KR102083797B1 - Method for producing polyclonal antibody against GIGANTEA protein from Arabidopsis thaliana - Google Patents

Abstract

According to the present invention, the amino and carboxy terminal cleavage peptides of the gygenthia protein are produced in Escherichia coli, respectively, and the recombinant recombinant gentogen cleavage peptides produced are mixed and injected into rabbits to obtain polyclonal antibodies directed against the gygentia protein. The present invention relates to a composition and kit for detecting gygenthia protein comprising a polyclonal antibody prepared by the method as an active ingredient.

Description

Method for producing polyclonal antibody against GIGANTEA protein from Arabidopsis thaliana

The present invention relates to the production of an antibody against a plant bioclock major factor gygenthia protein using protein cleavage, and more particularly, to produce the amino acid and carboxy terminal cleavage peptides of gygenthia protein in E. coli. The present invention relates to a method of producing a polyclonal antibody against gygentia protein by injecting a recombinant recombinant gentogen cleavage peptide produced in rabbits.

Arabidopsis GIGANTEA (GI) protein is encoded by a single gene, is well preserved in vascular plants and is controlled by a biological clock that mediates photoperiodic flowering. GI binds to ZTL (ZEITLUPE), an F-box protein that induces targeted proteosome-related degradation of Timing of Cab expression 1 (TOC1) and Pseudo-Response Regulator 5 (PRR5) proteins in the evening clock To stabilize. GI is also resistant to various plant physiological reactions such as optical signaling, hypocotyl length elongation, sugar signaling, starch accumulation, chlorophyll accumulation, and transpiration and resistance to abiotic stresses such as salt, cold and drought. It is involved in various plant physiological reactions such as acquisition. However, function at the molecular and conserved domains of GI proteins has not yet been identified. Interestingly, several gi mutant alleles with altered expression of GI transcripts showed different flowering periods and circadian differences. In a recent study, we found that the amino terminus (1-858aa) of the GI showed the same activity as the full length GI protein as a molecular chaperone for stabilization of the ZTL protein, and furthermore, unlike the carboxy terminus (920-1,173aa) In combination with the molecular chaperone HSP90, it has been reported to promote the cyclic protein refolding (refolding) of ZTL. In Arabidopsis, the active day cycle consists of transcription, translation and post-translational processes with complex feedback loops between morning- and evening-phase gene-proteins. GI shows proteasome dependent degradation in cancer conditions, repeats expression changes in transcription and translation levels daily, and increased GI protein during the day is essential for regulating ZTL protein homeostasis in post-translational loops of the Arabidopsis active cycle. In our previous study, we reported that the degradation of GI by salts in a proteasome dependent manner induces the activity of the Salt-Overly Sensitive (SOS) signaling pathway, a key mechanism for salt resistance, which suggests that the deficiency of GI proteins results in salts. It imparts resistance to stress. Although GI performs a variety of pleiotropic functions not only in physiological and developmental processes but also in biological and abiotic stress responses, it is difficult to identify the unique molecular function of GI due to the lack of antibodies specific for GI. . Therefore, to identify post-transcriptional modification of GI proteins regulated by various environmental factors, epitope tags such as GFP and HA are fused to GI, but there are disadvantages in that it takes several months to produce transgenic plants.

Meanwhile, Korean Patent No. 1406740 discloses a method for producing polyclonal antibody against E. coli expression and recombinant 2 protein of E. coli of tomato sulfide leaf curl virus, and Zygenthia is disclosed in Korean Patent No. 1348131. Although a 'Zigogenia protein regulating salinity stress resistance' regarding a recombinant protein expressing only the amino-terminus of the protein (1 to 391aa) is disclosed, the preparation of a polyclonal antibody against Arabidopsis gyogentia protein of the present invention There is no description of the method.

The present invention is derived from the above requirements, the inventors of the present invention to solve the problem of difficult expression of full-length gygenthia protein, two amino acid and carboxy-terminally cleaved zigenthia peptide in E. coli Using a mixture of the two cleaved gygentia peptides expressed and produced as antigen, polyclonal antibodies against gygentia proteins were produced in rabbits, wild-type Arabidopsis, cabbage, rapeseed, tomato, corn, and tolsque plants The present invention was completed by confirming that the full length Zygenthia protein can be successfully detected.

In order to solve the above problems, the present invention is to administer the amino-terminal and carboxy-terminal peptide of Arabidopsis derived GIGANTEA protein to the animal to produce antiserum, and to separate and purify the polyclonal antibody in antiserum It provides a method for producing a polyclonal antibody against a gygenthia protein comprising.

In another aspect, the present invention provides a composition for detecting gygenthia (GIGANTEA) protein comprising a polyclonal antibody prepared by the above method as an active ingredient.

In another aspect, the present invention provides a composition for detecting gygenthia (GIGANTEA) binding protein comprising a polyclonal antibody prepared by the above method as an active ingredient.

In another aspect, the present invention provides a kit for detection of gygenthia (GIGANTEA) protein comprising a polyclonal antibody prepared by the above method as an active ingredient.

The method for preparing a polyclonal antibody against gygenthia protein of the present invention facilitates expression and separation by recombinantly expressing a full-length gyogentia protein, which is difficult to express and separate, into a peptide in the form of a cleavage. Polyclonal antibodies against gyogentia proteins produced by injecting single peptides were found to be able to effectively recognize genicogen proteins in immunoblotting experiments on plant samples. Therefore, the polyclonal antibody against the gygenthia protein of the present invention may be usefully used to elucidate the molecular mechanisms of various gyogentia proteins that are not known.

1 is a result of cloning the GI full-length cDNA to the pGEX-KG vector for the expression of the whole GI protein, transformed into E. coli to confirm the expression of the recombinant protein. S: water soluble fraction in cell lysate, P: insoluble fraction in cell lysate.
2 is a result of confirming the expression of the recombinant GI fragment recombinant protein in Escherichia coli, (A) is a 6% SDS-PAGE results confirming the expression pattern of the GI ^cleavage (GI ^1-858 ) consisting of amino acids 1 ~ 858 , (B) is the 12% SDS-PAGE results confirming the expression pattern of the GI ^cleavage (GI ^920-1173 ) consisting of ^920-1,173 th amino acid. T: total extract, P: pellet, S: supernatant.
FIG. 3 shows SDS-PAGE results of cleaved GI ^1-858 and GI ^920-1173 , wherein (A) and (B) are pellets (P) and supernatant of cells expressing protein by IPTG at 50 ml culture level. (S) was confirmed by 8% and 12% SDS-PAGE, respectively, and (C) was extracted only from each gel of the GI fragments in the pellet (P) fraction isolated from (A) and (B), and then Electro- This is the result of reexamination by reextracting only the cleavage protein through eluter.
Figure 4 (A) is a schematic of the experimental procedure for the production of polyclonal antibodies to the gygentia protein, (B) is the total serum obtained from the immunized rabbit, the serum balance after purification, and purified Zygen Antibodies against thiagen (α-GI, 1: 500) were used to detect zigenthia protein in total protein samples extracted from GI :: GI-HA , 35S :: GI-HA and gi-201 plants. Western blot results confirmed, (C) is a result of Western blot by varying the antibody dilution ratio to confirm the binding capacity of the purified α-GI antibody. GI :: GI-HA ; GI promoter induced HA epitope tag binding GI gene expression Arabidopsis plants, 35S :: GI-HA ; HA epitope tagged GI gene overexpression Arabidopsis plant, gi-201 ; GI gene deficiency Arabidopsis plants.
5 (A) is a result of confirming the primary binding capacity for immunoprecipitation of the purified α-GI antibody, the input is Zeitgeber time (light time) 13 (ZT 13, light enters 13 Wild type (Col-0) and 35S :: HA-GI Arabidopsis plants harvested after time) and GI proteins present in extracts of Arabidopsis plants, IP by α-GI, wild type (Col) harvested at Zitgeber time 13 -0) and 35S :: HA-GI refers to GI proteins in which extracts of Arabidopsis plants have been detected with α-GI antibody-binding protein A-agarose bead mixture. (B) expresses ZTL, a GI binding protein according to the presence or absence of GI-HA in tobacco epidermal cells, induces primary binding for immunoprecipitation with α-GI antibody in the same manner as A, and binds ZTL protein. This is the result of checking.
Figure 6 shows the results of confirming the change in the expression of cyclic gygenthia protein in wild type Arabidopsis plants using purified α-GI antibody. CBB: Coomassie Billiant Blue Dye Blot.
7 is a result of confirming the cross-reactivity of the α-GI antibody in other plants using the purified Arabidopsis α-GI antibody. A. thaliana : Arabidopsis, B. rapa : Chinese cabbage, B. napus : rapeseed, S. lycopersicum : tomato, Z. mays : maize, F. arundinacea : tolsfescue.

In order to achieve the object of the present invention, the present invention is to administer the amino-terminal and carboxy-terminal peptide of Arabidopsis-derived GIGANTEA protein to animals to produce antiserum, and to isolate and purify polyclonal antibody in antiserum It provides a method for producing a polyclonal antibody to the gygenthia protein comprising the step.

In the method for producing a polyclonal antibody according to an embodiment of the present invention, the amino-terminal peptide and the carboxy-terminal peptide of the Arabidopsis derived GIGANTEA protein are SEQ ID NO: 1 and SEQ ID NO: 2, respectively Specifically, SEQ ID NO: 1 is composed of the 1 to 858th amino acid of the total gygenthia protein (GenBank accession no. AT1G22770.1; Protein: NP_564180.1), SEQ ID NO: 2 is a total gygenthia It may be composed of 920-1,173th amino acid of the protein, but is not limited thereto.

Specifically, the method for preparing a polyclonal antibody against gygenthia protein according to one embodiment of the present invention,

(a) Arabidopsis preparing a recombinant vector comprising a polynucleotide encoding an amino-terminal peptide of thaliana ) derived GIGANTEA protein;

(b) preparing a recombinant vector comprising a polynucleotide encoding a carboxy-terminal peptide of Arabidopsis derived gygenthia protein;

(c) transforming the recombinant vectors prepared in the above steps (a) and (b) into host cells to express the amino-terminal and carboxy-terminal peptides of the gygenthia protein, respectively;

(d) purifying the amino-terminal and carboxy-terminal peptides of each expressed zigenthia protein of step (c);

(e) administering the amino-terminal and carboxy-terminal peptides of the purified gygenthia protein of step (d) to an animal to produce antiserum; And

(f) isolating and purifying the polyclonal antibody from the antiserum produced in step (e); but may not be limited thereto.

As used herein, the term 'polyclonal antibody' refers to an antibody made by activating several clones simultaneously with respect to one antigen. In other words, a collection of antibodies that recognize various epitopes (epitopes) present in one antigen is a polyclonal antibody.

The term "antibody" is a term known in the art and means a molecule or active fragment of a molecule that binds a known antigen. Examples of active fragments that bind known antigens include Fab and F (ab ′) 2 fragments. Such active fragments can be derived from the antibodies of the invention by a number of techniques. For a description of general techniques for active fragment isolation of antibodies, see, eg, Khaw, B.A. et al., (1982) J. Nucl. Med. 23: 1011-1019. The term “antibody” also includes bispecific and chimeric antibodies.

In the method for producing a polyclonal antibody according to an embodiment of the present invention, the host cell of step (c) may be Escherichia coli, but is not limited thereto.

In the method for producing a polyclonal antibody according to an embodiment of the present invention, the amino-terminal and carboxy-terminal peptides of the gygenthia protein of step (e) may be administered in admixture with an immunostimulant. The adjuvant is used to enhance a mammal's immune response to antigens such as antibody formation before or when administering (inject) an antigen (immunogen). Freund's, Alum, the Ribi Adjuvant System or Titermax may be used, but is not limited thereto.

In the method for preparing a polyclonal antibody according to an embodiment of the present invention, the animal of step (e) may be preferably a rabbit, a mouse or a goat, but is not limited thereto. In general, animals used for the production of antibodies may be preferentially selected from relatively large mammals with larger amounts of serum capable of separating more antibodies. When the animal is injected with an immunogen, it stimulates B cells of the immune system to produce immunoglobulins (Ig) specific for the immunogen.

The term 'antiserum' as used herein refers to a serum having an antibody specific for an antigen from outside. Antigens that enter the body of humans and laboratory animals produce antibodies that react specifically to them. The newly produced antibodies are present in the serum components of the blood. Thus, the serum containing the antibodies is added to the antiserum or immune serum. serum). The antibody is any of IgG, IgM, IgA, IgE, IgD immunoglobulin, but is usually a mixture of them and mixed simultaneously with many other serum proteins in serum.

In the method for producing a polyclonal antibody according to an embodiment of the present invention, the polyclonal antibody of step (f) is obtained by separating from the antiserum of an animal immunized with amino-terminal and carboxy-terminal peptides of Zygenthia protein Can be. The antibody can be purified from antiserum using methods known in the art such as filtration, dialysis, ion exchange chromatography, size exclusion chromatography or affinity chromatography.

In the method for producing a polyclonal antibody according to an embodiment of the present invention, the polyclonal antibody of step (f) may be an IgG, but is not limited thereto.

The present invention also provides a composition for detecting gygenthia (GIGANTEA) protein and a composition for detecting gygenthia binding protein comprising a polyclonal antibody prepared by the method of the present invention as an active ingredient.

The present invention also provides a kit for detecting a ZIGENTEA protein comprising a polyclonal antibody prepared by the method of the present invention as an active ingredient.

The polyclonal antibody is as described above. By using the detection composition comprising a polyclonal antibody against the gygenthia protein of the present invention, it is possible to confirm the presence or absence of a protein binding to the gygenthia protein and / or gygenthia protein in the sample.

Antibodies of the invention can be coupled with a detectable substance, i.e., a detection labeling agent, to facilitate detection. Examples of detection markers include various enzymes, prosthesis molecules, fluorescent materials, luminescent materials, bioluminescent materials or radioactive materials, and the like. Detection markers can be directly coupled or coupled to the PINK1 polyclonal antibodies of the invention using techniques known in the art, or indirectly coupled or bound through intermediates (eg, linkers known in the art). Or a second polyclonal antibody that recognizes the PINK1 protein or a secondary antibody that is an antibody that recognizes the PINK1 antibody. Examples of suitable enzymes are horseradish peroxidase, acetylcholine esterase, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, malate dehydrogenase, glucose-6-phosphate Dehydrogenase or invertase and the like; Examples of suitable submolecular complexes include streptavidin / biotin or avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin or picobiliprotein, and the like; Examples of luminescent materials include luminol, isoleucinol or lucigenin; Examples of bioluminescent materials include luciferase, luciferin or aequorin; Examples of suitable radioactive materials may include, but are not limited to, ¹²⁵ I, ¹³¹ I, ¹¹¹ In, ⁹⁹ Tc, ¹⁴ C or ³ H, and the like.

The kit for detecting gygenthia protein of the present invention may further comprise a composition, solution or device having one or more other components suitable for the assay method.

The kit of the present invention is an immunoassay kit, and in general, the immunoassay has excellent advantages in both sensitivity and specificity as compared to enzyme immunoassay.

The term "immunoassay" refers to a method of detecting an analyte using specific immune responses, such as antigen-antibody responses.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Materials and methods

1. Plant Materials and Growth Conditions

Arabidopsis plants used Columbia-0 (Col-0) as wild type and used HA- tagged GI :: HA-GI , 35S: HA-GI , and gi-201 variants (David KM et al., 2006, FEBS Lett. 580: 1193-1197; Kim YW et al., 2007, Nature 449 (7160): 356-360; Martin-Tryon et al., 2007, Plant Physiol. 143: 473-486. Sterile seeds were placed in MS solid medium and cultured at 23 ° C. in a growth chamber under long-term conditions (16 hours light / 8 hours dark) of 100-120 μmol · m ⁻² · s ⁻¹ white light. In order to determine the cross-reactivity of the antibodies, Arabidopsis thaliana (Col-0), Chinese cabbage (Brassica rapa L. ssp. Pekinensis), rapeseed (Brassica napus L.), tomato (Lycopersicon lycopersicum L. Hongkwang), corn (Zea mays L .) And Festuca arundinacea Schreb. Seeds were surface sterilized, prepared in MS or B5 (tomato) solid medium and incubated for 2 weeks in a growth chamber at 23 ° C., 12 hours light / 12 hours dark. Corn and tolsfeque cue were planted directly into the soil without seed sterilization and incubated as above for 3 weeks.

2. Construction of Cleaved Zygenthia (GI) cDNA and Recombinant Protein Expression

Construction of the cleaved GI variant was made according to the previous method (Cha JY et al., 2017, Nature Commun. 8 (1): 3). A simple cloning one to 858th amino acid GI section groups (GI ^1-858) and the GI section groups (GI ^920-1173) made of 920-1173 amino acid consisting of pDONR-zeo vector after the recombinant-based gateway The cloning system (Invitrogen, USA) was used to transfer to the gREST vector with the 6xHis tag. For recombinant protein production, the plasmid was transduced with E. coli BL21 (DE3) pLysS strain, and the transduced cells were incubated at 37 ° C. until OD ₆₀₀ = 0.8, followed by 1 mM IPTG (isoprophyl-1-thio). -β-D-galactopyranoside) was added to induce expression and incubated at 30 ° C. for 3 hours. After incubation, cells were harvested by centrifugation at 6,000 rpm for 10 minutes, and the cell pellet was resuspended in 1 × Phosphate-buffered saline (PBS) and stored at −20 ° C. until use.

3. Preparation of Antigens and Production of Polyclonal Antibodies

After thawing the cryopreserved cells, and reacted for 20 minutes by adding 1% (v / v) Triton-100, the cells were disrupted by ultrasonic grinding. His-GI ^{1 -858} and a water-soluble and insoluble fractions of the His-GI ^{920 -1173} is 4 ℃, 12,000rpm, nanueotgo by centrifugation under the conditions of 10 minutes, it was confirmed via 12% SDS-PAGE. Protein bands expressing His-GI ^1-858 and His-GI ^920-1173 were cut from the insoluble fraction and the protein was recovered using Electro-Eluter (Bio-Rad, USA). 500 μg of each cleaved protein at 1: 1 ratio was mixed with Complete Freund's Adjuvant at 1: 1 ratio and then rabbit (New Zealand White species, male, 10 weeks old, 3 kg). Immunization was performed by injection into subcutaneous fat of, and immunization was performed three times at 2 week intervals. By collecting blood samples from the immunized rabbits, 4 ℃, 1,000x g, performing centrifugation under the conditions of 10 minutes, and the anti-serum is antigen using recombinant His-GI ^1-858 and His-GI ^{920 -1173} - specific Separation was performed using the red affinity separation method. All experimental animals were reared according to ethical principles, and the experimental procedure was conducted in compliance with the standard protocol of the Institutional Animal Care and Use Committee (IACUC). Antibody purification was performed using membrane-based affinity separation, insoluble His-GI ^1-858 and His-GI ^920-1173 were developed with 12% SDS-PAGE, then transferred to polyvinylidene difluoride (PVDF) membrane and nonspecific. To reduce the response, only the band portions of His-GI ^1-858 and His-GI ^920-1173 were cut out. The cut membrane was blocked with 1 × TBS buffer containing 1% (w / v) bovine serum albumin (BSA), and then the antisera was mixed and reacted at 4 ° C. overnight. After washing the membrane strip three times with 1 × TBS, bound polyclonal α-GI antibody was eluted with 0.1 M glycine, pH 2.5, and immediately neutralized with 2 M Tris-HCl, pH 8.0. Purified antibodies were stored at -20 ° C until use.

4. Western blot using purified α-GI antibody

Total protein from plant tissue was 8M urea, 5% (w / v) SDS, 100 mM Tris-HCl (pH 6.8), 1 mM EDTA, 2% (v / v) β-mercaptoethanol, 0.00125% ( w / v) bromophenol blue and protease / phosphatase / proteasome inhibitors (1 mM PMSF, 5 μg / ml leupeptin, 1 μg / ml aprotinin, 1 μg / ml pepstatin, 5 μg / ml antipain, 5 μg / Extracted using a buffer containing ml chymostatin, 2 mM Na ₂ VO ₃ , 2 mM NaF and 50 μM MG132). After centrifugation at 4 ° C., 12,000 rpm, 10 minutes, the supernatant was recovered, separated by 6% SDS-PAGE, transferred to PVDF membrane, and then immunoblotted using polyclonal α-GI antibody. GI protein was detected by chemiluminescence using ECL detection reagent (Thermo, USA).

5. Immunoprecipitation

Extracts of two-week-old Arabidopsis (Col-0 and 35S: HA-GI ) plants were extracted with 100 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 0.5% (v / v) NP-40, 1 mM EDTA, 3 mM DTT and After extraction using the extraction buffer containing the mentioned protease / phosphatase / proteasome inhibitor, the supernatant was collected by centrifugation at 4 ° C., 12,000 rpm, for 5 minutes. After the polyclonal α-GI antibody was reacted with Protein-A agarose (Sigma, USA) for 1 hour at 4 ° C, the supernatant was mixed and mixed at 4 ° C for 3 hours. The beads were washed three times with the above extraction buffer (without inhibitor) and then resuspended in cold 1 × PBS. The input (supernatant) and immunoprecipitated protein were mixed with loading buffer respectively and separated by 6% SDS-PAGE. Immunoblot performance is as described above.

6. Co-immunoprecipitation

After injecting Agrobacterium containing ZTL and GI-HA overexpression under the epidermal cells of 3.5-week-old tobacco ( Nicotiana Benthamiana ) to express ZTL and GI-HA proteins, the leaves were harvested on Day 2 ZT13 and described above. The whole protein was extracted in the same manner as the method. In addition, after binding the tobacco protein extract to a resin combined with α-GI antibody and protein A-agarose as described in the immunoprecipitation method, washing, SDS-PAGE separation and immunoblot were carried out in the same manner as above. Each protein was detected with a -ZTL antibody and an α-GI antibody to confirm ZTL co-immunoprecipitated with GI-HA.

Example 1 Expression of Cleaved Zygenthia (GI) Peptides

The present inventors cloned the entire cDNA sequence of GI into pGEX-KG, which is a protein expression vector, and then transformed into Escherichia coli BL21 (DE3) pLysS, which is a host cell. After incubation at 37 ° C. until OD ₆₀₀ = 0.8 in the medium, 1 mM IPTG was added and protein expression was induced. After induction of protein expression, it was transferred to 24, 30 or 37 ° C. incubator for further culture. The cultured cells were crushed and centrifuged at 4 ° C., 12,000 rpm, and 10 minutes, and divided into aqueous (S) and insoluble (P) fractions and examined for expression of total GI protein through 8% SDS-PAGE electrophoresis. saw. As a result, the total size of the GI protein produced by the pGEX KG :: GI clone was expected to be 155 kDa, but no increased protein band was detected under the 1 mM IPTG induction condition compared to the IPTG no addition condition (FIG. 1). Such attempts to express full-length GI proteins have also been carried out by Dr. Jo Putterill at the University of Auckland, New Zealand, but failed to extract full-length GI proteins, and has been reported in international journals that the GI proteins are highly unstable and readily fragmented into fragments. (Black et al., 2010, Protein Expression and Purification Vol. 76, p197-204). In this regard, the present inventors attempted to express a fragment of the GI protein.

1-858 amino acid group consisting of a GI section (GI ^1-858), and confirming the expression pattern in the 920-1173 amino acid group consisting of a GI section (GI ^{920 -1173)} in a small peptide of E. coli culture conditions (3㎖) As a result, it was confirmed that a significant amount of recombinant GI cleavage is expressed in the insoluble fraction (FIG. 2). In order to increase the amount of recombinant GI cleavage to be used as antigen, after expressing the GI cleavage in large-scale culture conditions (50 ml) in the same manner as in the small-scale culture conditions, the expression of the recombinant GI cleavage in the insoluble fraction was high. Peptide bands corresponding to the size of each GI cleavage were cut out, each GI peptide was recovered using Electro-Eluter, and electrophoresed with a known concentration of bovine serum albumin (BSA) for protein quantification (FIG. 3). The amount of each recovered GI ^cleavage was 1 μg / μl for GI ^1-858 and 3 μg / μl for GI ^920-1173 . Each recovered GI peptide was mixed and injected into rabbits for immunization. Antisera were obtained from the immunized rabbits and used for the experiment (FIG. 4A).

Example 2. Detectability Analysis of Polyclonal α-GI Antibodies

From the antiserum obtained, polyclonal α-GI antibodies were purified by membrane-based affinity separation, and in order to confirm that the purified α-GI antibodies could effectively detect Zygenthia protein, GI :: GI-HA , 35S: Western blots were performed using protein samples extracted from HA-GI and gi-201 variants. First, the presence of unpurified antiserum, supernatant after purely purifying α-GI antibody in the antiserum, and purified α-GI antibody were used to confirm the detection of Arabidopsis Zygenthia protein. In both blots using supernatant after purification, nonspecific bands of about 80 kDa and 130 kDa in all GI :: HA-GI , 35S: HA-GI , and gi-201 variant samples were identified, but purely purified α- GI antibodies In the used blot, a protein band of about 130 kDa size was found only in the samples of GI :: GI-HA and 35S: HA-GI , and the detection band did not appear in the sample of gi-201 variant, which is a Zygenthia gene deficient mutant plant (FIG. 4B). ). The difference in GI protein between GI :: GI-HA and 35S: HA-GI plants was that the GI protein in 35S: HA-GI , a GI overexpressing plant, was somewhat larger than that of GI :: HA-GI . . In order to confirm the sensitivity of the purified polyclonal α-GI antibody, the dilution ratio was adjusted to 1: 250, 1: 500, and 1: 1,000 to confirm the detection ability of the Zygenthia protein. It was confirmed that gentia protein was detected (FIG. 4C). In addition, both GI :: GI-HA and 35S: HA-GI plants were tagged with HA in the GI gene, so the GI protein bands detected using the α-GI antibody were cross-checked with the α-HA antibody. The band could be detected. Through the above results, it was confirmed that the purified polyclonal α-GI antibody can effectively detect the gygenthia protein in the plant.

Example 3. Analysis of protein-protein binding detectability of polyclonal α-GI antibody

Antibodies are not just for protein detection in vivo, but are also known for co-immunoprecipitation and protein-to-DNA binding that can identify protein-protein binding in vivo. It can be usefully used for immunoprecipitation. To do this, it is necessary to check whether the antibody can detect the target protein in vivo through an antibody binding resin such as protein A-agarose. FIG. 5A shows purified a-GI antibody from protein A-agarose resin (1 hour at 4 ° C.), followed by Arabidopsis wild-type (Col-0) and 35S :: HA-GI (GI overexpressing plants) plants. The extracted whole protein was mixed with antibody and protein A-agarose binding resin and reacted at 4 ° C. for 3 hours to induce binding with plant GI protein. To see only the specific binding of purified α-GI antibody to GI protein in the plant, washing with plant protein extraction buffer three times to remove nonspecific binding protein, separating by 6% SDS-PAGE and immunizing with α-GI antibody Blots were performed and it was confirmed that GI protein in plants was immunoprecipitated through α-GI antibody. Furthermore, the binding to ZTL protein, which binds to GI protein in Arabidopsis and regulates stability, was confirmed by co-immunoprecipitation using purified α-GI antibody. To this end, overexpression of GI-HA and ZTL on tobacco epidermal cells at the same time was detected using the α-GI antibody in the same way as the immunoprecipitation method, and the binding between GI-ZTL proteins was confirmed. It was confirmed that ZTL protein expressed in plants was detected by binding to α-GI antibody in the case of plants expressing GI-HA because there was no GI-HA protein bound to α-GI antibody. 5B). Through this, the α-GI antibody of the present invention can not only confirm the expression of GI protein in the plant, but also detect the partner protein which binds to the GI protein in the plant through various immunoprecipitation methods and regulate transcription through GI protein-DNA binding. It is inferred that it could be widely used for verification.

The GI gene is a genetically well-conserved gene in all plant species with land plant development. In addition, GI genes of all plant species reported to date have been reported to change photoperiod-dependent circadian expression at the transcription level. However, since there is no GI antibody, circadian expression changes at the protein level have only been observed through HA and TAP tags. In this regard, the cyclic expression of GI protein in wild-type (Col-0) Arabidopsis larvae was observed using the α-GI antibody prepared in the present invention. It was confirmed that the highest expression amount in the time zone of Zitgeber time 12 (ZT12), it was confirmed that the decrease thereafter (Fig. 6).

Example 4. Cross Reactivity Analysis of Polyclonal α-GI Antibodies

In order to analyze the cross-reactivity of the polyclonal α-GI antibody of the present invention prepared using Arabidopsis gygentia protein as an antigen, western blot was performed using cabbage, rapeseed, tomato, corn and tolsque cue samples. Each plant used a total protein extract of Zeitgeber time 1 (ZT1) and 13 (ZT13) as a sample and confirmed that the same amount of sample was loaded by Coomassie Brilliant Blue (CBB) staining.

As a result of the cross-reactivity experiments, the polyclonal α-GI antibody of the present invention was able to effectively detect not only Arabidopsis plants but also Zygenthia proteins of Chinese cabbage, rapeseed, tomato, corn, and tol pescue plants, and showed cyclic expression changes. It could be seen (Fig. 7). The result of the study is the result of the cyclic expression of GI protein, which is reported for the first time in the world in other crops than Arabidopsis. Through these results, the polyclonal α-GI antibody of the present invention can be used as an excellent detection means for GI research. It was predicted.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Method for producing polyclonal antibody against GIGANTEA protein          from Arabidopsis thaliana <130> PN17197 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 858 <212> PRT <213> Arabidopsis thaliana <400> 1 Met Ala Ser Ser Ser Ser Ser Glu Arg Trp Ile Asp Gly Leu Gln Phe   1 5 10 15 Ser Ser Leu Leu Trp Pro Pro Pro Arg Asp Pro Gln Gln His Lys Asp              20 25 30 Gln Val Val Ala Tyr Val Glu Tyr Phe Gly Gln Phe Thr Ser Glu Gln          35 40 45 Phe Pro Asp Asp Ile Ala Glu Leu Val Arg His Gln Tyr Pro Ser Thr      50 55 60 Glu Lys Arg Leu Leu Asp Asp Val Leu Ala Met Phe Val Leu His His  65 70 75 80 Pro Glu His Gly His Ala Val Ile Leu Pro Ile Ile Ser Cys Leu Ile                  85 90 95 Asp Gly Ser Leu Val Tyr Ser Lys Glu Ala His Pro Phe Ala Ser Phe             100 105 110 Ile Ser Leu Val Cys Pro Ser Ser Glu Asn Asp Tyr Ser Glu Gln Trp         115 120 125 Ala Leu Ala Cys Gly Glu Ile Leu Arg Ile Leu Thr His Tyr Asn Arg     130 135 140 Pro Ile Tyr Lys Thr Glu Gln Gln Asn Gly Asp Thr Glu Arg Asn Cys 145 150 155 160 Leu Ser Lys Ala Thr Thr Ser Ser Gly Ser Pro Thr Ser Glu Pro Lys Ala                 165 170 175 Gly Ser Pro Thr Gln His Glu Arg Lys Pro Leu Arg Pro Leu Ser Pro             180 185 190 Trp Ile Ser Asp Ile Leu Leu Ala Ala Pro Leu Gly Ile Arg Ser Asp         195 200 205 Tyr Phe Arg Trp Cys Ser Gly Val Met Gly Lys Tyr Ala Ala Gly Glu     210 215 220 Leu Lys Pro Pro Thr Ile Ala Ser Arg Gly Ser Gly Lys His Pro Gln 225 230 235 240 Leu Met Pro Ser Thr Pro Arg Trp Ala Val Ala Asn Gly Ala Gly Val                 245 250 255 Ile Leu Ser Val Cys Asp Asp Glu Val Ala Arg Tyr Glu Thr Ala Thr             260 265 270 Leu Thr Ala Val Ala Val Pro Ala Leu Leu Leu Pro Pro Pro Thr Thr         275 280 285 Ser Leu Asp Glu His Leu Val Ala Gly Leu Pro Ala Leu Glu Pro Tyr     290 295 300 Ala Arg Leu Phe His Arg Tyr Tyr Ala Ile Ala Thr Pro Ser Ala Thr 305 310 315 320 Gln Arg Leu Leu Leu Gly Leu Leu Glu Ala Pro Pro Ser Trp Ala Pro                 325 330 335 Asp Ala Leu Asp Ala Ala Val Gln Leu Val Glu Leu Leu Arg Ala Ala             340 345 350 Glu Asp Tyr Ala Ser Gly Val Arg Leu Pro Arg Asn Trp Met His Leu         355 360 365 His Phe Leu Arg Ala Ile Gly Ile Ala Met Ser Met Arg Ala Gly Val     370 375 380 Ala Ala Asp Ala Ala Ala Ala Leu Leu Phe Arg Ile Leu Ser Gln Pro 385 390 395 400 Ala Leu Leu Phe Pro Pro Leu Ser Gln Val Glu Gly Val Glu Ile Gln                 405 410 415 His Ala Pro Ile Gly Gly Tyr Ser Ser Asn Tyr Arg Lys Gln Ile Glu             420 425 430 Val Pro Ala Ala Glu Ala Thr Ile Glu Ala Thr Ala Gln Gly Ile Ala         435 440 445 Ser Met Leu Cys Ala His Gly Pro Glu Val Glu Trp Arg Ile Cys Thr     450 455 460 Ile Trp Glu Ala Ala Tyr Gly Leu Ile Pro Leu Asn Ser Ser Ala Val 465 470 475 480 Asp Leu Pro Glu Ile Ile Val Ala Thr Pro Leu Gln Pro Pro Ile Leu                 485 490 495 Ser Trp Asn Leu Tyr Ile Pro Leu Leu Lys Val Leu Glu Tyr Leu Pro             500 505 510 Arg Gly Ser Pro Ser Glu Ala Cys Leu Met Lys Ile Phe Val Ala Thr         515 520 525 Val Glu Thr Ile Leu Ser Arg Thr Phe Pro Pro Glu Ser Ser Arg Glu     530 535 540 Leu Thr Arg Lys Ala Arg Ser Ser Phe Thr Thr Arg Ser Ala Thr Lys 545 550 555 560 Asn Leu Ala Met Ser Glu Leu Arg Ala Met Val His Ala Leu Phe Leu                 565 570 575 Glu Ser Cys Ala Gly Val Glu Leu Ala Ser Arg Leu Leu Phe Val Val             580 585 590 Leu Thr Val Cys Val Ser His Glu Ala Gln Ser Ser Gly Ser Lys Arg         595 600 605 Pro Arg Ser Glu Tyr Ala Ser Thr Thr Glu Asn Ile Glu Ala Asn Gln     610 615 620 Pro Val Ser Asn Asn Gln Thr Ala Asn Arg Lys Ser Arg Asn Val Lys 625 630 635 640 Gly Gln Gly Pro Val Ala Ala Phe Asp Ser Tyr Val Leu Ala Ala Val                 645 650 655 Cys Ala Leu Ala Cys Glu Val Gln Leu Tyr Pro Met Ile Ser Gly Gly             660 665 670 Gly Asn Phe Ser Asn Ser Ala Val Ala Gly Thr Ile Thr Lys Pro Val         675 680 685 Lys Ile Asn Gly Ser Ser Lys Glu Tyr Gly Ala Gly Ile Asp Ser Ala     690 695 700 Ile Ser His Thr Arg Arg Ile Leu Ala Ile Leu Glu Ala Leu Phe Ser 705 710 715 720 Leu Lys Pro Ser Ser Val Gly Thr Pro Trp Ser Tyr Ser Ser Ser Glu                 725 730 735 Ile Val Ala Ala Ala Met Val Ala Ala His Ile Ser Glu Leu Phe Arg             740 745 750 Arg Ser Lys Ala Leu Thr His Ala Leu Ser Gly Leu Met Arg Cys Lys         755 760 765 Trp Asp Lys Glu Ile His Lys Arg Ala Ser Ser Leu Tyr Asn Leu Ile     770 775 780 Asp Val His Ser Lys Val Val Ala Ser Ile Val Asp Lys Ala Glu Pro 785 790 795 800 Leu Glu Ala Tyr Leu Lys Asn Thr Pro Val Gln Lys Asp Ser Val Thr                 805 810 815 Cys Leu Asn Trp Lys Gln Glu Asn Thr Cys Ala Ser Thr Thr Cys Phe             820 825 830 Asp Thr Ala Val Thr Ser Ala Ser Arg Thr Glu Met Asn Pro Arg Gly         835 840 845 Asn His Lys Tyr Ala Arg His Ser Asp Glu     850 855 <210> 2 <211> 254 <212> PRT <213> Arabidopsis thaliana <400> 2 Leu Ile Ala Ala Pro Glu Ile Gln Pro Thr Ala Glu Ser Thr Ser Ala   1 5 10 15 Gln Gln Gly Trp Arg Gln Val Val Asp Ala Leu Cys Asn Val Val Ser              20 25 30 Ala Thr Pro Ala Lys Ala Ala Ala Ala Val Val Leu Gln Ala Glu Arg          35 40 45 Glu Leu Gln Pro Trp Ile Ala Lys Asp Asp Glu Glu Gly Gln Lys Met      50 55 60 Trp Lys Ile Asn Gln Arg Ile Val Lys Val Leu Val Glu Leu Met Arg  65 70 75 80 Asn His Asp Arg Pro Glu Ser Leu Val Ile Leu Ala Ser Ala Ser Asp                  85 90 95 Leu Leu Leu Arg Ala Thr Asp Gly Met Leu Val Asp Gly Glu Ala Cys             100 105 110 Thr Leu Pro Gln Leu Glu Leu Leu Glu Ala Thr Ala Arg Ala Ile Gln         115 120 125 Pro Val Leu Ala Trp Gly Pro Ser Gly Leu Ala Val Val Asp Gly Leu     130 135 140 Ser Asn Leu Leu Lys Cys Arg Leu Pro Ala Thr Ile Arg Cys Leu Ser 145 150 155 160 His Pro Ser Ala His Val Arg Ala Leu Ser Thr Ser Val Leu Arg Asp                 165 170 175 Ile Met Asn Gln Ser Ser Ile Pro Ile Lys Val Thr Pro Lys Leu Pro             180 185 190 Thr Thr Glu Lys Asn Gly Met Asn Ser Pro Ser Tyr Arg Phe Phe Asn         195 200 205 Ala Ala Ser Ile Asp Trp Lys Ala Asp Ile Gln Asn Cys Leu Asn Trp     210 215 220 Glu Ala His Ser Leu Leu Ser Thr Thr Met Pro Thr Gln Phe Leu Asp 225 230 235 240 Thr Ala Ala Arg Glu Leu Gly Cys Thr Ile Ser Leu Ser Gln                 245 250

Claims (4)

Amino-terminal and carboxy-terminal peptides of Arabidopsis-derived GIGANTEA protein are administered to animals other than humans to produce antiserum, and to separate and purify polyclonal antibodies from antiserum. As a method for producing a polyclonal antibody against
The amino-terminal and carboxy-terminal peptides of the Arabidopsis derived gygenthia protein,
(a) preparing a recombinant vector comprising a polynucleotide encoding an amino-terminal peptide of Arabidopsis derived gygenthia protein consisting of the amino acid sequence of SEQ ID NO: 1;
(b) preparing a recombinant vector comprising a polynucleotide encoding the carboxy-terminal peptide of the Arabidopsis derived gygenthia protein consisting of the amino acid sequence of SEQ ID NO: 2;
(c) transforming the recombinant vectors prepared in the above steps (a) and (b) into host cells to express the amino-terminal and carboxy-terminal peptides of the gygenthia protein, respectively; And
(d) purifying the amino-terminal and carboxy-terminal peptides of each expressed gygenthia protein of step (c); polyclonal for gygentia protein, characterized in that it is produced by a method comprising the Method for producing an antibody. delete delete The method of claim 1, wherein the polyclonal antibody against the gygenthia protein is IgG.

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