WO2001098523A2

WO2001098523A2 - Detection method of genetic recombinant food and detection kit of that

Info

Publication number: WO2001098523A2
Application number: PCT/KR2001/001054
Authority: WO
Inventors: Hee-Young Park
Original assignee: Gd Biotech Co., Ltd
Priority date: 2000-06-20
Filing date: 2001-06-20
Publication date: 2001-12-27
Also published as: WO2001098523A3; AU2001274652A1

Abstract

The present invention relates to a method of detecting food included genetically modified organism and detection kit of same, and more particularly, to a detecting method of food included genetically modified organism by detecting recombinant protein expressed from transgenic plants. The present invention provides antibodies for recombinant proteins, thereby can be selected food included genetically modified organism with easy.

Description

DETECTION METHOD OF GENETIC RECOMBINANT FOOD AND DETECTION KIT OF THAT

BACKGROUND OF THE INVENTION (a) Field of the Invention

The present invention relates to a method for detecting foodstuff that

has been made from a genetically modified organism and a its detection, and

more particularly to a method and a kit for detecting whether a foodstuff product

was prepared from genetically modified organism or from organisms.

(b) Description of the Related Art

Genetically modified organism (GMO) is a common ter m for a plant

developed by a genetic engineering technology having genes or characteristics

that are not generated by its original reproduction system, in order to enhance

the convenience of distribution and process and to increase of output.

Characteristics of generally modified crops include increased output, resistance

to damage by harmful insects, a high concentration of agricultural chemicals,

deterioration during a long period of distribution, and improved shape, color and

taste, and genetically modified organisms having more various characteristics

can be developed in the future.

Commercialization of genetically modified organisms has rapidly

progressed since the US company Calgene developed tomatoes that are not easily crushed in 1994, and GMO soybeans of the Monsanto company and corn

of Novartis were fully commercialized in 1996. Although genetically modified

organism seeds were initially much more expensive than natural seeds, the

culture of genetically modified organisms rapidl y increased with a lack of

legislative regulations and expectations about its effect on people, because

farmers could save expenses on agricultural chemicals and fertilizers, and

damage by harmful insects was reduced, and then commercially available

genetically modified organisms inspected by the FDA from 1994 to 1998

numbered 39, including corns, tomatoes, potatoes, and soybeans, and another

30 are expected to be commercially available within 6 years.

In support of GMO's are the fact that there are econo mic advantages to

both companies and farmers in their production and they have the potential to

solve the food shortage and environmental problems, because they save on

herbicide use and produce more output with less labor and expense, however

the opposite opinion says that genetically modified organisms are not confirmed

safe as food, and the culture of genetically modified organisms over a long

period of time will cause a disturbance of the ecosystem and destruction of

species diversity such that ultimately there will be a harmful influence on people,

and also introduction of labeling that indicates the presence of genetically

modified organisms is also under dispute. In Korea, notification was given of an impending standard for genetically

modified organisms, within one-year grace period elapsed, in July of 2001 it was

established that food and food additives that are made or processed by using at

least one genetically modified agricultural or marine product as a main material

should indicate this fact, under Article 16 of the Agricultural and Marine

Products Quality Administration Law, and foodstuff that does not contain

recombinant DNA or foreign protein in the final product was excluded from

indication of the presence of genetically modified organisms because

recombinant DNA or exterior protein is removed during production or processing.

Therefore, detection method for recombinant DNA and protein should be

systemized.

Methods employed for the confirmation of gene manipulation of a plant

include a method for detecting DNA and a method for detecting protein in

genetically modified organisms. The representative example of the DNA

detection method is a genetically modified organism test kit of the Takara

Company and it carries out PCR by way of a specific primer that amplifies

foreign genes within a genetically modified Organism. However, it takes a long

time to test, has low reproducibility and cannot confirm whether foreign protein

is actually expressed or not. The protein detection method uses an EPSPS(5-

enolpyruvylshikimate-3-phosphate synthase) detection kit developed by the US company SDI, but it cannot detect processed foodstuff. A lthough the protein

detection method is relatively quick and has good reproducibility for seed in

general, it is difficult to detect foreign protein in processed foodstuff prepared

from a genetically modified organism.

Thus, a new technology to detect genetically modified organisms and

foodstuff prepared from genetically modified organisms is needed.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an antibody for

detecting foodstuff prepared from a genetically modified organism containing a

glyphosate-resistant gene.

It is another object of the present invention to provide an antibody for

detecting foodstuff prepared from a genetically modified organism containing a

resistant gene to harmful insects.

It is another object of the present invention to provide a method for

detecting foodstuff that is prepared from a genetically modified organism.

It is still another object of the present invention to provide a kit for

detecting foodstuff prepared froirta genetically modified organism.

In order to accomplish these objects, the present invention provides an

antibody that combines specifically with a re combinant protein, wherein the

antibody is prepared by the method comprising: analyzing a protease cleavage map of recombinant proteins expressed in transformed DNA in genetically

modified organisms; selecting peptides that do not have homology with peptid es

prepared from the same protease cleavage map for a wild -type plant protein

among peptides analyzed from the cleavage map ; preparing synthetic peptides

from amino acids of the selected peptides; and preparing an antibody from the

synthetic peptides.

The present invention also provides a method for detecting foodstuff

prepared from genetically modified organisms, comprising reacting the antibody

that combines specifically with a recombinant protein with food, and confirming

an antigen -anti body reaction to distinguish foodstuff prepared from a wild -type

plant from foodstuff prepared from a genetically modified organism.

The present invention also provides a kit for detecting foodstuff prepared

from a genetically modified organism, comprising: a protein extr action solution

for extracting protein from test foodstuff; an enzyme reaction solution containing

an enzyme that digests the extracted protein; an antibody that can combine .

digested recombinant protein; a strip smeared with the antibody at a certain part

of its -surfaGe;„a coloring antibody conjugated with a chromophore that. indicates--.,

the complex of the antibody and the recombinant protein; and a vessel wherein

the protein is digested by the enzyme.

The present invention also provides a kit for detecting foodstuff prepared from a genetically modified organism, comprising: a protein extraction

solution for extracting protein from test foodstuff; an enzyme reaction solution

containing an enzyme that digests the extracted protein; an antibody that can

combine digested recombinant; a 10 -well plate coated with the antibody on its

surface; a coloring antibody conjugated with a chromophore that indicates the

complex of the antibody and the recombinant protein; and a vessel wherein the

protein is digested by the enzy me.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig.1 is a photograph of western blotting of genetically modified rice and

wild-type rice with an antibody for PAT protein of the present invention.

Fig. 2 is a photograph showing results of testing genetically modified

rice and wild-type rice by using a detection kit of the present invention.

Fig. 3 is a photograph of western blotting of genetically modified beans

and wild-type beans with an antibody for EPSPS protein of the present invention.

Fig. 4 is a photograph of western blotting of genetically modified corn

and wild-type corn with an antibody for BT protein of the present invention.

_^. . ., „ . DETAILED DESCRIPTION OF THE INVENTION , .... ..

Hereinafter, the present invention will be explained in detail.

The detection method of food stuff prepared from genetically modified

organisms of the present invention is to confirm gene manipulation by preparing an antibody for recombinant protein expressed in genetically modified

organisms and reacting it with foodstuff or plants.

The recombinant protein of the present invention is expressed from

genes that generally transformed agricultural crops, and it includes

characteristics to resist herbicides, damages by harmful insects, cold -weather

damage, to improve storage stability, and to increase output, and recombinant

protein expressed from genetically modified organisms is preferable.

In present invention, a detection method for EPSPS, PAT and BT

recombinant protein was developed.

EPSPS is a 5-enolpyruvylshikimate-3-phosphate synthase that shows

resistance to glyphosate, a chemical having a weed -killing effect. Glyphosate

is a main component of Roundup, which is the most popular herbicide in the

world, and it is also available in Korea under the name "Kunsami". As

glyphosate-resistant genetically modified organisms, glyphosate

resistant/harmful-insect-resistant corn (Agrobacteήum sp. CP4 EPSPS gene

and glyphosate oxidaoreductase gene of Ochrobactrum anthropi among

.glyphosate resistant species) of the Monsanto company, glyphosate resistant

cottonseed (Agrobactaήum spp. CP4 EPSPS gene), and glyphosate resistant

canola (Agrobactarium spp. CP4 EPSPS gene) have been developed and sold.

PAT includes a phosphinothricin acetyltransferase genes derived from Streptomyces viridochromogenes , and a phosphinothricin acetylhydrolase

genes derived from Streptomyces hygroscopicus, and it has resistance to the

herbicide glyphosate. The genetically modified organisms transformed with

PAT include glyphosate -resistant corn (phosphinothricin acetyl hydrolase gene

of Streptomyces hygroscopicus) of Dekalb Genetics Corp, gluphosinate-

resistant canola (phosphinothricin acetyl transferase of Steptomyces

virdochromogenes) and gluphosinate- resistant corn (phosphinothricin acetyl

transferase of Steptomyces virdochromogenes) of Agevo Inc., and the Korea

Rural Development Administration has developed a genetically modified rice

transformed with PAT.

BT induces the resistance to harmful insects by transferring crylA and

crylllA genes of Bacillus thuringiensis to plants. BT plants include harmful -

insect-resistant corn (crylA(b) gene) and harmful-insect-resistant potatoes (cylll

A gene) of the Monsanto company, harmful-insect-resistant corn (crylA(b) gene)

of the Northrup King company, and harmful- insect-resistant corn (crylA(b) gene)

of Ciba-Geigy Corp. These plants are circulated as genetically modified and

are exported to Korea. • .< ^{• ,} . , ...,*--...

In the present invention, protein sequences of genes transferred to

plants were analyzed, peptides generated by digesting recombinant proteins

with various proteases were compared to peptides generated by digesting other proteins with the same proteases, and specific peptides that are distinguished from amino acid sequences were selected.

The protease is preferably at least one selected from the group consisting of trypsin, chymotrypsin, pepsin, submaxillarus protease and St. aureus V8 protease , and the specific peptide among the peptides generated by the proteases is preferable a peptide comprising at least ten amino acids.

Trypsin digests carboxyl terminals of arginine (R) and lysine (K) residue; chymotrypsin digests carboxyl terminals of phenylalanin (F), tyrosin (Y) and tryptopan (W) residue; submaxillarus digests carboxyl terminals of arginine (R) residue; and St. aureus V8 protease digests carboxyl terminals of aspartic acid

(D) and glutamic acid (E) residue. After synthetic peptides for the specific peptide that lacks homology with other proteins are synthesized, antibodies for the synthetic peptides are prepared.

The selected specific peptides of the present invention are shown below. Peptides selected from EPSPS (Sequence No.163) were compiled on sequence-listing software, Sequence No. of each peptide is numbered and the peptide sequences are shown in Table.!. Table 1

PAT was specified as a phosphinothricin acetyl transferase gene of Streptomyces viridochromogenes (Sequence No. 164) (PAT1), and a gene of

Streptomyces hygroscopicus (Sequence No. 165) (PAT2). Each selected

peptide was compiled on sequence -listing software, Sequence No. of each peptide is numbered, and the peptide seq uences are shown in Table 2. Table 2

BT was specified as a peptide from the MonBT gene (sequence No.166), Cry1 gene (sequence No.167), CryBI gene (sequence No.168), CryHD-1 gene

(sequence No.169) and Crylil gene (sequence No.170). Each selected peptide was compiled on sequence -listing software, Sequence No. of each

peptide is numbered, and the peptide sequences are shown in Table 3. Table 3

Antibodies for the selected peptides were reacted to test food or processed food for antigen -antibody reactivity. In order to improve reliability, at

least one of the antibodies used for the test were selected from the group

consisting of the prepared antibodies.

Confirmation of antigen-antibody reaction of recombinant protein can be

achieved by a conventional method performed in a laboratory, and the present

invention preferably comprises a method using coloring protein or showing

fluorescence by preparing a secondary antibody conjugated with an inactive

fluorescent protein that binds only with the antigen -antibody complex, binding

the secondary antibody to the antigen -antibody complex, adding an enzyme and

activating the inactive florescence protein, and a method using an antibody

conjugated with a coloring protein. Particularly, it is more preferable to show

the coloring reaction by conjugating colloidal gold with the antibody and binding

the antibody-colloidal gold conjugate to an antigen- antibody complex.

In addition, a detection kit is developed for detecting foodstuff prepared

from genetically modified organisms, so as to easily discriminate gene

modification in a laboratory of an official office. In the present invention a

detection kit for each. of EPSPS, PAT and BT was developed and it is possible,

to confirm whether foodstuffs contain expressed recombinant protein in these

three genes. Although components comprised in the detection kit for each of

EPSPS, and PAT and BT are different as to the kind of recombinant protein, the protocol is similar.

The detection kit for the recombinant protein of the present invention is

preferably a conventional detection kit such as a strip kit and an ELISA kit, and

it is also possible to use other kinds of detection kits based on methods for

detecting foodstuff prepared from genetically modified organisms of the present

invention.

When a testing specimen is a plant or a unprocessed food, the specimen

is ground, extracted with buffer (Tris- HCI), and the supernatant obtained by

centrifugation is reacted on a strip of a strip kit or is added to a well of an ELISA

kit, and then the gene modification of the specimen is determined by coloring

reaction by a conventional method.

In addition when a specimen is processed food, bean curd, Korean bean

paste, and other, it is pretreated with protease and centrifuged, and the

supernatant is used in the same way as above. The protease used is

preferable a conventional one.

The strip kit of the present invention comprises a protein extraction

solution. that.can.extract the protein of a specimen, an enzyme, reaction solution.,

(trypsin, chymotrypsin, pepsin, submaxi llarus protease, St. aureus V8 protease),

a detection strip containing an antibody, and an antibody conjugated with a

chromophore. The kit further comprises a recombinant protein (protein group comprising EPSPS, BT, and PAT) and a protein linked with a sy nthetic peptide

(BSA-, ovalbumin-, KLH-recombinant protein) as a control. The protocol of the

detection kit is as follows: protein of a foodstuff prepared from a genetically

modified organism is extracted with the protein extraction solution, centrifuged,

5 and is reacted with an enzyme. The reactant is wet on a strip containing the

antibody, and the strip is then reacted with the antibody conjugated with the

chromophore and the existence of the recombinant protein is determined by

color.

. . The ELISA kit of the present invention comprises a protein extraction

0 solution that can extract the protein of a specimen, an enzyme reaction solution

(trypsin, chymotrypsin, pepsin, submaxillarus protease, St. aureus V8 protease),

a 10-well plate that is coated with antibodies for peptides that are not

complementary with other protein prepared in the present invention, and an

antibody conjugated with a chromophore. The kit further comprises a

5 recombinant protein (protein group comprising EPSPS, BT, and PAT) and a

protein-linked synthetic peptide (BSA- , ovalbumin-, KLH-recombinant protein) as

,..*- ,. , a control. . .. . .._J.. ,,.,... „ , .,....,..,..

The process of the detection method using the ELISA kit comprises

extracting protein of a test sample, digesting it with an enzyme reaction and

0 placing it in a plate pre -coated with an antibody. After a predetermined time, the plate is washed with a washing solution and an antibody conjugated with a

chromophore is placed in the plate to confirm coloring. If the color occurs, the

test sample is determined to be a genetically modified food, and if color does

not occur, the sample is natural. Also the recombinant protein and protein -

linked synthetic peptide are analyzed by the same method at the same time as

a control. The well wherein the recombinant protein is placed shows a coloring

reaction, whereas the well wherein synthetic peptide is placed does not.

The detection kit of the present invention further comprises a buffer

solution in addition to the essential components mentioned above.

Hereinafter, preferred examples are presented for the sake of clarity.

These examples, however, are merely provided to facilitate understanding and

the present invention is not limited thereto.

Example 1

Analysis of PAT amino acid sequence

For the amino acid sequence of PAT-1 (Phosphinothricin-N-

acetyltransferase, Streptomyces viridochromogenes) of Sequence No. 164 and

PAT-2 (Phosphinothricin acetyl transferase, Streptomyces hygroscopicus) of

Sequence No. 165, cleavage regions for trypsin, chymotrypsin, pepsin, St.

aureus V8 protease, Submaxillarus protease were analyzed and peptides of

Sequence Nos. 30 to 54 having low homology were obtained. Synthesis of peptide

Among the above peptides of Sequence Nos. 30 to 54, peptides of Sequence Nos. 53 and 54 were synthesized by the Peptron Company and antibodies were prepared. The antibody for Sequence No.53 is referred to as P2-3 and the antibody for Sequence No.54 is referred to as P2-6.

Test l

Immunity of antibodies of Example 1 was confirmed.

Genetically modified rice and wild -type rice were individually dried for 12

hours in a dry oven at 60 ^°C , and the dried specimens were ground by a mixer

and screened with a mesh with a size greater than 150. The specimens were dissolved in buffer solution (0.6 ml of 1 M Tris-HCI (pH 6.8), 5 ml of 50% glycerol, 2 ml of 10% SDS, 0.5 ml of 2-mercaptoethanol, 1 ml of 1% bromophenol blue, and 0.9 ml H₂0) for SDS-electrophoresis at a concentration of 10 mg/ml, and

boiled at 100 ^°C . The samples were centrifuged and the supernatant was used

as the specimen solutions. The supernatant was subjected to SDS - electrophoresis on 10% acrylamide gel, and the protein band on the gel was transferred (Trans blot kit, BioRad Mini Trans- Blot Electrophretic Transfer Cell, 170-3930) to PVDF (BioRad, Immuno-Blot PVDF Membrane 162-0177). After the PVDF was immersed in a buffer solution, it was coated with 5% (w/v) of a skim milk solution for 1 hour, and it was reacted in a reaction solution (P2-3, P2-

6, PAT protein antibody: 5(w/v)% skim milk solution = 1 :1000) containing the

first antibody for more than 4 hours. After the reaction, the PVDF was washed

three times with a TSBT buffer solution (10 mM Tris, 0.15 M NaCI, 0.1% Tween

20, pH7.4) and once with a TBS buffer solution (10 mM Tris, 0.15 M NaCI,

pH7.4). The secondary antibody (HRP-anti rabbit IgG, SIGMA 9169) was

diluted by 1000 times with 5% skim milk and the PVDF was reacted in the

secondary antibody solution for 1 hour. It was then washed three times with

the TBST buffer solution and once with TBS buffer solution. After washing, it

was reacted in a dark room with a reaction substrate solution (Supersignal West

Pico Chemiluminescent substrate, Pierce 34080ZZ) and then developed with x-

ray film (Fuji, Supra RX) and confirmed. As a result, the PAT protein antibody

and antibodies of Example 1 showed an immune reaction with the pr otein of the

genetically modified rice and were sensitized, but no immune reaction occurred

with the wild-type rice.

Fig. 1 is a photograph of western blotting of genetically modified rice and

wild-type rice with the antibody of the PAT protein of the present invention, and

it was confirmed that P2-3 and P2-6 antibodies of the present invention could

detect the PAT protein successfully.

The P2-3 and P2-6 antibodies were purified. After the antibodies were applied onto the Protein A gel (Affi-GEL Protein A GEL, BioRad 153-6153), they were washed with a bonding buffer solution (pH8.0), and eluted with an elution buffer solution (pH3.0). The elution fractions were measured for absorbance (280nm) and fractions that had more than OD 0.2 were collected and concentrated, and the salt was removed with sephadex G- 25. The purified antibody was quantified with a protein quantification kit (BioRad 500-0006).

Example 2: strip kit for genetically modified rice

Preparation of strip

An upper end of the strip was coated with goat anti-rabbit IgG and a lower end was coated with rabbit anti -PAT2. Also, after preparing the rabbit anti-PAT2 conjugated with colloidal gold that shows coloring with an immune reaction, it was attached to a glass fiber and the glass fiber was atta ched to the upper end and the lower end of the strip. The goat anti -rabbit IgG serves as a control and it shows coloring by binding with the anti -rabbit IgG conjugated with the colloidal gold, and the PAT protein of genetically modified rice binds to the colloidal gold-anti PAT2 antibody, then it diffuses along the strip and shows a red line by binding with the rabbit anti -PAT2 on a detection line. .. .. .. ... . .

Preparation of a specimen

Genetically modified rice and wild-type rice were dried for 12 hours in a dry oven at 60 ^°C, and the dried specimens were ground by a mixer and then

screened with a mesh with a size greater than 150. The specimen was placed in an extraction buffer (50 mM Tris-HCI, pH 7.5 0.01% SDS containing; 6.06g Tris/1 L + 28mg SDS; pH is set by 1 N HCI) (specimen :buffer = 1 :4 (v:w)), mixed and extracted by revolving stirrer for more than 1 hour. The extract was centrifuged (12000rpm, 10min.) and the supernatant was used as the specimen solution.

Detection

(1) A strip was placed in the specimen solution to react for 3 min. When there is no centrifuge, the strip is placed directly in the extract to react.

(2) Coloring was confirmed on the detection line.

The result of the test is shown in Fig. 2. The coloring part of the upper end of the strip is that of the control, and coloring part of the lower end shows the existence of PAT protein. Nos.1 to 5 of Fig. 2 show the test results of wild- type rice and Nos. 6 to 10 show the results for detecting genetically modified rice. As seen from Fig. 2, the PAT detection kit of the present invention can

- - detect a genetically modified organism successfully. . ^,.*. .^,.^.. ^.. .. ^„,.-^,. -.

Example 3: ELISA kit

A 10-well plate for ELISA testing was coated with 5 ug each of P2 -3 and P2-6 antibodies of Example 1 and treated with 5% skim milk solution so that the part not combined with antibodies was coated.

After preparing a specimen by the same method of Example 2, 100 μl of

the specimen was placed in an ELISA plate well and reacted for 30 min. After the reaction, the specimen was removed and the we II was washed three times

with washing buffer solution, and 100 μl of the reaction solution containing the

secondary antibody was placed therein and reacted for 10 min. The reaction solution was removed again and the well was washed three times, and the coloring reaction solution was placed in the well and the coloring reaction was induced for 5 min. As a result, the well with the genetically modified rice specimen showed a coloring reaction, but the well with the wild-type rice specimen did not show a coloring reaction.

Example 4

Analysis of EPSPS amino acid sequence For the amino acid sequence of EPSPS of Sequence No.163 , cleavage regions for trypsin, chymotrypsin, pepsin, St. aureus V8 protease, ^{, ,}- ^: ., ^,,. ^Submaxillarus protease were analyzed, and peptides of Sequence Nos.1 to 29

that have low homology were obtained. Synthesis of peptide Among the above peptides of Sequence Nos. 1 to 29, peptides of

Sequence Nos. 28 and 29 were synthesized by the Peptron Company and

antibodies were prepared. The antibody for Sequence No.28 is referred to as

E-6b, and the antibody for Sequence No.29 is referred to as E- 9.

Test 2

Immunity of antibodies of the Example 4 was confirmed.

Western blot was performed for genetically modified beans and wild-type

beans. The experimental method was the same as in Example 1. As a result,

the EPSPS protein antibody and antibodies of Example 4 underwent an immune

reaction with the protein of the genetically modified beans, but wild-type beans

did not undergo an immune reaction.

Fig. 3 is a photograph of western blotting of genetically modified beans

with the antibody for EPSPS protein of the present invention, wherein it is

confirmed that E-6b and E-9 antibodies of the present invention can detect

EPSPS protein.

Example 5: strip kit for genetical ly modified beans

A strip kit was prepared by using the antibody of Example 4 by the same

method as Example 2. The genetically modified beans and wild-type beans

were tested with the strip kit. As a result, the strip kit showed a coloring

reaction for the genetically modified beans, but it did not show the reaction for the wild-type beans. Therefore, it was confirmed that the EPSPS detection kit

of the present invention could detect genetically modified organisms

successfully.

Example 6: ELISA kit

An ELISA kit was prepared by using the antibody of Example 4 by the

same method as Example 3. As a result, the well reacted with the specimen of

genetically modified beans showed a coloring reaction, but the well reacted with

the specimen of wild-type beans did not show the coloring reaction. Therefore,

it was confirmed that the EPSPS detection kit of the present invention could

detect genetically modified organism successfully.

. Example 7

Analysis of BT amino acid sequence

For MonBT genes (Sequence No.166), Cryl genes (Sequence No.1 67),

CryBI genes (Sequence No.168), CryHD- 1 genes (Sequence No.169), and

Crylil genes (Sequence No.170) , the cleavage region for trypsin, chymotrypsin,

pepsin, St. aureus V8 protease, and Submaxillarus protease was analyzed and

peptides of Sequence Nos. 55 to ,162 that have low homology were obtained.

Synthesis of peptide

Among the above peptides of Sequence Nos. 55 to 162, peptides of

Sequence Nos.160, 161 and 162 were synthesized by the Peptron Company, and antibodies were prepared. The antibody for Sequence No.160 is referred

to as Bt-1 , the antibody for Sequence No.161 is referred to as Bt- 2, and the

antibody for Sequence No.162 is referred to as Bt- 3.

Test 3

Immunity of antibodies prepared in Example 7 was confirmed.

Western blot was performed for genetically modified beans and wild-type

beans. The experimental method was the same as in the Example 1. As a

result, the BT protein antibody and antibodies of Example 7 underwent an

immune reaction with the protein of the genetically modified corns, but wild-type

corns did not occur an immune reaction.

Fig. 4 is a photograph of western blotting of genetically modified corn with

the antibody for BT protein of the present invention, wherein it is was confirmed

that Bt-1 (G1), Bt-2(G2) and Bt-3(G3) antibodies of the present invention can

detect BT protein.

Example 8: strip kit for genetically modified corn

A strip kit was prepared by using the antibody of Example 7 by the same

method as in. Example,-?. A test of genetically modified corn and wild-type corn

specimens was carried out with the strip kit. As a result the strip kit showed a

coloring reaction for the genetically modified corn, but it did not show the

reaction for the wild-type corn. Therefore, it was confirmed that the BT detection kit of the present invention could detect genetically modified organisms successfully.

Example 9: ELISA kit

An ELISA kit was prepared by using the antibody of Example 7 by the same method as in Example 3. As a result of an ELISA test being performed on genetically modified corn and wild- type corn, the well reacted with the genetically modified corn specimen showed a coloring reaction, but the well reacted with the wild-type corn specimen did not show the coloring reaction.

Therefore, it was confirmed that the BT detection kit of the present invention can detect genetically modified organisms successfully.

Example 10

For a bean curd made from genetically modified beans or wild -type beans, EPSPS detection using a strip kit was performed. 1 g each of GMO bean, curd and natural bean curd was ground and reacted in an enzyme reaction solution (trypsin solution 10 mg/ml (buffer solution; 100 mM Tris HCI

(pH 7.5)) or chymotrypsin solution 10 mg/ml (buffer solution; 100 mM Tris HCI

and the supernatant was tested by the strip detection method of Example 2.

As a result, GMO bean curd showed a red line on the strip so it was confirmed to be prepared from genetically modified beans tr ansformed with EPSPS, but the bean curd made from natural beans did not show coloring.

Example 11

For GMO Korean bean paste made from genetically modified beans and Korean bean paste made from wild -type beans, EPSPS detection using a strip kit was performed. Each 1 g of GMO Korean bean paste and natural Korean bean paste were reacted in an enzyme reaction solution (trypsin solution 10 mg/ml (buffer solution; 100 mM Tris HCI (pH 7.5)) or chymotrypsin solution 10

mg/ml (buffer solution; 100 mM Tris HCI (pH 7.5)) for 3 hours at 37 °C . The

next process was the same as described in Example 10. As a result, GMO Korean bean paste showed a coloring line on the strip so it was confirmed to be prepared from genetically modified beans transformed with EPSPS, but the natural Korean bean paste did not show coloring.

Example 12

For GMO Chinese bean paste made from genetically modified beans and

Chinese bean paste made from wild -type beans, EPSPS detection using a strip ^ιkit- was performed. Each 1 g of GMO Chinese bean -paste- nd regular Chinese bean paste were collected and tested by the same method as described in

Example 11. As a result, GMO Chinese bean paste showed a coloring line on the strip so it was confirmed to be prepared from genetically modified bean transformed with EPSPS, but the natural Chinese bean paste did not show coloring.

As mentioned above, the genetically modified organism examination method of the present invention can easily determine whether recombinant protein is expressed in genetically modified organisms, and whether processed food contains a recombinant protein.

Claims

WHAT IS CLAIMED IS:

1. An antibody that combines specifically with a recombinant protein,

wherein the antibody is prepared by the method comprising:

(a) analyzing a protease cleavage map of recombinant proteins

expressed in transformed DNA in genetically modified organisms;

(b) selecting peptides that do not have homology with peptides prepared from the same protease cleavage map for a wild -type plant protein among peptides analyzed from the cleavage map;

(c) preparing synthetic peptides from amino acids of the selected peptides; and

(d) preparing an antibody from the synthetic peptides.

2. The antibody that combines specifically with a recombinant protein

according to claim 1 , wherein the transformed DNA is EPSPS

(enoIpyruvylshikimate-3-phosphate synthase) DNA.

3. The antibody that combines specifically with a recombinant protein

according to claim 1 , wherein the synthetic peptides are peptides prepared by digesting a recombinant protein expressed in the EPSPS DNA of claim 2 with

protease and are selected from the group consisting of peptides of Sequence No.1 to Sequence No.29 compiled by sequence listing software.

4. The antibody that combines specifically with a recombinant protein

according to claim 1 , wherein the transformed DNA is PAT (phosphinothricin acetyl transferase) DNA.

5. The antibody that combines specifically with a recombinant protein

according to claim 1 , wherein the synthetic peptides are peptides prepared by digesting recombinant proteins expressed in the PAT DNA of claim 4 with protease and are selected from the group consisting of peptides of Sequence No.30 to Sequence No.53 compiled by sequence listing software.

6. The antibody that combines specifically with a recombinant protein

according to claim 1 , wherein the transformed DNA is BT (Bacillus thuringiensis) DNA.

7. The antibody that combines specifically with a recombinant protein

according to claim 1 , wherein the synthetic peptides are peptides prepared by digesting recombinant proteins expressed in the BT DNA of claim 6 with protease and are selected from the group consisting of peptides of Sequence

No.55 to Sequence No.162 compiled by base sequence compiler.

8. A method for detecting foodstuff prepared from a g enetically modified

organism comprising reacting the antibody of claim 1 that combines specifically with a recombinant protein with food and confirming an antigen- antibody reaction to distinguish foodstuff prepared from a wild -type plant from foodstuff prepared from a genetically modified organism.

9. The method for detecting foodstuff prepared from a genetically

modified organism according to claim 8, wherein the foodstuff is a plant or a

processed food.

10. A kit for detecting foodstuff prepared from a genetically modified

organism, comprising:

(a) a protein extraction solution for extracting protein from test foodstuff;

(b) an enzyme reaction solution containing an enzyme that digests the extracted protein;

(c) an antibody of claim 1 that can combine reco mbinant protein among the digested protein by the enzyme;

(d) a strip smeared with the antibody at a certain part of its surface;

(e) a coloring antibody conjugated with a chromophore that indicates complex of the antibody and the recombinant protein; and

(f) a vessel wherein the protein is digested by the enzyme.

¹ 11. The^* kit^' for detecting foodstuff prepared from a genetically modified

organism according to claim 10, wherein the kit further comprises recombinant

proteins selected from the group consisting of protein group containing EPSPS,

BT and PAT or proteins linked to synthetic peptides as a control.

12. A kit for detecting foodstuff prepared from a genetically modified

organism, comprising:

(a) a protein extraction solution for extracting protein from test foodstuff; (b) an enzyme reaction solution containing an enzyme that digests the extracted protein;

(c) an antibody of claim 1 that can combine recombinant protein among the digested protein by the enzyme;

(d) a 10-well plate coated with the antibody on its surface; . . (e) a coloring antibody conjugated with a chromophore that indicates the complex of the antibody and the recombinant protein; and

(f) a vessel wherein the protein is digested by the enzyme.

13. The kit for detecting foodstuff prepared from a genetically modified

organism according to claim 12, wherein the kit further comprises recombinant proteins selected from the group consisting of protein group containing EPSPS,

BT and PAT or proteins linked to synthetic peptides as a control.