is a cell supernatant after ultrasonic disruption, and 4 which is a cell precipitate after ultrasonic disruption, so that the Cry1Ie protein is expressed in a large amount in the cell.

5. Protein purification (inclusion body)

(1) 20-30ml of 10 mmol/L Tris-HCl (pH8.0) solution is used for resuspending the sediment obtained by ultrasonic centrifugation and standing for 10 min.

(2)12000rpm, centrifugal 10min, supernatant transfer to another tube for preservation.

(3) 20-30ml of 10 mmol/L Tris-HCl (pH8.0) solution are used for resuspending the precipitate and standing for 10 min.

(4)12000rpm, centrifugation for 10min, and discarding the supernatant.

(5) Repeating the steps (3) and (4) once.

(6) Adding a small amount of 10 mmol/L Tris-HCl (pH8.0) solution to resuspend the precipitate, and adding 5-10ml of 10 mmol/L Tris-HCl (pH8.0) solution containing 8mol urea to dissolve protein.

(7)12000rpm, centrifugation for 10min, collecting supernatant, and taking 50. mu.l electrophoresis. The results are shown in FIG. 3, for each band: m is Marker; 1-5 are purified Cry1Ie protein bands.

EXAMPLE 2 hybridoma cell line preparation

1. Animal immunization

4 SPF-grade Balb/c female mice were subjected to abdominal subcutaneous primary immunization using "Cry 1Ie (full length)" as immunogen in the following amounts: 60 μ g protein/mouse, numbered: 1.2, 3 and 4. Two weeks later, a first subcutaneous boost was performed at 30 μ g protein/mouse. Two weeks later, a second subcutaneous boost was performed at an amount of 30 μ g protein/mouse. Two additional weeks later, a third subcutaneous boost was performed at 30 μ g protein/mouse. Blood was collected from the orbit 7 days later, and serum titer was measured and shown in Table 1. After 3 days, a No. 2 mouse with higher titer is selected, 50 mu g of immunogen is used for carrying out abdominal cavity impact, and cell fusion is carried out after 3 days.

TABLE 1 serum titers

Dilution factor	No.1 mouse	No. 2 mouse	No. 3 mouse	No. 4 mouse
					200	0.865	1.367	1.180	0.942
400	0.530	1.291	1.007	0.669
					800	0.320	1.155	0.712	0.396
1600	0.210	0.909	0.630	0.204
					3200	0.073	0.687	0.427	0.119
6400	0.045	0.494	0.281	0.050
					12800	0.041	0.395	0.191	0.041
25600	0.038	0.247	0.112	0.024
					51200	0.031	0.147	0.071	0.014
102400	0.028	0.100	0.053	0.013
					Blank space	0.029	0.021	0.021	0.017
Negative of	0.077	0.078	0.085	0.079

2. Cell fusion

(1) Well conditioned sp2/0 cells were gently blown off the flask wall and aspirated into a 50ml centrifuge tube.

(2) The mice were bled from the eyeballs and then sacrificed by pulling the neck, and soaked in 75% ethanol for 5 min.

(3) A small amount of serum-free IMDM was poured into the dish, and the cell sieve and plunger were placed in the dish. The spleen of the mouse was removed with scissors and forceps and placed on the cell sieve. The spleen was gently crushed sufficiently with the inner core of the syringe, and the crushed cells were aspirated into a centrifuge tube containing sp2/0 and centrifuged at 1500rad/min for 5 min.

(4) The thymus of the mouse was removed with scissors and forceps and ground. The milled thymocytes were put into a 15ml centrifuge tube, and 1ml HAT was added to the tube, and the tube was put into an incubator for use.

(5) The centrifuged cells were decanted, the supernatant was removed, the cells were gently and gently blown down with serum-free IMDM, and centrifuged at 1500rad/min for 5 min.

(6) The centrifuged cell supernatant was discarded as much as possible. Beating the bottom of the centrifuge tube to suspend the cells completely, putting the centrifuge tube into warm water at 37 ℃, slowly adding 1ml of PEG1450 in 1 minute, and standing in warm water for 1min after the addition is finished. Then 2ml serum free IMDM was added slowly over 2min followed by 8ml serum free IMDM over 2min and centrifuged at 1000rad/min for 5 min.

(7) Pouring out the supernatant, adding 10ml of newborn bovine serum, blowing the cells evenly, pouring the thymus cells prepared in the step (4), adding 25ml of sterilized semisolid culture medium (2.2% of carboxymethyl cellulose +2 × IMDM + HT), fully mixing, pouring the mixture into 30 cell culture dishes uniformly, putting the cell culture dishes into a wet box, and then putting the wet box into an incubator for culture.

3. Picking clone

7-10 days after the fusion, the clones were pipetted into 96-well plates and × 93 cell monoclonals were picked into 10 plates under a stereomicroscope, and cultured on 96-well cell culture plates (previously plated with 100. mu.l/IMDM supplemented with thymocytes).

4. Hybridoma cell line screening

4.1 screening, selecting clones, wrapping with Cry1Ie (full length) plate after 3 days, and screening the selected clones for the first time by adopting an E L ISA method to obtain 37 positive hybridoma cell strains.

(1) "Cry 1Ie (full length)" was diluted with coating solution (sodium carbonate-sodium bicarbonate buffer, pH9.6) to a final concentration of 2. mu.g/ml, 100. mu.l/well, 4 ℃ overnight; then washed 3 times with PBS-T wash (PBS containing 0.05% Tween).

(2) Sealing with PBS containing 2% milk as sealing solution at 200 μ l/well, incubating at 37 deg.C for 2 hr; then washed 3 times with PBS-T wash.

(3) Adding primary antibody (cell culture supernatant), negative control (SP2/0 culture supernatant), blank control (PBS) and positive control (positive serum PBS diluted by 200 times) 100 μ l/well, incubating at 37 deg.C for 1 h; then washed 3 times with PBS-T wash.

(4) Adding a secondary antibody (goat anti-mouse IgG/HRP) which is diluted 20000 times by PBS (100 mu l/hole), and incubating for 1h at 37 ℃; after removal, the cells were washed 3 times with PBS-T wash.

(5) Adding 100 μ l/hole of color developing solution, and developing for about 5 min. The developing solution is 1% solution A and 10% solution B (solution A: DMSO containing 1% TMB; solution B: 0.1% H₂O₂Citrate buffer of (4).

(6) Each well was stopped by adding 50. mu.l of stop solution (2 mol/L sulfuric acid).

(7) Absorbance was measured at two wavelengths (450nm, 630 nm).

And 4.2 secondary screening, namely 3 days after the primary screening, coating 37 positive cell strains screened out by the primary screening with Cry1Ie (full length) again, and performing secondary screening by adopting an E L ISA method to obtain 21 positive hybridoma cell strains.

(5) Adding 100 μ l/hole of color developing solution, and developing for about 5 min. The developing solution was 1% solution A + 10% solution B (solution A: DMSO containing 1% TMB; solution B: citric acid buffer solution containing 0.1% H2O 2).

(7) Absorbance was measured at two wavelengths (450nm, 630 nm).

5. Hybridoma cell strain subclass identification

And carrying out subclass identification on the 21 screened positive cell strains to obtain 15 IgG type positive hybridoma cell strains.

(1) The coated antibody was diluted to 0.5. mu.g/ml with 100 mmol/L PBS (pH7.4), 0.1ml per well and left overnight at 4 ℃.

(2) PBS-T wash 2 times (PBS with 0.05% Tween), add 200. mu.l of blocking solution (PBS with 2% BSA and 3% sucrose) per well, incubate 2h at 37 ℃.

(3) PBS-T washing 3 times; mu.l hybridoma supernatant was added to each well and incubated for 1h at 37 ℃.

(4) PBS-T washing 3 times; using a confining liquid 1: 10000(κ, λ) or 1:20000 (classes of subclasses IgM, IgG1, IgG2a, IgG2b, IgA) diluted HRP-labeled antibody 0.1ml per well, added to the appropriate wells individually, incubated at 37 ℃ for 1 h.

(5) PBS-T washing 3 times; mu.l of developing solution (1% solution A + 10% solution B; solution A: DMSO containing 1% TMB; solution B: 0.1% H) was added to each well₂O₂Citric acid buffer (g), 50. mu.l of stop buffer (2 mol/L sulfuric acid), absorbance at two wavelengths (450nm, 630nm) was measured within 10-20 min.

6. Mass culture and cryopreservation

Transferring the identified cell strain into a 6-well plate, and carrying out amplification culture until the cell strain is 2 × 10⁶Cells were preserved in liquid nitrogen using 2.5ml of the cryopreservative.

EXAMPLE 3 monoclonal antibody preparation

1. Recovery and expanded culture of hybridoma cells

(1) About 2ml of cell culture medium was added to each well of a 6-well cell culture plate.

(2) The cells were removed from the liquid nitrogen and placed in a 37 ℃ water bath for rapid thawing.

(3) The cells were aspirated into 5-10ml complete medium, centrifuged at 1000rpm for 5min, and the supernatant was discarded.

(4) And (3) sucking up the centrifuged cell supernatant, sucking 1ml of cell culture solution, uniformly mixing the cell culture solution with the precipitated cells, sucking out the cell culture solution, uniformly mixing the cell culture solution with the precipitated cells to corresponding culture plate holes, and putting the culture plates into a cell culture box for incubation.

2. Preparation of mice

Before the hybridoma cells are knocked out, mice are beaten with paraffin oil by an intraperitoneal injection mode to prepare ascites, each Balb/c mouse is beaten with the paraffin oil of 500 mu l, and the corresponding mice are prepared according to the prepared ascites volume.

3. Hybridoma cell collection and injection

Collecting the cultured cells in logarithmic phase, sucking 1ml of supernatant, blowing the cells to make them fall off, collecting in 15ml centrifuge tube, centrifuging at 1000rmp for 5min, resuspending the cell precipitate with proper amount of physiological saline, adjusting cell concentration to 5 × 10⁵-9×10⁵One per ml.

The hybridoma was injected intraperitoneally, and 1ml was injected into each mouse.

4. Ascites collection

The abdominal cavity of the mouse begins to bulge about 7 days after the hybridoma cells are applied. If the abdomen is large enough (generally 7-10 days), ascites can be collected. Before ascites were collected, the cell line name was noted on a tube (15ml centrifuge tube) containing ascites fluid.

The left hand holds the mouse firmly, and the right hand holds the needle (for taking ascites) parallel to the mouse. The needle is inserted through the triangle formed by the groin and the two lowest nipples. One end of the needle is placed in a spare centrifuge tube. The needle was not inserted too deeply into the abdomen of the mice to avoid injuring the internal organs. The ascites can flow into the centrifuge tube along the needle tube. During fetching, the gesture of the hand is properly changed, and as many fetches and caches as possible are realized. (preferably once every two days)

And centrifuging the taken ascites at 5000rpm for 10min, sucking the ascites supernatant into a corresponding centrifuge tube after centrifugation, and marking and recording. Placing the mixture at a designated position with the temperature of-20 ℃ for storage.

5. Antibody purification

(1) Sample pretreatment by diluting with coupling buffer (20 mmol/L sodium phosphate buffer, pH7.0) at 1:3, balancing, centrifuging at 10000rpm at 4 deg.C for 20min, filtering the supernatant with 0.22 μm filter membrane, and removing fat, cell residue and small particulate matter.

(2) Balancing: the pre-packed affinity column (HiTrap Protein A FF, GE) was equilibrated with 5-10 column volumes of coupling buffer, maintaining a flow rate of 4 s/drop.

(3) Loading: the sample was injected into the upper port of the column by syringe, maintaining a flow rate of 5 s/drop.

(4) Impurity washing: the column was run with 5 column volumes of coupling buffer, maintaining a flow rate of 4 s/drop. Excessive washing is avoided, which reduces the yield if the interaction between the protein of interest and the ligand is weak.

(5) Elution the antibody was eluted with 5 column volumes of elution buffer (0.1 mol/L sodium citrate buffer) and collected in an EP tube.

(6) The concentration of the purified antibody is measured by a K5500 micro ultraviolet spectrophotometer, and the antibody is recorded and then subpackaged and stored at the temperature of 20 ℃ below zero.

Example 4 relative affinity assay for antibodies

1. Coating: 2.0. mu.g/ml "Cry 1Ie (full length)" (sodium carbonate-sodium bicarbonate buffer, pH9.6) coated 96-well plates overnight; the plate was washed 1 time.

2. And (3) sealing: blocking solution (2% skimmed milk powder dissolved in PBS, pH7.4), 200. mu.l/well, blocked at 37 ℃ for 2 h. The plate was washed 1 time.

3. A first antibody: purified antibody was added, and a 2-fold gradient dilution was started at 1:200, a blank was set, and incubation was performed at 37 ℃ for 1 h. The plate was washed 3 times.

4. Adding a goat anti-mouse secondary antibody into the secondary antibody, diluting the secondary antibody at a ratio of 1:20000, and incubating the secondary antibody at 37 ℃ for 1 h. The plate was washed 3 times.

5. Color development: 100 mul/hole of color developing solution is added.

6. And (4) terminating: stop solution was added at 50. mu.l/well.

7. And (3) determination: and (3) measuring a light absorption value by using a microplate reader, and finding out a dilution multiple A corresponding to the 'platform OD value' of not less than 1/2.

The affinity constant measurement data are shown in table 2.

Table 2 affinity constant assay data

The size of the affinity constant reflects the strength of the antibody affinity, i.e., the quality of the antibody is high and low, the affinity constant of the antibody generally reaches 1.0E +7, which is the level available for E L ISA, and the current antibody can reach 1.23E +10, which indicates that the antibody has high affinity and very good quality.

Example 5 recognition reaction of monoclonal purified antibodies on Positive and negative Natural materials at the level of Western blot

1. Experimental materials: antigen: cry1Ie corn leaf positive samples;

one negative corn leaf sample: nongda 108 is non-Bt.

A first antibody: cry1Ie monoclonal antibody.

Secondary antibody: HRP-labeled goat anti-mouse antibody.

2. The experimental method comprises the following steps: western blot

3. The experimental principle is as follows: western blotting is based on SDS polyacrylamide gel electrophoresis, and transfers the protein component separated by electrophoresis from the gel to a solid phase support, and uses the characteristic that the antibody can produce specific reaction with target protein attached to the solid phase support to make identification and quantification of specific protein.

4. Primary reagent

(1) Animal and plant tissue lysate 50 mmol/L Tris (pH7.4), 150 mmol/L NaCl, 1% Triton-100, 0.2% SDS, 1 mmol/L EDTA, 1% Sodium deoxycholate (Sodium deoxycholate), 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 5% β -mercaptoethanol

(2) 30% acrylamide (4 ℃ photophobic storage)

(3) 10% SDS solution

(4) Isolation gel buffer (1.5 mol/L pH8.8Tris-HCl stock, 4 ℃ storage)

(5) Concentrated gel buffer (1.0 mmol/L pH6.8Tris-HCl stock, 4 ℃ storage)

(6) TEMED (to be packaged 1ml in advance, attention light-shading)

(7) SDS-PAGE sample loading buffer (5 × loading) 1 mol/L pH6.8Tris-HCl, 0.1% SDS, 0.05% bromophenol blue, 0.5% glycerol, 0.01% mercaptoethanol

(8)10％AP

(9) Transfer buffer 0.5M Glycine, 60 mmol/L Tris, 10% SDS

(10) Confining liquid (5% skimmed milk powder)

(11) TBST 50 mmol/L Tris (pH7.4), 150 mmol/L NaCl, 2ml Tween-20

5. Operating procedures

Animal and plant tissue sample preparation protocol

The whole experiment needs to be kept in an environment of about 4 ℃, the operation is strictly limited in ice bath, 1g of tissue sample is weighed, 2-3ml of lysis solution is added after grinding, the tissue lysis solution is absorbed into a 1.5ml precooled EP tube, vortex and uniformly mix for 1min every 5min, repeat for 3 times, after full lysis, 12000r, centrifuge for 10min at 4 ℃, supernatant is collected, 5 × loading Buffer is added, 100 ℃ water bath is carried out for 10min, and low-temperature storage is carried out for later use.

6. Preparation of SDS-PAGE gels

(1) Preparing a sample: the loading was 0.1ug recombinant protein or 100ug whole protein (native)/well and the sample was boiled in a water bath for 10 min.

(2) Loading: 20 μ l/well.

(3) Glue running: and stopping electrophoresis when the bromophenol blue front reaches the bottom of the glass plate, and taking out the gel. The gel was soaked in transfer buffer for 20 min.

(4) Treating the film: 30min before electrophoresis, soaking the PVDF membrane in 100% methanol solution for 5min, rinsing with triple distilled water for 5min, finally soaking the membrane in a transfer buffer solution for 20min, and soaking filter paper in the transfer buffer solution for 20 min.

(5) Film transfer: the time is adjusted according to the molecular weight.

(6) And (3) sealing: rinsing the electrotransformed membrane with TBST for 5min, placing in 5% skimmed milk powder sealing solution, sealing overnight, and taking out the membrane the next day and shaking for 1 h.

(7) Incubating primary antibody: primary antibody is diluted (monoclonal antibody cell culture supernatant 1: 5-1: 10, polyclonal antibody supernatant 1:500-1:1000, purified antibody 1: 1000-1: 5000, blocking solution is diluted). Incubate with shaking at room temperature for 2 h.

(8) Rinsing the membrane for 4 × 5min with TBST, diluting the secondary antibody with the blocking solution 1: 5000, and incubating for 1.5 h.

(9) EC L color development-TBST rinse membrane for 4 × 5min, take out GE Amersham EC L prime color development kit for color development, observe the color development effect and record and store.

Specific results are shown in table 3:

TABLE 3 WB data

WB results are shown in FIGS. 4-6, where M in FIG. 4 is Marker; 2-10 is a positive sample, and the purposive band of 2-10 in the range of 70-100KD in the figure shows that the purified Cry1Ie monoclonal antibody can detect Cry1Ie protein in the positive sample. In FIG. 5, M is a Marker mark; the negative sample in the figure has no band in the range of 70-100KD, which indicates that the Cry1Ie protein is not contained in the negative sample. In FIG. 6, 1, 3, 5 and 7 are positive samples, and 2, 4, 6 and 8 are negative samples, and it can be seen that the positive samples have bands in the range of 70-100KD, and the negative samples have no corresponding bands in the range of 70-100 KD.

According to the invention, through the antigenicity, hydrophilicity and epitope analysis of the amino acid sequence of the Cry1Ie protein, an epitope is selected, and a specific peptide segment is artificially synthesized, so that a monoclonal hybridoma cell strain resisting the Cry1Ie protein is obtained, a foundation is laid for developing a sensitive and rapid Bt Cry1Ie crystal protein detection kit (test strip), and the monoclonal hybridoma cell strain has a wide prospect in the qualitative and quantitative detection aspects of the Cry1Ie protein. The monoclonal antibody of the invention has high affinity to Bt Cry1Ie protein expressed in plants, is used for qualitatively or quantitatively detecting Bt Cry1Ie protein in transgenic plants such as cotton, corn and the like, and has the advantages of strong specificity, rapidness and sensitivity

As used in the specification and claims, certain terms are used to refer to particular components or methods. As one skilled in the art will appreciate, different regions may refer to a component by different names. The present specification and claims do not intend to distinguish between components that differ in name but not in name. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect. The following description is of the preferred embodiment for carrying out the invention, and is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.

It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a commodity or system that includes the element.

The foregoing description shows and describes several preferred embodiments of the invention, but as aforementioned, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The Cry1Ie monoclonal antibody cell strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO.12297 in 2016, 5 and 19 days.

2. The monoclonal antibody is produced by a Cry1Ie monoclonal antibody cell strain with the preservation number of CGMCC NO.12297, and is a monoclonal antibody specific to Cry1Ie protein.

3. The use of the Cry1Ie monoclonal antibody cell strain of claim 1 for detecting a Cry1Ie protein.

4. Use of a monoclonal antibody according to claim 2 for detecting the Cry1Ie protein.

5. The Cry1Ie protein detection test paper is characterized by comprising a monoclonal antibody secreted by a Cry1Ie monoclonal antibody cell strain with the preservation number of CGMCC NO. 12297.

6. A Cry1Ie protein detection kit is characterized by comprising a monoclonal antibody secreted by a Cry1Ie monoclonal antibody cell strain with the preservation number of CGMCC NO. 12297.