KR100979907B1 - Protein Marker Related with the CW for Improving economic traits of Hanwoo - Google Patents

Abstract

The present invention relates to biomarker proteins related to economic traits, including carcass weight, etc., for the production of high-quality meat, and to methods for selecting Korean beef having excellent economic traits using them.

According to the present invention, the biomarker proteins related to economic traits including the carcass weight of Hanwoo at the protein level can be selected using proteome analysis technology and then used to establish a new concept of high-quality meat production technology. While minimizing phenotypic competence testing, they can be replaced by low-cost assays that allow for early genetic and early prediction of growth potential or embryonic status.

Korean beef, carcass weight, economic trait, biomarker, proteome

Description

Protein Marker Related with the CW for Improving economic traits of Hanwoo}

The present invention relates to the development of a protein that can be used as a biomarker related to carcasses, which is one of the important factors in the grading of livestock grading. The present invention relates to a biomarker protein which is relatively expressed in Hanwoo loin tissue and a diagnostic kit for predicting the initial growth rate.

Hanwoo has a strong adaptability to the climate climate, but has the disadvantage of low meat production capacity due to low growth rate and poor development of the rear. Also, in order to enhance the competitiveness of domestic Korean cattle by increasing the autonomy of foreign beef imports, Korean beef production was converted into high-quality meat production. Since 1992, the improvement goal of Korean cattle has been decided on the direction of improvement of meat quality from the center of breeding capacity and the selection of breeding cattle. In the domestic livestock sector, a large-scale analysis of livestock genes is underway using this research direction, but research on protein bodies is still inadequate.

Recently, as the investigation of carcass traits for the production of high-quality meat has been actively conducted, interest in judging carcass traits in living bodies is increasing. Carcass traits include carcass weight, backfat thickness, dorsum length, intramuscular fat and carcass rate. Although there is great interest in improving carcass quality, that is, marbling score, which can improve production efficiency, carcass weight, longest root cross-sectional area, and back fat thickness, which are objective indicators of meat grade determination in livestock grading, It is also a related factor to increase production efficiency.

Therefore, the main object of the present invention is to provide a new biomarker protein for the production of high-quality beef cattle.

Another object of the present invention to provide an antibody and a diagnostic kit using the biomarker protein.

According to an aspect of the present invention, the present invention provides a biomarker for selecting high-quality beef cattle comprising the following proteins whose expression is relatively increased in the loin tissue with a higher estimate of carcass value than the loin tissue with a lower estimated carcass weight.

Glyceraldehyde-3-phosphate dehydrogenase having a molecular weight of 35-37 kDa comprising a trypsin digested peptide sequence of SEQ ID NOs: 1, 2 and 3 and a pi of 8.3-8.7.

In the present invention, the trypsin digested peptide refers to a peptide of proteins digested with trypsin for MS / MS analysis. In addition, the molecular weight and pi are values determined on two-dimensional electrophoresis and include generally accepted experimental error ranges.

According to another aspect of the present invention, the present invention provides a diagnostic agent for the selection of high-quality beef cattle, including an antibody that specifically binds to the biomarker protein or immunogenic fragment thereof.

In the diagnostic agent for screening high-quality beef cattle of the present invention, the antibody may be a polyclonal antibody, but is preferably a monoclonal antibody. Polyclonal antibodies can be prepared by injecting a biomarker protein or fragment thereof that is an immunogen into an external host according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, rabbits. Immunogens are injected by intramuscular, intraperitoneal or subcutaneous injection and are usually administered with an adjuvant to increase antigenicity. Blood is collected periodically from the external host to collect serum that shows improved titers and specificity for the antigen, or to separate and purify antibodies therefrom.

Monoclonal antibodies can be prepared by techniques of generation of immortalized cell lines by fusions known to those skilled in the art (Koeher and Milstein 1975, Nature, 265: 495). The manufacturing method is briefly described as follows. First, 20 μg of pure protein is immunized to Balb / C mice or peptides are synthesized and combined with bovine serum albumin to immunize mice. The antigen-producing lymphocytes isolated from mice are then fused with myeloma in humans or mice to produce immortalized hybridomas, and hybridoma cells producing the desired monoclonal antibodies using ELISA methods. Only by propagation by selection, monoclonal antibodies are isolated and purified from the culture.

According to another aspect of the present invention, the present invention provides a method for selecting high-quality meat of Hanwoo, comprising detecting the presence of the biomarker protein in the loin tissue of Hanwoo.

In the screening method of the present invention, the detecting step directly detects the presence of the biomarker protein by 2-D electrophoresis (2-DE) from the loin tissue of Hanwoo, or makes the antigen antibody reaction by contacting the loin tissue with the antibody of the present invention. Indirectly through the presence of a biomarker protein. Currently known immunoassays (immunoassay) as an antigen antibody reaction include enzyme immunoassay (ELISA, Coated tube), magnetic particles using antibody-bound magnetic particles, latex particles using antibody-bound latex particles.

According to another aspect of the present invention, the present invention provides a diagnostic kit for selection of high-quality beef cattle, comprising an antibody that specifically binds to the biomarker protein or immunogenic fragment thereof.

Diagnostic kits of the present invention are prepared by conventional methods known to those skilled in the art, and typically include lyophilized antibodies, buffers, stabilizers, inactive proteins and the like. The antibody can be labeled by radionuclides, fluorescors, enzymes, and the like.

Monoclonal antibodies of the present invention can be used in a variety of immunoassay kits (ELISA, antibody coated tube test, lateral-flow test, potable biosensor), as well as various economic traits through the development of antibodies showing higher specificity and sensitivity It can also be used to develop protein chips with a spectrum.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Protein Extraction in Korean Beef Loin

The thirteenth rib area, which was graded, was biopsyed using a Spring-loaded biopsy instrument (Biotech Nitra, Slovak), immediately frozen in liquid nitrogen, and stored at -80 ° C until analysis. As shown in Fig. 1, the top 20 and bottom 20 of the total 146 Korean beef loin tissues were ranked according to the ranking of carcass weight (EBV) of carcass weight (CW) in the loin samples of 146 reared black cattle. Two-dimensional electrophoresis was performed as a group.

Breeding value (BV) is the value of a gene that can be genetically donated to the next generation, that is, the genetic value of an individual as a parent. The estimated value of these breeders is also called estimated breeding value (EBV). In the present invention, the breeder was analyzed by MTDFREML Package using the following livestock individual model.

Where Y: observation, μ: overall mean, YS: year-seasonal effect, L: birth-effect, D: co-variate for slaughter age, A: Animal Effects, e: Random Error.

Based on the carcass weight-related EBV values, the top 20 (26.639 ~ 13.866) and the bottom 20 (26.639 ~ 13.866) and lower 20 (5) were ranked according to the ranking of breeding stock values (CW) such as carcass weight (CW). -7.364 ~ -41.865) were selected as high and low meat groups, respectively, and two-dimensional electrophoresis was performed. The average of the upper group EBV is (18.233), and the average of the lower group EBV is (-14.361) (see FIG. 5).

Example 2. IEF (1-D electrophoresis) and 2-D electrophoresis

(1) First-dimension isoelectric focusing (IEF)

100mM protein extracted from the tissue was put into 80mM DTT, 0.5% IPG buffer, and the mixture was prepared so that the total volume of the tissue and serum to 340㎛, 250㎛, respectively, as a rehydration solution. The composition of rehydration soluion used was as follows: Urea 7M, Thiourea 2.8M, CHAPS 4%, Bromophenol blue 0.002% (w / v). Pharmalyte 3-10 was used as an IPG buffer instead and was not added when the solution was prepared. However, IPG buffer was added immediately upon loading by adding the sample to the rehydration solution for each pH. After loading the prepared mixture in a clean holder, isoelectrofocusing was performed using IPG strips (tissue: pH3 ~ 10 and 18cm, serum: pH3 ~ 10 and 13cm) under the following conditions. After 12 hours of rehydration, 1 hour at 500V gradient condition, then 1 hour development at 8000V gradient, and finally 8000V steady-n-state level for 13 hours.

(2) Second-dimension SDS-PAGE

After completion of isoelectrofocusing, each strip was placed in 1% DTT (Dithiotheitol) and 2.5% IAA (iodoacetamide) for 15 minutes, respectively. Bromophenol blue) was used for the reaction while mixing well. Equilibration strips were loaded on 12.5% SDS-PAGE, filled with agrose sealing solution, and developed at 2 Watt per sheet. The developed SDS-PAGE was fixed for about 30 minutes in a fixing solution (acetate acid 10%, ethanol 40%).

Example 3 Silver Staining and Image Scanning, Analysis

The immobilized gel was reacted with sensitizing solution (30% ethanol, 0.125% (w / v glutardialdehyde, 0.2% (w / v) sodium thiosulphate, 6.8% sodium acetate) for 30 minutes and washed with distilled water 0.25% (wv) silver After 20 minutes of reaction with nitrate solution and washing with distilled water, the immobilized gel was reacted with developing solution (2.5% (w / v) sodium carbonate) for 8 minutes and finally stopping solution 1.46% (w / v) EDTA? Na 2H ₂ O. The stained gel was scanned using an ImageScanner flatbed scanner, and each protein spot was analyzed using ImageMaster 2-D melanie software (Amersham Bioscience Inc.).

3 is a graph comparing the intensity of expression of spots in which carcass weight (CW) related breeding estimate (EBV) is changed.

Example 4 Examination of Association Between Phenotype and Genetic Ability and Labeled Protein

(Correlation analysis and significance test between expression level of candidate marker protein and economic trait)

T-test was performed on the loin of the top 20 cows and the bottom 20 cows selected according to the ranking of carcass weight estimate (EBV) for the spot density of the image such as two-dimensional electrophoresis. In addition, correlation analysis between relative density of spots and economic traits was performed using SAS 8.12 package. Table 1 below shows the carcass weight-related significance test results and correlation coefficients for sirloin proteins.

TABLE 1

Example 5 Protein Identification Using Mass Spectrometry

The spots showing differences in protein expression patterns were selected through image analysis, and the spots were separated from the gel using a blue tip. The separated spots were destained by soaking in a 1: 1 mixture of 30 mM potassium ferricyanide and 100 mM sodium thiosulfat. Destained spots were pretreated with 100 mM ammomium bicarbonate, pH 7.8 and acetonitrile solution and reacted with trypsin.

After ingel trypic digestion method, the extract was analyzed using nano-ESI-Q-TOF mass spectrometer (MS / MS). Using the analyzed peptide information, the error range was set to the minimum in the MASCOT search program, and finally, the matching protein was searched and identified. Table 2 below is a list of sirloin candidate marker proteins identified using ESI-Q-TOF.

TABLE 2

The amino acid sequence of the identified proteins as described above is NCBInr database (http; // www.ncbi.nlm.nih.gov /) the Cleveland Sergio aldehyde-3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase) to search the It was confirmed that it had the amino acid sequence of SEQ ID NO: 4.

As described above, according to the present invention, the biomarker protein related to economic traits including the carcass weight of Hanwoo at the protein level is selected by using proteome analysis technology, and then, it is possible to establish a new concept of high-quality meat production technology. have. In addition, it can be replaced by a low-cost test method that can minimize the cost-based phenotypic capacity test and allow for early genetic growth and early prediction.

1 is a 2-D electrophoresis image showing muscle proteins expressed in the sirloin and low Hanwoo sirloin with high estimated carcass weight.

FIG. 2 is an enlarged view of spots in the high CW group and the low CW group of the carcass weight (CW) related breeding estimate (EBV) in the arrow region of FIG. 1.

3 is a graph comparing the expression intensity (intensity) of the spots identified in FIG.

Figure 4a is a result of protein identification using the MASCOT search program ESI-Q-TOF mass spectrometry of the spot 1758, Figure 4b, 4c, 4d are MS / MS fragmentation spectrum of the peptide of SEQ ID NO: 1, 2, 3, respectively to be.

Figure 5 is the result of selecting the high-quality and low-grade meat group of the total 40 black cattle cows based on the carcass weight-related EBV value.

<110> Industry Academic Cooperation Foundation of KyungHee University <120> Protein Marker Related with the CW for Improving economic traits          of Hanwoo <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 15 <212> PRT <213> Bos taurus <400> 1 Gly Ala Ala Gln Asn Ile Ile Pro Ala Ser Thr Gly Ala Ala Lys   1 5 10 15 <210> 2 <211> 14 <212> PRT <213> Bos taurus <400> 2 Val Pro Thr Pro Asn Val Ser Val Val Asp Leu Thr Cys Arg   1 5 10 <210> 3 <211> 14 <212> PRT <213> Bos taurus <400> 3 Leu Ile Ser Trp Tyr Asp Asn Glu Phe Gly Tyr Ser Asn Arg   1 5 10 <210> 4 <211> 333 <212> PRT <213> Bos taurus <400> 4 Met Val Lys Val Gly Val Asn Gly Phe Gly Arg Ile Gly Arg Leu Val   1 5 10 15 Thr Arg Ala Ala Phe Asn Ser Gly Lys Val Asp Ile Val Ala Ile Asn              20 25 30 Asp Pro Phe Ile Asp Leu His Tyr Met Val Tyr Met Phe Gln Tyr Asp          35 40 45 Ser Thr His Gly Lys Phe Asn Gly Thr Val Lys Ala Glu Asn Gly Lys      50 55 60 Leu Val Ile Asn Gly Lys Ala Ile Thr Ile Phe Gln Glu Arg Asp Pro  65 70 75 80 Ala Asn Ile Lys Trp Gly Asp Ala Gly Ala Glu Tyr Val Val Glu Ser                  85 90 95 Thr Gly Val Phe Thr Thr Met Glu Lys Ala Gly Ala His Leu Lys Gly             100 105 110 Gly Ala Lys Arg Val Ile Ile Ser Ala Pro Ser Ala Asp Ala Pro Met         115 120 125 Phe Val Met Gly Val Asn His Glu Lys Tyr Asn Asn Thr Leu Lys Ile     130 135 140 Val Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys 145 150 155 160 Val Ile His Asp His Phe Gly Ile Val Glu Gly Leu Met Thr Thr Val                 165 170 175 His Ala Ile Thr Ala Thr Gln Lys Thr Val Asp Gly Pro Ser Gly Lys             180 185 190 Leu Trp Arg Asp Gly Arg Gly Ala Ala Gln Asn Ile Ile Pro Ala Ser         195 200 205 Thr Gly Ala Ala Lys Ala Val Gly Lys Val Ile Pro Glu Leu Asn Gly     210 215 220 Lys Leu Thr Gly Met Ala Phe Arg Val Pro Thr Pro Asn Val Ser Val 225 230 235 240 Val Asp Leu Thr Cys Arg Leu Glu Lys Pro Ala Lys Tyr Asp Glu Ile                 245 250 255 Lys Lys Val Val Lys Gln Ala Ser Glu Gly Pro Leu Lys Gly Ile Leu             260 265 270 Gly Tyr Thr Glu Asp Gln Val Val Ser Cys Asp Phe Asn Ser Asp Thr         275 280 285 His Ser Ser Thr Phe Asp Ala Gly Ala Gly Ile Ala Leu Asn Asp His     290 295 300 Phe Val Lys Leu Ile Ser Trp Tyr Asp Asn Glu Phe Gly Tyr Ser Asn 305 310 315 320 Arg Val Val Asp Leu Met Val His Met Ala Ser Lys Glu                 325 330  

Claims (5)

A molecular weight of 35-37 kDa and 8.3-8.7 comprising trypsin digested peptide sequences of SEQ ID NOs: 1, 2 and 3, with increased expression in lumber tissue with higher carcass value estimates than carcass tissue with lower carcass weight estimates Biomarker composition for screening Hanwoo beef meat comprising a glyceraldehyde-3-phosphate dehydrogenase protein having a pI of. A diagnostic agent composition for screening Hanwoo beef meat comprising an antibody that specifically binds to the biomarker protein of claim 1 or an immunogenic fragment thereof. According to claim 2, wherein the antibody is a monoclonal antibody diagnostic diagnostic composition, characterized in that the monoclonal antibody. delete The diagnostic kit for selecting high-quality beef from Hanwoo, comprising an antibody that specifically binds to the biomarker protein of claim 1 or an immunogenic fragment thereof.

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