KR20140058010A - Biomarker protein specific for environmental stress of poultry - Google Patents

Abstract

The present invention relates to a biomarker protein specific to environmental stress of poultry, and more specifically, to developing proteins differentially expressed in eggs produced from poultry receiving stress and eggs produced from poultry which does not receive stress as biomarker proteins, a kit for analyzing environmental stress of poultry using the same, a method for analyzing environmental stress of the poultry, and a method for finally evaluating the quality of eggs and the welfare of the poultry. According to the present invention, because it is possible to analyze the increase/decrease of expression of protein components of specific eggs through the differential expression analysis of the egg proteins, it is possible to determine the presence or absence of stress of the poultry while spawning without sacrifice of the poultry and the protein can be used as a novel marker (biomarker protein) in egg quality analysis.

Description

Biomarker protein specific environmental stress of poultry.

The present invention relates to a biomarker protein specific to environmental stress of poultry. More particularly, the present invention relates to a biomarker protein specific for environmental stress of poultry, and more particularly, to a method for producing a biomarker protein which is differentially expressed from eggs produced by stressed poultry and eggs produced by unstressed poultry A biomarker protein, a kit for evaluating environmental stress of poultry using the same, a method of analyzing environmental stress of poultry, and a method of evaluating egg quality and poultry welfare finally.

Stress reduces animal immunity and decreases quantitative and qualitative productivity (Choi, Y.-H. 2007. Management of Stress in Poultry Production. Korean Journal of Poultry Science 34: 295-300. (Henriksen, R., T. G. Groothuis, and S. Rettenbacher. 2011. Elevated plasma corticosterone reduced yolk testosterone and progesterone in chickens: Linking maternal stress and hormone-mediated maternal effects. PloS one 6: e23824. doi 10.1371 / journal.pone.0023824; Mashaly, M. M., G. L. Hendricks, 3rd, M. A. Kalama, A. E. Gehad, A. O. Abbas, and P. H. Patterson. 2004. Effect of heat stress on the production parameters and immune responses of commercial laying hens. Poult Sci 83: 889-894. It is known that stress on the laying hens changes the concentration of corticosterone, glucose, and cholesterol in the blood and changes the weight of the spleen, liver and F-sac {Mumma, JO, JP Thaxton, Y. Vizzier-Thaxton , and WL Dodson. 2006. Physiological stress in laying hens. Poult Sci 85: 761-769 .; Puvadolpirod, S., and J. P. Thaxton. 2000. Model of physiological stress in chickens 1. Response parameters. Poult Sci 79: 363-369 .; Rochester, J. R., K. C. Klasing, L. Stevenson, M. S. Denison, W. Berry, and J. R. Millam. 2009. Dietary red clover (Trifolium pratense) induces oviduct growth and decreases ovary and testes growth in Japanese quail chicks. Reproductive toxicology (Elmsford, N.Y.) 27: 63-71. doi 10.1016 / J.Repirotox.2008.11.056; Shini, S., P. Kaiser, A. Shini, and W. L. Bryden. 2008. Biological response of chickens (Gallus gallus domesticus) induced by corticosterone and a bacterial endotoxin. Comp Biochem Physiol B Biochem Mol Biol 149: 324-333.

Stress has been shown to delay the aging period of laying hens and shorten the spawning peak period (Shini, S., Shini, and G. R. Huff. 2009. Effects of chronic and repeated corticosterone administration in rearing chickens on physiology, the onset of lay and egg production of hens. Physiology & behavior 98: 73-77.), Reducing the weight of the egg-producing organs and ovaries {Shini, S., A. Shini, and G. R. Huff. 2009. Effects of chronic and repeated corticosterone administration in rearing chickens on physiology, the onset of lay and egg production of hens. Physiology & behavior 98: 73-77; Mumma, J. O., J. P. Thaxton, Y. Vizzier-Thaxton, and W. L. Dodson. 2006. Physiological stress in laying hens. Poult Sci 85: 761-769. Consistent with these results, stress reduces the number of large follicles and increases the number of obturator follicles in the ovary, thereby reducing plasma levels of reproductive-related steroid hormones (testosterone, progesterone and estrogen) {Henriksen, R., TG Groothuis, and S. Rettenbacher. 2011. Elevated plasma corticosterone reduced yolk testosterone and progesterone in chickens: Linking maternal stress and hormone-mediated maternal effects. PloS one 6: e23824. doi 10.1371 / journal.pone.0023824}. Stress reduces the expression of ovarian genes {Rozenboim, I., E. Tako, O. Gal-Garber, J. A. Proudman, and Z. Uni. 2007. The effect of heat stress on ovarian function of laying hens. Poult Sci 86: 1760-1765.), It has been reported that it can affect protein expression in tubo-oviducts and ovaries (Rozenboim, I., E. Tako, O. Gal-Garber, J. Proudman, and Z. Uni. 2007. The effect of heat stress on ovarian function of laying hens. Poult Sci 86: 1760-1765 .; Sargan, D. R., M. J. Tsai, and B. W. O'Malley. 1986. hsp108, a novel heat shock inducible protein of chicken. Biochemistry 25: 6252-6258. However, these stress markers require the sacrifice of the laying hens, but if eggs are used, stress markers can be measured without sacrificing the laying hens.

Recently, proteome analysis has been introduced as a means of analyzing differential gene expression at the protein level and identifying biomarkers {Matt, P., Z. Fu, Q. Fu, and JE Van Eyk . 2008. Biomarker discovery: proteome fractionation and separation in biological samples. Physiol Genomics 33: 12-17. However, proteomics studies have been developed mainly in disease-related studies (Burgess, S. C. 2004. Proteomics in the chicken: tools for understanding immune responses to avian diseases. Poult Sci 83: 552-573.}, There is very little research on the proteome of environmental stress.

Therefore, it is necessary to study the effects of diverse environmental physiological and genetic differences of poultry including laying hens on the expression of egg protein and use it as a stress marker.

Burgess, S. C. 2004. Proteomics in the chicken: tools for understanding immune responses to avian diseases. Poult Sci 83: 552-573. Choi, Y.-H. 2007. Management of Stress in Poultry Production. Korean J Poult Sci 34: 295-300. Henriksen, R., T. G. Groothuis, and S. Rettenbacher. 2011. Elevated plasma corticosterone reduced yolk testosterone and progesterone in chickens: Linking maternal stress and hormone-mediated maternal effects. PloS one 6: e23824. doi 10.1371 / journal.pone.0023824 Mashaly, M. M., G. L. Hendricks, 3rd, M. A. Kalama, A. E. Gehad, A. O. Abbas, and P. H. Patterson. 2004. Effect of heat stress on the production parameters and immune responses of commercial laying hens. Poult Sci 83: 889-894. Matt, P., Z. Fu, Q. Fu, and J. E. Van Eyk. 2008. Biomarker discovery: proteome fractionation and separation in biological samples. Physiol Genomics 33: 12-17. Mumma, J. O., J. P. Thaxton, Y. Vizzier-Thaxton, and W. L. Dodson. 2006. Physiological stress in laying hens. Poult Sci 85: 761-769. Puvadolpirod, S., and J. P. Thaxton. 2000. Model of physiological stress in chickens 1. Response parameters. Poult Sci 79: 363-369. Rochester, J. R., K. C. Klasing, L. Stevenson, M. S. Denison, W. Berry, and J. R. Millam. 2009. Dietary red clover (Trifolium pratense) induces oviduct growth and decreases ovary and testes growth in Japanese quail chicks. Reproductive toxicology (Elmsford, N.Y.) 27: 63-71. Rozenboim, I., E. Tako, O. Gal-Garber, J.A. Proudman, and Z. Uni. 2007. The effect of heat stress on ovarian function of laying hens. Poult Sci 86: 1760-1765. Sargan, D. R., M. J. Tsai, and B. W. O'Malley. 1986. hsp108, a novel heat shock inducible protein of chicken. Biochemistry 25: 6252-6258. Shini, S., P. Kaiser, A. Shini, and W. L. Bryden. 2008. Biological response of chickens (Gallus gallus domesticus) induced by corticosterone and a bacterial endotoxin. Comp Biochem Physiol B Biochem Mol Biol 149: 324-333. Shini, S., A. Shini, and G. R. Huff. 2009. Effects of chronic and repeated corticosterone administration in rearing chickens on physiology, the onset of lay and egg production of hens. Physiology & behavior 98: 73-77.

Accordingly, it is an object of the present invention to provide a biomarker protein specific to environmental stress of poultry.

 It is also an object of the present invention to provide an antibody against the biomarker protein and a diagnostic kit using the same.

It is another object of the present invention to provide a method for analyzing the environmental stress of poultry which is characterized by detecting the change in the biomarker protein expression level.

It is also an object of the present invention to provide a method for analyzing the quality of a egg, characterized in that the biomarker protein expression level is changed.

In order to accomplish the above object, the present invention provides a biomarker protein specific for environmental stress of poultry, which comprises an egg protein or a fragment thereof specifically expressed in environmental stress of poultry.

The present invention also provides a composition for diagnosing environmental stress of poultry containing the antibody specifically binding to the biomarker protein as an active ingredient or a diagnostic kit.

The present invention also provides a method for analyzing the environmental stress of poultry which is characterized by detecting the change in the expression level of an egg protein or a fragment thereof specifically expressed in environmental stress of poultry.

In addition, the present invention provides a method for analyzing egg quality, which comprises detecting the change in the expression level of an egg protein or a fragment thereof specifically expressed in environmental stress of a poultry.

Hereinafter, the present invention will be described in detail with reference to the drawings, examples, and the like.

As described above, according to the present invention, it is possible to analyze whether the expression of a specific egg protein component is increased or decreased through differential expression analysis of egg protein, so that it is possible to determine the stress of poultry at the time of scattering without sacrifice of poultry, Can be used as a new indicator (biomarker protein) in quality analysis of eggs.

In addition, antibodies prepared on the basis of the biomarker protein of the present invention can be used for diagnosis of environmental stress of poultry and immunoassay kit (ELISA, antibody-coated tube test, lateral-flow test, potable biosensor, etc.) Not only can it be used, but it can also be used to develop a protein chip having a variety of environmental stress diagnosis and egg quality analysis spectrum through the development of antibodies showing higher specificity and sensitivity.

FIG. 1 shows the result of SDS-PAGE analysis of the expression amount of egg white protein according to the stress (corticosterone) treatment of laying hens.
FIG. 2 is a result of two-dimensional electrophoresis analysis of egg white proteins of eggs laid on the day of feeding start (Day 0) in a control laying hens.
FIG. 3 shows the result of two-dimensional electrophoresis analysis of the egg white protein of eggs laid on the 5th day of feeding (Day 5) in the control laying hens.
FIG. 4 shows the result of two-dimensional electrophoresis analysis of the egg white protein of the egg laid on the day of feeding start (Day 0) in the experimental group.
FIG. 5 shows the result of two-dimensional electrophoresis analysis of the egg white protein of the egg laid on the fifth day of feeding (Day 5) in the experimental group.
FIG. 6 is a graph showing the change of the egg white protein spot number 9 (ovoinhibitor protein) in the control.
FIG. 7 is a graph showing changes in egg white protein spot number 21 (hemopexin protein) in the control. FIG.
8 is a graph showing changes in egg white protein spot number 28 (TENP protein) in the control.
9 is a graph showing changes in ovalbumin-related protein X 12 in the experimental group.
FIG. 10 is a graph showing the change of egg white protein spot number 18 (TENP, ovalbumin-related protein X) in the experimental group.
FIG. 11 is a graph showing the change of egg white protein spot number 19 (chondrogenesis-associated lipocalin) in the experimental group.
FIG. 12 is a graph showing changes in egg white protein spot number 22 (hemopexin) in the experimental group.
13 is a graph showing changes in ovalbumin-related protein X 24 in the experimental group.
14 is a graph showing changes in egg white protein spot number 27 (TENP, IGY FCU 3-4) in the experimental group.
15 is a graph showing the change in egg white protein spot number 28 (TENP) in the experimental group.
FIG. 16 is a graph showing changes in extracellular fatty acid-binding protein 29 in the experimental group.

Hereinafter, the composition and diagnostic kit for environmental stress diagnosis of poultry including the biomarker protein specific to environmental stress of the poultry according to the present invention and the antibody specifically binding to the protein, and the environmental stress of the poultry using the biomarker protein And how to analyze the quality of eggs.

The present invention provides a biomarker protein specific to environmental stress of poultry, which comprises an egg protein or a fragment thereof specifically expressing environmental stress of poultry.

The present invention also provides a composition for diagnosing environmental stress of poultry containing the antibody specifically binding to the biomarker protein as an active ingredient or a diagnostic kit.

"Fragment" in the present invention means a fragment of an egg protein having at least one epitope that can be recognized by an antibody to the egg protein of the present invention.

The egg protein of the present invention refers to a protein expressed in all parts of eggs such as egg white, egg yolk, eggshell egg, egg shell, and the like, but the egg white protein is used in the embodiment of the present invention.

The antibody of the present invention may be a polyclonal antibody, a monoclonal antibody or a recombinant antibody, but the antibody of the present invention is preferably a monoclonal antibody.

Polyclonal antibodies can be prepared by injecting an immunogen-causing biomarker protein into an external host according to conventional methods known to those skilled in the art. External hosts include, but are not limited to, mammals such as mice, goats, rabbits, sheep, monkeys, horses, pigs, cows, dogs and the like. Immunogens are injected by intramuscular injection, intraperitoneal injection, subcutaneous injection, etc., and are generally administered together with an adjuvant to increase antigenicity. Blood is routinely collected from an external host to collect the sera showing improved titer and specificity for the antigen or isolating and purifying the antibody therefrom.

Monoclonal antibodies can be produced by immortalized cell line generation techniques (Koeher and Milstein (1975) Nature, 256: 495 and Kohler and Milstein (1976) European Jounal of Immunology 6: 511) by fusion well known to those skilled in the art . More specifically, about 20 μg of a pure biomarker protein is first obtained and immunized with a suitable host animal such as a Balb / C mouse, or the peptide is synthesized and bound to bovine serum albumin and immunized with the host animal. The antibody-producing lymphocytes isolated from host animals such as mice are then fused by methods well known in the art, such as the use of human or mouse microRNAs and polyethylene glycols to produce immortalized hybridomas. Thereafter, only the hybridoma cells producing the desired monoclonal antibody are selected and proliferated using the ELISA method, and then the monoclonal antibody is separated and purified from the culture. The monoclonal antibody produced by the hybridoma may be used without purification. However, in order to obtain the best results, it is preferable to purify the antibody by high purity according to a method well known in the art.

Monoclonal antibodies may also be prepared by techniques known in the art, such as the phage antibody library (Clackson et al, Nature, 352: 624-628, 1991; Marks et al, J. Mol. Biol., 222: 58, . ≪ / RTI > More specifically, a phage antibody library method comprises obtaining an antibody gene (single chain fragment variable, scFv form) for a biomarker protein and expressing it in the form of a fusion protein on the surface of a phage, thereby preparing an antibody library in vitro And separating and preparing the monoclonal antibody binding to the biomarker protein.

The antibody prepared from the above method can be separated by methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography and the like.

Antibodies included in compositions or kits of the invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2, Fv and the like.

The diagnostic kit of the present invention is produced by a conventional manufacturing method known to a person skilled in the art, and includes a reagent capable of detecting an antigen-antibody complex in addition to the freeze-dried type antibody, buffer, stabilizer, For example, a labeled secondary antibody, chromophores, an enzyme (e.g., conjugated to an antibody) and its substrate, or other material capable of binding to the antibody, and the like. In addition, the antibody according to the present invention can be labeled with radionuclides, fluorescors, enzymes, and the like.

According to another aspect of the present invention, there is provided a method for analyzing environmental stress of a poultry that is characterized by detecting the change in the expression level of an egg protein or a fragment thereof that specifically expresses environmental stress of poultry .

In addition, the present invention provides a method for analyzing egg quality, which comprises detecting the change in the expression level of an egg protein or a fragment thereof specifically expressed in environmental stress of a poultry.

In the analysis methods of the present invention, the step of detecting the change of the expression level of the egg white protein may be performed by directly detecting the presence of the biomarker protein by two-dimensional electrophoresis (2-DE) from the eggs of the poultry, The antibody is specifically contacted with a biomarker protein to indirectly confirm the presence of the biomarker protein through an antigen-antibody reaction.

In order to measure protein levels using antibodies, Western blot, enzyme linked immunosorbent assay (ELISA) and antibody-conjugated magnetic particles were bound to the tube, and then antigen-tracer and degradation-resistant contaminants were competitively reacted with each other, (RIA), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket immunoassay, which can be used to quantitate and quantitate the response of an antibody- But are not limited to, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS, protein chips, and the like.

Through the above analysis methods, it is possible to analyze the environmental stress of the poultry and the egg quality by judging the egg protein produced by the stressed poultry and the egg protein which is differentially expressed in the egg produced by the unstressed poultry.

In particular, the advantage of using egg white for such analysis is that it takes 7-10 days to produce mature egg yolk from the ovary of a hen, but since it takes only 2-3 hours to produce egg white, (Ie, the stress state) of the plant.

In the present invention, the change in the expression level of the egg protein means that the expression level of the egg protein is increased or decreased.

In the present invention, expression of OVA and OVA protein X was increased while expression of TENP, hemopexin, IGY-FCU3-4 and extracellular fatty acid-binding protein (Ex-FABP) was decreased.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited by the following examples.

Example 1: Animal experiment (stress model of laying hens)

Twenty-four-week-old single-comb brown Hy-Line Leghorn hens were individually housed in a three-row upright cage with four chambers in each of two chambers of equal size. The temperature of the breeding room was maintained at around 20 ± 2 ℃ and turned on at 6:00 am and turned off at 9:00 pm (lights up for 15 hours). During the test period feed and water were supplied without restriction. Layers were randomly divided into two treatments (control and experimental groups) and divided into two groups. After 2 weeks of adaptation period, control diet (containing 1 mg of corticosterone in 1 kg of feed) In the other treatments (experimental group), experimental feeds (including 1 kg of feed and 30 mg of corticosterone) were fed for 2 weeks.

Control feeds were prepared by dissolving 500 ml of ethanol (EtOH) in 500 ml of 99.5% EtOH and 815 mg of corticosterone (92%, C2505, Sigma, USA) in a total volume of 99.5% EtOH. .

Example 2: Egg filling

Control Eggs or experimental feeds Eggs were collected at the start of day 0 (Day 0) and 5 days of feeding (Day 5), and only egg white was collected, lyophilized and stored at -80 ° C until analysis.

Example 3: egg white protein extraction and SDS - PAGE  analysis

After lyophilized egg white was crushed into a mortar, the lysate buffer A (1% sodium dodecyl sulfate (SDS), 1 mM phenylmethanesulfonyl fluoride (PMSF), protease inhibitor cocktail, 100 mM Tris-HCl (pH 6.8)). Same dissolution buffer B as dissolution buffer A (7 M urea, 2 M thiourea, 4% CHAPS, 0.1 M dithiothreitol (DTT), 1 mM PMSF, protease inhibitor cocktail, 40 mM Tris-HCl (pH 6.8)) was added and mixed at room temperature for 1 hour. The mixture was sonicated for a while and incubated at room temperature for 1 hour. The cells were centrifuged at 15,000 × g at 4 ° C. for 20 minutes, cultured at 4 ° C. for 1 hour, and further centrifuged at 15,000 × g at 4 ° C. for 20 minutes. The supernatant was subjected to quantification by using 2DE Quant kit (GE Healthcare Life Sciences, Uppsala, Sweden). SDS-PAGE analysis was performed as follows. 5 μg of soluble protein was mixed with sample buffer (2% SDS, 10% glycerol, 500 mM Tris-HCl, pH 6.8) and electrophoresed in 12% SDS polyacrylamide gel at 20 mA for 3 hours. (silver stain).

Example 4: Two-dimensional electrophoresis (2 DE ) analysis

200 μg of the water soluble protein was mixed with a rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2.5% DTT, 10% isopropanol, 5% glycerol and 2% IPG buffer). Protein was rehydrated for 18 hours using an 18 cm IPG strip of pH 4-10, non-linear (NL). In order to firstly separate the proteins by pH, the voltage was gradually increased from 30 V at 50 V for 30 minutes, 100 V for 30 minutes, 250 V for 1 hour and 8,000 V for 9 hours using an IPGphor 3 system (GE amersharm) And isoelectric focusing for 6 hours. The IPG strips subjected to isoelectric point electrophoresis were equilibrated in equilibration buffer (6M urea, 50 mM Tris-HCl pH 8.8, 30% glycerol, 2% SDS and bromophenol blue) and 1% DTT for 15 minutes equilibration was performed and secondary equilibration was performed with 2.5% iodoacetamide. SDS-PAGE gels were prepared using 7.5% SDS polyacrylamide gels using Gradi-Gel ^™ II gradient PAGE analysis kit (EBA-1015, Elpis, Daejeon, Korea)

SDS-PAGE gels were electrophoresed at 20 mA for 18 hours and silver stained. Protein expression between two treatments (control and experimental) was compared and analyzed using image master (Image Master 2D Plantinum v7.0, GE) and the spot intensity of three gel images per treatment area was measured. In the same treatment, the difference between the day of onset of feeding (Day 0) and day 5 of feeding (Day 5) was tested by t-test and significance was recognized when the difference was P ≤0.05. All experimental data were expressed as mean ± standard error.

Example 5 Mass Spectrometry ( Matrix - Assisted Laser Desorption - Ionization  Time-of-Flight Mass Spectrometry  ( MALDI - TOF / MS ) Wow MALDI - TOF / TOF / MS / MS  analysis)

The desired spot was cut out using a pipette tip and collected in a tube. 30mM potassium ferricyanide and 100mM sodium thiosulfate pentahydrate were added to remove the silver dye from the gel and dried completely. 50-100 μl of 10 mM DTT / 0.1 M ABC was added and reacted at 56 ° C for about 45 minutes. The supernatant was removed and 50-100 μl of 55 mM lodoacetamide / 0.1 M ABC was added, followed by reaction for about 30 minutes and then completely dried. 3 μl of trypsin digestion buffer (0.0012 μg / μL in 25 mM NH 4 HCO 3, pH 7.8) was added and left overnight at 37 ° C. The sample was extracted with an extraction solution (acetonitrile: water: TFA = 66: 33: 0.1) to extract the peptide from the gel. The extracted peptide solution was mixed with a matrix solution (α-cyano-4-hydroxy-cinnamic acid), and the mixture was dispensed into a MALDI TOF / TOF sample plate. The ABI 4800 Plus TOF-TOF Mass Spectrometer (Applied Biosystems, Framingham, Mass., USA) and analyzed as follows. The amino acid sequences of these proteins were searched using the NCBInr database for MS / MS spectra.

Spot number protein
name Accession No. Molecular Weight PI Score
(MOWSE) Sequence coverage
(%) Matched peptides mass
analysis Database 4 Ovoinhibitor P10184 54,394 6.16 58 32 14 MS Uni / Swiss 5 Ovoinhibitor P10184 54,394 6.16 47 23 14 MS Uni / Swiss 6 Ovoinhibitor P10184 54,394 6.16 46 22 13 MS Uni / Swiss 7 Ovoinhibitor P10184 54,394 6.16 362 33 19 MS / MS Uni / Swiss 8 Ovoinhibitor P10184 54,394 6.16 46 17 10 MS Uni / Swiss 11 Ovoinhibitor P10184 54,394 6.16 37 20 11 MS Uni / Swiss 12 Ovalbumin-related protein X P01013 26,331 5.1 79 30 7 MS / MS Uni / Swiss 17 TENP O42273 47,804 5.53 39 13 6 MS / MS Uni / Swiss 18 TENP O42273 47,804 5.53 147 23 10 MS / MS Uni / Swiss 18 Ovalbumin-related protein X P01013 26,331 5.1 103 27 8 MS / MS Uni / Swiss 21 Hemopexin Q90WR3 29,765 5.92 35 24 9 MS / MS Uni / Tr 22 Hemopexin Q90WR3 29,765 5.92 340 48 18 MS / MS Uni / Tr 24 Ovalbumin-related protein X P01013 26,331 5.1 108 27 8 MS / MS Uni / Swiss 24 Ovotransferrin P02789 79,551 6.69 63 21 10 MS / MS Uni / Swiss 27 TENP O42273 47,804 5.53 246 24 10 MS / MS Uni / Swiss 27 IGY FCU3-4 UPI00018BF9C1 25,466 5.57 112 30 4 MS / MS UniParc 28 TENP O42273 47,804 5.53 232 20 13 MS / MS Uni / Swiss 29
EX-FABP P21760 20,359 5.56 351 52 17 MS / MS Uni / Swiss

* Spot number corresponds to two-dimensional electrophoresis (2DE) gel image label.

* Spot numbers 1, 2, 3, 9, 10, 13, 14, 15, 16, 19, 20, 23, 25, 26 and 30 were not found in the database or were not identified due to detection limits.

* The source of accession number is the Uniprot (universal protein resource) database.

* Score (MOWSE) is used for MASCOT PMF (peptide mass fingerprint) and represents MS data of NCBInr database. Significance when score is greater than 84 ( P ≤ 0.05).

The results of the above embodiments are shown in the following figures and tables.

Firstly, the expression of egg white protein in the eggs of the laying hens (experimental group) treated with corticosterone for 5 days in SDS-PAGE analysis was significantly lower than the day of feeding (Day 0). However, these changes were not observed in the control (Fig. 1).

After the start of the test, secondary electrophoresis was carried out using the egg white of the egg packed on the day of the pay (Day 0) and the 5th day of the feeding (Day 5), and 2DE gel was analyzed with Image Mater. The spots matched. Among them, 30 spots in egg protein of laying hens fed corticosterone for 5 days were increased or decreased 1.2 times compared to the control.

The spot intensities of three gel images were measured using an image master and analyzed statistically by t-test. In the group treated with Corticosterone (experimental group), three protein spots (numbers 12, 18 and 24) were significantly increased (P? 0.05) and five spots (numbers 19, 22, 27, 28 and 29) Respectively. On the other hand, in the control, two spots (numbers 21 and 28) increased and one spot (spot number 9) decreased in comparison with day 0 on the pay day (Day 5) ).

Treatment
Spot number showing a significant expression change of P < 0.05 decrease increase Control 9 21, 28 Experimental Section 19, 22, 27, 28, 29 12, 18, 24

Protein spots were analyzed by MALDI TOF / MS or MALDI TOF-TOF / MS / MS and classified as MASCOT database (taxonomy) by gallus gallus. In the MASCOT database, scoring based on the MOWSE algorithm was performed and a score of 84 or more was defined as a statistically significant sequence (P <0.05). The results are shown in Figs. 2 to 5 and Table 3 below. The results are shown in Figs. 2 to 5 and Table 3 below.

Protein name

Spot number
Protein identification or estimation Protein differential expression between Day 1 (Day 0) and Day 5 (Day 5) Control Experimental Section Probability of significance
(p-value) Probability of significance
(p-value)



Ovoinhibitor 3 calculation - 0.24 - 0.23 4 Sympathy - 0.30 - 0.19 5 Sympathy - 0.21 - 0.24 6 Sympathy - 0.28 ↑ 0.07 7 Sympathy - 0.23 ↑ 0.07 8 Sympathy - 0.33 ↑ 0.07 9 calculation ↓ 0.03 ↑ 0.07 10 calculation - 0.22 ↑ 0.08 11 Sympathy ↓ 0.07 ↑ 0.07
Ovalbumin-related protein X

12 Sympathy - 0.36 ↑ 0.05 18 Sympathy - 0.41 ↑ 0.01 23 calculation - 0.08 - 0.19 24 Sympathy - ↑ 0.06 26 calculation - 0.27 - 0.13
Hemopexin 20 calculation - 0.33 - 0.35 21 Sympathy ↑ 0.05 - 0.12 22 Sympathy - 0.11 ↓ 0.05

TENP



13 calculation ↑ 0.06 ↓ 0.07 14 calculation - - 0.28 15 calculation - 0.46 ↓ 0.09 16 calculation - 0.15 - 0.16 17 Sympathy - 0.41 - 0.20 18 Sympathy - 0.41 ↑ 0.01 27 Sympathy - 0.06 ↓ 0.01 28 Sympathy ↑ 0.02 ↓ 0.03 Chondrogenesis-associated lipocalin 19 calculation ↑ 0.09 ↓ 0.05 30 calculation - 0.36 - 0.18 IGY-FCU 3-4 27 Sympathy - 0.06 ↓ 0.01 Extracellular fatty acid-binding protein 29 Sympathy - 0.33 ↓ 0.01

* All spots showed a significance of P <0.09, -, no change; ↑, increase; ↓, decrease

15 spots were identified as ovoinhibitor (spot numbers 4-8, 11), ovalbumin-related protein X (spot numbers 12, 18 and 24), hemopexin (spot numbers 21 and 22), TENP (spot numbers 17, ), IGY FCU3-4 (spot number 27), and extracellular fatty acid-binding protein (spot number 29).

The number of ovoinhibitor proteins (spot number 9) decreased significantly ( P <0.05) in the eggs of the control laying hen compared to the day of feeding (Day 0) (Spot number 11) also decreased ( P <0.07). hemopexin (spot number 21) and the protein estimated by TENP (spot number 28) were significantly increased (FIGS. 6 to 8).

On the other hand, ovalbumin-related protein X (spot numbers 12, 18 and 24) and TENP (spot number 18) were observed on the 5th day of feeding (Day 5) in the eggs of the corticosterone- (Spot number 22), TENP (spot numbers 27 and 28), IGY-FCU 3-4 (spot number 27), extracellular fatty acid-binding protein (spot number 29), and chondrogenesis- The protein estimated to be associated lipocalin (spot number 19) was significantly reduced but the difference was small (Figs. 9 to 16). Estimated (spot numbers 9 and 10) (P = 0.08) protein spots were identified as Ovoinhibitor (spot numbers 6, 7, 8 and 11) (P = 0.07) and tended to be increased by treatment with corticosterone. On the other hand, proteins estimated to be TENP (spot numbers 13 and 15) tended to decrease ( P <0.07, P <0.09, respectively).

Hemopexin (HPX) (spot numbers 21 and 22) is a glycoprotein that is mainly expressed in the liver and secreted into the blood. HPX has a very strong affinity for heme and binds at the same molar ratio and protects the body from oxidative damage that can be caused by freezing ham by removing the ham from hemoglobin { Tolosano, E., and F. Altruda. 2002. Hemopexin: structure, function, and regulation. DNA and cell biology 21: 297-306. doi 10.1089 / 104454902753759717}. The binding with ham confers resistance to proteolytic digestion {Goldfarb, V., RB Trimble, M. De Falco, HH Liem, SA Metcalfe, D. Wellner, and U. Muller-Eberhard. 1986. An avian serum alpha 1-glycoprotein, hemopexin, differing significantly in amino acid and carbohydrate composition from mammalian (beta-glycoprotein) counterparts. Biochemistry 25: 6555-6562. HPX belongs to the acute phase protein, which increases the synthesis of this protein by inflammation and stress (Tolosano, E., and F. Altruda. 2002. Hemopexin: structure, function, and regulation. DNA and cell biology 21: 297-306. doi 10.1089 / 104454902753759717}. HPX is found in serum of chicken is a kind of glycoprotein, molecular weight _{52 kDa (α 1 -glycoprotein) {} Goldfarb, V., RB Trimble, M. De Falco, HH Liem, SA Metcalfe, D. Wellner, and U. Muller -Eberhard. 1986. An avian serum alpha 1-glycoprotein, hemopexin, differing significantly in amino acid and carbohydrate composition from mammalian (beta-glycoprotein) counterparts. Biochemistry 25: 6555-6562. In chickens, HPX begins to be measured several days before hatching, and the concentration of plasma increases rapidly after hatching, and the concentration at 4 days is about half of the adult (3.5 months) level. Plasma concentrations are rapidly increased by stresses such as terpentine administration (Grieninger, G., TJ Liang, G. Beuving, V. Goldfarb, S. Metcalfe, and U. Muller-Eberhard. 1986. Hemopexin is a developmentally regulated, acute-phase plasma protein in the chicken. The Journal of Biological Chemistry 261: 15719-15724. Similarly, an increase in hemopexin synthesis has been reported by immune challenge such as intraperitoneal administration of lipopolysaccharide or subcutaneous administration of human serum albumin (Barnes, DM, Z. Song, KC Klasing, and W. Bottje. 2002. Protein metabolism during an acute phase response in chickens. Amino acids 22: 15-26 .; Buyse, J., Q. Swennen, TA Niewold, KC Klasing, GP Janssens, M. Baumgartner, and BM Goddeeris. 2007. Dietary L-carnitine supplementation enhances the lipopolysaccharide-induced acute phase protein response in broiler chickens. Veterinary immunology and immunopathology 118: 154-159. doi 10.1016 / J.Vehim.2007.04.014; Buyse, J., Q. Swennen, F. Vandemaele, K. Klasing, TA Niewold, M. Baumgartner, and BM Goddeeris. 2009. Dietary beta-hydroxy-beta-methylbutyrate supplementation influences performance differently after immunization in broiler chickens. Journal of animal physiology and animal nutrition 93: 512-519. doi}. When administered intravenously to uninfected ducks, full-length IgY and short IgY isolated from E. coli-infected ducks, the concentration of plasma HPX was increased by the administration of E. coli alone (100: 0 or 0: 100) (50:50) at the same ratio (Humphrey, BD, CC Calvert, and KC Klasing. 2004. The ratio of full length IgY to truncated IgY in immune complexes affects macrophage phagocytosis and the acute phase response of mallard ducks (Anas platyrhynchos). Developmental and comparative immunology 28: 665-672. doi 10.1016 / j.dci.2003.11.003}.

In the present study, HPX was significantly ( P <0.05) low (spot number 22) and significantly increased ( P <0.05) in the control egg compared with egg white eggs produced from the corticosterone-treated laying hens (Spot number 21) suggests the following fact. First, these spots (spot numbers 21 and 22) may be proteins with different functions. Although these spots (spot numbers 21 and 22) were determined to be HPX through MALDI-TOF / TOF analysis and bioinformatis, these two spots showed the opposite response to treatment with corticosterone. Second, corticosterone reduces the secretion of HPX in the fallopian tubes. In contrast to the slight but significant increase in HPX at the control, treatment with corticoseterone significantly reduced HPX expression. Although the concentration of HPX in the blood was not measured in this study, documented evidence suggests that a decrease in the expression of HPX in the egg white is a notable change, considering that serum HPX concentrations are increased by acute stress such as inflammation.

Ovoinhibitor (Spot Nos. 4, 5, 6, 7, 8 and 11) is one of the four proteinase inhibitors (or also known as antiproteinases) found in egg white and has a molecular weight of 48.6-49 kDa {Saxena, I., and S. Tayyab. 1997. Protein proteinase inhibitors from avian egg whites. Cell Mol Life Sci 53: 13-23. }, The gene of this protein is strongly expressed in the liver, fallopian tube and egg yolk of algae {Bourin, M., J. Gautron, M. Berges, S. Attucci, G. Le Blay, V. Labas, Y. Nys, and S. Rehault-Godbert. 2011. Antimicrobial potential of egg yolk ovoinhibitor, a multidomain Kazal-like inhibitor of chicken egg. Journal of Agricultural and Food Chemistry 59: 12368-12374. doi 10.1021 / jf203339t; Yen, CF, EC Lin, YH Wang, PH Wang, HW Lin, JC Hsu, LS Wu, YN Jiang, and ST Ding. 2009. Abundantly expressed hepatic genes and their differential expression in liver of prelaying and laying geese. Boulin, M., J. Gautron, M. Berges, S. Attucci, G., < / RTI &gt; Poult Sci 88: 1955-1962. Le Blay, V. Labas, Y. Nys, and S. Rehault-Godbert. 2011. Antimicrobial potential of egg yolk ovoinhibitor, a multidomain Kazal-like inhibitor of chicken egg. Journal of Agricultural and Food Chemistry 59: 12368-12374 .; Yen, CF, EC Lin, YH Wang, PH Wang, HW Lin, JC Hsu, LS Wu, YN Jiang, and ST Ding. 2009. Abundantly expressed hepatic genes and their differential expression in liver of prelaying and laying geese. Poult Sci 88: 1955-1962. Ovoinhibitor has also been reported to be expressed in F-sac {Moore, RW, BM Hargis, TE Porter, DY Caldwell, CM Oubre, F. Vandesande, and LR Berghman. 2004. Ovoinhibitor in the chicken bursa of Fabricius: identification, isolation, and localization. Cell and tissue research 317: 247-251. doi 10.1007 / s00441-004-0910-x}. The concentration of ovoinhibitor in a protein is about 1.5% of the total protein {Kinoshita, K., T. Shimogiri, S. Okamoto, K. Yoshizawa, H. Mannen, HR Ibrahim, HH Cheng, and Y. Maeda. 2004. Linkage mapping of chicken ovoinhibitor and ovomucoid genes to chromosome 13. Animal genetics 35: 356-358. doi 10.1111 / j.1365-2052.2004.01159.x}, trypsin, chymotrypsin elastase and the like (Feeney, RE, FC Stevens, and DT Osuga. 1963. The specificities of chicken ovomucoid and ovoinhibitor. The Journal of Biological Chemistry 238: 1415-1418 .; Rehault, S. 2007. Antiproteases. Pages 85-92 in Bioactive Egg Compounds. R. Huopalahti, R. L., M. Anton, and R. Schade eds. Springer Berlin Heidelberg. The synthesis and secretion of this protein is controlled by estrogen and progesterone {Saxena, I., and S. Tayyab. 1997. Protein proteinase inhibitors from avian egg whites. Cell Mol Life Sci 53: 13-23 .; Zhu, Y., M. Wang, H. Lin, Z. Li, and J. Luo. 2001. Identification of estrogen-responsive genes in chick liver. Cell and tissue research 305: 357-363. In this study, the expression of ovoinhibitor was increased in eggs of corticosterone-treated laying hens (experimental group) (spot numbers 6, 7, 8 and 11) ( P <0.07). However, in the control laying hens, the number of spots (number 9) estimated as ovoinhibitor decreased significantly, but the expression of another protein spot (number 11) tended to decrease ( P <0.07). These results suggest that stress increases the expression of ovoinhibitor in the egg white.

The TENP protein was presumed to be involved in the early neural development of the embryo, since it is initially transiently expressed only in the retina and brain of the hatching chicken embryo and not in other organs (i.e., heart, liver and kidney) {Yan, RT , and SZ Wang. 1998. Identification and characterization of tenp, a gene transiently expressed before overt cell differentiation during neurogenesis. Journal of neurobiology 34: 319-328. However, TENP protein is expressed in amniotic white eggs {Guerin-Dubiard, C., Pasco, D. Molle, C. Desert, T. Croguenec, and F. Nau. 2006. Proteomic analysis of hen egg white. Journal of Agricultural and Food Chemistry 54: 3901-3910 .; Mann, K. 2007. The chicken egg white proteome. Proteomics 7: 3558-3568 .; D'Ambrosio, C., S. Arena, A. Scaloni, L. Guerrier, E. Boschetti, M. E. Mendieta, A. Citterio, and P. G. Righetti. 2008. Exploring the chicken egg white proteome with combinatorial peptide ligand libraries. J Proteome Res 7: 3461-3474.} And yolk {Mann, K., and M. Mann. 2008. The chicken egg yolk plasma and granule proteomes. Proteomics 8: 178-191 .; Farinazzo, A., U. Restuccia, A. Bachi, L. Guerrier, F. Fortis, E. Boschetti, E. Fasoli, A. Citterio, and P. G. Righetti. 2009. Chicken egg yolk cytoplasmic proteome, mined via combinatorial peptide ligand libraries. Journal of chromatography. A 1216: 1241-1252.}, It was found that these estimates were not true. In recent years, TENP has also been found in oocytes of ostrich and amorphous nematodes {Maehashi, K., M. Matano, T. Irisawa, M. Uchino, Y. Itagaki, K. Takano, Y. Kashiwagi, and T. Watanabe. 2010. Primary Structure of Potential Allergenic Proteins in Emu (Dromaius novaehollandiae) Egg White. Journal of agricultural and food chemistry. doi 10.1021 / jf103239v; Takeuchi, J., K. Maehashi, Y. Yasutake, Y. Muramatsu, K. Miyata, T. Watanabe, and T. Nagashima. 2012. Properties of emu (Dromaius novaehollandiae) albumen proteins. Food Research International 49: 567-571. doi 10.1016 / j.foodres.2012.07.045}. TENP accounts for 0.1-0.5% of the dried egg white protein {Mann, K. 2007. The chicken egg white proteome. Proteomics 7: 3558-3568.} A protein with a molecular weight of about 47-50 kDa {Guerin-Dubiard, C., M. Pasco, D. Molle, C. Desert, T. Croguennec, and F. Nau. 2006. Proteomic analysis of hen egg white. Journal of agricultural and food chemistry 54: 3901-3910. Although the function of this protein is still unknown, it is presumed to have antibacterial activity due to belonging to the family of bacterial permaability-increasing protein (BPI) {Guerin-Dubiard, C., M. Pasco, D. Molle, C. Desert, T Croguennec, and F. Nau. 2006. Proteomic analysis of hen egg white. Journal of agricultural and food chemistry 54: 3901-3910. TENP (spot number 28) was significantly decreased in the stressed laying hens (experimental group), but the expression of this protein (spot number 28) was significantly increased in the control group. In another spot (# 18) analyzed by TENP or OVAX, expression in the egg white was significantly increased after stress, but another spot (# 27) analyzed with TENP or IGY-FCU3-4 showed small but significant Respectively. However, no changes in these (spot numbers 18 and 27) were found in egg white derived from the control laying hens.

Ovalbumin-related protein X (OVAX) (spot number 12), also known as Gene X protein, is composed of 232 amino acids and the gene for this protein is named SERPINB14C (http://www.uniprot.org/uniprot/P01013) . According to the Serpin nomenclature, ovalbumin, gene Y and gene X are serpinb14, serpinb14b and serpinb14c, respectively (Benarafa, C., and E. Remold-O'Donnell. 2005. The ovalbumin serpins revisited: perspective from the chicken genome of clade B serpin evolution in vertebrates. Proceedings of the National Academy of Sciences of the United States of America 102: 11367-11372. doi 10.1073 / pnas.0502934102}. OVAX is present in canine vaginal epithelium and has 77% homology with ovalbumin {Poirier, J. C., V. Herve-Grepinet, Y. Nys, and S. Rehault-Godbert. Year. Development of an ELISA for quantifying egg white ovalbumin-related protein X (OVAX) [Conference poster]. Paris.}. In a recent study, OVAX was "similar to ovalbumin-related protein Y (IPI00585021)" {Mann, K. 2007. The chicken egg white proteome. Proteomics 7: 3558-3568 .; D'Ambrosio, C., S. Arena, A. Scaloni, L. Guerrier, E. Boschetti, M. E. Mendieta, A. Citterio, and P. G. Righetti. 2008. Exploring the chicken egg white proteome with combinatorial peptide ligand libraries. J Proteome Res 7: 3461-3474.}, The latter containing a complete sequence of the Ovalbumin-related protein X derived from a known cDNA {Mann, K. 2007. The chicken egg white proteome. Proteomics 7: 3558-3568. Also, ovalbumin-related protein Y is also present in the yolk sac {Farinazzo, A., U. Restuccia, A. Bachi, L. Guerrier, F. Fortis, E. Boschetti, E. Fasoli, A. Citterio, and P. G. Righetti. 2009. Chicken egg yolk cytoplasmic proteome, mined via combinatorial peptide ligand libraries. Journal of chromatography. A 1216: 1241-1252. The average content of OVAX in egg white is about 2.4 mg / mL and exhibits antimicrobial activity {Poirier, J. C., V. Herve-Grepinet, Y. Nys, and S. Rehault-Godbert. Year. Development of an ELISA for quantifying egg white ovalbumin-related protein X (OVAX) [Conference poster]. Paris.}. In this study, OVAX expression was significantly increased in stressed laying hens (spot numbers 12, 18, and 24), but these changes were not observed in control egg laying hens. In another spot (No. 18) analyzed by OVAX or TENP, expression in the egg white was significantly increased after stress.

Fatty acids are important not only as nutrients that provide energy, essential fatty acids, but also as a mediator of signal transduction for constitutive and metabolic regulation of cell membranes {Hotamisligil, G. S. 2006. Inflammation and metabolic disorders. Nature 444: 860-867 .; Kalish, B. T., E. M. Fallon, and M. Puder. 2012.A tutorial on fatty acid biology. JPEN. Journal of parenteral and enteral nutrition 36: 380-388. doi 10.1177 / 0148607112449650; Saltiel, A. R., and C. R. Kahn. 2001. Insulin signaling and regulation of glucose and lipid metabolism. Nature 414: 799-806. Since fatty acids are hydrophobic, they are believed to exist in the state of being bound to fatty acid-binding proteins rather than being present in the state of free fatty acids in order to dissolve in biological fluids and migrate for metabolism. Extracellular fatty acid-binding protein (Ex-FABP) (spot number 29) was first found in chickens and was identified as protein Ch21, quiescence-specific protein or p20K (http://www.uniprot.org/uniprot/P21760) or and is called chondrogenesis-associated lipocalin {Cancedda, FD, M. Malpeli, C. Gentili, V. Di Marzo, P. Bet, M. Carlevaro, S. Cermelli, and R. Cancedda. 1996. The developmentally regulated avian Ch21 lipocalin is an extracellular fatty acid-binding protein. The Journal of Biological Chemistry 271: 20163-20169.), Belonging to the superfamily of chondrogenesis-associated lipocalin. These members have a molecular weight of about 20 kDa and are 20-30% homologous (Larsen et al., 1999). This protein was first identified in bone and cartilage of developing chicken embryos. {Cancedda, F. D., B. Dozin, F. Rossi, F. Molina, R. Cancedda, A. Negri, and S. Ronchi. 1990. The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins. In addition, granulocytes have been found in muscle fibers and blood {Descalzi Cancedda, F., B. Dozin, B. Zerega, S. Cermelli, and R. Cancedda . 2000. Ex-FABP: a fatty acid binding lipocalin developmentally regulated in chicken endochondral bone formation and myogenesis. Biochimica et biophysica acta 1482: 127-135. Ex-FABP can bind and transport long-chain unsaturated fatty acids (not conjugated to short chain fatty acids) in the extracellular fluid and serum {Cancedda, FD, M. Malpeli, C. Gentili, V. Di Marzo, P Bet, M. Carlevaro, S. Cermelli, and R. Cancedda. 1996. The developmentally regulated avian Ch21 lipocalin is an extracellular fatty acid-binding protein. The Journal of Biological Chemistry 271: 20163-20169. Ex-FABP is involved in cardiac development, fatty acid transport and lipid metabolism and is likely to function as a cell viable protein that protects cells from lipid toxicity from fat accumulation {Gentili, C., G. Tutolo, B. Zerega, E Di Marco, R. Cancedda, and FD Cancedda. 2005. Acute phase lipocalin Ex-FABP is involved in heart development and cell survival. Journal of cellular physiology 202: 683-689. doi 10.1002 / jcp.20165}. Ex-FABP is expressed in chicken liver and its expression is increased by inflammation inducer (LPS; IL6), and this expression is reduced by the treatment of anti-inflammatory agent and plays the role of acute phase protein {Cermelli, S., B Zerega, M. Carlevaro, C. Gentili, B. Thorp, C. Farquharson, R. Cancedda, and FD Cancedda. 2000. Extracellular fatty acid binding protein (Ex-FABP) modulation by inflammatory agents: "physiological" acute phase response in endochondral bone formation. European Journal of Cell Biology 79: 155-164 .; Di Marco, E., N. Sessarego, B. Zerega, R. Cancedda, and F. D. Cancedda. 2003. Inhibition of cell proliferation and induction of apoptosis by ExFABP gene targeting. Journal of cellular physiology 196: 464-473. doi 10.1002 / jcp.10310}. The expression of Ex-FABP was reduced by antibody treatment to Ex-FABP, and when the expression of Ex-FABP was inhibited, cell death was increased, cell activity was decreased, and differentiation and proliferation were inhibited {Di Marco, E. , N. Sessarego, B. Zerega, R. Cancedda, and FD Cancedda. 2003. Inhibition of cell proliferation and induction of apoptosis by ExFABP gene targeting. Journal of cellular physiology 196: 464-473. doi 10.1002 / jcp.10310}. Thus, Ex-FABP has been suggested as a constitutive survival protein (Di Marco, E., N. Sessarego, B. Zerega, R. Cancedda, and F. D. Cancedda. 2003. Inhibition of cell proliferation and induction of apoptosis by ExFABP gene targeting. Journal of cellular physiology 196: 464-473. doi 10.1002 / jcp.10310}. Ex-FABP was associated with abdominal fat weight in chickens and was suggested as the main gene controlling abdominal fat characteristics by SNP analysis (Qiu, XM, N. Li, XM Deng, ZX Lian, QG Wang, XL Wang, and CX Wu. 2005. Study of Ex-FABP as a main candidate gene on abdominal fat traits in chicken. Prog Biochem Biophys 32: 429-434. In a study in which 3-week-old white leghorn was infected with E. coli, gene expression of Ex-FABP was significantly increased in lung, liver and spleen compared with control, especially in lung and liver (Garenaux, A. , S. Houle, B. Folch, G. Dallaire, M. Truesdell, F. Lepine, N. Doucet, and CM Dozois. 2012. Avian lipocalin expression in chickens following Escherichia coli infection and inhibition of avian pathogenic Escherichia coli growth by Ex-FABP. Veterinary immunology and immunopathology. doi 10.1016 / j. Similar results have also been reported by other investigators {Cermelli, S., B. Zerega, M. Carlevaro, C. Gentili, B. Thorp, C. Farquharson, R. Cancedda, and F. D. Cancedda. 2000. Extracellular fatty acid binding protein (Ex-FABP) modulation by inflammatory agents: "physiological" acute phase response in endochondral bone formation. European Journal of Cell Biology 79: 155-164 .; Di Marco, E., N. Sessarego, B. Zerega, R. Cancedda, and F. D. Cancedda. 2003. Inhibition of cell proliferation and induction of apoptosis by ExFABP gene targeting. Journal of cellular physiology 196: 464-473. doi 10.1002 / jcp.10310}. In addition, Ex-FABP inhibited the growth of E. coli in in vitro studies with limited iron utilization (Garenaux, A., S. Houle, B. Folch, G. Dallaire, M. Truesdell, F. Lepine, N. Doucet, and CM Dozois. 2012. Avian lipocalin expression in chickens following Escherichia coli infection and inhibition of avian pathogenic Escherichia coli growth by Ex-FABP. Veterinary immunology and immunopathology. doi 10.1016 / j.

In this study, expression of Ex-FABP was significantly ( P <0.01) decreased in eggs of egg produced from corticosterone-treated laying hens (experimental group) but no such changes were detected in the control group. Although no causal relationship between Ex-FABP expression in the egg white and Ex-FABP gene expression in the lung and liver has been studied, the results of this study suggest that the organ of the infected chicken (eg lung, liver, ), The expression of Ex-FABP gene and the expression of Ex-FABP in egg white may be inversely proportional. From another point of view, current results suggest that stress may decrease the expression of Ex-FABP gene in the fallopian tube and result in decreased secretion of Ex-FABP into the egg white. From this viewpoint, recent proteomics studies that the content of the precursor of Ex-FABP decreases as the egg storage temperature increases {Qiu, N., M. Ma, L. Zhao, W. Liu, Y. Li, and Y Mine. 2012. Comparative Proteomic Analysis of Egg White Proteins under Various Storage Temperatures. Journal of Agricultural and Food Chemistry 60: 7746-7753. doi 10.1021 / jf302100m} suggest that Ex-FABP can be used to evaluate the quality of the egg.

Chondrogenesis associated lipocalin (CAL) was also reported as Lipocalin-type prostaglandin D synthase and was first detected in chickens. This protein has 185 amino acid sequences and belongs to the calycin superfamily / Lipocalin family (http://www.uniprot.org/uniprot/Q8QFM7). The lipocalin family of proteins includes Ex-FABP, chondrogenesis-related lipocalin beta (CAL beta) and chondrogenesis-related lipocalin gamma (CAL gamma) {Pagano, A., P. Giannoni, A. Zambotti, N. Randazzo, B. Zerega, R. Cancedda, and B. Dozin. 2002. CALbeta, a novel lipocalin associated with chondrogenesis and inflammation. European journal of cell biology 81: 264-272 .; Pagano, A., R. Crooijmans, M. Groenen, N. Randazzo, B. Zerega, R. Cancedda, and B. Dozin. 2003. A chondrogenesis-related lipocalin cluster includes a third new gene, CALgamma. Gene 305: 185-194.}, Divided into individual genes in a single bundle of genomes. Chondrogenesis associated lipocalin is specifically expressed during the hypertrophic phase of chondrocyte maturation during intra-osteochondral ossification, can be induced by immunostimulation and is expressed in liver and brain (http://www.ebi.ac.uk/ena/data / view / AAL99254). Pagano et al. Found CALβ in chicken embryos {Pagano, A., P. Giannoni, A. Zambotti, N. Randazzo, B. Zerega, R. Cancedda, and B. Dozin. 2002. CALbeta, a novel lipocalin associated with chondrogenesis and inflammation. European journal of cell biology 81: 264-272.}. CALβ is highly expressed in the heart of chicken embryos and is expressed to a lesser extent in skeletal muscles, lungs and liver and is the lowest expression in stomach, brain and skin. In addition, CALβ was scarcely expressed in the differentiated cells during cartilage production, but ex-FABP was weakly expressed. On the other hand, CALβ was weakly expressed in the hypertrophic phase, but Ex-FABP was strongly expressed. Ex-FABP was strongly expressed in the differentiated chondrocytes and more strongly expressed in the non-expanded phase than in the LPS treatment, but CALβ was weakly expressed in the former and more strongly expressed in the latter (about half of the Ex-FAB) {Pagano, A., P. Giannoni, A. Zambotti, N. Randazzo, B. Zerega, R. Cancedda, and B. Dozin. 2002. CALbeta, a novel lipocalin associated with chondrogenesis and inflammation. European journal of cell biology 81: 264-272.}. This suggests that the responses of Ex-FABP and CALβ to inflammatory stimuli are different. CALγ is expressed at high concentrations in liver, brain and bone of chicken embryos and is known to synergize with Ex-FABP in bone formation of chicken embryos {Pagano, A., Crooijmans, M. Groenen, N. Randazzo , B. Zerega, R. Cancedda, and B. Dozin. 2003. A chondrogenesis-related lipocalin cluster includes a third new gene, CALgamma. Gene 305: 185-194. The presence of this protein has been reported for the first time in the ovulation of daunting {Guerin-Dubiard, C., M. Pasco, D. Molle, C. Desert, T. Croguennec, and F. Nau. 2006. Proteomic analysis of hen egg white. Journal of agricultural and food chemistry 54: 3901-3910. On the other hand, the expression of CALβ and CALγ gene in the 3 week-old white leghorn infected with E. coli was significantly increased in the lung, liver and spleen compared to the control, but their expression patterns were different according to the tissues {Garenaux, A. , S. Houle, B. Folch, G. Dallaire, M. Truesdell, F. Lepine, N. Doucet, and CM Dozois. 2012. Avian lipocalin expression in chickens following Escherichia coli infection and inhibition of avian pathogenic Escherichia coli growth by Ex-FABP. Veterinary immunology and immunopathology. doi 10.1016 / j. That is, CALβ increased more than 15-fold in the lung and liver, but not in the spleen. Changes in CALγ expression were not detected in the lung but increased by at least 4.8 fold in the lung and liver.

In the present study, the expression of protein (spot number 19) estimated to be CAL in egg white of corticosterone-treated laying hens (experimental group) decreased significantly ( P <0.05) but increased in control group ( P <0.09) . However, there was no change in control and experiment in another protein spot (No. 30). These results suggest that stress may affect bone formation in chicken embryos. However, in this study, proteins were not identified because of the limit of detection of analytes that could detect trace amounts of proteins contained in these spots (Nos. 19 and 30), and therefore they were not tested for CALβ or CALγ.

Egg yolk protein in fresh eggs accounts for about 16% of total yolk weight. The Livetin fraction of the egg yolk is a type of egg yolk protein and contains α-, β- and γ-livetin. Egg yolk α-livetin and chicken serum albumin are the same, and β-livetin is known as α-2-glycoprotein with a molecular mass of 45 kDa. The third protein, γ-livetin, is IgY and is known to have a molecular weight of about 167 kDa. Algae IgY is closely related to the ancestors of IgG and IgE to mammals {Taylor, A. I., S. M. Fabiane, B. J. Sutton, and R. A. Calvert. 2009. The crystal structure of an avian IgY-Fc fragment reveals conservation with both mammalian IgG and IgE. Biochemistry 48: 558-562. doi 10.1021 / bi8019993}. IgY is the form of passive immunity conferred by the egg in the mother, transported by the receptor in the yolk to the blood, and this amount seems to be linked to the concentration of yolk in the blood. In the structure of IgY, the normal Fc portion and linkage region are indispensable for IgY to be transported from the serum to yolk. The human IgG administered in the vein of the laying hens takes about 5 days to be maximally secreted from the egg yolk {Mohammed, S. M., Morrison, L. Wims, K. R. Trinh, A. G. Wildeman, J. Bonselaar, and R. J. Etches. 1998. Deposition of genetically engineered human antibodies into the egg yolk of hens. Immunotechnology 4: 115-125. These authors did not detect IgG in egg white, but in this study the presence of IGY-FCU3-4 protein was confirmed in egg white. In chicken, the fragment of IGY-FCU3-4 consists of two chains (A and B), 231 amino acids each (PDB: 2W59_A & 2W59_B) {Taylor, AI, SM Fabiane, BJ Sutton, and RA Calvert. 2009. The crystal structure of an avian IgY-Fc fragment reveals conservation with both mammalian IgG and IgE. Biochemistry 48: 558-562. doi 10.1021 / bi8019993}. Thus, the low expression of IGY-FCU3-4 in egg white suggests that although the IgG content in egg yolk was not identified in this study, the amount of immunized antibody in stressed chicken eggs may be small, It can be a cause of chick production that is less resistant to disease.

The results of this study first provide evidence that treatment with corticosterone (stress) may affect the expression of proteins. The quality of eggs can be affected by various factors. The results of this study are summarized as follows: 1) the storage conditions of eggs {Qiu, N., M. Ma, L. Zhao, W. Liu, Y. Li, and Y. Mine. 2012. Comparative Proteomic Analysis of Egg White Proteins under Various Storage Temperatures. Journal of Agricultural and Food Chemistry 60: 7746-7753. doi 10.1021 / jf302100m}, nutritional factors such as feed {Kirunda, D. F., S. E. Scheideler, and S. R. McKee. 2001. The efficacy of vitamin E (DL-alpha-tocopheryl acetate) supplementation in hen diets to alleviate egg quality deterioration associated with high temperature exposure. Poult Sci 80: 1378-1383.} It also suggests that other specification conditions, environmental stress, may affect the expression of direct egg protein. This means that the content of the individual proteins constituting the protein by the environment can change, thus indicating that the environmental conditions can directly affect the quality of the egg.

These proteins not only can be used for the evaluation of egg quality, but also can be used as an index for estimating the stress state of laying hens at the time of scattering. Therefore, it can be used as a marker to estimate the animal welfare for the environment at the post-breeding stage by assaying the egg protein of the egg.

Therefore, the egg protein of the present invention can be utilized as a biomarker protein specific to environmental stress of poultry.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Biomarker protein specific for environmental stress of poultry <130> P12-0044 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 472 <212> PRT <213> Ovoinhibitor <400> 1 Met Arg Thr Ala Arg Gln Phe Val Gln Val Ala Leu Ala Leu Cys Cys   1 5 10 15 Phe Ala Asp Ile Ala Phe Gly Ile Glu Val Asn Cys Ser Leu Tyr Ala              20 25 30 Ser Gly Ile Gly Lys Asp Gly Thr Ser Trp Val Ala Cys Pro Arg Asn          35 40 45 Leu Lys Pro Val Cys Gly Thr Asp Gly Ser Thr Tyr Ser Asn Glu Cys      50 55 60 Gly Ile Cys Leu Tyr Asn Arg Glu His Gly Ala Asn Val Glu Lys Glu  65 70 75 80 Tyr Asp Gly Glu Cys Arg Pro Lys His Val Met Ile Asp Cys Ser Pro                  85 90 95 Tyr Leu Gln Val Val Arg Asp Gly Asn Thr Met Val Ala Cys Pro Arg             100 105 110 Ile Leu Lys Pro Val Cys Gly Ser Asp Ser Phe Thr Tyr Asp Asn Glu         115 120 125 Cys Gly Ile Cys Ala Tyr Asn Ala Glu His His Thr Asn Ile Ser Lys     130 135 140 Leu His Asp Gly Glu Cys Lys Leu Glu Ile Gly Ser Val Asp Cys Ser 145 150 155 160 Lys Tyr Pro Ser Thr Val Ser Lys Asp Gly Arg Thr Leu Val Ala Cys                 165 170 175 Pro Arg Ile Leu Ser Pro Val Cys Gly Thr Asp Gly Phe Thr Tyr Asp             180 185 190 Asn Glu Cys Gly Ile Cys Ala His Asn Ala Glu Gln Arg Thr His Val         195 200 205 Ser Lys Lys His Asp Gly Lys Cys Arg Gln Glu Ile Pro Glu Ile Asp     210 215 220 Cys Asp Gln Tyr Pro Thr Arg Lys Thr Thr Gly Gly Lys Leu Leu Val 225 230 235 240 Arg Cys Pro Arg Ile Leu Leu Pro Val Cys Gly Thr Asp Gly Phe Thr                 245 250 255 Tyr Asp Asn Glu Cys Gly Ile Cys Ala His Asn Ala Gln His Gly Thr             260 265 270 Glu Val Lys Lys Ser His Asp Gly Arg Cys Lys Glu Arg Ser Thr Pro         275 280 285 Leu Asp Cys Thr Gln Tyr Leu Ser Asn Thr Gln Asn Gly Glu Ala Ile     290 295 300 Thr Ala Cys Pro Phe Ile Leu Gln Glu Val Cys Gly Thr Asp Gly Val 305 310 315 320 Thr Tyr Ser Asn Asp Cys Ser Leu Cys Ala His Asn Ile Glu Leu Gly                 325 330 335 Thr Ser Val Ala Lys Lys His Asp Gly Arg Cys Arg Glu Glu Val Pro             340 345 350 Glu Leu Asp Cys Ser Lys Tyr Lys Thr Ser Thr Leu Lys Asp Gly Arg         355 360 365 Gln Val Val Ala Cys Thr Met Ile Tyr Asp Pro Val Cys Ala Thr Asn     370 375 380 Gly Val Thr Tyr Ala Ser Glu Cys Thr Leu Cys Ala His Asn Leu Glu 385 390 395 400 Gln Arg Thr Asn Leu Gly Lys Arg Lys Asn Gly Arg Cys Glu Glu Asp                 405 410 415 Ile Thr Lys Glu His Cys Arg Glu Phe Gln Lys Val Ser Pro Ile Cys             420 425 430 Thr Met Glu Tyr Val Pro His Cys Gly Ser Asp Gly Val Thr Tyr Ser         435 440 445 Asn Arg Cys Phe Phe Cys Asn Ala Tyr Val Gln Ser Asn Arg Thr Leu     450 455 460 Asn Leu Val Ser Ser Ala Ala Cys 465 470 <210> 2 <211> 232 <212> PRT <213> Ovalbumin-related protein X <400> 2 Gln Ile Lys Asp Leu Leu Val Ser Ser Ser Thr Asp Leu Asp Thr Thr   1 5 10 15 Leu Val Leu Val Asn Ale Ile Tyr Phe Lys Gly Met Trp Lys Thr Ala              20 25 30 Phe Asn Ala Glu Asp Thr Arg Glu Met Pro Phe His Val Thr Lys Gln          35 40 45 Glu Ser Lys Pro Val Gln Met Met Cys Met Asn Asn Ser Phe Asn Val      50 55 60 Ala Thr Leu Pro Ala Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala  65 70 75 80 Ser Gly Asp Leu Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Asp                  85 90 95 Leu Glu Arg Ile Glu Lys Thr Ile Asn Phe Glu Lys Leu Thr Glu Trp             100 105 110 Thr Asn Pro Asn Thr Met Glu Lys Arg Arg Val Lys Val Tyr Leu Pro         115 120 125 Gln Met Lys Ile Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala     130 135 140 Leu Gly Met Thr Asp Leu Phe Ile Pro Ser Ala Asn Leu Thr Gly Ile 145 150 155 160 Ser Ser Ala Glu Ser Leu Lys Ile Ser Gln Ala Val His Gly Ala Phe                 165 170 175 Met Glu Leu Ser Glu Asp Gly Ile Glu Met Ala Gly Ser Thr Gly Val             180 185 190 Ile Glu Asp Ile Lys His Ser Pro Glu Ser Glu Gln Phe Arg Ala Asp         195 200 205 His Pro Phe Leu Phe Leu Ile Lys His Asn Pro Thr Asn Thr Ile Val     210 215 220 Tyr Phe Gly Arg Tyr Trp Ser Pro 225 230 <210> 3 <211> 440 <212> PRT <213> TENP <400> 3 Met Gly Ala Leu Leu Ala Leu Leu Asp Pro Val Gln Pro Thr Arg Ala   1 5 10 15 Pro Asp Cys Gly Gly Ile Leu Thr Pro Leu Gly Leu Ser Tyr Leu Ala              20 25 30 Glu Val Ser Lys Pro His Ala Glu Val Val Leu Arg Gln Asp Leu Met          35 40 45 Pro Lys Glu Pro Gln Thr Cys Ser Leu Ala Pro Trp Ser Pro Ala Gly      50 55 60 Thr Glu Leu Pro Ala Val Lys Val Ala Asp Leu Trp Leu Ser Val Ile  65 70 75 80 Pro Glu Ala Gly Leu Arg Leu Gly Ile Glu Val Glu Leu Arg Ile Ala                  85 90 95 Pro Leu His Thr Val Pro Met Pro Val Arg Ile Ser Ile Arg Ala Asp             100 105 110 Leu His Val Asp Met Gly Pro Asp Gly Asn Leu Gln Leu Leu Thr Ser         115 120 125 Ala Cys Arg Pro Thr Val Gln Ala Gln Ser Thr Arg Glu Ala Glu Ser     130 135 140 Lys Ser Ser Arg Ser Ser Leu Asp Lys Val Val Asp Val Asp Lys Leu 145 150 155 160 Cys Leu Asp Val Ser Lys Leu Leu Leu Phe Pro Asn Glu Gln Leu Met                 165 170 175 Ser Leu Thr Ala Leu Phe Pro Val Thr Pro Asn Cys Gln Leu Gln Tyr             180 185 190 Leu Ala Leu Ala Ala Pro Val Phe Ser Lys Gln Gly Ile Ala Leu Ser         195 200 205 Leu Gln Thr Thr Phe Gln Val Ala Gly Ala Val Val Pro Val Val     210 215 220 Ser Pro Val Pro Phe Ser Met Pro Glu Leu Ala Ser Thr Ser Thr Ser 225 230 235 240 His Leu Ile Leu Ala Leu Ser Glu His Phe Tyr Thr Ser Leu Tyr Phe                 245 250 255 Thr Leu Glu Arg Ala Gly Ala Phe Asn Met Thr Ile Pro Ser Met Leu             260 265 270 Thr Thr Ala Thr Leu Ala Gln Lys Ile Thr Gln Val Gly Ser Leu Tyr         275 280 285 His Glu Asp Leu Pro Ile Thr Leu Ser Ala Ala Leu Arg Ser Ser Pro     290 295 300 Arg Val Val Leu Glu Glu Gly Arg Ala Ala Leu Lys Leu Phe Leu Thr 305 310 315 320 Val His Ile Gly Ala Gly Ser Pro Asp Phe Gln Ser Phe Leu Ser Val                 325 330 335 Ser Ala Asp Val Thr Arg Ala Gly Leu Gln Leu Ser Val Ser Asp Thr             340 345 350 Arg Met Met Ile Ser Thr Ala Val Ile Glu Asp Ala Glu Leu Ser Leu         355 360 365 Ala Ala Ser Asn Ale Leu Leu Glu Leu     370 375 380 Phe Leu Ala Pro Val Cys Gln Gln Val Pro Ala Trp Met Asp Asp Val 385 390 395 400 Leu Arg Glu Gly Val His Leu Pro His Met Ser His Phe Thr Tyr Thr                 405 410 415 Asp Val Asn Val Val Val His Lys Asp Tyr Val Leu Val Pro Cys Lys             420 425 430 Leu Lys Leu Arg Ser Thr Met Ala         435 440 <210> 4 <211> 383 <212> PRT <213> Hemopexin <400> 4 Met Leu Phe Phe Arg Gly Gly Asp Val Trp Glu Ile Ser Gly Glu Gly   1 5 10 15 Pro Gln Pro His Ser Arg Pro Leu Ala Glu Ser Trp Pro Glu Leu Glu              20 25 30 Gly Pro Val Asp Ala Ala Leu Arg Ile His Arg Gln Asp His Pro Glu          35 40 45 Glu His Gln Ser Leu Tyr Leu Phe Gln Asp Glu Lys Val Trp Ser Tyr      50 55 60 Ala Gly Gly Gln Leu Arg Pro Gly Phe Pro Arg Leu Ile Gly Asp Glu  65 70 75 80 Phe Pro Gly Val Pro Gly Gly Leu Asp Ala Ala Val Glu Cys His Pro                  85 90 95 Glu Glu Cys Gly Gly Glu Thr Val Leu Phe Phe Lys Gly Asp Lys Val             100 105 110 Phe Ser Phe Asp Leu Glu Leu Arg Val Thr Lys Glu Arg Pro Trp Leu         115 120 125 Asp Ala Gly Pro Cys Asp Ala Ala Leu Arg Trp Leu Glu Arg Tyr Tyr     130 135 140 Cys Leu Gln Gly Thr Gln Phe Tyr Arg Phe Arg Pro His Ser Trp Glu 145 150 155 160 Val Leu Pro Gly Tyr Pro Arg Asp Leu Arg Asp Tyr Phe Ile Pro Cys                 165 170 175 Pro Gly Arg Gly His Arg His Gly Asn Thr Ser Trp Gly Asn Ala Gly             180 185 190 Asp Arg Cys Ser Gly Glu Pro Phe Gln Ala Ile Thr Ser Asp Asp Ser         195 200 205 Gly Arg Ile Tyr Ala Phe Arg Gly Gly Leu Ser Phe Arg Leu Asp Ser     210 215 220 Trp Arg Asp Gly Trp His Ala Trp Pro Gln Ala His Ser Trp Pro Gly 225 230 235 240 Leu Gln Gly Asp Val Asp Ala Phe Ser Trp Asp Lys Arg Met Tyr                 245 250 255 Leu Ile Gln Gly Ser Gln Val Ser Ile Tyr Val Ser Gly Arg Gly Gly             260 265 270 His Gln Leu Val Glu Gly Tyr Pro Arg Ala Leu Gln Glu Glu Leu Gly         275 280 285 Val Pro Lys Ala Asp Ala Phe Thr Cys Pro Gly Ser Ala Glu Leu     290 295 300 Tyr Val Ile Thr Gly Asp Arg Met Gln Arg Val Asp Leu Thr Lys Ser 305 310 315 320 Pro Arg His Ala Asp Glu Pro Gln Pro Leu Pro Tyr Asp Gly Val Asp                 325 330 335 Gly Ala Met Cys Thr Ala Asp Gly Ile Tyr Leu Leu Arg Gly Asp Arg             340 345 350 Tyr His Arg His Arg Asp Val Ala Glu Leu Leu Ala Ala His Pro Pro         355 360 365 Ala Asp Pro Pro Ser Ile Ala Val Asp Leu Phe His Cys Ala Gln     370 375 380 <210> 5 <211> 231 <212> PRT <213> IGY-FCU3-4 <400> 5 Asp Ile Ser Pro Asp Gly Ala Gln Ser Cys Ser Pro Ile Gln Leu Tyr   1 5 10 15 Ala Ile Pro Pro Ser Gly Glu Leu Tyr Ile Ser Leu Asp Ala Lys              20 25 30 Leu Arg Cys Leu Val Val Asn Leu Pro Ser Asp Ser Ser Leu Ser Val          35 40 45 Thr Trp Thr Arg Glu Lys Ser Gly Asn Leu Arg Pro Asp Pro Met Val      50 55 60 Leu Gln Glu His Phe Asn Gly Thr Tyr Ser Ala Ser Ser Ala Val Pro  65 70 75 80 Val Ser Thr Gln Asp Trp Leu Ser Gly Glu Arg Phe Thr Cys Thr Val                  85 90 95 Gln His Glu Glu Leu Pro Leu Pro Leu Ser Lys Ser Val Tyr Arg Asn             100 105 110 Thr Gly Pro Thr Thr Pro Pro Leu Ile Tyr Pro Phe Ala Pro His Pro         115 120 125 Glu Glu Leu Ser Leu Ser Arg Val Thr Leu Ser Cys Leu Val Arg Gly     130 135 140 Phe Arg Pro Arg Asp Ile Glu Ile Arg Trp Leu Arg Asp His Arg Ala 145 150 155 160 Val Pro Ala Thr Glu Phe Val Thr Ala Val Leu Pro Glu Glu Arg                 165 170 175 Thr Ala Asn Gly Asp Gly Asp Gly Asp Thr Phe Phe Val Tyr Ser             180 185 190 Lys Met Ser Val Glu Thr Ala Lys Trp Asn Gly Gly Thr Val Phe Ala         195 200 205 Cys Met Ala Val His Glu Ala Leu Pro Met Arg Phe Ser Gln Arg Thr     210 215 220 Leu Gln Lys Gln Ala Gly Lys 225 230 <210> 6 <211> 178 <212> PRT <213> Extracellular fatty acid-binding protein <400> 6 Met Arg Thr Leu Ala Leu Ale Leu Ale Leu Leu Cys Leu Leu   1 5 10 15 His Thr Glu Ala Ala Thr Val Pro Asp Arg Ser Glu Val Ala Gly              20 25 30 Lys Trp Tyr Ile Val Ala Leu Ala Ser Asn Thr Asp Phe Phe Leu Arg          35 40 45 Glu Lys Gly Lys Met Lys Met Val Met Ala Arg Ile Ser Phe Leu Gly      50 55 60 Glu Asp Glu Leu Glu Val Ser Tyr Ala Ala Pro Ser Pro Lys Gly Cys  65 70 75 80 Arg Lys Trp Glu Thr Thr Phe Lys Lys Thr Ser Asp Asp Gly Glu Leu                  85 90 95 Tyr Tyr Ser Glu Glu Ala Glu Lys Thr Val Glu Val Leu Asp Thr Asp             100 105 110 Tyr Lys Ser Tyr Ala Val Ile Phe Ala Thr Arg Val Lys Asp Gly Arg         115 120 125 Thr Leu His Met Met Arg Leu Tyr Ser Arg Ser Ser Glu Val Ser Pro     130 135 140 Thr Ala Met Ala Ile Phe Arg Lys Leu Ala Arg Glu Arg Asn Tyr Thr 145 150 155 160 Asp Glu Met Val Ala Val Leu Pro Ser Glu Glu Glu Cys Ser Val Asp                 165 170 175 Glu Val        

Claims (18)

A biomarker protein specific to environmental stress of a poultry characterized by comprising an egg protein or a fragment thereof specifically expressing environmental stress of poultry. The biomarker protein of claim 1, wherein the egg protein is an egg white protein. 3. The method of claim 2, wherein the egg white protein is selected from the group consisting of Ovoinhibitor of SEQ ID NO: 1, Ovalbumin-related protein X of SEQ ID NO: 2, TENP of SEQ ID NO: 3, Hemopexin of SEQ ID NO: 4, IGY-FCU3-4 of SEQ ID NO: (Ex-FABP). &Lt; Desc / Clms Page number 13 &gt; 6. A biomarker protein specific for environmental stress of a poultry. The biomarker protein according to any one of claims 1 to 3, wherein the poultry is chicken. A composition for diagnosing environmental stress in birds comprising an antibody that specifically binds to the biomarker protein of claim 4 as an active ingredient. 6. The composition for diagnosing environmental stress of poultry according to claim 5, wherein the antibody is a monoclonal antibody. An environmental stress diagnostic kit for poultry comprising an antibody that specifically binds to the biomarker protein of claim 4 as an active ingredient. 8. The environmental stress diagnosis kit for poultry according to claim 7, wherein the antibody is a monoclonal antibody. As a method for analyzing environmental stress of poultry,
A method for analyzing the environmental stress of a poultry, the method comprising detecting whether the expression level of an egg protein or a fragment thereof specifically expressing environmental stress of poultry is changed. 10. The method according to claim 9, wherein the egg protein is an egg white protein. 11. The method of claim 10, wherein the egg white protein is selected from the group consisting of Ovoinhibitor of SEQ ID NO: 1, Ovalbumin-related protein X of SEQ ID NO: 2, TENP of SEQ ID NO: 3, Hemopexin of SEQ ID NO: 4, IGY-FCU3-4 of SEQ ID NO: 6, &lt; / RTI &gt; and an extracellular fatty acid-binding protein (Ex-FABP). 12. The method of claim 11, wherein the increased expression of the Ovoinhibitor of SEQ ID NO: 1 or the Ovalbumin-related protein X of SEQ ID NO: 2 is used as a marker indicating a high probability of stress of the poultry. 12. The method according to claim 11, wherein at least one egg white selected from TENP of SEQ ID NO: 3, Hemopexin of SEQ ID NO: 4, IGY-FCU3-4 of SEQ ID NO: 5 and Extracellular fatty acid- Wherein the decrease in expression of the protein is used as a marker indicating a high possibility of stress of the poultry. As a method of analyzing the quality of eggs,
A method for analyzing egg quality characterized by detecting whether the expression level of an egg protein or a fragment thereof specifically expressing environmental stress of poultry is changed. 15. The method of claim 14, wherein the egg protein is an egg white protein. 16. The method of claim 15, wherein the egg white protein comprises an Ovoinhibitor of SEQ ID NO: 1, Ovalbumin-related protein X of SEQ ID NO: 2, TENP of SEQ ID NO: 3, Hemopexin of SEQ ID NO: 4, IGY- 6. The method for analyzing egg quality according to claim 1, wherein the extract is selected from the group consisting of Extracellular fatty acid-binding protein (Ex-FABP). 17. The method of claim 16, wherein the increased expression of the Ovoinhibitor of SEQ ID NO: 1 or of Ovalbumin-related protein X of SEQ ID NO: 2 is the egg produced by highly stressed poultry. The method according to claim 16, wherein the TENP of SEQ ID NO: 3, Hemopexin of SEQ ID NO: 4, IGY-FCU3-4 of SEQ ID NO: 5 and the Extracellular fatty acid-binding protein of SEQ ID NO: 6 (Ex-FABP) A method for analyzing egg quality characterized in that the reduction of protein expression is the egg produced by highly stressed poultry.

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