CN104497142B - The monoclonal antibody of CP4 EPSPS albumen - Google Patents
The monoclonal antibody of CP4 EPSPS albumen Download PDFInfo
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- CN104497142B CN104497142B CN201410770732.9A CN201410770732A CN104497142B CN 104497142 B CN104497142 B CN 104497142B CN 201410770732 A CN201410770732 A CN 201410770732A CN 104497142 B CN104497142 B CN 104497142B
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Abstract
It is related to the monoclonal antibody for detecting resistance glyphosate CP4 EPSPS albumen the present invention relates to the technical field of antigen-antibody protein, particularly one of present invention.The monoclonal antibody includes weight chain variable district and light chain variable district, and the amino acid sequence such as SEQ ID NO of weight chain variable district:Shown in 1, the amino acid sequence such as SEQ ID NO of light chain variable district:Shown in 2；Two provide a kind of application of said monoclonal antibody；Three provide and a kind of be used to detect the kit of CP4 EPSPS albumen；Four provide a kind of application of the kit；Five provide a kind of DNA sequence dna, the DNA sequence dna includes the DNA sequence dna for encoding the weight chain variable district of above-mentioned monoclonal antibody, and the light chain variable district of the above-mentioned monoclonal antibody of coding DNA sequence dna；And six provide a kind of cell, above-mentioned DNA sequence dna is contained in the cell.
Description
Technical field
The present invention relates to the technical field of Ag-Ab protein, the more particularly to CP4- for detecting resistance glyphosate
The monoclonal antibody of EPSPS albumen.
Background technology
With the development of transgenic technology, substantial amounts of genetically modified crops enter commodity circulation.Wherein, with Meng Shandou
(Monsanto) patent genes of company, CP4-EPSPS genes are most widely used.At present to be widely used in corn and soybean, cotton
The crops such as flower.CP4-EPSPS gene sources are in soil Agrobacterium strain (Agrobacterium sp) CP4 strains, CP4-
EPSPS gene code CP4-EPSPS synzyme, the enzyme has resistance to herbicide glyphosate.
Because various transgene agricultural product by trade largely enters international market, the security of GM food is standby
Paid close attention to by people.Countries in the world have larger dispute to the security of GM food, and the area such as European Union, Japan is
Relevant laws and regulations are formulated and strict management have been carried out to transgene agricultural product.
At present, China is the fourth-largest Soybean production state of the world, and annual production is only second to the U.S., Brazil and Argentina.2010,
About 14,500,000 tons of Chinese soybean yield, and imported soybean reaches 47,500,000 tons, has exceeded as many as 3 times of output in domestic.Soybean is
One of China's main body entrance agricultural product, but the genetically engineered soybean of the mostly antiweed of imported soybean, thus to turning
The management of gene modified food becomes an important topic of China.
And to the management of transgene agricultural product, be since being detected transgene agricultural product.At present, for transgenic product
Detection mainly has using DNA as mesh object detection method, such as PCR methods, Southern blot methods and biology by target of DNA
Chip method etc.；And using albumen as mesh object detection method, such as ELISA or colloidal gold fast detecting test paper strip.Wherein,
Although DNA detection sensitivity is high, be due to different crops CP4-EPSPS detection without general primer, to sample
Acquisition process and extracting method poor repeatability, cause PCR results false positive and false negative rate high, the cumbersome time-consuming, inspection of detection method
Survey cost height and need to be equipped with more advanced expensive instrument, testing staff will could also be competent at by professional training.These shortcomings
Its a large amount of or conventional detection for importing and exporting product in international and national trade from undertaking is seted to work, it is also difficult to be pushed away in grass-roots unit
Extensively use.Using albumen as mesh object detection method, because sensitivity is not high enough, also occur that false positive and/or false negative rate are high
The problem of.
For this reason, it may be necessary to develop, one kind can save time, reduction testing cost and operation more simply can be accurate
Detection turns the product of the genetically modified plants of CP4-EPSPS genes.
The content of the invention
One of present invention provides a kind of monoclonal antibody of CP4-EPSPS albumen, and the monoclonal antibody includes heavy chain
Variable region and light chain variable district, and the amino acid sequence such as SEQ ID NO of the weight chain variable district:Shown in 1, the light chain variable
The amino acid sequence in area such as SEQ ID NO:Shown in 2.
The affinity constant of the monoclonal antibody is 2.61 × 1012.That is, itself and the antigenic determinant of antigen
Specifically bind intensity very high, better than the bond strength of the monoclonal antibody of CP4-EPSPS albumen of the prior art, because
And, the application of the antibody is more more advantageous than the application of the antibody of the prior art.Utilizing double crush syndrome detection
During target protein, CP4-EPSPS monoclonal antibodies of the invention and other antibody for example can be Beijing Hua Da protein research and development
The article No. of centered finite company is AbM59705-3-PU monoclonal antibody, during pairing antibody, can detect content as little as
1ng/ml transgene protein, and with 100% specificity.This is absolutely proved, uses the CP4-EPSPS Dan Ke of the present invention
Grand antibody is at a relatively high as sensitivity and specificity when detecting the genetically modified plants containing CP4-EPSPS genes, thus more holds
Target gene easily is detected, and is unlikely to missing inspection or false retrieval.This is by identifying that the genetically modified plants containing CP4-EPSPS genes have
There is important meaning, be that the management of the transgene agricultural product of China has stepped solid step forward.
Moreover, can accurately detect turning containing CP4-EPSPS genes using CP4-EPSPS proteantigens-antibody response
Gene plant, and the time can be saved, testing cost is reduced, is operated more simple.
In a specific embodiment, the monoclonal antibody can resist selected from univalent antibody, single-chain antibody, double-strand
One kind in body, bispecific antibody and chimeric antibody.
In a specific embodiment, the monoclonal antibody is CGMCC NO by deposit number:10099 hybridoma
Cell line secretion is produced.
The two of the present invention provide a kind of application of monoclonal antibody as described above.
The three of the present invention provide a kind of kit for being used to detect CP4-EPSPS albumen, and the kit is included as above
Described monoclonal antibody.
The kit for being used to detect CP4-EPSPS albumen of the present invention includes but is not limited to the immune layer of ELISA kit, enzyme
Analyse detection kit, chemiluminescence detection kit and immunofluorescence detection agent box.
It is ELISA detection kit in a specific embodiment, sample dilution can be included in the kit
Liquid, cleaning solution, nitrite ion, terminate liquid, positive control and negative control.Positive control for example can be protokaryon or eukaryotic expression
Purifying CP4-EPSPS albumen；Negative control for example can be the holoprotein of nontransgenic soy seeds.
In a specific embodiment, the sample diluting liquid in the ELISA detection kit is containing de-
The phosphate buffer of fat milk, the preferably phosphate buffer containing 10% (W/V) defatted milk；
The cleaning solution is the phosphate buffer containing Tween-20, preferably containing 0.05% (V/V) Tween-20
Phosphate buffer；
The nitrite ion includes nitrite ion A and nitrite ion B, the nitrite ion A contain 3,3', 5,5'- tetramethyl benzidines
(TMB) 20mg, absolute ethyl alcohol 10ml, and use ddH2O is settled to 100ml；It is nitrite ion B 2.1g containing citric acid, anhydrous
Na2HPO42.82g, 0.75% (V/V) hydrogen peroxide urea 0.64ml, and use ddH2O is settled to 100ml；
The terminate liquid is 2mol/L H2SO4。
Term " phosphate buffer " refers to containing phosphoric acid or its salt and the solution for being brought to desired pH, is biochemistry
The most widely used a kind of buffer solution in research.Usually, phosphate buffer (including but is not limited from phosphoric acid or phosphate
In sodium and sylvite) prepare.Some phosphate, such as sodium dihydrogen phosphate and potassium dihydrogen phosphate, phosphoric acid hydrogen have been known in the art
Disodium and dipotassium hydrogen phosphate, sodium phosphate and potassium phosphate.Having known phosphate is present with the hydrate forms of salt.Due to slow
Two grades of dissociations of fliud flushing, the pH value range of buffering is very wide, e.g., from about pH 4 to about pH 10 scope, preferably from about pH 5 to
PH 9 scope, the scope for more having choosing about pH 6 to about pH 8, most preferably from about pH 7.4.It is further preferred that the phosphate
Buffer solution is the phosphate buffer of sodium chloride-containing and/or potassium chloride.
The preparation method of ELISA detection kit for detecting CP4-EPSPS albumen may comprise steps of：
(1) CP4-EPSPS holoproteins (such as SEQ ID NO are cloned:Sequence shown in 6) DNA (such as SEQ ID NO:
Sequence shown in 5), and carry out protein expression；
(2) hybridoma is produced using the protein immunization mouse, then by substantial amounts of screening, obtains function admirable
The anti-CP4-EPSPS albumen of secretion monoclonal antibody CP4-EPSPS AS5102；Wherein, monoclonal antibody CP4-EPSPS
AS5102 is CGMCC NO by preserving number:10099 hybridoma cell strain secretion is produced.
In a specific embodiment, the preparation method of the ELISA detection kit can also include above-mentioned step
Suddenly after (2) the step of (3)：Monoclonal antibody CP4-EPSPS AS5102 obtained by step (2) are carried out on ELISA Plate
Coating；Commercially available monoclonal antibody is subjected to enzyme mark；The commercially available monoclonal antibody for example can be Beijing Hua Da protein research and development
The article No. of centered finite company is AbM59705-3-PU monoclonal antibody；
(4) prepare sample diluting liquid, cleaning solution, nitrite ion A liquid, nitrite ion B liquid, terminate liquid, prepare positive control and
Negative control；
(5) component for preparing step (3), and/or optional component prepared by (4) are assembled into ELISA detection kit.
As a kind of preferred embodiment of the present invention, in the preparation method of the ELISA detection kit of the present invention
In, coating buffer used can be carbonate buffer solution, preferably pH9.6 carbonate buffer when being coated with the step (3)
Liquid, 4 DEG C of coatings are overnight or 37 DEG C are coated with 2 hours, and confining liquid used is 1% (W/V) bovine serum albumin(BSA) (BSA) during closing
Phosphate buffer (PBS), the phosphate buffer of 5% (W/V) skimmed milk power, the phosphate buffer of 1.5% (V/V) gelatin
Or 5% (V/V) calf serum FCS any of phosphate buffer, 37 DEG C are closed 2 hours, cleaning solution used during washing
For the phosphate buffer containing Tween-20, the preferably phosphate buffer containing 0.05% (V/V) Tween-20.
The application method of the ELISA detection kit of the present invention may comprise steps of：
(1) serum to be checked is added to the ELISA Plate for being coated with monoclonal antibody CP4-EPSPS AS5102 by 100 μ l/ holes
On, 37 DEG C of reaction 60min add 200 μ l/ holes cleaning solutions and washed 3-5 times, 1min/ times, patted dry after washing on blotting paper.
(2) by the 1 of 100 μ l/ holes:Horseradish peroxidase (the Horseradish of 10000 times (V/V) dilution
Peroxidase, HRP) mark commercially available monoclonal antibody add to the enzyme for being coated with monoclonal antibody CP4-EPSPS AS5102
On target, 37 DEG C of reaction 60min add 200 μ l/ holes cleaning solutions and washed 3-5 times, 1min/ times, clapped after washing on blotting paper afterwards
It is dry.The commercially available monoclonal antibody for example can be that the article No. of BeiJing HuaDa protein Research Center Co., Ltd is AbM59705-
3-PU monoclonal antibody.
(3) the substrate nitrite ion A and nitrite ion B μ l/ holes of mixed liquor 100, room temperature avoid light place 15min are added.
(4) 50 μ l/ holes terminate liquids are added with terminating reaction.
(5) OD is read on ELIASA450Value, and calculated and judged according to criterion.
In a specific embodiment, the application method of the ELISA detection kit can also include：With it is above-mentioned
Step (1) is while the step of carrying out (1 ')：Negative control, positive control are added by 100 μ l/ holes and are coated with monoclonal antibody
On CP4-EPSPS AS5102 ELISA Plate, 37 DEG C of reaction 60min add 200 μ l/ holes cleaning solutions and washed 3-5 times, 1min/ times, wash
Patted dry after complete on blotting paper.The same step of remaining step (2) arrives (5).
The four of the present invention provide a kind of application of kit as described above.
The five of the present invention provide a kind of DNA sequence dna, and the DNA sequence dna includes encoding monoclonal antibody as described above
The DNA sequence dna of weight chain variable district, and encode the DNA sequence dna of the light chain variable district of monoclonal antibody as described above.
In a specific embodiment, the DNA sequence dna for encoding the monoclonal antibody heavy variable region is such as SEQ ID
NO:Sequence shown in 3, the DNA sequence dna for encoding the monoclonal antibody light chain variable district is such as SEQ ID NO:Sequence shown in 4
Row.
The six of the present invention, which provide, contains DNA sequence dna as described above in a kind of cell, the cell.
In a specific embodiment, the DNA sequence dna can be endogenous or external source DNA sequence dna, and can
To express monoclonal antibody as described above.
In a specific embodiment, the cell can be selected from eucaryotic cell strain or prokaryotic strain；It is described thin
The preferred Bacillus coli expression cell line of born of the same parents, Yeast expression cell line, the strain of insect expression cell, mouse hybridoma cell strainses；It is especially excellent
It is CGMCC NO to select deposit number:10099 hybridoma cell strain.
Term " CP4-EPSPS albumen " in the present invention refers to resistance glyphosate resistance protein -5- enolpyruvyls acyl-thick grass
Acid -3- phosphate synthases (5-enolpyruvyl-shikimate-3-phosphate synthase, EPSPS) holoprotein, base
Because of coded sequence such as Genbank Accession No.AF464188 sequence.
Term " monoclonal antibody (MAb) " in the present invention refers to containing by unique light chain gene product and unique heavy chain base
Because of the antibody population for the only one species that product is constituted.Specifically, in all molecules of colony monoclonal antibody it is mutual
Benefit property determining area (CDR) is identical.Mab, which contains, can be immunized the specific of the antigen that binding characteristic is to have it unique affinity
The antigen binding site of epitope.It includes natural or genetic modification form, such as single-stranded, chimeric, synthesis, restructuring
, heterozygosis, mutation, grafting and external preparation antibody.It can also include as Fab, F (ab ') 2, Fv, scFv, Fd,
DAb, Fc and other compositions, are especially to maintain the antibody fragment of antigen binding function, or containing with antigen-binding domains
Any polypeptide, and be fused to another polypeptide, also wrap comprising the chimer molecules or equivalent with antigen-binding domains
Include wherein.It can also include introducing the DNA of the immune globulin variable region of encoding antibody or complementarity-determining region (CDRs)
The constant region or constant region of different immunoglobulins add framework region.In addition, also to hybridoma or producing the other thin of antibody
Born of the same parents carry out genetic mutation or other changes, thus it is possible to vary or the binding specificity of antibody produced by not changing.Specifically,
" monoclonal antibody " can also be obtained with recombinant DNA method, for example according to DNA disclosed by the invention it is artificial synthesized or with PCR methods expand
Increasing is obtained, and the sequence is connected into suitable expression vector, under conditions of antibody expression of the present invention is adapted to, culture conversion gained
Host cell, then those skilled in the art's application is well known conventional isolates and purifies the monoclonal that means purifying obtains the present invention
Antibody.
Phrase " specific binding " in the present invention refers to such association reaction, in the association reaction combine to
The compatibility that two members's (for example, antibody and molecule for including the antibody target epitope) are bonded to each other is than the member to heterogeneous
The compatibility of mixture other components (for example, mixture of hybridoma culture supemates or other albumen) is high at least 100 times.
In the present invention without in the case of specified otherwise, the term in the present invention belongs to signified logical in the prior art
Use term.
In this manual, the deformation of term "comprising" or such as " comprising " is understood to mean that what is specified including one
The group of element, integer or step or element, integer or step, but it is not excluded for any other element, integer or step or member
The group of element, integer or step.Term " can with " or its deformation are understood to mean that the element, integer or step that can select to specify
Or the group of element, integer or step, but it is also possible to be not element, integer or the step specified of selection or element, integer or
The group of step, but carry out equivalent substitution or other corresponding selections according to actual conditions.
Brief description of the drawings
The SDS-PAGE of the CP4-EPSPS albumen in expression in escherichia coli after Fig. 1 is purified, wherein M is
Albumen Marker.
Monoclonal antibody CP4-EPSPS AS5102 and CP4-EPSPS albumen that Fig. 2 is prepared using the application Western
Blot, wherein M are albumen Marker.
Fig. 3 monoclonal antibody CP4-EPSPS AS5102 and transgenic line and the Western of non-transgenic material
blot。
Cyropreservation
Mouse bone marrow cells hybridoma cell strain AS5102 is preserved in China General Microbiological culture presevation administrative center, and preservation is compiled
Number be CGMCC NO:10099, preservation date is on December 4th, 2014.The ground of China General Microbiological culture presevation administrative center
Location is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode 100101.What mouse bone marrow cells hybridoma cell strain AS5102 was produced
Monoclonal antibody is CP4-EPSPS AS5102.
Embodiment
The present invention is described below with reference to embodiment and accompanying drawing.But these embodiments be only it is exemplary,
Any limitation is not constituted to the scope of the present invention.It will be understood by those skilled in the art that in the essence without departing from the present invention
The details and form of technical solution of the present invention can be modified and replaced under god and scope, but these modifications and replacement fall
Enter in protection scope of the present invention.
Embodiment 1
The preparation of antigen
Design CP4-EPSPS (sequence such as SEQ ID NO:5) primer, NdeI and XhoI are separately added at the two ends of primer
Site, then by CP4-EPSPS gene clonings to pET30a expression vectors, resistance is kanamycins.The final table in Bl21
Reach, primary condition is that 25 DEG C of low temperature inductions are stayed overnight under the conditions of resistance.
The above-mentioned albumen produced using His labels to induction is purified, and condition elutes for 15mM imidazoles, and 60mM imidazoles is washed
Wash, the elution of 150mM imidazoles, the elution of 300mM imidazoles.150mM imidazoles is eluted to the component obtained to be dialysed with 10mM Tris-HCl,
Measure protein concentration is 0.9mg/ml.CP4-EPSPS albumen (sequence such as SEQ ID NO after purification:6) SDS-PAGE electrophoresis
Figure is shown in Fig. 1.
Embodiment 2
The preparation of monoclonal antibody
1) animal immune
The amount of 60 μ g/ mouse is pressed with CP4-EPSPS albumen, subcutaneous 4 SPF BALB/c female mices of initial immunity are compiled
Number it is：1、2、3、4.Subcutaneous first time booster immunization, the immune amount of albumen is 30 μ g/；Subcutaneous second of booster immunization, albumen
Immune amount for 30 μ g/ only；Subcutaneous third time booster immunization, the immune amount of albumen is 30 μ g/；Subcutaneous 4th reinforcement is exempted from
Epidemic disease, the immune amount of albumen is 30 μ g/；Eye socket takes blood, surveys serum titer.
2) immunizing potency is detected
Step：By CP4-EPSPS albumen coating buffers, 2 μ g/ml, 4 DEG C of coatings are stayed overnight；2% skim milk, 37 DEG C of closings
2h；Serum 2 times of gradient dilutions since 200 times, blank control (blank) is PBS, and negative control (negative) is negative blood
Clear 200 times of dilutions.As a result：Choose immunizing potency No. 3 mouse of highest and do abdominal cavity impact cell fusion experiment.
3) cell fusion is tested
With the μ g of CP4-EPSPS protein immunogens 50 of above-mentioned purifying, No. 3 mouse are impacted in abdominal cavity.Take mouse boosting cell with
SP2/0 cells, are merged using PEG methods.Merge cell and carry out screening and culturing with semisolid culturemedium (containing HAT).
Experiment equipment
The operating theater instruments of sterilizing：Three scissors, three put down tweezers, cell sieve, the inner core of syringe, one
Ware, wet box, 2 500ml beakers, 2 50ml centrifuge tubes, 3 15ml centrifuge tubes.
Experiment reagent
IMDM culture mediums
IMDM complete mediums (contain 15% serum)
2.2% methylcellulose：Producer：SIGMA；Article No.：M0262-100G
NBCS：10ml
PEG1500：Producer：Roche；Article No.：78364
HAT：Producer：Sigma；Article No.：H0262-10VL
HT：Producer：Sigma；Article No.：H0137-10VL
Fusion experiment step
I. blow and beat sp2/0 cells in good condition are soft, be drawn into 50ml centrifuge tubes from culture bottle wall.
Ii. mouse plucks eyeball and takes blood, then draws neck to put to death, and 5min is soaked in the alcohol for being put into 75%.
Iii. the IMDM culture mediums of a small amount of serum-free are poured into plate, cell sieve and plunger are put into plate
In.The spleen of mouse is removed with scissors and tweezers, is put into cell sieve.Lightly spleen is fully pulverized with the inner core of syringe,
In the centrifuge tube that the cell ground is drawn into dress sp2/0,1500rad/min, 5min are centrifuged.
Iv. the thymus gland of mouse is removed with scissors and tweezers, is pulverized.By the thymocyte ground into 15ml centrifuge tubes, then
Addition 2ml HAT, 2ml HT are placed on standby in incubator.
V. by the cell centrifuged, supernatant is outwelled, cell is carefully gently blown even with the IMDM culture mediums of serum-free, from
The heart (1500rad/min, 5min).
Vi. the cell conditioned medium centrifuged is tried one's best and outwelled.The abundant suspension cell in centrifuge tube bottom is patted, centrifuge tube is put into 37
In DEG C warm water, 1ml PEG was slowly added in 1 minute, after adding, 1min is stood in warm water.Then it is slowly added in 2min
The IMDM culture mediums of 2ml serum-free, are then slowly added to the IMDM culture mediums of 8ml serum-frees in 2min.Centrifuge 1000rad/
Min, 5min.
Vii. supernatant is outwelled, 10ml serum is added, careful blows even by cell, pours into above ready thymus gland thin
Born of the same parents.The sterilized semisolid culturemediums of 25ml are added, are fully mixed.Then uniformly pour into 30 Tissue Culture Dish.Will be thin
Born of the same parents' culture dish is put into wet box, is then placed in incubator and is cultivated.
4) hybridoma is screened
When cell culture to be fused was to the 7th day, i.e. cell culture draws 96 well culture plates to when covering 10% bottom hole
The culture supernatant for occurring clone cell cluster in hole detects antibody content with indirect elisa method, and secondary sub- gram is carried out through limiting dilution
Grand screening, the cell line of high-titer, high specific is filtered out according to the secretion situation of antibody, expands culture, and by high-titer, Gao Te
The cell line of the opposite sex freezes.A strain of hybridoma finally is filtered out, mouse bone marrow cells hybridoma cell strain AS5102 is named as, will
It is preserved in China General Microbiological culture presevation administrative center, and deposit number is CGMCC NO:10099.Its Dan Ke produced
Grand antibody is named as CP4-EPSPS AS5102.
Western blot using monoclonal antibody CP4-EPSPS AS5102 and CP4-EPSPS albumen are shown in Fig. 2.
Utilize monoclonal antibody CP4-EPSPS AS5102 and the Western of transgenic line and non-transgenic material
Blot is shown in Fig. 3.
Embodiment 3
The feature of monoclonal antibody
1) monoclonal cell subgroup identification
Experiment reagent includes：PBS-T is the PBS solution added with 0.05% polysorbas20.Purchased from Southern Biotech
Coated antibody；Confining liquid, is dissolved in PBS 2%BSA and 3% sucrose；Nitrite ion, 0.2ml A liquid and 10 μ l 30%H2O2It is dissolved in
(A liquid in 10ml B liquid：15mg/ml ABTS are dissolved in H2O；B liquid：Citrate buffer solution, pH4.0)；Purchased from Southern
Biotech various subclass secondary antibody.
Experimental procedure
A. coated antibody is diluted to 0.5 μ g/ml with 100mM PBS (pH7.4), 0.1ml is added per hole, 4 DEG C, is stayed overnight.
B.PBS-T is washed 2 times, and 200 μ l confining liquids, 37 DEG C of incubation 2h are added per hole.
C.PBS-T is washed 3 times；100 μ l hybridoma supematants, 37 DEG C of incubation 1h are added per hole.
D.PBS-T is washed 3 times；With confining liquid 1：1000 (κ, λ) or 1：The every holes of antibody 0.1ml of the HRP marks of 2000 dilutions,
It is separately added into appropriate hole, 37 DEG C of incubation 1h.
E.PBS-T is washed 3 times；Add per hole in 50 μ l substrate solutions, 10-20min in dual wavelength (450nm, 630nm) survey extinction
Value, record preserves data.
Data results show that monoclonal antibody CP4-EPSPS AS5102 subclass is IgG1 (being shown in Table 1).
Table 1
2) identification of monoclonal antibody specificity
Determine monoclonal antibody CP4-EPSPS AS5102 and genetically engineered soybean seed respectively using indirect ELISA method
Total protein, the total protein of transgenic corn seed, the total protein of transgenic cotton flower seed, total egg of nontransgenic soy seeds
In vain, the total protein atopic of the total protein of non-transgenic corn seed and non-transgenic cotton seeds, test material is common
1000 parts.
Measurement result is shown：Monoclonal antibody and transgenic line have 100% specific reaction, and and non-transgenic
The no cross reaction of material 100%, it is the monoclonal antibody specific of anti-CP4-EPSPS albumen to show monoclonal antibody.
3) monoclonal antibody affinity constant Kd measure
Coating restructuring EP4-EPSPS albumen, coating concentration is 2 μ g/ml, and 100 μ l/ holes, 4 DEG C of coatings are stayed overnight, and phosphate is told
Warm buffer solution (PBST) is washed 3 times.Adding 200 μ l to close 37 DEG C of closings of liquid per hole, (hour, h), PBST were washed 3 times in 2 hours.Monoclonal is resisted
Body 2 times of gradient dilutions since 1: 200, last 1 hole is blanked control, and 37 DEG C of incubation 1h, PBST is washed 3 times.The goat-anti of HRP marks
Mouse secondary antibody 1: 20000 dilutes, and per the μ l of hole 100,37 DEG C are incubated 1h, and PBST is washed 3 times.100 μ l are added per hole containing 0.1% tetramethyl to join
Aniline (TMB) (Sigma companies) and 0.03%H2O2Citrate phosphate buffer develop the color 10 minutes (minutes, min), plus
50 μ l 0.5M sulfuric acid solution terminating reactions.Wavelength 450nm light absorption value is determined with ELIASA.Draw OD450Value correspondence antibody is dilute
The curve of multiple is released, >=1/2 " platform OD values " corresponding extension rate A is found out.Calculating affinity costant using following equation is
2.61×1012。
4) outward appearance of monoclonal antibody purification
Under light-illuminating, visible antibody purification is visually observed in achromaticity and clarification state, has no that floccule is precipitated.
5) sterility test
Using Sterility Test, monoclonal antibody raw material after purification is taken to be seeded to the nutrient agar slopes culture of 10ml/ pipes
Base, improvement Martin's culture medium and each 2 pipe of sulphur glycollate culture medium (inoculum concentration is managed for 0.5ml/).Improvement Martin training after inoculation
Foster base puts 20-25 DEG C of culture.Each pipe of sulphur glycollate culture medium and nutrient agar slant medium after inoculation is placed in 30-35
DEG C culture, another pipe in 20-25 DEG C culture.Negative control is done with method operation with 0.9% sterile NaCl solution simultaneously.Culture 7 days
Do not observe in culture medium there is microorganism growth afterwards, show that monoclonal antibody raw material meets sterility requirements.
6) potency of monoclonal antibody
A) the μ g/ml of CP4-EPSPS albumen 2 are diluted with coating buffer, 100 μ l/ holes, are stayed overnight by 4 DEG C；Wash liquid is used afterwards 3 times.
B) 2% skim milk confining liquid is closed, 200 μ l/ holes, 37 DEG C of incubation 2h；Wash liquid is used afterwards 3 times.
C) addition primary antibody (cells and supernatant), negative control (SP2/0 culture supernatants), blank control (PBS), the positive are right
It is 100 μ l/ holes, 37 DEG C of incubation 1h according to (200 times of dilutions of PBS of positive serum)；Wash liquid is used afterwards 3 times.
D) secondary antibody that PBS dilutes 20000 times, 100 μ l/ holes, 37 DEG C of incubation 1h are added；Wash liquid is used after taking-up 3 times.
E) develop the color, the μ l/ holes of nitrite ion 100, developing time is 5min or so.
F) 50 μ l terminate liquids are added per hole to terminate.
G) dual wavelength (450nm, 630nm) surveys light absorption value, and record preserves data.
The potency for measuring monoclonal antibody CP4-EPSPS AS5102 is 1:10000.
7) sensitivity of monoclonal antibody
Commercially available monoclonal antibody is (limited using Beijing Hua Da protein research and development centre in this experiment with CP4-EPSPS
The article No. of company is AbM59705-3-PU monoclonal antibody) pairing, it is standard items with the CP4-EPSPS albumen of restructuring, detection
CP4EPSPS monoclonal antibodies are used for the effect of double crush syndrome.Experimental procedure is as follows：
By CP4-EPSPS monoclonal antibodies (2 μ g/ml), 100 μ l/ holes, 4 DEG C of coatings are stayed overnight.With 1%BSA at 37 DEG C, envelope
Close 2h.The CP4-EPSPS albumen for taking after purification be diluted to phosphate Tween buffer (PBST) 10 μ g/ml, 1 μ g/ml,
100ng/ml, 10ng/ml, 1ng/ml, 100pg/ml, 10pg/ml diluted protein solution, are added with 100 μ l/ holes (2 repetitions)
Different holes, 37 DEG C of incubation 1h.The commercially available monoclonal antibodies of CP4-EPSPS of 100 μ l dilutions, 37 DEG C of incubation 1h are added per hole.Plus
Enter the goat-anti rabbit (1: 10000) of HRP marks, 100 μ l/ holes, 37 DEG C of incubation 0.5h.0.05%PBST is used after the completion of often walking above
Clean ELISA Plate 5-8 times, blotting paper is thoroughly patted dry.Add 100 μ l tetramethyl benzidines (TMB) nitrite ions per hole, react 3 to 5 points
Clock, adds 50 μ l terminate liquid terminating reactions.Determine and read each hole OD under 450nm wavelength450Value.
Sandwich ELISA experimental result shows, what CP4-EPSPS pairing antibody can detect content as little as 1ng/ml turns base
Because of albumen.
Embodiment 4
Variable region of mab is sequenced
Write according to G.C. Howards etc. are waited《Antibody preparation is with using experiment guide》And Shen is put forth energy again, Chen Zhinan, Liu Minpei is compiled
Write《Recombinant antibodies》Middle associated description carries out the clone of antibody variable gene, and main process is as follows.
First, Total RNAs extraction
The 10 of the fresh collection of liquid nitrogen grinding6After above hybridoma, move in Trizol, acutely vibration, room temperature is placed
10 minutes, 4 DEG C centrifuged 10 minutes.Supernatant is fully mixed with isometric isopropanol after phenol chloroform, is precipitated 30 minutes on ice.
Precipitation after centrifugation is washed with 70% ethanol of precooling, dried.RNA dried objects are dissolved in DEPC water.
2nd, reverse transcription PCR
Take 9 μ L total serum IgEs, 2.5 μ L oligo (dT) 12-18primer (10mM), and 5 μ L dNTPs mixing, 70 DEG C of insulations 5
Rearmounted 5 minutes on ice of minute.5 μ L RT buffer (5 ×), 2.5 μ L DTT (0.1M) and 1 μ L reverse transcriptases are added, 42 DEG C anti-
Answer 1 hour.70 DEG C are incubated 15 minutes with terminating reaction, and the cDNA of acquisition is stored in -20 DEG C.1 μ L cDNA are taken to enter performing PCR amplification
(95 DEG C of denaturation 30s, annealing temperature time 1min extend 72 DEG C of 40s, 30 circulations).
3rd, PCR things are sequenced
Take 10 μ L PCR primers carry out electrophoretic analysis (1.5% agarose gel), the length of heavy chain between 360-420bps,
The length of light chain (κ light chains) is connected conversion between 350-390bps with carrier T after gel extraction.Blue hickie selects the positive
Clone, send sequencing, according to the DNA sequence dna (heavy chain DNA sequences measured such as SEQ ID NO after bacterium colony PCR checkings are positive:Shown in 3,
Light chain DNA sequences such as SEQ ID NO:Shown in 4) with the corresponding relation of amino acid coding, determine the amino acid sequence of variable region
Row.
Sequencing analysis obtain the amino acid sequence such as SEQ ID NO of the heavy chain of CP4-EPSPS clonal antibodies:Shown in 1, light chain
Amino acid sequence such as SEQ ID NO:2.
Claims (12)
1. a kind of monoclonal antibody of CP4-EPSPS albumen, the monoclonal antibody includes weight chain variable district and light chain variable district,
Characterized in that, the amino acid sequence of the weight chain variable district such as SEQ ID NO:Shown in 1, the amino acid of the light chain variable district
Sequence such as SEQ ID NO:Shown in 2.
2. monoclonal antibody according to claim 1, it is characterised in that the monoclonal antibody is selected from univalent antibody, list
One kind in chain antibody, double-chain antibody, bispecific antibody and chimeric antibody.
3. monoclonal antibody according to claim 1, it is characterised in that the monoclonal antibody is by deposit number
CGMCC NO:10099 hybridoma cell strain secretion is produced.
4. the application of the monoclonal antibody according to claims 1 to 3 any one.
5. a kind of kit for being used to detect CP4-EPSPS albumen, it is characterised in that the kit includes such as claim 1
To the monoclonal antibody described in 3 any one.
6. the application of kit according to claim 5.
7. a kind of DNA molecular, it is characterised in that the DNA molecular includes the list described in coding claims 1 to 3 any one
The DNA sequence dna of the weight chain variable district of clonal antibody, and the monoclonal antibody described in coding claims 1 to 3 any one
The DNA sequence dna of light chain variable district；And the DNA molecular can express the monoclonal as described in claim 1-3 any one
Antibody.
8. DNA molecular according to claim 7, it is characterised in that the DNA of the coding monoclonal antibody heavy variable region
Sequence is such as SEQ ID NO:Sequence shown in 3, the DNA sequence dna for encoding the monoclonal antibody light chain variable district is such as SEQ ID
NO:Sequence shown in 4.
9. a kind of cell, it is characterised in that contain DNA molecular as claimed in claim 7 or 8 in the cell.
10. cell according to claim 9, it is characterised in that the cell is selected from eucaryotic cell strain or prokaryotic strain.
11. cell according to claim 10, it is characterised in that the cell is selected from Bacillus coli expression cell line, ferment
Matrix reaches cell line, the strain of insect expression cell, mouse hybridoma cell strainses.
12. cell according to claim 11, it is characterised in that the cell is that deposit number is CGMCC NO:10099
Hybridoma cell strain.
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| CN104946598B (en) * | 2015-06-19 | 2018-02-13 | 中国科学院遗传与发育生物学研究所 | A kind of monoclonal antibody of anti-XA21 albumen and its application |
| CN105154408B (en) * | 2015-09-18 | 2018-10-09 | 中国农业科学院生物技术研究所 | A kind of monoclonal antibody and application thereof of detection antiweed glyphosate albumen |
| CN105717295B (en) * | 2016-01-15 | 2018-05-29 | 北京市农林科学院 | Test strip for rapidly detecting CP4-EPSPS transgenic plant and derivative thereof |
| CN109929034B (en) * | 2019-03-15 | 2020-10-30 | 杭州贤至生物科技有限公司 | CP4-EPSPS monoclonal antibody and preparation method thereof |
| CN113109562B (en) * | 2021-04-09 | 2022-03-11 | 安徽省农业科学院水稻研究所 | ELISA quantitative detection method of exogenous EPSPS protein in plant |
| CN117230021B (en) * | 2023-11-14 | 2024-01-26 | 中国农业科学院生物技术研究所 | CP4 EPSPS monoclonal antibody hybridoma cell strain, antibody produced by same and application thereof |
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| CN101042403A (en) * | 2007-04-25 | 2007-09-26 | 华南农业大学 | ELISA kit for detecting EPSPS gene in herbicide-tolerance soybeans and method of use thereof |
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| CN101042403A (en) * | 2007-04-25 | 2007-09-26 | 华南农业大学 | ELISA kit for detecting EPSPS gene in herbicide-tolerance soybeans and method of use thereof |
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| Title |
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| 转基因靶分子CP4-EPSPS单克隆抗体的制备及其ELISA检测方法研究;李忠鹏;《中国优秀硕士学位论文全文数据库 农业科技辑》;20131215;正文第22页2.3.2节，图2.1，第23页2.3.3节，表2-2，第23页2.3.4节，图2.2，第23页2.3.5节，图2.3，第24页2.3.6节，表2-3，图2.4，图2.5，第25页2.4节第2段第4-5行，正文第29-30页，3.2.9节 * |
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