CN108396012A

CN108396012A - Applications of the monoclonal antibody 1DB4 in detecting IrrE transgenic crops

Info

Publication number: CN108396012A
Application number: CN201810118344.0A
Authority: CN
Inventors: 张维; 周正富; 刘奇; 张君; 陈明; 林敏�
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2018-02-06
Filing date: 2018-02-06
Publication date: 2018-08-14
Anticipated expiration: 2038-02-06
Also published as: CN108396012B

Abstract

The present invention provides a kind of monoclonal antibodies, the antibody is secreted to obtain by the hybridoma that deposit number is CGMCC NO.12268, while providing purposes and a kind of method that detects the transgenic crop that turns IrrE of the antibody in detection turns the transgenic crop of IrrE.The monoclonal antibody of the present invention can be used in the WESTERN BLOT detections of drought resistance and salt tolerance stress regulatory protein I rrE, can detect recombination IrrE albumen and turns IrrE crop seeds；Cross reaction is not also presented to the various non-IrrE genetically modified crops of 7 kinds of separate sources.

Description

Applications of the monoclonal antibody 1DB4 in detecting IrrE transgenic crops

Technical field

The present invention relates to agricultural biological technical fields, and in particular, to a kind of hybridoma cell strain, a kind of monoclonal antibody And its detection turn purposes in the transgenic crop of IrrE and detection turn IrrE transgenic crop method.

Background technology

What Deinococcus radiodurans (Deinococcus radiodurans) were acknowledged as finding so far has most forceful electric power Bacterium bacterial strain from radioresistance.Activation reparation of the bacterium of this non-sporogenesis to its lesioned gene group, can be Existence in Extreme dosages ionising radiation, and will not be by fatal damage and gene mutation.Deinococcus radiodurans are gathered around Some repair mechanisms make its 200 times higher than Escherichia coli to the resistance of ionising radiation or more.Deinococcus radiodurans are not only Passive protection cell resistance ionising radiation, while also there is thundering accurate DNA damage repair ability.Radiation hardness is abnormal Coccus has very high resistance to the oxidative stress of diversified forms.Deinococcus radiodurans have pole for long-term drought stress High resistance.Relative humidity is less than 5% drought environment and handles 6 weeks and can not generate shadow to the survival ability of Deinococcus radiodurans It rings, Deinococcus radiodurans R1 only loses 15% by such Osmotic treatment cell survival.Even there is relevant report to point out Deinococcus radiodurans are subjected to Osmotic treatment 6 years, thalline still retains 10% viability.This survival efficiency is not for forming It is extraordinary for the bacterium of spore.

IrrE genes are located on No. 1 chromosome of Deinococcus radiodurans genome, and ORF numbers are DR0167.Full length gene 987-bp encodes the 35kDa albumen being made of 328 amino acid.IrrE albumen is not found in existing database Albuminoid, identify that it is deinococcus differential protein.Recent study finds, irrE genes in Deinococcus radiodurans It is the switch gene of a distinctive, extremely important DNA restoration and protection approach, plays an important role to DNA damage reparation.It grinds Study carefully and think that irrE genes are a kind of promotor genes of novel responsible extreme radioresistance, in various DNA damage reparations and protection It radiates in the approach of Stress responses and plays central regulator.Meanwhile the gene can be significantly improved it in expression in escherichia coli The ability of radioresistance and scavenging activated oxygen, and irrE genes significantly improve the height of bacterial strain in expression in escherichia coli The resistances such as ooze, be with high salt, is arid.Being transferred to the Escherichia coli of irrE genes and rape has high salt resistance ability, therefore the gene It is with a wide range of applications on cultivating drought resistance and salt tolerance new varieties crop

With the development of the society, more and more based on the genetically modified crops commercial growth of non-transgenic elite characteristic by state Family's approval, the food and environmental security that thus may be brought cause the great attention of people.To genetically modified crops or turn The detection and identification of gene modified food is not only the inevitable requirement of relevant laws and regulations, and meets the necessary technology of public right to information.Cause This, establishes effective, sensitivity, fast and accurately detection method, is both the important research content of transgenosis safe evaluation, and Further strengthen important technology guarantee of the China in terms of genetically modified crops and product supervision.

In the development of genetically modified plants or when carrying out screening or safety evaluatio to transgenic product, generally require Qualitative or semiquantitative determination is carried out to the allogenic gene being transferred to.And the use of monoclonal antibody is to implement immunology detection technology Necessary condition, hypersensitivity and specificity monoclonal antibody be then to advanced optimize the important guarantee of reaction.

Invention content

The object of the present invention is to provide the monoclonal antibodies of a kind of higher susceptibility and the anti-IrrE albumen of specificity.

To achieve the goals above, on the one hand, the present invention provides a kind of hybridoma cell strain, the hybridoma cell strain Deposit number is CGMCC NO.12268.

In another aspect, the present invention also provides a kind of monoclonal antibody, which is CGMCC by deposit number The hybridoma cell strain of NO.12268 generates.

In another aspect, the present invention also provides monoclonal antibodies as described above to detect the transgenic crop for turning IrrE In purposes.

On the other hand, the present invention also provides a kind of methods for the transgenic crop for detecting and turning IrrE, wherein this method Include the following steps：S1, holoprotein is extracted from crops to be detected；S2, list as described above is used to the holoprotein Clonal antibody carries out immuning hybridization；If there is the positive band of IrrE in S3, immuning hybridization result, it indicates that agriculture to be detected Crop is the transgenic crop for turning IrrE.

Through the above technical solutions, the present invention can be detected IrrE albumen by WESTERN BLOT methods, and And cross reaction is not also presented to the various non-IrrE genetically modified crops of 7 kinds of separate sources.

Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.

Biomaterial preservation

The hybridoma cell strain of secretion monoclonal antibody 1DB4 of the present invention is voluntarily merged what screening obtained by inventor, is protected It is CGMCC NO.12268 to hide number, and the deposit date is on 04 07th, 2016, depositary institution was Chinese microorganism strain preservation Administration committee's common micro-organisms center, address are located at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism Research institute, Classification And Nomenclature are anti-IrrE monoclonal antibody hybridoma cells strain.

Description of the drawings

Attached drawing is to be used to provide further understanding of the present invention, an and part for constitution instruction, with following tool Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings：

Fig. 1 is the expression and purification result figure of His-Tag IrrE fusion proteins in embodiment 1.

Fig. 2 is the result figure that monoclonal antibody affinity chromatography purifies in embodiment 2.

Fig. 3 is the result figure that the dilution of monoclonal antibody 1DB4 low powers applies in Western Blot detections in embodiment 2.

Specific implementation mode

The specific implementation mode of the present invention is described in detail below in conjunction with attached drawing.It should be understood that this place is retouched The specific implementation mode stated is merely to illustrate and explain the present invention, and is not intended to restrict the invention.

On the one hand, the present invention provides a kind of hybridoma cell strains, and the deposit number of the hybridoma cell strain is CGMCC NO.12268。

Wherein, the method for monoclonal antibody being generated by the hybridoma cell strain that deposit number is CGMCC NO.12268 can be with For the method for this field routine, such as mouse ascites method.

Wherein, the IrrE albumen refers to the albumen of IrrE coded by said gene, and IrrE genes refer to that NCBI Gene ID are 1798483 gene (number by gene：DR_0167).

Wherein it is possible to using the protein immunization detection method of this field routine by monoclonal antibody as described above for examining Survey turns IrrE gene crops.For example, holoprotein can be extracted from crops to be detected, immuning hybridization is then used The method of (WESTERN BLOT) is detected, if the holoprotein of crops to be detected monoclonal antibody as described above There is the positive band of IrrE after carrying out immuning hybridization, it indicates that crops to be detected are the transgenic crop for turning IrrE.

Optionally, wherein the crops are cauliflower, rape, cotton, corn, rice or soybean.

In method as described above, optionally, the crops are cauliflower, rape, cotton, corn, rice or big Beans.

Hereinafter, present invention will be further described in detail through examples.In following embodiment, reagent used is commercially available obtains .

Embodiment 1

The induced expression of IrrE albumen and purifying.

According to the Deinococcus radiodurans Genomic sequence information announced, primer amplified target gene piece is designed Section.Deinococcus radiodurans genomic DNA is extracted using Tiangeng bacterial genomes extracts kit.To extract and after purification resistance to Radiation abnormal cocci genomic DNA is template, carries out PCR reactions using specific primer IrrE (F)/IrrE (R), expands resistance to spoke Abnormal cocci IrrE gene orders are penetrated, are expanded using exo+ polymerase in PCR reactions, to ensure the accurate of DNA fragmentation Property.Primer used in PCR is IrrE (F)：5 '-CACAGGAGGACCCCATATGCCCAGTGC-3 ' (SEQ ID NO.1) and IrrE(R):5'-ACCAAGCTTAGTTCACTGTG-3'(SEQ ID NO.2)。

PCR product upstream and downstream contains Nde I and Hind III restriction enzyme digestion sites respectively.By the PCR of acquisition After product purification recycling, carried out using restriction enzyme Nde I and Hind III double digested.It similarly utilizes restricted Restriction endonuclease Nde I and Hind III carry out double digested colibacillus high expression vector pET28a.By above-mentioned digestion products into Row purifying recycling, is attached under T4DNA connection enzyme effects, and connection product enters common protein expression place by heat-shock transformed In main bacterial strain BL21 (DE3), and recombinant bacterial strain is coated on the LB solid mediums containing kanamycins.Several single bacteriums of picking Same resistant panel of transferring in order is fallen, and is cultivated in the LB liquid medium containing antibiotic of transferring simultaneously, thalline is extracted In the Plasmid DNA that contains carry out digestion and PCR verifications；Recombinant bacterial strain is verified also with bacterium colony round pcr in experiment. Obtained correct recombinant plasmid pET-IrrE does further verification by DNA sequencing.Sequencing result shows the coding of IrrE genes Sequence is as shown in SEQ ID NO.3, and the protein amino acid sequence of coding is as shown in SEQ ID NO.4.

SEQ ID NO.4 are：MPSANVSPPCPSGVRGGGMGPKAKAEASKPHPQ IPVKLPFVTAPDALAAAKARMRDLAAAYVAALPGRDTHSLMAGVPGVDLKFMPLGWRDGAFDPEHNVILINSAARPE RQRFTLAHEIGHAILLGDDDL LSDIHDAYEGERLEQVIETLCNVAAAAILMPEPVIAEMLERFGPTGRALAELAKRAEVSASSALYALTEQTPVPVIY AVCAPGKPPREQAASDEDAGPSTEKVLTVRASSSTRGVKYTLASGTPVPADHPAALALATGMEVREESYVPFRSGRK MKAEVDAYPSRGIVAVSFEFDPARLGRKDSEQADRDEPQDAAQ；Referring specifically to sequence table.

SEQ ID NO.3 are：ATGCCCAGTGCCAACGTCAGCCCCCCTTGCCC CTCTGGGGTAAGGGGCGGGGGGATGGGGCCAAAAGCTAAAGCTGAAGCCTCCAAGCCCCACCCCCAAATCCCTGTTA AGCTCCCATTCGTGACCGCCCCCGACGCCCTCGCCGCCGCCAAAGCCAGGATGCGCGACCTGGCGGCGGCCTACGTG GCGGCCCTGCCCGGACGCGACACCCACAGCCTGATGGCGGGGGTGCCCGGCGTAGACCTCAAATTCATGCCGCTCGG CTGGCGCGACGGGGCGTTCGACCCCGAGCACAACGTCATCCTCATCAACTCGGCGGCCCGCCCCGAACGCCAGCGCT TCACCCTCGCCCACGAAATCGGGCACGCGATTTTACTCGGCGACGACGACCTGCTCTCCGACATCCACGACGCCTAC GAGGGCGAGCGGCTCGAACAGGTCATCGAAACGCTGTGCAACGTGGCGGCGGCGGCGATTTTGATGCCCGAACCCGT CATCGCGGAAATGCTGGAACGCTTCGGCCCCACCGGGCGCGCCCTCGCCGAACTCGCCAAGCGGGCCGAAGTCAGCG CGTCGTCGGCGCTCTACGCCCTGACCGAGCAGACCCCGGTGCCCGTCATCTACGCGGTCTGTGCGCCGGGCAAGCCT CCGCGTGAGCAGGCCGCAAGCGACGAGGACGCTGGCCCAAGCACAGAAAAAGTCCTGACGGTCCGCGCCAGCAGCTC GACGCGGGGCGTCAAGTACACCCTGGCGAGCGGCACGCCGGTACCCGCCGACCACCCGGCGGCGCTTGCCCTCGCCA CGGGCATGGAAGTGCGCGAGGAAAGCTACGTGCCCTTTCGCTCGGGCCGGAAAATGAAGGCGGAGGTGGACGCCTAC CCGTCGCGCGGCATCGTGGCCGTCAGTTTCGAGTTCGACCCCGCCCGCCTGGGCCGCAAGGACAGCGAGCAGGCCGA CCGGGACGAGCCGCAGGACGCTGCACAGTGA；Referring specifically to sequence table.

Gene in the carrier pET-IrrE of the high efficient expression IrrE albumen of structure contains terminator codon, the fusion of expression The aminoterminal of IrrE albumen carries 6His-Tag and can be used for isolating and purifying for albumen.It will contain recombinant plasmid pET-IrrE's Switching is in fresh culture medium after the activation of BL21 (DE3) bacterial strain.Strain growth is 0.6 or so to OD values, is added into culture medium Enter the IPTG that final concentration is respectively 0.5mM, 16 DEG C of culture 12h, inducing cell expresses IrrE albumen.Utilize IrrE fusion protein ammonia The His-Tag that base end is contained, using the NTA resins progress affinitive layer purification of the Ni-NTA couplings fusion protein.It collects 40mM imidazole elutions take off the destination protein in liquid, are detected by SDS-PAGE, the results are shown in Figure 1.In Fig. 1, swimming lane M is point Son amount label, swimming lane 1 are control strain whole-cell protein；Swimming lane 2 is expression IrrE cell soluble proteins after IPTG inductions；Swimming Road 3 is expression IrrE cell insoluble albumens after IPTG inductions；Swimming lane 4 is soluble protein loading efflux；Swimming lane 5 is NTA-0 Eluent；Swimming lane 6 is His-Tag IrrE fusion proteins after purification, it is seen that purity is higher.The IrrE albumen of purifying can be used for Prepare its monoclonal antibody.

Embodiment 2

The preparation and identification of IrrE protein monoclonal antibodies

By 6-8W⁺The female mouse of BALB/c be according to dosage divided into four groups, according to dosage 50,100,150,200 μ g/ only (are implemented The IrrE albumen that example 1 obtains) it is immunized, it is immunized 3 times altogether.Take blood as negative control before initial immunity, when initial immunity Add the subcutaneous multi-point injection of isometric Freund's complete adjuvant；Primary immunization is carried out every surrounding later, is added with same dose recombinant antigen The subcutaneous multi-point injection of isometric freund 's incomplete adjuvant.It is coated with ELISA with recombinant antigen within 10 days or so after third time is immune Plate measures the antibody titer of mice serum with indirect ELISA；Merge first three day highest to antibody titer (1:10⁵More than) small 50 μ g of tail vein injection recombinate IrrE albumen booster immunizations.Booster immunization carries out cell fusion after three days.

Cell fusion the previous day, feeder cells are prepared in accordance with the following methods：1) by 8W⁺Healthy male BALB/c mouse, After drawing neck to put to death, 3-5min is impregnated in 75% ethyl alcohol；2) it moves in super-clean bench, skin is cut off with sterile scissors, expose peritonaeum, and With its peritonaeum of 75% ethanol disinfection；3) with haemostatic clamp gently pull-up peritonaeum, 1640 fluid nutrient mediums of 10mL pre-temperatures are injected Device injects abdominal cavity, and abdominal cavity 1-2min is gently rubbed with cotton balls, and cell suspension is sucked out, is put into centrifuge tube；4) it centrifuges：1000 × g, 5min abandons supernatant；5) with 10mL HAT containing serum culture mediums by cell mixing, cell count, adjustment cell density is in 2 × 10⁵/ mL；6) 96 porocyte culture plates are added by 100 holes μ L/ in this cell suspension, then cell density is 2 × 10⁴/ hole；7) 37 DEG C are set, 5%CO₂Incubator culture, for next day fusion experiment.

The preparation of myeloma cell SP2/0：1) exponential phase SP2/0 cells are harvested, 3 are washed with 1640 fluid nutrient mediums Secondary, 1000 × g centrifuges 5min, abandons supernatant；2) cell is resuspended with 1640 fluid nutrient mediums.100 μ L cell suspensions are taken, with 0.2% Trypan Blue carries out cell count, it is desirable that cell viability>95%, and it is for use to adjust cell density.

The preparation of splenocyte suspension：1) BALB/c mouse being immunized through overbump before 3 days is extractd into eyeball bloodletting, at the neck that breaks Extremely, 2min is impregnated in 75% ethyl alcohol；2) it moves in super-clean bench, abdominal cut skin is removed to both sides, exposure stomach wall；Change scissors, Tweezers cut off peritonaeum, take out spleen, remove fat and connective tissue, are rinsed with 1640 culture mediums；3) spleen is placed in 200 mesh Sieve on, lightly ground with piston on one side；It is rinsed on one side with culture solution, collection splenocyte suspension, centrifugation 1000 × G, 5min abandon supernatant；4) cell, centrifuge washing 2 times is resuspended with 1640 culture mediums：1000 × g, 5min abandon supernatant；5) 10mL is used Incomplete culture medium is resuspended.100 μ L cell suspensions are taken, with 0.2% Trypan Blue, it is desirable that cell viability>95%, and count spleen Cell.It is for use that remaining cell adjusts cell density.

Cell fusion and culture：1) by splenocyte and SP2/0 myeloma cell with 5:1 ratio is mixed in 50mL centrifuge tubes In, 1000 × g centrifuges 5min, abandons supernatant, flicks centrifugation bottom of the tube, keeps cell precipitation loose；2) one side uniform rotation centrifuge tube, The 50%PEG4000 1mL of 37 DEG C of pre-temperatures are added dropwise with suction pipe on one side, in being completed in 1min；3) add 1640 culture mediums of 37 DEG C of preheatings 1mL is completed in 1min；4) the 1640 culture medium 10mL for adding 37 DEG C of preheatings, are completed in 5min；5) 800 × g centrifuges 8min； Cell precipitation is resuspended with 100mL HAT culture mediums；6) cell suspension is transferred to by 100 holes μ L/ and is vaccinated with the thin of feeder cells In born of the same parents' culture plate, while staying 2 holes that not fused SP2/0 cells is added to compare, sensibility of the observation cell to HAT.Culture plate It is placed in 37 DEG C, 5%CO₂It is cultivated in incubator；7) after merging 3 days, the fresh HAT culture solutions of 100 μ L are added per hole.Later per 3-5 It is primary that its half amount replaces HAT culture solutions；8) after 2 weeks, HT culture mediums half, which are measured, changes liquid；9) it after 3-4 weeks, changes complete medium and maintains training It supports.

ELISA screens positive hybridoma cell：1) merge after cell growth arrive culture hole floor space 1/4 when (cultivate About 12-15 days or so), supernatant indirect elisa method detection specific reaction and cross reaction are taken, hybridoma is sieved Choosing.Recombinant protein IrrE is envelope antigen, and 5 μ g/mL of peridium concentration are routinely coated with elisa plate.Add 100 μ into coating elisa plate The cell culture supernatant in the holes L/, with PBS 1:100 diluted immune mouse serums are positive control, in SP2/0 cell hole cultures It is negative control clearly.37 DEG C of incubation 30min of cell conditioned medium and coating elisa plate, are fully added the sheep anti mouse of HRP labels after washing IgG antibody (1:10000 dilutions) 100 holes μ L/, 37 DEG C are incubated 30min, abandon secondary antibody.Add 100 μ L of TMB aobvious per hole after abundant board-washing Color 15min adds 50 μ L 1N H per hole₂SO₄Terminate reaction.Measure OD₄₅₀Value.2) ELISA screenings obtain 98 plants of secretion IrrE The cell strain of monoclonal antibody；Monoclonal antibody secreted by the cell strain of above-mentioned 98 plants of IrrE monoclonal antibodies has sun to recombination IrrE albumen Property reaction.The selection wherein strongest 37 plants of cells of positive reaction are further subcloned culture, remaining cell strain directly expands culture, It freezes and produces ascites on a small quantity.

Limiting dilution assay carries out colonized culture：1) cell to be cloned is blown and beaten into mixing with pipettor, with containing 20% blood Clear HT selection culture solutions are diluted to the density of 1 cells/well, and the cell plates for having feeder cells are added, set 5%CO₂、37℃ Incubator in cultivated；2) when culture was to the 4th day, cell monoclonal growth hole is observed and recorded under inverted microscope； Culture 1 week or so draws the cell culture supernatant that 100 μ L have turned yellow, with above-mentioned ELISA method detection cells and supernatant；3) The hole inner cell for being detected as strong positive carries out 2-3 subclone, the training of to the last primary all only one cell colony growths Until foster hole supernatant ELISA testing results are the positive；4) by best miscellaneous of ELISA testing results after last time limiting dilution It hands over oncocyte clone to expand culture, and freezes.

The preparation of ascites：1) 10 week old BALB/c mouses are chosen, intraperitoneal injection atoleine 0.5mL/ is only；2) after 7 days, abdomen Chamber inoculation is through the diluted cultures of PBS to the positive hybridoma cell of logarithmic phase, every mouse 5 × 10⁵/ mL hybridomas；3)5 It is observed after it, when mouse web portion obviously expands, collects ascites with No. 12 injection needles, collected once, until mouse every 3 days It is dead；4) ascites is centrifuged into 10min with 7 000 × g；- 80 DEG C of refrigerators preserve after staying supernatant to dispense.

The purifying (protein A affinity chromatographys) of monoclonal antibody：1) column is filled：With Equilibration Buffer (50mM Tris-HCl, 150mM NaCl, pH8.6) moistens pillar, checks whether pillar blocks, and adds 5mL Protein AAgarose is in column；2) the Equilibration Buffer that 10 times of column volumes are added balance pillar；It 3) slowly will be in ascites Sample；4) the Equilibration Buffer of 10 times of column volumes are added, liquid is penetrated until OD by the collection of 4-5mL/ pipes₂₈₀<0.1；5) Prepare collecting pipe, neutralization buffer (20mM phosphate buffers, pH 7.7) is added by 500 μ L/ pipes in the test tube for collecting eluent； 6) it is eluted with the Elution Buffer of 5 times of column volumes (50mM glycine, 0.5M NaCl, pH 2.3), is received by 1.5mL/ pipes Collect eluent until OD₂₈₀<0.1；7) column and balance pillar are washed with the Equilibration Buffer of 5 times of column volumes.

It is adapted to the monoclonal antibody screening of WESTERN BLOT：

By obtained 37 plants of purified monoclonal antibodies, using WESTERN BLOT methods, by IrrE recombinant antigens and non- The specificity screening of IrrE genetically modified crops finally filters out the monoclonal antibody just for IrrE high specific high sensitivities.

The operating procedure of WESTERN BLOT monoclonal antibodies screening：Albumen is separated by electrophoresis in 12%SDS-PAGE.Swimming lane 1 is Albumen Marker；Swimming lane 2 is recombination IrrE albumen (1mg/mL)；Swimming lane 3 is to turn IrrE rape seeds extract (0.2g/mL)； Swimming lane 4 is to turn NPTII rice paddy seeds extract (0.2g/mL)；Swimming lane 5 is to turn BT Cry1Ac corn seed extracts (0.2g/ mL)；Swimming lane 6 is to turn phy corn seeds extract (0.2g/mL)；Swimming lane 7 is to turn CP4-EPSPS soya seeds extracts (0.2g/mL)；Swimming lane 8 is to turn G2-EPSPS corn seeds extract (0.2g/mL)；Swimming lane 9 is to turn PAT/bar corn seeds pumping Extract (0.2g/mL)；Swimming lane 10 is to turn BT/CPTI cotton seeds extract (0.2g/mL)；Albumen in half-dried transfer gel is extremely Nitrocellulose membrane (NC films), 20 volts of constant pressure 1h；5% skimmed milk power will be added in PBST as with confining liquid, electricity takes after the completion of turning Go out NC films to be put into confining liquid, overnight, PBST washs 5min × 3 time for 4 DEG C of closings；With 37 plants filtered out anti-IrrE monoclonal antibodies difference For primary antibody, 1 is pressed respectively with confining liquid:200 dilution proportions, shaken at room temperature abandon reaction solution after being incubated 2h, and PBST washs NC films 5min × 3 times；It marks sheep anti-mouse igg as secondary antibody using HRP, 1 is pressed with confining liquid:10000 dilution proportions are abandoned after shaking table incubation 2h at room temperature Reaction solution, PBST wash 5min × 4 time；DAB developing solutions develop the color, and observe result.

The identification of monoclonal antibody subclass：Using immunoglobulin standard subgroup identification kit (Sigma companies) to each Strain monoclonal antibody carries out subgroup identification, and specific test method is as follows：1 is separately added into ELISA Plate:1000 times of diluted sheep anti mouses of PBS IgG(IgM、IgA、IgG₁、IgG_2a、IgG_2bAnd IgG₃), 100 holes μ L/, 37 DEG C of placement 1h；Liquid in ELISA Plate is discarded, PBST is used Washing 3 times；The monoclonal antibody purification (antibody concentration 2-5 μ g/mL) after PBS dilutions is added in 100 holes μ L/, is incubated at room temperature 1h； PBST is washed 3 times；The sheep anti-mouse igg antibody (1 of HRP labels is added:10000 dilutions) 100 holes μ L/, it is incubated at room temperature 30min；It fills Add 100 μ L TMB after point board-washing per hole, develop the color 15min, adds 1N H₂SO₄50 holes μ L/ terminate reaction.Measure OD₄₅₀Value.

The qualification result of monoclonal antibody purity and subclass：Experiments have shown that monoclonal antibody 1DB4 is suitable for WESTERN BLOT, monoclonal antibody 1DB4 purification result such as Fig. 2 show that purity is more than 95%.

Monoclonal antibody 1DB4 subgroup identification results are IgG1 hypotypes.

Monoclonal antibody specificity and sensitivity evaluation：WESTERN BLOT experiment screenings to monoclonal antibody 1DB4 press 1:200、1:500、1:1000、1:2000、1:When 5000 dilution, recombination IrrE albumen is detected.Testing result shows, no Monoclonal antibody 1DB4 with extension rate is able to detect that recombination IrrE albumen, illustrates that monoclonal antibody 1DB4 has good detection spirit Sensitivity.In table 1 ,+represent there is the positive band concentration of quantity representative band (+) ,-to represent band invisible, ± represent item Band and background difference be not notable or has miscellaneous band, it is difficult to judge；It is same as below.

Table 1

The hybridoma cell strain for producing 1DB4 monoclonal antibodies is subjected to preservation, deposit number is CGMCC NO.12268.

Monoclonal antibody 1DB4 is pressed 1:200 dilutions to recombination IrrE albumen, turn IrrE crop seeds and 7 kinds of differences The various non-IrrE genetically modified crops in source carry out WESTERN BLOT detections.As a result as shown in Table 2, monoclonal antibody 1DB4 can Specific detection is to recombination IrrE albumen and turns IrrE crop seeds, as a result as Fig. 3 shows.To the various non-of 7 kinds of separate sources Cross reaction is not also presented in IrrE genetically modified crops.Illustrate that monoclonal antibody 1DB4 has good detection sensitivity.

Table 2

	1DB4(1:200 dilutions)
		Recombinate IrrE albumen (1mg/mL)	+++
Turn IrrE rape seeds extract (0.2g/mL)	++
		Turn NPTII rice paddy seeds extract (0.2g/mL)	-
Turn BT Cry1Ac corn seeds extracts (0.2g/mL)	-
		Turn phy corn seeds extract (0.2g/mL)	-
Turn CP4-EPSPS soya seeds extract (0.2g/mL)	-
		Turn G2-EPSPS corn seed seed extracts (0.2g/mL)	-
Turn PAT/bar maize seed extracts (0.2g/mL)	-
		Turn BT/CPTI cotton seeds extract (0.2g/mL)	-

According to embodiment 2 as can be seen that the present invention has screened a kind of monoclonal antibody 1DB4, it can be used in WESTERN BLOT is detected.It can detect recombination IrrE albumen and turn IrrE crop seeds；To the various non-IrrE transgenosis of 7 kinds of separate sources Cross reaction is not also presented in crop.Monoclonal antibody 1DB4 has very excellent sensibility and specificity.

Comparative example 1

Remaining 10 plants in 37 plants of purified monoclonal antibodies that embodiment 2 is obtained in addition to 1DB4, use WESTERN BLOT Method, by 1:200 dilutions, carry out the detection of specificity and sensitivity, the results are shown in Table 3.

Table 3

According to table 3 as can be seen that monoclonal antibody 1DB4 has very excellent sensibility compared to other monoclonal antibodies And specificity.

The preferred embodiment of the disclosure is described in detail above in association with attached drawing, still, the disclosure is not limited to above-mentioned reality The detail in mode is applied, in the range of the technology design of the disclosure, a variety of letters can be carried out to the technical solution of the disclosure Monotropic type, these simple variants belong to the protection domain of the disclosure.

It is further to note that specific technical features described in the above specific embodiments, in not lance In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the disclosure to it is various can The combination of energy no longer separately illustrates.

In addition, arbitrary combination can also be carried out between a variety of different embodiments of the disclosure, as long as it is without prejudice to originally Disclosed thought equally should be considered as disclosure disclosure of that.

Sequence table

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cagaccccgg tgcccgtcat ctacgcggtc tgtgcgccgg gcaagcctcc gcgtgagcag 660

gccgcaagcg acgaggacgc tggcccaagc acagaaaaag tcctgacggt ccgcgccagc 720

agctcgacgc ggggcgtcaa gtacaccctg gcgagcggca cgccggtacc cgccgaccac 780

ccggcggcgc ttgccctcgc cacgggcatg gaagtgcgcg aggaaagcta cgtgcccttt 840

cgctcgggcc ggaaaatgaa ggcggaggtg gacgcctacc cgtcgcgcgg catcgtggcc 900

gtcagtttcg agttcgaccc cgcccgcctg ggccgcaagg acagcgagca ggccgaccgg 960

gacgagccgc aggacgctgc acagtga 987

<210> 4

<211> 328

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 4

Met Pro Ser Ala Asn Val Ser Pro Pro Cys Pro Ser Gly Val Arg Gly

1 5 10 15

Gly Gly Met Gly Pro Lys Ala Lys Ala Glu Ala Ser Lys Pro His Pro

20 25 30

Gln Ile Pro Val Lys Leu Pro Phe Val Thr Ala Pro Asp Ala Leu Ala

35 40 45

Ala Ala Lys Ala Arg Met Arg Asp Leu Ala Ala Ala Tyr Val Ala Ala

50 55 60

Leu Pro Gly Arg Asp Thr His Ser Leu Met Ala Gly Val Pro Gly Val

65 70 75 80

Asp Leu Lys Phe Met Pro Leu Gly Trp Arg Asp Gly Ala Phe Asp Pro

85 90 95

Glu His Asn Val Ile Leu Ile Asn Ser Ala Ala Arg Pro Glu Arg Gln

100 105 110

Arg Phe Thr Leu Ala His Glu Ile Gly His Ala Ile Leu Leu Gly Asp

115 120 125

Asp Asp Leu Leu Ser Asp Ile His Asp Ala Tyr Glu Gly Glu Arg Leu

130 135 140

Glu Gln Val Ile Glu Thr Leu Cys Asn Val Ala Ala Ala Ala Ile Leu

145 150 155 160

Met Pro Glu Pro Val Ile Ala Glu Met Leu Glu Arg Phe Gly Pro Thr

165 170 175

Gly Arg Ala Leu Ala Glu Leu Ala Lys Arg Ala Glu Val Ser Ala Ser

180 185 190

Ser Ala Leu Tyr Ala Leu Thr Glu Gln Thr Pro Val Pro Val Ile Tyr

195 200 205

Ala Val Cys Ala Pro Gly Lys Pro Pro Arg Glu Gln Ala Ala Ser Asp

210 215 220

Glu Asp Ala Gly Pro Ser Thr Glu Lys Val Leu Thr Val Arg Ala Ser

225 230 235 240

Ser Ser Thr Arg Gly Val Lys Tyr Thr Leu Ala Ser Gly Thr Pro Val

245 250 255

Pro Ala Asp His Pro Ala Ala Leu Ala Leu Ala Thr Gly Met Glu Val

260 265 270

Arg Glu Glu Ser Tyr Val Pro Phe Arg Ser Gly Arg Lys Met Lys Ala

275 280 285

Glu Val Asp Ala Tyr Pro Ser Arg Gly Ile Val Ala Val Ser Phe Glu

290 295 300

Phe Asp Pro Ala Arg Leu Gly Arg Lys Asp Ser Glu Gln Ala Asp Arg

305 310 315 320

Asp Glu Pro Gln Asp Ala Ala Gln

325

Claims

1. a kind of hybridoma cell strain, which is characterized in that the deposit number of the hybridoma cell strain is CGMCC NO.12268.

2. a kind of monoclonal antibody, which is characterized in that the monoclonal antibody is by the hybridization that deposit number is CGMCC NO.12268 Tumor cell strain generates.

3. purposes of the monoclonal antibody in detection turns the transgenic crop of IrrE described in claim 2.

4. purposes according to claim 3, wherein the crops are cauliflower, rape, cotton, corn, rice or big Beans.

5. purposes according to claim 3 or 4, wherein if the holoprotein of crops to be detected claim 2 institute There is the positive band of IrrE after carrying out immuning hybridization in the monoclonal antibody stated, it indicates that crops to be detected are to turn IrrE bases The crops of cause.

6. a kind of method detecting the transgenic crop for turning IrrE, wherein this method includes：

S1, holoprotein is extracted from crops to be detected；

S2, immuning hybridization is carried out using the monoclonal antibody described in claim 2 to the holoprotein；

If there is the positive band of IrrE in S3, immuning hybridization result, it indicates that crops to be detected are to turn turning for IrrE Gene crops.

7. according to the method described in claim 6, wherein, the crops are cauliflower, rape, cotton, corn, rice or big Beans.