CN107513521A - Hybridoma cell strain, secreted monoclonal antibody and its application in bar/PAT albumen is detected - Google Patents

Abstract

The invention discloses a pair of hybridoma cell lines, the secreted monoclonal antibody and the application thereof in detecting bar/PAT protein. The present invention firstly discloses a hybridoma cell strain secreting monoclonal antibody Ab1, and its microorganism preservation number is CGMCC No.11094. The invention also discloses a hybridoma cell strain secreting the monoclonal antibody Ab2, and its microbial preservation number is CGMCC No.11095. The invention further discloses an immune colloidal gold test paper for detecting bar/PAT protein. The immune colloidal gold test paper prepared by using the monoclonal antibody Ab1 and the monoclonal antibody Ab2 secreted by the hybridoma cell line of the present invention has high detection sensitivity and specificity for the bar/PAT protein. The hybridoma cell line and the monoclonal antibody secreted by the invention have application prospects in the detection of herbicide-resistant transgenic plants.

Description

Hybridoma cell lines, secreted monoclonal antibodies and their ability to detect bar/PAT protein application in

technical field

The invention relates to a pair of hybridoma cell lines, and also relates to a pair of paired monoclonal antibodies secreted by the hybridoma cell lines and their application in detecting bar/PAT protein, belonging to the field of agricultural biotechnology.

Background technique

Since human farming, we have been exploring ways to effectively control weeds, and spraying herbicides in the field is currently the most efficient way. But because herbicides can kill weeds and crops at the same time, the commercialization of genetically modified crops with herbicide resistance genes has become one of the most advantageous traits in agricultural production. Herbicide-resistant transgenic crops can be planted with narrow row spacing methods, so that more crops can be planted on the same cultivated area, and the yield of crops can be increased. In addition, the planted herbicide-resistant genetically modified crops do not need to deal with the residues of field crops after using herbicides, which reduces the amount and intensity of labor. As of 2003, herbicide-resistant crops accounted for more than 80% of the world's genetically modified crops. From the perspective of development trend, the proportion of genetically modified crops is still increasing. Since herbicide resistance markers are widely used in commercial transgenic plants, and with the rapid development of genetically modified foods, a large number of foreign genetically modified crops and foods have entered China. Genetically modified food detection technology is an important technical platform for the safety evaluation of genetically modified foods. Significance.

Bialaphos is one of many antibiotics produced by Streptomyces, and its chemical structure includes glutamic acid isomers (phosphinothricin (PPT)) and two alanine residues (-alanyle-alanine). Bialaphos is an inhibitor of glutamine synthetase. The active glufosinate is generated after the removal of the alanine residue by the action of the endonuclease, thereby inhibiting the action of glutamine synthetase, which eventually leads to the accumulation of high concentrations of ammonia in the organism (including plants and bacteria) and is harmful to the organism. Toxicological effects. Therefore, glufosinate has become a widely used non-specific selective herbicide. Certain microorganisms are able to produce an enzyme that detoxifies this effect by acetylating the amino group. This resistance gene is named Bar, after bialaphos resistance. The protein encoded by this gene can acetylate phosphinothricin into a non-toxic form of protein phosphinothricin acetyl transferase (phosphinothricin acetyl transferase, PAT), and the protein expressed by this gene is also called bar/PAT. Bar gene is widely used in genetic engineering breeding, and it is also a marker gene in genetic transformation, which makes plants resist herbicides with phosphinothricin as the active ingredient.

The detection of transgenic exogenous proteins, including the research and development of transgenic plants or the screening or safety evaluation of transgenic products, the main methods are Western, enzyme-linked immunosorbent assay (ELISA), etc. These methods are limited by factors such as instruments and sites, and the detection time is long and the detection cost is high, so it is difficult to popularize and apply. Therefore, in practice, there is a need for a device that can quickly detect the bar/PAT protein in transgenic plants, and is fast, easy to operate, and reliable.

Immunobinding colloidal gold chromatography is an application form formed by combining immunogold labeling technology and antigen-antibody reaction. It is similar to ELISA, except that the markers are different. The method uses a microporous membrane as a solid-phase carrier, and includes implementation methods such as double-antibody sandwich method, double-antigen sandwich method, capture method, and competitive inhibition method. The double-antibody sandwich method is mainly used to detect biomolecules with multiple antigenic sites. Its technical scheme includes: first, a certain amount of known specific antibodies is coated on the membrane as a detection zone, and a secondary antibody that can bind to a gold standard is used as a quality control zone. Another monoclonal antibody gold-labeled conjugate paired with the coated antibody is adsorbed on the gold-labeled pad and dried. The dry gold standard pad is connected to the membrane at one end and the sample pad at the other end. Stick the absorbent pad on the other side of the membrane. During testing, a certain amount of liquid sample is added to the sample pad, and by capillary action, the sample moves along the direction from the sample pad to the absorbent pad. It first passes through the dry gold label pad to redissolve the gold label conjugate. If the specimen contains the antigen to be tested, an antigen-antibody reaction occurs to form complex A (gold particle-antibody-antigen); complex A continues to move to the detection If the position of the detection band is detected, antigen-antibody reaction occurs again with the coated antibody to form a complex B (gold particles-antibody-antigen-coated antibody), which gathers at the detection zone and finally reaches the level visible to the naked eye. Antigens to be detected cannot form red bands visible to the naked eye in the detection zone. The free gold-labeled conjugate or uncaptured complex A crosses the detection zone to the quality control zone, and reacts with the secondary antibody that can bind to the gold label to form complex C (gold particle-antibody-secondary antibody), aggregated In the quality control band and produce a red band visible to the naked eye. Regardless of whether the sample contains the substance to be tested, the quality control strip will show a red band.

Immune binding colloidal gold chromatography has the advantages of simple operation, rapidity, single-part inspection, and no need for special equipment, but it generally has the defect of low sensitivity. In order to make up for the shortcomings of immuno-binding colloidal gold chromatography, the emphasis is on improving the sensitivity and specificity of anti-monoclonal antibody pairs. Therefore, the development of anti-PAT protein monoclonal antibodies with high specificity and sensitivity and no cross-reactivity with other genetically modified materials or expressed proteins will be useful for the rapid and effective detection of genetically modified ingredients in plants, food and other agricultural products, as well as for public health emergencies. detection is of great significance.

Contents of the invention

The first technical problem to be solved by the present invention is to provide a pair of hybridoma cell lines, the monoclonal antibodies secreted by the hybridoma cell lines constitute a monoclonal antibody pair, which has high sensitivity and specificity for bar/PAT protein detection;

The second technical problem to be solved by the present invention is to apply the above-mentioned pair of hybridoma cell lines and the monoclonal antibody secreted thereto to the detection of bar/PAT protein.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:

The present invention utilizes expressed and purified recombinant bar/PAT protein (its amino acid sequence is shown in SEQ ID NO: 2, and the nucleotide sequence of the coding gene is shown in SEQ ID NO: 1) to immunize Balb/c female mice, and the immunized Splenocytes were fused with SP2/0 myeloma cells, and 111 positive hybridoma cell lines secreting bar/PAT monoclonal antibody were obtained by screening. The monoclonal antibodies secreted by the above-mentioned 111 cell lines all had positive reactions to the recombinant PAT protein. In the present invention, the purified 111 strains of monoclonal antibodies are tested in pairs, and a total of 12,321 paired monoclonal antibodies are obtained, and colloidal gold test strips are prepared respectively to eliminate cross-reactions, and screen only for high-specificity and high-activity pairs of bar/PAT monoclonal antibody. Finally, the present invention obtains the first monoclonal antibody Ab1 and the second monoclonal antibody Ab2 with optimal sensitivity and specificity.

The identification results of monoclonal antibody subclasses showed that the heavy chain type of monoclonal antibody Ab1 was IgG2a, and the heavy chain type of monoclonal antibody Ab2 was IgG3; the light chains of monoclonal antibodies Ab1 and Ab2 were both Kappa chains.

In the present invention, the hybridoma cell line 1BE6 secreting monoclonal antibody Ab1 is submitted to a patent-recognized institution for preservation, and its microorganism preservation number is: CGMCC No. 11094; the classification name is: anti-Bar monoclonal antibody hybridoma cell line. Preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee; preservation time is August 12, 2015; preservation address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

In the present invention, the hybridoma cell line 2CH12 secreting the monoclonal antibody Ab2 is submitted to a patent-recognized institution for preservation, and its microorganism preservation number is: CGMCC No. 11095; the classification name is: anti-Bar monoclonal antibody hybridoma cell line. Preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee; preservation time is August 12, 2015; preservation address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

The pair of hybridoma cell lines obtained in the present invention, as well as the monoclonal antibody Ab1 or the monoclonal antibody Ab2 can be applied to the detection of herbicide-resistant transgenic plants or their derivative products (that is, agricultural products such as food and drink containing transgenic ingredients). Wherein, the herbicide-resistant transgenic plant is a transgenic bar/pat plant.

The pair of hybridoma cell lines obtained in the present invention can be applied to detect bar/PAT protein, especially to prepare reagents for detecting bar/PAT protein.

The monoclonal antibody Ab1 or the monoclonal antibody Ab2 of the present invention can be applied to the detection of bar/PAT protein, especially for the preparation of reagents for detecting bar/PAT protein.

The invention further discloses an immune colloidal gold test paper for detecting bar/PAT protein, comprising: a basement membrane, an absorption pad, a colloidal gold pad, a sample pad and a substrate plate; the basement membrane is provided with a C line (quality control line) ) and T line (detection line), the C line is coated with goat anti-mouse IgG, and the T line is coated with the second monoclonal antibody; the colloidal gold pad contains the first monoclonal antibody labeled with colloidal gold Clonal antibody; the first monoclonal antibody and the second monoclonal antibody constitute a monoclonal antibody pair; wherein, the first monoclonal antibody is monoclonal antibody Ab1; the second monoclonal antibody is monoclonal antibody Ab2.

The reaction membrane has a great influence on the detection sensitivity, detection time, specificity and stability of immobilized adsorption. The present invention selects nitrocellulose membrane (NC) as the base membrane because it has greater advantages in terms of protein binding ability, activity maintenance and production cost compared with other membranes.

The assembly of the immune colloidal gold test paper of the present invention includes: adhering the base film coated with the C-line and the T-line, the absorption pad, the colloidal gold pad and the sample pad to the substrate board in sequence, to obtain the result.

The sensitivity detection results of paired antibodies showed that the detection sensitivity of the first monoclonal antibody Ab1 and the second monoclonal antibody Ab2 to the recombinant bar/PAT protein was 1 ng/mL; High detection sensitivity. The results of paired antibody specificity detection showed that the first monoclonal antibody Ab1 and the second monoclonal antibody Ab2 had no cross-reactivity to 25 non-transgenic crops and 15 non-bar/PAT transgenic crops from different sources, and had high specificity . The above results prove that the monoclonal antibodies Ab1 and Ab2 of the present invention have application prospects in the detection of herbicide-resistant transgenic products and the preparation of transgenic safety evaluation reagents.

Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

The anti-bar/PAT protein monoclonal antibodies Ab1 and Ab2 obtained by screening in the present invention have high specificity and sensitivity, no cross-reaction with other transgenic materials or expressed proteins, and high titer. Colloidal gold test strips prepared by paired monoclonal antibodies Ab1 and Ab2 can be used to quickly and effectively detect bar/PAT genetically modified components of agricultural products such as plants and food, as well as be applied to the detection of public health emergencies, and will be used for the detection of herbicide resistance Provide technical support for the detection of genetically modified products.

Description of drawings

Figure 1 is a schematic diagram of the assembly structure of the immunocolloidal gold test paper; wherein, 1 is the sample pad; 2 is the substrate plate; 3 is the colloidal gold pad; 4 is the T line; 5 is the C line; pad;

Figure 2 is the result of qualitative or semi-quantitative detection using the color depth of the signal generated by colloidal gold; wherein, A is the detection sensitivity of the paired monoclonal antibody to the recombinant bar/PAT protein; B is the detection sensitivity of the paired monoclonal antibody to the recombinant bar/PAT protein; Detection sensitivity of PAT rice seeds;

Fig. 3 is a graph showing the results of specific detection experiments on 25 non-transgenic crops and 15 non-bar/PAT transgenic crops (No 1-40) from different sources using the signal color shades generated by colloidal gold.

detailed description

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, it should be understood that the described embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

Preparation of embodiment 1 recombinant bar/PAT protein

According to the bar/pat gene expression sequence in pCAMBIA-3301 released by LabLife (https://www.lablife.org/ll), specific primers were designed according to the nucleic acid sequence expressed by the entire protein. The start codon of the gene was retained and the stop codon was deleted. Utilize PRIMER 5.0 to design a pair of primers (Table 1) for amplifying this fragment, the full length of the amplified sequence is 552bp (the nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence encoded is shown in SEQ ID NO: 2 ).

Table 1 Primer Sequence

name Primer sequence Introducing enzyme cutting sites upstream primer 5'- AAAGGATCC ATGAGCCCAGAACG-3' BamHI downstream primer 5'- GGCAAGCTT AATCTCGGTG-3' Hind III

Using the pCAMBIA-3301 plasmid as a template, the PCR amplified product was recovered and purified after BamH I/Hind III double digestion, and it was directional ligated into the pET-32a expression vector that was cut by BamH I/Hind III and transformed into the expression vector The recombinant plasmid was named pET-32a-bar after extracting the plasmid from strain BL21(DE3) cells, identified by PCR, identified by enzyme digestion and sequenced.

The PCR reaction system is: Ex Taq (5U/μL) 0.5μL, 10×Ex Taq Buffer 5μL, dNTP (2.5mmol/L) 5μL, upstream and downstream primers (25nmol/μL) 1μl, template 5μL, add water to 50μL; The reaction conditions are: 94°C for 1 min; 94°C for 45s, 60°C for 30s, 72°C for 1min, 30 cycles; 72°C for 5min. Reaction system for enzyme digestion identification: 10×Buffer for BamH I 2 μL, BamH I and Hind III enzymes 0.5 μL each, plasmid 7 μL, add water to 20 μL.

Transform the competent cells of the expression strain BL21(DE3) with the positive recombinant plasmid pET-32a-bar, pick a single colony, add it to 10 mL of LB liquid medium containing ampicillin, and culture it overnight at 37°C for enrichment. Take 1 mL of the bacterial culture and inoculate it into 50 mL of LB containing ampicillin, and continue culturing at 37°C until the OD ₆₀₀ is 0.6-1.0, then add the inducer IPTG to a final concentration of 0.5 mmol/L. The culture was continued at 37°C, and after 4 hours, the bacterial solution was collected by centrifugation to collect the bacterial precipitate. The N-terminus of the fusion protein contained 6 His-Tags, so Ni-NTA-coupled NTA resin was used for affinity chromatography purification. Firstly, the fusion protein supernatant was gradually added dropwise to the chromatography column, and the solution was filtered by gravity. Then the chromatographic column was used to elute the protein with NTA eluent containing different imidazole gradients (10, 50, 100, 150, 200, 250 mM), and the eluate was collected in sections. After detecting the purity of the collected protein by polyacrylamide gel electrophoresis, the purified protein was dialyzed with dialysate (20mM Hepe, pH7.5, 1mM DTT, 200mM KCl and 50% glycerol), and the purified protein was packed at -70°C save.

Example 2 Preparation and identification of bar/PAT protein monoclonal antibody

1. Experimental method

1.1 Immunization of mice

Select 8W+ Balb/c female mice to be immunized in groups according to doses (eye blood is taken as a negative control before the initial immunization), and the immunization doses are 50, 100, 150, 200 μg/only (i.e. the recombinant bar/PAT protein obtained in Example 1 ). Recombinant antigen plus equal volume of Freund's complete adjuvant was injected subcutaneously at multiple points for immunization; after that, immunization was performed once every two weeks, with the same dose of antigen plus equal volume of Freund's incomplete adjuvant subcutaneously injected at multiple points, followed by two additional immunizations. About 10 days after the third immunization, the recombinant bar/PAT protein was used to coat the ELISA plate, and the mouse serum titer was measured by indirect ELISA. Three days before the fusion, the mice with high antibody titer (above 10 ⁵ ) were given booster immunization (50 μg) by tail vein injection. Cell fusion was carried out 72 hours after booster immunization.

1.2 Cell Fusion

1.2.1 Preparation of feeder cells

Unimmunized BALB/c mice were sacrificed by orbital bloodletting. Negative serum was collected and soaked in 75% alcohol for 5 minutes for disinfection. Fix the sterilized mouse on the dissecting board, clamp the skin of the lower abdomen with tweezers, cut a small mouth, tear the skin to expose the peritoneum, change a set of tweezers and scissors, cut a small mouth on the peritoneum (in the center of the abdomen), and then Use a straw to draw 3.0mL 1640 liquid into the abdominal cavity of the mouse, gently blow and suck several times, then transfer the liquid into a sterile 50.0mL centrifuge tube, and repeat once. Mix the cells with 10 mL serum-containing HAT medium, count, and adjust the cell density. Cultured in a 96-well cell culture plate in a 37°C, 5% CO ₂ incubator for fusion experiments the next day.

1.2.2 Preparation of myeloma cell SP2/0

Mice with myeloma cells growing on their backs were sacrificed by cervical dislocation. Soak in 75% ethanol for 2 minutes to 5 minutes to disinfect the fur. The mice were fixed on their backs, and the tumor tissues were taken out under aseptic conditions, homogenized, and suspended in 20 mL of basal culture medium. Slowly add the suspension into the centrifuge tube that already has 20mL lymphocyte separation medium. Centrifuge at 1,800r/min for 10min, and absorb the dense white cell layer between the culture medium and the separation medium. Wash the cells twice with 1640 basal culture medium, resuspend the cells in 10 mL of basal culture medium, stain with 0.2% trypan blue, and count the cells. The cell viability should be >95%, and the cell density should be adjusted for use.

1.2.3 Preparation of splenocyte suspension

The BALB/c mice that had undergone immune shock were taken, the orbits were bled, and the positive serum was collected. The peritoneum of the mouse was cut open, the adventitia of the spleen was cut to expose the spleen, and the adipose tissue adhered to the spleen was removed with scissors. Put the spleen on a 200-mesh stainless steel cell mesh, squeeze the spleen with a needle core of a 5ml syringe, and make the dispersed spleen cells fall through the mesh into a sterilized homogenizer. Wash with an appropriate amount of 1640 base fluid and blow evenly. Centrifuge at 1200r/min for 10min to remove the supernatant, and resuspend the cells for counting.

1.2.4 Cell fusion and culture

Centrifuge the feeder cells at 1000r/min for 5min, remove the supernatant, suspend with an appropriate amount of HAT medium, and place them at 37°C for later use. Mix 1×10 ⁷ -2×10 ⁷ SP2/0 myeloma cells and 10 ⁸ immune splenocytes (1:10-1:5) in a 50 mL centrifuge tube, and centrifuge at 1500 r/min for 10 min. Discard the supernatant, turn it upside down on sterilized filter paper, and blot the liquid in the tube as dry as possible. Gently tap the bottom of the tube to loosen the cell pellet, and place the centrifuge tube in a 37°C water bath. Then slowly add 0.8 mL of 50% PEG pre-warmed to 37°C dropwise within 1 min, stirring gently with a suction tip while adding. Continue stirring for 1 min, and then slowly add 10 mL of 1640 base solution pre-warmed at 37 °C. The specific method is: add 1.0mL drop by drop at the first minute, add 1.0mL at the second minute, add 3.0mL at the third minute to 4min, add the remaining 5.0mL at the fifth minute, add slowly each time you add, and keep stirring gently , and finally slowly add 30mL 1640 solution. Centrifuge at 1000r/min for 5min, remove the supernatant. After washing once with 1640 base solution as above, suspend the mixed cells with HAT medium, add feeder splenocytes suspended in HAT medium, add appropriate amount of HAT medium as needed, mix evenly, and divide into 96-well culture plates, About 250 μL/well. After the cells were fused, the culture plate was placed in a 37°C, 5% CO ₂ saturated humidity incubator and cultivated. After 5 days, HAT was used to select the culture medium and put 1/2 of the culture medium in the well. Thereafter, they were cultured with HT medium, and the HT content was gradually reduced. After 7-10 days, when the cell colony is 2 mm in size, calculate the fusion rate (number of hybrid cell growth wells/total number of culture wells×100%), and prepare for detection.

1.3 ELISA screening positive hybridoma cells

The fused cells were cultured for about 12-15 days, and when they grew to 1/4 of the bottom area of the culture well, the supernatant was taken to detect the specific reaction and cross-reaction by indirect ELISA method, and the hybridoma cells were screened. Add 100μl supernatant to each well, use immune mouse serum as positive control (1:50 dilution, diluent is PBS), SP2/0 as negative control, recombinant protein bar/PAT-Ag as coating antigen, conventional coating ELISA plate. The cell supernatant was incubated with the coated ELISA plate for 1 hour, and after thorough washing, 100 μl/well of HRP-labeled goat anti-mouse IgG antibody (1:10,000 dilution) was added, incubated at 37°C for 30 minutes, and the secondary antibody was discarded. After fully washing the plate, add 100 μl of TMB to each well for color development for 15 minutes, and then add 50 μl/well of 1N H ₂ SO ₄ to terminate the reaction. Determine the _OD450 value.

111 cell lines secreting bar/PAT monoclonal antibody were screened by ELISA; the monoclonal antibody secreted by the above 111 cell lines all had positive reaction to recombinant PAT protein. The 30 cell lines with the strongest positive reaction were selected for further subcloning culture, and the remaining cell lines were directly expanded for culture, cryopreserved and produced a small amount of ascites.

1.4 Cloning culture by limiting dilution method

The hybridoma cells detected as positive in the cell supernatant were cloned by the limiting dilution method, specifically as follows: before cloning, mouse feeder cells were prepared according to the above-mentioned method, tiled in a cell culture plate, and placed in an incubator for subsequent use. Use a 200 μL pipette tip with a snipped tip to resuspend the cells several times in the well to be cloned, count the cells with a hemocytometer, and dilute the cells with complete medium to a density of 1 cell/well. After the addition, place in a CO ₂ incubator for cultivation. Change the medium once on the 4th day of culture, observe and record the cell monoclonal growth well under an inverted microscope; culture for about 1 week, when the cell culture medium turns yellow, make a mark. On the 7th to 9th day after cloning, when the cell clones cover 1/4 to 1/3 of the visual field, pipette 100 μL of supernatant, use the above-mentioned ELISA method to detect the cell culture supernatant, and calculate the ratio of positive wells of hybridoma cells (number of positive wells/hybridization Cell growth wells × 100%). If it is detected that there is specific antibody in the cell growth well, the cell well with high antibody titer, single clone growth and good shape can be selected, and the same method can be used to continue to clone or expand the culture. Cells in positive wells can be moved to 24-well culture plates, and another batch of medium should be added to the original wells to prevent simultaneous contamination of the two or cell death. When the cells in the 24-well plate grow well, transfer the hybridoma cell clone with the best ELISA test result after the last limited dilution to a 6-well culture plate, and finally collect the cells in a 100mL culture flask, and freeze more than 2 tubes of cells .

Cloning generally needs to be carried out more than 2-3 times until the positive rate of cell colony growth wells reaches 100%, then it can be considered that a monoclonal hybridoma cell line capable of secreting a single antibody has been obtained.

1.5 Large-scale preparation of monoclonal antibodies

1.5.1 Preparation of ascites

BALB/c mice aged 8 to 10 weeks were injected intraperitoneally with 0.5 mL of sterilized liquid paraffin, and after 7 days to 10 days, the logarithmic growth phase hybridoma cells were adjusted to a cell density of about 10 ⁷ /mL with basal culture medium. Inject hybridoma cells at 0.5 mL/mouse intraperitoneally. After the abdominal cavity of the mouse expands, the ascites of the mouse is extracted with a 12-gauge needle, and the supernatant is obtained by centrifugation. The titer is determined according to the above method and frozen.

1.5.2 Purification of monoclonal antibodies

Add 5ml Protein A Agarose to the column. After adding 10 times the column volume of Equilibration Buffer to equilibrate the column, add ascites water equal to the volume of the column, and slowly flow through the gel bed; and put the collected effluent on the column again. Use Equilibration Buffer to wash away impurity proteins, and collect the permeate at 4-5ml/tube until OD ₂₈₀ <0.1. Finally, add ElutionBuffer (pH 2.3) to elute the antibody. Add 500 μl/tube of phosphate buffer solution with pH 7.7 to the collection tube before collecting the antibody. Collect the eluate at 2ml/tube until OD ₂₈₀ <0.1. Finally, wash and equilibrate the column with 5 column volumes of Equilibration Buffer.

1.6 Monoclonal antibody pair screening

The obtained 111 strains of purified monoclonal antibodies were respectively coated with nitrocellulose membrane and labeled colloidal gold to prepare colloidal gold test strips, and then tested in pairs. A total of 111×111 (12321) pairs of combinations were obtained, cross-reaction was excluded, and screening A paired monoclonal antibody with high specificity and high activity that only targets bar/PAT.

1.6.1 Gold label and membrane preparation of monoclonal antibody

Colloidal gold was prepared by trisodium citrate reduction method. The specific operation was as follows: measure 100mL of three-distilled deionized water with a volumetric flask, pour it into a flat-bottomed flask with a specification of 500.0mL, and place the flask on a magnetic heating stirrer for heating. Put a magnetic stirrer in the cover, turn on the stirring knob to an appropriate speed, turn on the heating knob, and heat to boiling. Add 1.0mL 1% HAuCL ₄ solution, continue heating for 2min, then quickly add 1% trisodium citrate solution at one time, and continue heating. The light yellow chloroauric acid aqueous solution gradually turns from yellow to gray to black and finally to red or orange red within 2 minutes after the addition of trisodium citrate. After the solution turns bright red or orange red, continue heating and stirring for 15 min. Turn off the heating knob and let it cool down to room temperature naturally.

Preparation of colloidal gold-antibody conjugate (conjugate): add purified monoclonal antibody at an appropriate concentration to the colloidal gold solution with adjusted pH and ionic strength, and let it react at room temperature for 2 minutes. Add PEG4000 with a final concentration of 0.2% and centrifuge at 12,000rpm for 30min, suck the red precipitate into another 12ml centrifuge tube, and centrifuge at 8000rpm for 30min at night to take the precipitate. Suspend the precipitate with a colloidal gold protectant, dilute to the working concentration (usually OD ₅₃₂ = 30-40) and store at 4°C, or spray it on a non-woven film, dry it at 37°C, put it in an aluminum foil bag, and put it in a dry place agent, sealed, and stored at 4°C.

Coating of nitrocellulose membrane (NC): The reaction membrane affects detection sensitivity, detection time, specificity and stability of immobilized adsorption. The present invention selects NC membrane because it has greater advantages in terms of protein binding ability, activity maintenance and production cost compared with other membranes. Coating of nitrocellulose membrane: Dilute the purified monoclonal antibody to 2 mg/mL with 0.01 M pH7.2 PBS buffer for coating T line, dilute goat anti-mouse with 0.01 M pH7.2 PBS buffer IgG to 1 mg/mL for coating C wire. Use XYZ3050 working system of BIODOT company to spray on the nitrocellulose membrane at a speed of 30mm/s to form parallel detection T lines and quality control C lines, and dry at 37°C.

1.6.2 Assembly of immunocolloidal gold test paper

The nitrocellulose membrane 6 coated with the C line 5 and the T line 4, the absorbent pad 7, the colloidal gold pad 3 and the sample pad 1 are adhered to the non-absorbent PVC backing plate 2 in sequence, as shown in Figure 1. Colloidal gold test paper.

1.6.3 Antibody Pair Screening

A total of 111×111 (12321) pairs of monoclonal antibodies were obtained from 111 strains of monoclonal antibodies, which were combined into 12321 kinds of colloidal gold test strips. For the detection, ddH ₂ O was used as a negative sample, recombinant PAT protein 1ng/mL, and transbar/PAT rice seed extract 0.2g/mL were used as positive samples. A specific screening was performed on 25 non-GM crops and 15 various non-bar/PAT GM crops from different sources. The experiment finally obtained the first monoclonal antibody Ab1 and the second monoclonal antibody Ab2 with the best sensitivity and specificity.

1.7 Identification of monoclonal antibody subclasses

The subclass identification of each monoclonal antibody was carried out by using the immunoglobulin standard subclass identification kit (Southern Biotech Company). First, the bar/PAT protein was subjected to SDS-PAGE electrophoresis, and the target gel strip was cut off, crushed as much as possible in an EP tube, and an appropriate amount of PBS was added, and left overnight at 4°C. The next day, after centrifugation at 5000rpm for 5min, the supernatant was taken to measure the protein concentration. Then coat the microtiter plate according to 4 μg/mL, 100 μL per well, overnight at 37°C. After washing with PBST three times, 100 μL of 0.5% BSA was added to each well to block, and placed at 37°C for 1 h. Then, add the supernatant of each cell culture solution to the sealed ELISA plate, 100 μL/well, incubate at 37° C. for 1 h; wash with PBST 3 times, 5 min each time. Finally, add HRP-labeled goat anti-mouse secondary antibody (respectively anti-mouse κ, λ, IgM, IgA, IgG ₁ , IgG _2a , IgG _2b and IgG ₃ ) diluted with PBS 1:250 in sequence, 100 μL /well, placed at 37°C for 1h, washed 3 times with PBST, 5min each time. After fully washing the plate, add 100 μl of TMB to each well for color development for 15 minutes, and then add 50 μl/well of 1N H ₂ SO ₄ to terminate the reaction. Determine the _OD450 value.

2. Experimental results

2.1 Identification results of monoclonal antibody subclasses

The identification results of the heavy chains of each monoclonal antibody are shown in Table 2; the light chains are all Kappa chains.

Table 2 Identification of monoclonal antibody subclasses

Antibody Ab1 Ab2 Antibody subclass IgG2a IgG3

2.2 Sensitivity evaluation of paired antibodies

The paired monoclonal antibodies screened by the present invention: the first monoclonal antibody Ab1 and the second monoclonal antibody Ab2, the detection sensitivity to recombinant bar/PAT protein can reach 1ng/mL; the detection sensitivity to trans-bar/PAT rice seeds can reach 0.2 μg/mL (Figure 2).

2.3 Evaluation of paired antibody specificity

The paired monoclonal antibodies screened by the present invention: the first monoclonal antibody Ab1 and the second monoclonal antibody Ab2, have done specific detection experiments on 25 kinds of non-transgenic crops and 15 kinds of various non-bar/PAT transgenic crops from different sources , the test results are shown in Figure 3 and Table 3.

It can be seen that the paired monoclonal antibodies Ab1 and Ab2 have no cross-reaction to all detected non-transgenic and non-bar/PAT transgenic crops, and have high specificity. However, the specificity of other paired monoclonal antibodies screened by the present invention (screening control example 1 and screening control example 2) is obviously worse than that of the paired antibody Ab1/Ab2.

The present invention finally obtains the first monoclonal antibody Ab1 and the second monoclonal antibody Ab2 with optimal sensitivity and specificity, which can be used to prepare reagents for detecting transgenic crops and evaluating transgenic safety.

In the present invention, the hybridoma cell strain 1BE6 producing Ab1 monoclonal antibody is submitted to the General Microbiology Center of China Microbiological Culture Collection Management Committee for preservation, and its microbial preservation number is: CGMCC No.11094; the hybridoma cell strain producing Ab2 monoclonal antibody 2CH12 was submitted to the General Microbiology Center of China Committee for the Collection of Microorganisms for preservation, and its microorganism preservation number is: CGMCC No.11095.

Table 3 specificity detection results

SEQUENCE LISTING

<110> Liu Qi, Institute of Biotechnology, Chinese Academy of Agricultural Sciences

<120> Hybridoma cell line, secreted monoclonal antibody and its application in detection of bar/PAT protein

<130> BJ-2002-161113A

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 552

<212>DNA

<213> artificial sequence

<400> 1

atgagcccag aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga catgccggcg 60

gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg taccgagccg 120

caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta tccctggctc 180

gtcgccgagg tggacggcga ggtcgccggc atcgcctacg cgggcccctg gaaggcacgc 240

aacgcctacg actggacggc cgagtcgacc gtgtacgtct ccccccgcca ccagcggacg 300

ggactgggct ccacgctcta cacccacctg ctgaagtccc tggaggcaca gggcttcaag 360

agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca cgaggcgctc 420

ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa ctggcatgac 480

gtgggtttct ggcagctgga cttcagcctg ccggtaccgc cccgtccggt cctgcccgtc 540

accgagattt ga 552

<210> 2

<211> 183

<212> PRT

<213> artificial sequence

<400> 2

Met Ser Pro Glu Arg Arg Pro Ala Asp Ile Arg Arg Ala Thr Glu Ala

1 5 10 15

Asp Met Pro Ala Val Cys Thr Ile Val Asn His Tyr Ile Glu Thr Ser

20 25 30

Thr Val Asn Phe Arg Thr Glu Pro Gln Glu Pro Gln Glu Trp Thr Asp

35 40 45

Asp Leu Val Arg Leu Arg Glu Arg Tyr Pro Trp Leu Val Ala Glu Val

50 55 60

Asp Gly Glu Val Ala Gly Ile Ala Tyr Ala Gly Pro Trp Lys Ala Arg

65 70 75 80

Asn Ala Tyr Asp Trp Thr Ala Glu Ser Thr Val Tyr Val Ser Pro Arg

85 90 95

His Gln Arg Thr Gly Leu Gly Ser Thr Leu Tyr Thr His Leu Leu Lys

100 105 110

Ser Leu Glu Ala Gln Gly Phe Lys Ser Val Val Ala Val Ile Gly Leu

115 120 125

Pro Asn Asp Pro Ser Val Arg Met His Glu Ala Leu Gly Tyr Ala Pro

130 135 140

Arg Gly Met Leu Arg Ala Ala Gly Phe Lys His Gly Asn Trp His Asp

145 150 155 160

Val Gly Phe Trp Gln Leu Asp Phe Ser Leu Pro Val Pro Pro Arg Pro

165 170 175

Val Leu Pro Val Thr Glu Ile

180

<210> 3

<211> 23

<212>DNA

<213> artificial sequence

<400> 3

aaaggatcca tgagcccaga acg 23

<210> 4

<211> 19

<212>DNA

<213> artificial sequence

<400> 4

ggcaagctta atctcggtg 19

Claims (10)

1. one plant of secrete monoclonal antibody Ab1 hybridoma cell strain, it is characterised in that its microbial preservation, which is numbered, is：CGMCC No.11094。

2. one plant of secrete monoclonal antibody Ab2 hybridoma cell strain, it is characterised in that its microbial preservation, which is numbered, is：CGMCC No.11095。

3. the monoclonal antibody Ab1 that the hybridoma cell strain as described in claim 1 is secreted.

4. the monoclonal antibody Ab2 that the hybridoma cell strain as described in claim 2 is secreted.

5. application of the hybridoma cell strain of claim 1 or 2 in antiweed genetically modified plants are detected.

6. monoclonal antibody Ab2 described in monoclonal antibody Ab1 described in claim 3 or claim 4 turns in detection antiweed Application in gene plant.

7. according to the application described in claim 5 or 6, it is characterised in that：The antiweed genetically modified plants are to turn bar/pat Gene plant.

8. application of the hybridoma cell strain of claim 1 or 2 in bar/PAT albumen is detected.

9. monoclonal antibody Ab2 described in monoclonal antibody Ab1 described in claim 3 or claim 4 is in detection bar/PAT albumen In application.

10. a kind of immune colloid gold test paper of detection bar/PAT albumen, including：Basement membrane, absorption pad, colloidal gold pad, sample pad and By underboarding；C lines and T lines are provided with the basement membrane, sheep anti-mouse igg is coated with the C lines, is coated with the T lines Two monoclonal antibodies；The first monoclonal antibody containing colloid gold label in the colloidal gold pad；It is characterized in that：Described first Monoclonal antibody is the monoclonal antibody Ab1 described in claim 3；The second monoclonal antibody is described in claim 4 Monoclonal antibody Ab2.