CN107513521A - Hybridoma cell strain, secreted monoclonal antibody and its application in bar/PAT albumen is detected - Google Patents
Hybridoma cell strain, secreted monoclonal antibody and its application in bar/PAT albumen is detected Download PDFInfo
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- CN107513521A CN107513521A CN201710782130.9A CN201710782130A CN107513521A CN 107513521 A CN107513521 A CN 107513521A CN 201710782130 A CN201710782130 A CN 201710782130A CN 107513521 A CN107513521 A CN 107513521A
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- C—CHEMISTRY; METALLURGY
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- G01N2333/91057—Acyltransferases other than aminoacyltransferases (general) (2.3.1) with definite EC number (2.3.1.-)
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Abstract
The invention discloses a pair of hybridoma cell strains, secreted monoclonal antibody and its application in bar/PAT albumen is detected.The present invention discloses one plant of secrete monoclonal antibody Ab1 hybridoma cell strain first, and its microbial preservation numbering is CGMCC No.11094.The invention also discloses one plant of secrete monoclonal antibody Ab2 hybridoma cell strain, its microbial preservation numbering is CGMCC No.11095.The present invention further discloses a kind of immune colloid gold test paper of detection bar/PAT albumen.The immune colloid gold test paper prepared using the monoclonal antibody Ab1 and monoclonal antibody Ab2 of hybridoma cell strain of the present invention secretion, the detection sensitivity and specificity to bar/PAT albumen are high.Hybridoma cell strain of the present invention and its monoclonal antibody of secretion have application prospect in the detection of antiweed genetically modified plants.
Description
Technical field
The present invention relates to a pair of hybridoma cell strains, further relate to a pair of the pairing Dan Ke secreted by the hybridoma cell strain
Grand antibody and their purposes in bar/PAT albumen is detected, belong to agricultural biological technical field.
Background technology
Since mankind's farming, the approach of effective management of weeds just is explored, sprinkling herbicide is presently the most in field
Efficient mode.But because herbicide can also result in weeds and plant death, the genetically modified crops with anti-herbicide gene
Commercialization, become as most one of character of advantage in agricultural production.Planting anti-fecundi-t can be using between narrow row
Away from method, so as to plant more crops on equivalent farming area, the yield of crop is added.In addition, the antiweed of plantation
Transgenic crop need not handle the residue of field crops after using herbicide, reduce the quantity of labour
And dynamics.By 2003, Resistant Herbicide Crops accounted for more than the 80% of global genetically modified crops.From development trend, turn base
The crop ratio of cause is still being continuously increased.Because antiweed mark is widely used in commercialization genetically modified plants, with turning base
Because of the fast development of food, the crop of external transgenosis and food largely enter China, and the food inspection technology of transgenosis is to turn
The important technological platform of gene modified food safety evaluatio, its research have great importance.
Bialaphos is one kind of numerous antibiotic of streptomycete production, and its chemical constitution includes glutamic acid isomers
(phosphinothricin (PPT)) and two alanine residues (- alanyle-alanine).Bialaphos is glutamine
The inhibitor of synzyme (glutamine synthetase).Generated after removing alanine residue by the effect of cell restriction endonuclease
Active glufosinate ultimately results in the ammonia of high concentration in vivo so as to suppress the effect of glutamine synthelase
(including plant and bacterium) assembles and produces toxicological effect to organism.Thus, glufosinate turns into a kind of wide variety of
Non-specificity selection herbicide.Certain micro-organisms can produce a kind of enzyme by carrying out acetylation to amino, so as to this effect of detoxifying
Should, this resistant gene is named as Bar, and title is taken from bialaphos resistance.The albumen of the gene code can
So that phosphinothricin acetylations to be become to the albumen phosphinothricin acetyl transferase of avirulent form
(phosphinothricin acetyl transferase, PAT), the albumen for also having the gene expression is referred to as bar/PAT.Bar
Gene is widely used in genetic engineering breeding, and the marker gene in genetic transformation, it make plant resistant with
Phosphinothricin is the herbicide of active component.
Carry out the detection of transgenosis foreign protein, be included in the development of genetically modified plants or enter to transgenic product
When row screening or safety evaluatio, main method has Western, enzyme linked immunosorbent assay (ELISA) etc..These methods all by
The limitation of the factors such as instrument, place, and detection time is grown, and testing cost is higher, it is difficult to popularization and application.Therefore, needed in practice
Have one kind can bar/PAT albumen in quick detection genetically modified plants, and quick detection, device easy to operate, reliable results.
Immune one kind that association colloid gold chromatography is immuno-gold labeling technology and antigen-antibody reaction is combined and formed
Application form, similar with ELISA, simply label is different.The method using microporous barrier as solid phase carrier, including double antibody sandwich method,
Dual-antigen sandwich method, prize law and the implementations such as suppression method are striven unexpectedly.Double antibody sandwich method is mainly for detection of there is multiple antigens
The biomolecule in site.Its technical scheme includes：First by known specific antibody using a certain amount be coated on film as
Detection band, the secondary antibody that can be combined with gold mark thing is as quality control band.Another monoclonal antibody gold mark conjugate to match with coated antibody is then
It is adsorbed in gold standard pad and is dried.Dry gold standard pad one end connects with film, and one end is connected with sample pad.The opposite side of film
Paste adsorptive pads.During detection, a certain amount of liquid sample is added in sample pad, by capillarity, sample is along sample pad to suction
The direction movement of water cushion.It first passes around dry gold standard pad, redissolves gold mark conjugate, as contained determined antigen in sample, then
Generation antigen-antibody reaction, form compound A (gold grain-antibody-antigene)；Compound A continues to move to reach detection band position
Put, then antigen-antibody reaction occurs again with coated antibody, forming compound B, (gold grain-antibody-antigene-coating resists
Body), and assemble in the position of detection band, macroscopic degree is finally reached, as that without antigen to be checked, then can not examined in sample
Measuring tape forms macroscopic red stripes.Free gold mark conjugate or the compound A not being captured cross detection band arrival
Quality control band, reacted with the secondary antibody that can be combined with gold mark thing, form compound C (gold grain-antibody-secondary antibody), be gathered in
Quality control band simultaneously produces macroscopic red stripes.Whether no matter material to be checked is contained in sample, red can be all presented in quality control band
Band.
Immune association colloid gold chromatography have it is simple to operate, quick, can single part examine, do not need the excellent of special installation
Point, but the defects of generally existing sensitivity is low.Law limitation is analysed to make up immune association colloid layer gold, it is preferred that emphasis is improves anti-Dan Ke
The sensitivity and specificity of grand antibody pair.Therefore, exploitation has high specific and sensitivity, and with other transgenic lines or table
Up to the anti-PAT protein monoclonal antibodies of albumen no cross reaction, will turn to fast and effeciently detecting the agricultural product such as plant, food
Gene element, and detection to public health emergency event etc. are significant.
The content of the invention
First technical problem to be solved by this invention is to provide a pair of hybridoma cell strains, and this is to hybridoma cell strain
Secreted monoclonal antibody forms monoclonal antibody pair, high to the sensitivity and specificity of bar/PAT Protein Detections；
Second technical problem to be solved by this invention is the Dan Ke by above-mentioned a pair of hybridoma cell strains and its secretion
Grand antibody is applied to detection bar/PAT albumen.
In order to solve the above technical problems, the technical solution used in the present invention is：
(its amino acid sequence is SEQ ID NO to restructuring bar/PAT albumen of the invention using expression and purification：Shown in 2, compile
The nucleotides sequence of code gene is classified as SEQ ID NO：Shown in 1) the female mouse of immune Balb/c, by immune spleen cell and SP2/0 marrow
Oncocyte merges, and screening obtains the positive hybridoma cell strain of 111 plants of secretion bar/PAT monoclonal antibodies.Above-mentioned 111 plants of cell lines divide
The monoclonal antibody secreted has positive reaction to restructuring PAT albumen.The present invention is by 111 plants of monoclonal antibodies of purifying group two-by-two
Detection is closed, 12321 is obtained altogether and assembles to monoclonal antibody, prepare colloidal gold strip respectively, exclude cross reaction, screen a pin
To the pairing monoclonal antibody of bar/PAT high specific high activity.Finally, the present invention is obtained with optimum sensitivity and specific the
One monoclonal antibody Ab1 and second monoclonal antibody Ab2.
The qualification result of monoclonal antibody subclass shows, monoclonal antibody Ab1 heavy chain types are IgG2a, monoclonal antibody
Ab2 heavy chain types are IgG3；Monoclonal antibody Ab1 and Ab2 light chain are Kappa chains.
Secrete monoclonal antibody Ab1 hybridoma cell strain 1BE6 is submitted the mechanism of patent accreditation to be protected by the present invention
Hide, its microbial preservation numbering is：CGMCC No.11094；Classification And Nomenclature is：Anti- Bar monoclonal antibody hybridoma cells strain.
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center；The preservation time is on 08 12nd, 2015；
Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Secrete monoclonal antibody Ab2 hybridoma cell strain 2CH12 is submitted the mechanism of patent accreditation to be protected by the present invention
Hide, its microbial preservation numbering is：CGMCC No.11095；Classification And Nomenclature is：Anti- Bar monoclonal antibody hybridoma cells strain.
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center；The preservation time is on 08 12nd, 2015；
Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
A pair of hybridoma cell strains that the present invention is obtained, and monoclonal antibody Ab1 or monoclonal antibody Ab2 can be answered
For detecting antiweed genetically modified plants or its derived product (i.e. the agricultural product such as the food containing transgene component, drink).
Wherein, the antiweed genetically modified plants are to turn bar/pat gene plants.
A pair of hybridoma cell strains that the present invention is obtained can be applied to detection bar/PAT albumen, be applied especially to make
The reagent of standby detection bar/PAT albumen.
Monoclonal antibody Ab1 or monoclonal antibody Ab2 of the present invention can be applied to detection bar/PAT albumen, especially
Applied to the reagent for preparing detection bar/PAT albumen.
The present invention further discloses 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 (nature controlling line) and T lines (detection line), the C lines are provided with the basement membrane
On be coated with sheep anti-mouse igg, be coated with second monoclonal antibody on the T lines；Contain colloid gold label in the colloidal gold pad
The first monoclonal antibody；First monoclonal antibody and the second monoclonal antibody form monoclonal antibody pair；Wherein,
First monoclonal antibody is monoclonal antibody Ab1；The second monoclonal antibody is monoclonal antibody Ab2.
Stability that reaction film adsorbs to detection sensitivity, detection time, specificity and immobilization etc. has considerable influence.
The present invention is basement membrane from nitrocellulose filter (NC), because its binding ability to albumen, the holding of activity and production cost
Etc. compare other film classes there is greater advantage.
The assembling of immune colloid gold test paper of the present invention includes：The basement membrane, absorption pad, colloid of C lines and T lines will be coated with
Gold pad and sample pad are adhered to by underboarding successively, are produced.
Match antibody sensitivity technique result to show, the first monoclonal antibody Ab1 and second monoclonal antibody Ab2, counterweight
Group bar/PAT Protein Detections sensitivity reaches 1ng/mL；Detection sensitivity to turning bar/PAT rice paddy seeds is examined up to 0.2 μ g/mL
Survey high sensitivity.Pairing antibody specificity testing result shows that the first monoclonal antibody Ab1 and second monoclonal antibody Ab2 are right
25 kinds of non-transgenic crops and the non-bar/PAT genetically modified crops of 15 kinds of separate sources do not have cross reaction, have higher spy
The opposite sex.Result above proves, monoclonal antibody Ab1 and Ab2 of the present invention antiweed transgenic product detection and
In terms of prepare transgenosis safety evaluation reagent, there is application prospect.
Technical solution of the present invention compared with prior art, has the advantages that：
Anti- bar/PAT the protein monoclonal antibodies Ab1 and Ab2 that present invention screening obtains, have high specific and sensitivity,
It is high with other transgenic lines or expressing protein no cross reaction, potency.The glue prepared by pairing monoclonal antibody Ab1 and Ab2
Body gold test paper strip can be used in quickly and efficiently detecting the bar/PAT transgene components of the agricultural product such as plant, food, Yi Jiying
For the detection of public health emergency event, the detection for antiweed transgenic product is provided to technical support.
Brief description of the drawings
Fig. 1 is the assembly structure diagram of immune colloid gold test paper；Wherein, 1 is sample pad；2 be by underboarding；3 be glue
Body gold pad；4 be T lines；5 be C lines；6 be cellulose membrane；7 be absorption pad；
Fig. 2 is using the signal colour depth caused by collaurum, carries out qualitative or half-quantitative detection result figure；Wherein, A
Detection sensitivity for pairing monoclonal antibody to restructuring bar/PAT albumen；B is pairing monoclonal antibody to turning bar/PAT water
The detection sensitivity of rice；
Fig. 3 is using the signal colour depth caused by collaurum, to 25 kinds of non-transgenic crops and 15 kinds of separate sources
The result figure of various non-bar/PAT genetically modified crops (No 1-40) specific detection experiments.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.It should be understood that the embodiment is only exemplary, any restrictions are not formed to the scope of the present invention.This area
Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and
Form is modified or replaced, but these modifications or substitutions each fall within protection scope of the present invention.
Embodiment 1 recombinates the preparation of bar/PAT albumen
According to LabLife (https://www.lablife.org/ll) issue pCAMBIA-3301 in bar/pat
Expressed sequence, according to the nucleotide sequence of its whole protein expression, separately design specific primer.The starting for retaining gene is close
Numeral, delete terminator codon.The pair of primers (table 1) for expanding this fragment, extension increasing sequence are devised using PRIMER 5.0
(nucleotides sequence is classified as SEQ ID NO to total length 552bp：Shown in 1, the amino acid sequence of coding is SEQ ID NO：Shown in 2).
The primer sequence of table 1
| Title | Primer sequence | Introduce restriction enzyme site |
| Sense primer | 5’-AAAGGATCCATGAGCCCAGAACG-3’ | BamHI |
| Anti-sense primer | 5’-GGCAAGCTTAATCTCGGTG-3’ | HindIII |
Using pCAMBIA-3301 plasmids as template, pcr amplification product reclaims pure after BamH I/Hind III double digestions
Change, direct it and be connected in the pET-32a expression vectors through BamH I/Hind III double digestions and be transformed into expression bacterial strain
In BL21 (DE3) cell, extraction plasmid is identified, after digestion identification and sequencing through PCR, and recombinant plasmid is named as into pET-
32a-bar。
PCR reaction systems are：Ex Taq(5U/μL)0.5μL、10×Ex Taq Buffer 5μL、dNTP(2.5mmol/
L) 5 μ L, each 1 μ l of upstream and downstream primer (25nmol/ μ L), the μ L of template 5, add water to 50 μ L；PCR reaction conditions are：94℃1min；94
DEG C 45s, 60 DEG C of 30s, 72 DEG C of 1min, 30 circulations；72℃5min.Digestion identification reaction system is：10×Buffer for
μ L, the BamH I of BamH I 2 and each 0.5 μ L of Hind III enzymes, the μ L of plasmid 7, add water to 20 μ L.
Bacterial strain BL21 (DE3) competent cell is reached with positive recombinant plasmid pET-32a-bar translation tables, picking single bacterium colony,
It is added in LB fluid nutrient mediums of the 10mL containing ampicillin, 37 DEG C of overnight incubations carry out increasing bacterium.1mL bacterial cultures is taken to connect
For kind into LBs of the 50mL containing ampicillin, 37 DEG C are continued culture to OD600For 0.6~1.0 when add derivant IPTG to final concentration
For 0.5mmol/L.37 DEG C are continued to cultivate, and take bacterium solution that bacterial precipitation is collected by centrifugation after 4h, and the N-terminal of fusion protein contains 6
His-Tag, therefore affinitive layer purification is carried out using the NTA resins of Ni-NTA couplings.First by fusion protein supernatant progressively
It is added drop-wise in chromatographic column, utilizes Action of Gravity Field filtering solution.Then chromatographic column use containing different imidazole gradients (10,50,100,150,
200,250mM) NTA elution albumen, fraction collection eluent.The egg that polyacrylamide gel electrophoresis detection is collected into
Purifying protein is carried out with dialyzate (20mM Hepe, pH7.5,1mM DTT, 200mM KCl and 50% glycerine) after Bai Chundu
Analysis, the albumen of purifying is dispensed, -70 DEG C of preservations.
The preparation and identification of the bar/PAT protein monoclonal antibodies of embodiment 2
1st, experimental method
1.1 mouse are immunized
Immune (blood is taken before initial immunity as negative control) is according to dosage grouped from the 8W+ female mouse of Balb/c, is exempted from
Epidemic disease dosage is respectively 50,100,150,200 μ g/ (the restructuring bar/PAT albumen that i.e. embodiment 1 obtains).Recombinant antigen adds
The subcutaneous multi-point injection of volume Freund's complete adjuvant is immunized；Primary immune response was carried out every two weeks later, is added with same dose antigen
The subcutaneous multi-point injection of isometric freund 's incomplete adjuvant, supplementary immunization is twice.Restructuring is used after the immune end of third time within 10 days or so
Bar/PAT albumen is coated with elisa plate, and mice serum potency is determined with indirect ELISA.It is high to antibody titer to merge first three day
(105More than) mouse tail vein injection booster immunization (50 μ g).Cell fusion is carried out after supplementary immunization after 72h.
1.2 cell fusion
1.2.1 prepared by feeder cells
Non-immune BALB/c mouse is taken, eye socket sacrificed by exsanguination, collects negative serum, 5min is soaked in 75% alcohol and is disappeared
Poison.The mouse disinfected is fixed on dissection plate, lower abdomen skin is clamped with tweezers, cuts an osculum, tear skin and expose abdomen
Film, transducer set tweezers and scissors, an osculum (in belly center) is cut on peritonaeum, then the liquid of 3.0mL 1640 is drawn with suction pipe and notes
Enter mouse peritoneal, gently pressure-vaccum is transferred in a sterile 50.0mL centrifuge tubes several times, then by liquid, is repeated once.Contain blood with 10mL
Cell is mixed, counts, adjusts cell density by clear HAT culture mediums.Culture puts 37 DEG C, 5%CO in 96 porocyte culture plates2
Incubator culture, for next day fusion experiment.
1.2.2 myeloma cell SP2/0 preparation
The mouse cervical dislocation of back growth myeloma cell is put to death.2min~5min in 75% ethanol is immersed in disappear
Malicious fur.Mouse takes out tumor tissue backwards to fixing under aseptic condition, is homogenized, is hanged with basic culture solution 20mL.Suspension is slow
Adding oneself has in the centrifuge tube of 20mL lymphocyte separation mediums.1,800r/min centrifugation 10min, draws in nutrient solution and separating liquid
Between fine and close white cellular layer.Cell is washed with basic culture solution 1640 twice, and cell is resuspended with 10mL basic culture solutions, with
0.2% Trypan Blue, carry out cell count, it is desirable to cell viability>95%, and it is stand-by to adjust cell density.
1.2.3 the preparation of splenocyte suspension
Learnt from else's experience and the BALB/c mouse of impact is immunized, eye socket bloodletting, after collecting positive serum.Mouse peritoneum is cut off, cut
Broken spleen outer membrane, exposes spleen, removes the adipose tissue sticked on spleen with scissors.Spleen is placed on 200 mesh stainless steels
In cell screen clothes, spleen is extruded with the nook closing member of 5ml syringes, scattered splenocyte is fallen into the homogenizer of sterilizing by screen cloth
In.Cleaned, blown even with appropriate 1640 basal liquid.1200r/min centrifugations 10min removes supernatant, and it is standby that counting is resuspended in cell.
1.2.4 cell fusion and culture
Feeder cells 1000r/min is first centrifuged into 5min, removes supernatant, is suspended with appropriate HAT culture mediums, in 37 DEG C of placements
It is standby.By l × 107~2 × 107Individual SP2/0 myeloma cell and 108Individual immune spleen cell (l:10~l:5) in 50mL centrifuge tubes
Middle mixing, 1500r/min centrifugations 10min.Supernatant is abandoned, tipping upside down on the filter paper of sterilizing blots liquid feed in pipe.Gently strike
Ttom of pipe is hit, cell precipitation is loosened slightly, centrifuge tube is put in 37 DEG C of water-baths.Then pre-temperature is slowly instilled in 1min to 37
DEG C 50%PEG 0.8mL, side edged gently with inhale point stirring.Continue to stir 1min, be then slowly added into 37 DEG C of pre-temperatures
1640 basal liquid 10mL.Specific method is:L.0mL 1min is instilled dropwise, 2min adds 1.0mL, 3min~4min to add
3.0mL, 5min add remaining 5.0mL, and each added-time need to be slowly added to, and constantly lightly stir, and are finally slowly added into
The liquid of 30mL 1640.1000r/min centrifuges 5min, removes supernatant.After ibid being cleaned once with 1640 basal liquids, hanged with HAT culture mediums
The cell of floating mixing, the raising splenocyte that HAT culture mediums suspend is added, add appropriate HAT culture mediums as needed, mixing is equal
Even, point kind is in 96 well culture plates, about 250 μ L/ holes.Culture plate after cell fusion is placed in 37 DEG C, 5%CO2Saturated humidity is trained
Support and cultivated in case, 1/2 nutrient solution in hole is put with HAT selection nutrient solutions after 5d.Hereafter HT nutrient solution cultures are used, and gradually decrease HT
Content.After 7~10d cell colony calculated in 2mm sizes fusion rate (hybrid cell growth hole count/culture hole sum ×
100%) detection, is prepared.
1.3 ELISA screen positive hybridoma cell
Cell culture after fusion about 12-15 days or so, when growing into the 1/4 of culture hole floor space, supernatant is taken with indirectly
ELISA method detection specific reaction and cross reaction, are screened to hybridoma.Add 100 μ l supernatants per hole, with immune
Mouse serum is positive control (1:50 dilutions, dilution PBS), SP2/0 is negative control, and recombinant protein bar/PAT-Ag is bag
It is conventional to be coated with elisa plate by antigen.Cell conditioned medium is incubated 1h with coating elisa plate, and the sheep of HRP marks is fully added after washing
Dynamics (1:10000 dilutions) 100 μ l/ holes, 37 DEG C of incubation 30min, abandon secondary antibody.Add TMB 100 per hole after abundant board-washing
μ l colour developing 15min, add 1N H2SO450 μ l/ holes terminating reactions.Determine OD450Value.
ELISA screenings obtain the cell line of 111 plants of secretion bar/PAT monoclonal antibodies；Dan Ke secreted by above-mentioned 111 plants of cell lines
Grand antibody has positive reaction to restructuring PAT albumen.Selection wherein 30 plants of most strong cells of positive reaction are further subcloned
Culture, remaining cell line directly expand culture, freeze and produce ascites on a small quantity.
1.4 limiting dilution assays carry out colonized culture
The hybridoma of cell conditioned medium test positive is cloned using limiting dilution assay, it is specific as follows:Clone
It is preceding to prepare mouse feeder cells with reference to the above method, it is laid in Tissue Culture Plate, puts standby in incubator.With cutting tip
200 μ L pipette tips are blown and beaten in hole to be cloned is resuspended cell for several times, with blood counting chamber to cell count, by cell training completely
Foster base is diluted to the density of 1 cells/well.Add and put CO2Incubator culture.Cultivate 4d and change liquid once, in inverted microscope
It is lower to observe and record cell monoclonal growth hole；Culture 1 week or so, when cell culture fluid turns yellow, carries out mark.After cloning
When 7d~9d cell clones cover with 1/4~1/3 visual field, 100 μ L of supernatant are drawn, with above-mentioned ELISA method detection cell culture
Supernatant, calculate hybridoma positive boring ratio rate (positive hole count/hybrid cell growth hole count × 100%).Such as detect cell life
Elongated hole has specific antibody, and antibody titer height may be selected, and in single clonal growth, the good cell hole of form, continues with same
Culture is cloned or expanded to method again.Positive hole cell can be moved to 24 well culture plates, and another batch of culture medium is added in foramen primum, with
The two anti-pollution or cell death simultaneously.When the cell growth in 24 orifice plates is good, by ELISA after last time limiting dilution
The best hybridoma cell clone of testing result goes to 6 well culture plates, finally collects cell to 100mL blake bottles, and freeze 2
Cell above.
Clone need to typically be carried out more than 2-3 times, and the positive rate that hole is grown until cell colony reaches 100%, then can recognize
Obtain secreting the monoclonal hybridoma system of monospecific antibody for oneself.
A large amount of preparations of 1.5 monoclonal antibodies
1.5.1 the preparation of ascites
The BALB/c mouse of 8~10 week old is taken, atoleine 0.5mL/ of sterilizing is injected intraperitoneally, will be right after 7d~10d
Number growth period hybridoma adjusts cell density about 10 with basic culture solution7/mL.Hybridoma 0.5mL/ is injected intraperitoneally
Only, after waiting mouse peritoneal to expand, mouse ascites is extracted with No. 12 syringe needles, centrifuging and taking supernatant, carry out potency survey according to the method described above
Determine and freeze.
1.5.2 the purifying of monoclonal antibody
Add 5ml Protein A Agarose in post.The Equilibration Buffer for adding 10 times of column volumes are put down
Weigh after pillar, addition and the isometric ascites of pillar, slowly flow across gel bed；And by the efflux of collection upper prop again.With
Equilibration Buffer wash away foreign protein, are collected by 4-5ml/ pipes and penetrate liquid until OD280<0.1.Finally plus Elution
Buffer (pH 2.3) elutes to antibody.Collecting pipe is added pH 7.7 phosphoric acid buffer before antibody is collected by 500 μ l/ pipes
Liquid.Eluent is collected by 2ml/ pipes until OD280<0.1.Finally with the Equilibration Buffer of 5 times of column volumes wash post and
Balance pillar.
The pairing screening of 1.6 monoclonal antibodies
Obtained 111 plants of purified monoclonal antibodies are coated with nitrocellulose filter and mark collaurum respectively, prepare collaurum examination
Paper slip, then combination of two detect, be obtained 111 × 111 (12321) to combination, exclude cross reaction, filter out just for
The pairing monoclonal antibody of bar/PAT high specific high activity.
1.6.1 the gold mark of monoclonal antibody and film preparation
Collaurum is prepared using trisodium citrate reduction method, concrete operations are：Measure l00mL with volumetric flask three boil off
Ionized water, pour into the boiling flask that specification is 500.0mL, flask is placed in the heating mantle of magnetic force heating stirrer, be put into
Magnetic stir bar, stirring knob is opened to appropriate speed, heating knob is opened, is heated to seething with excitement.Add 1.0mL 1%HAuCL4
Solution, continue to heat 2min, be then disposably rapidly added 1% citric acid three sodium solution, continue to heat.Lurid gold chloride
Gradually by xanthochromia ash, blackening finally reddens or orange red the aqueous solution again in 2min after trisodium citrate addition.Treat that solution is changed into bright
After red or orange red, heating stirring 15min is further continued for.Turn off heating turn-knob, it is naturally cold really to room temperature.
The preparation of collaurum-antibody conjugates (conjugate)：By the purified monoclonal antibody of suitable concentration be added on set up pH and from
In the colloidal gold solution of sub- intensity, room temperature reaction 2min is placed.Add final concentration of 0.2% PEG4000 12,000rpm from
Heart 30min, red precipitate is sucked into another 12ml centrifuge tubes, the stillness of night centrifuges 30min with 8000rpm, takes precipitation.Precipitation colloid
Golden protective agent suspends, and it (is usually OD to be diluted to working concentration532=30-40) 4 DEG C preserve, or be sprayed at non-woven thin-film, 37 DEG C
After drying, it is put into aluminium foil bag, and is put into drier, seals, 4 DEG C of preservations.
The coating of nitrocellulose filter (NC)：Reaction film influences detection sensitivity, detection time, specificity and immobilization and inhaled
Attached stability.The present invention selects NC films, because its binding ability to albumen, the holding of activity and production cost etc. are compared
Other film classes have larger advantage.The coating of nitrocellulose filter:It is single with 0.0l M pH7.2 PBS dilution purifying
Resist to 2mg/mL, for being coated with T lines, sheep anti-mouse igg is diluted to l mg/mL with 0.0l M pH7.2 PBS, for wrapping
By C lines.It is sprayed on BIODOT companies XYZ3050 work systems with 30mm/s speed on nitrocellulose filter, formation is parallel to each other
Detection T lines and Quality Control C lines, 37 DEG C drying.
1.6.2 the assembling of immune colloid gold test paper
The nitrocellulose filter 6 of C lines 5 and T lines 4 will be coated with, absorption pad 7, colloidal gold pad 3 and sample pad 1 are glued successively
The PVC that does not absorb water is invested by underboarding 2, as Fig. 1 is assembled into immune colloid gold test paper.
1.6.3 antibody conjugates screen
111 plants of monoclonal antibodies, 111 × 111 (12321) are obtained altogether and are assembled to monoclonal antibody, are combined into 12321 kinds
Colloidal gold strip.Detection is with ddH2O recombinates PAT albumen 1ng/mL, turns the extracting of bar/PAT rice paddy seeds as negative sample
Thing 0.2g/mL is as positive sample.And to 25 kinds of non-transgenic crops and the various non-bar/PAT transgenosis of 15 kinds of separate sources
Crop has been cooked specificity screening.Experiment has finally given optimum sensitivity and specific first monoclonal antibody Ab1 and second
Monoclonal antibody Ab2.
The identification of 1.7 monoclonal antibody subclass
Each strain monoclonal antibody is carried out using immunoglobulin standard subgroup identification kit (Southern Biotech companies)
Subgroup identification.Bar/PAT albumen is cut purpose adhesive tape, pulverize, add as much as possible in EP pipes through SDS-PAGE electrophoresis first
Enter appropriate PBS, 4 DEG C overnight.Supernatant is taken after next day 5000rpm centrifugations 5min, surveys protein concentration.Enzyme mark is coated with according still further to 4 μ g/mL
Plate, per the μ L of hole 100,37 DEG C overnight.PBST adds 100 μ L 0.5%BSA closings, 37 DEG C of placement 1h per hole after washing 3 times.So
Each cell culture supernatant, 100 μ L/ holes, 37 DEG C of incubation 1h are separately added into the ELISA Plate closed afterwards；PBST is washed 3 times,
Each 5min.Sequentially added in most backward ELISA Plate with PBS 1:The sheep anti mouse secondary antibody of the HRP marks of 250 dilutions is (respectively anti-
Mouse κ, λ, IgM, IgA, IgG1、IgG2a、IgG2bAnd IgG3), 100 μ L/ holes, 37 DEG C are placed 1h, and PBST is washed 3 times, each 5min.
Add the μ l of TMB 100 colour developing 15min after abundant board-washing per hole, add 1N H2SO450 μ l/ holes terminating reactions.Determine OD450Value.
2nd, experimental result
The qualification result of 2.1 monoclonal antibody subclass
Each monoclonal antibody heavy chain qualification result is as shown in table 2；Light chain is Kappa chains.
The identification of the monoclonal antibody subclass of table 2
| Antibody | Ab1 | Ab2 |
| Subclass of antibody | IgG2a | IgG3 |
2.2 pairing antibody sensitivity evaluations
The pairing monoclonal antibody that the present invention screens：First monoclonal antibody Ab1 and second monoclonal antibody Ab2 is right
Restructuring bar/PAT Protein Detections sensitivity reaches 1ng/mL；To turning the detection sensitivity of bar/PAT rice paddy seeds up to 0.2 μ g/mL
(Fig. 2).
2.3 pairing antibody specificity evaluations
The pairing monoclonal antibody that the present invention screens：First monoclonal antibody Ab1 and second monoclonal antibody Ab2 is right
25 kinds of non-transgenic crops and the various non-bar/PAT genetically modified crops of 15 kinds of separate sources have done specific detection experiment, inspection
Survey result and see Fig. 3 and table 3.
It can be seen that pairing monoclonal antibody Ab1 and Ab2 does not have to all detection non-transgenics and non-bar/PAT genetically modified crops
There is cross reaction, there is higher specificity.And other pairing monoclonal antibody (screening Hes of reference examples 1 that the present invention screens
Screening reference examples 2) specificity be then significantly worse than pairing antibody A b1/Ab2.
The present invention finally gives to be resisted with optimum sensitivity and specific first monoclonal antibody Ab1 and the second monoclonal
Body Ab2, it can be used in preparing the reagent of detection genetically modified crops and Transgene-safty evaluation.
The hybridoma cell strain 1BE6 for producing Ab1 monoclonal antibodies is submitted Chinese microorganism strain preservation management committee by the present invention
Member's meeting common micro-organisms center carries out preservation, and its microbial preservation numbering is：CGMCC No.11094；Production Ab2 monoclonals are resisted
The hybridoma cell strain 2CH12 of body submits China Committee for Culture Collection of Microorganisms's common micro-organisms center to carry out preservation,
Its microbial preservation is numbered：CGMCC No.11095.
The specific detection result of table 3
SEQUENCE LISTING
<110>Biological Technology institute, Chinese Academy of Agricultural Sciences Liu Qi
<120>Hybridoma cell strain, secreted monoclonal antibody and its application in bar/PAT albumen is detected
<130> BJ-2002-161113A
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 552
<212> DNA
<213> artifical 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> artifical 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> artifical sequence
<400> 3
aaaggatcca tgagcccaga acg 23
<210> 4
<211> 19
<212> DNA
<213> artifical 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.
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| CN110133292A (en) * | 2019-06-04 | 2019-08-16 | 中国农业科学院生物技术研究所 | A kind of colloidal gold immunochromatographimethod quick measuring card of herbicide resistant protein Bar |
| CN110205300A (en) * | 2019-06-04 | 2019-09-06 | 中国农业科学院生物技术研究所 | Bar monoclonal antibody hybridoma cell strain, antibody of generation and preparation method thereof |
| CN110221078A (en) * | 2019-06-04 | 2019-09-10 | 中国农业科学院生物技术研究所 | A kind of enzyme linked immunological kit of quantitative detection herbicide resistant protein Bar |
| CN111733141A (en) * | 2020-06-19 | 2020-10-02 | 清华大学深圳国际研究生院 | Hybridoma cell capable of secreting monoclonal antibody against novel coronavirus N protein, monoclonal antibody and application |
| CN112326969A (en) * | 2020-10-26 | 2021-02-05 | 中国农业科学院生物技术研究所 | Enzyme linked immunosorbent assay kit for quantitatively detecting herbicide-resistant protein PAT/PAT |
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| CN115094042A (en) * | 2022-05-30 | 2022-09-23 | 中国农业科学院生物技术研究所 | PAT/PAT monoclonal antibody hybridoma cell strain, antibody produced by same and application of antibody |
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| CN108395477A (en) * | 2018-02-06 | 2018-08-14 | 中国农业科学院生物技术研究所 | Purposes of the monoclonal antibody FB9b in detecting GAT transgenic crops |
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| CN110133292A (en) * | 2019-06-04 | 2019-08-16 | 中国农业科学院生物技术研究所 | A kind of colloidal gold immunochromatographimethod quick measuring card of herbicide resistant protein Bar |
| CN110205300A (en) * | 2019-06-04 | 2019-09-06 | 中国农业科学院生物技术研究所 | Bar monoclonal antibody hybridoma cell strain, antibody of generation and preparation method thereof |
| CN110221078A (en) * | 2019-06-04 | 2019-09-10 | 中国农业科学院生物技术研究所 | A kind of enzyme linked immunological kit of quantitative detection herbicide resistant protein Bar |
| CN111733141A (en) * | 2020-06-19 | 2020-10-02 | 清华大学深圳国际研究生院 | Hybridoma cell capable of secreting monoclonal antibody against novel coronavirus N protein, monoclonal antibody and application |
| CN111733141B (en) * | 2020-06-19 | 2022-02-18 | 清华大学深圳国际研究生院 | Hybridoma cell capable of secreting monoclonal antibody against novel coronavirus N protein, monoclonal antibody and application |
| CN112326969A (en) * | 2020-10-26 | 2021-02-05 | 中国农业科学院生物技术研究所 | Enzyme linked immunosorbent assay kit for quantitatively detecting herbicide-resistant protein PAT/PAT |
| CN112798793A (en) * | 2020-12-30 | 2021-05-14 | 中国农业科学院油料作物研究所 | Test strip and test card for detecting PAT/bar protein, and preparation method and application thereof |
| CN115094042A (en) * | 2022-05-30 | 2022-09-23 | 中国农业科学院生物技术研究所 | PAT/PAT monoclonal antibody hybridoma cell strain, antibody produced by same and application of antibody |
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Application publication date: 20171226 |