KR100979907B1 - Protein Marker Related with the CW for Improving economic traits of Hanwoo - Google Patents
Protein Marker Related with the CW for Improving economic traits of Hanwoo Download PDFInfo
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- KR100979907B1 KR100979907B1 KR1020070106895A KR20070106895A KR100979907B1 KR 100979907 B1 KR100979907 B1 KR 100979907B1 KR 1020070106895 A KR1020070106895 A KR 1020070106895A KR 20070106895 A KR20070106895 A KR 20070106895A KR 100979907 B1 KR100979907 B1 KR 100979907B1
- Authority
- KR
- South Korea
- Prior art keywords
- hanwoo
- val
- protein
- antibody
- ala
- Prior art date
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- 239000012474 protein marker Substances 0.000 title description 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 36
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Abstract
본 발명은 고급육 생산을 위한 도체중 등을 포함한 경제형질과 관련된 바이오 마커 단백질과 이들을 이용하여 우수한 경제형질을 지닌 한우를 선발하는 방법에 관한 것이다.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.
본 발명에 따르면, 단백질 수준에서 한우의 도체중 등을 포함한 경제형질과 관련된 바이오마커 단백질을 프로테옴분석 기술을 이용하여 선발한 후 이를 활용하여 새로운 개념의 고급육 생산기술을 정착시킬 수 있다 또한, 고비용의 표현형 위주의 능력검정작업을 최소화함과 동시에 성장초기나 배아상태에서의 유전적 잠재력과 조기예측이 가능할 수 있는 저비용의 검정방법으로 대치할 수 있다.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.
한우, 도체중, 경제형질, 바이오 마커, 프로테옴 Korean beef, carcass weight, economic trait, biomarker, proteome
Description
본 발명은 축산물 등급판정에서의 육량등급 판정의 중요한 요소 중 하나인 도체중 관련 바이오 마커로 사용될 수 있는 단백질 개발에 관한 것으로, 더욱 상세하게는 도체중 관련 육종가가 낮은 한우 등심조직에서보다 육종가가 높은 한우 등심조직에서 상대적으로 많이 발현되는 바이오 마커 단백질과 이를 이용한 성장초기 산육량을 예측할 있는 진단키트에 관한 것이다.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.
한우는 기후풍토에 대한 적응력은 강하지만, 성장률이 낮고 후구 발달이 빈약하여 고기생산능력이 작은 단점이 있다. 또한 외국산 소고기 수입의 자율화에 따른 국내산 한우의 경쟁력 제고를 위하여 한우 생산을 고급육 생산으로 전환하게 됨에 따라 1992년부터 한우의 개량목표는 산육능력중심에서 육질중심 개량의 방향을 정하고 종모우를 선발하게 되었다. 국내의 축산분야에서는 현재 이러한 연구방향을 이용하여 가축유전자 대량 분석 사업이 진행 중에 있으나, 단백질체에 대한 연구는 아직 미비한 실정이다.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.
최근 고급육 생산을 위하여 도체형질에 대한 조사가 활발히 진행되면서 생체에서의 도체형질에 대한 판단에 관심이 고조되고 있다. 도체형질에는 도체중, 등지방두께, 배최장근단면적, 근내지방도 및 도체율이 포함된다. 현재 생산효율을 높일 수 있는 도체형질 즉 근내지방교잡도(marbling score)의 개량에 큰 관심을 두고 있지만, 축산물등급판정에서의 육량등급 판정의 객관적 지표인 도체중, 배최장근단면적, 등지방두께도 역시 생산효율을 높일 수 있는 관련 요소이다.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.
따라서, 본 발명의 주된 목적은 한우 고급육 생산을 위한 새로운 바이오 마커 단백질을 제공하는 데 있다.Therefore, the main object of the present invention is to provide a new biomarker protein for the production of high-quality beef cattle.
본 발명의 다른 목적은 상기 바이오 마커 단백질에 대한 항체 및 이를 이용한 진단키트를 제공하는데 있다.Another object of the present invention to provide an antibody and a diagnostic kit using the biomarker protein.
본 발명의 한 양태에 따르면, 본 발명은 도체중 관련 육종가 추정치가 낮은 등심조직보다 육종가 추정치가 높은 등심조직에서 발현이 상대적으로 증가되는 하기 단백질을 포함하는 한우 고급육 선별용 바이오 마커를 제공한다: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.
서열번호 1, 2 및 3의 트립신 분해된 펩타이드 서열을 포함하는 35-37 kDa의 분자량 및 8.3-8.7의 pI를 갖는 클리세르알데히드-3-포스페이트 탈수소효소(Glyceraldehyde-3-phosphate dehydrogenase).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.
본 발명에서, 상기 트립신 분해된 펩타이드란 MS/MS 분석을 위해 트립신으로 분해된 단백질들의 펩타이드를 의미한다. 또한 상기 분자량과 pI는 2차원 전기영동(Two-dimensional electrophoresis) 상에서 확인된 값으로서 일반적으로 허용되는 실험상의 오차범위를 포함한다.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.
본 발명의 다른 양태에 따르면, 본 발명은 상기 바이오마커 단백질 또는 그 면역원성 단편에 특이적으로 결합하는 항체를 유효성분으로 포함하는 한우 고급육 선별용 진단제를 제공한다.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.
본 발명의 한우 고급육 선별용 진단제에서, 상기 항체는 폴리클로날 항체일 수도 있으나, 모노클로날 항체인 것이 바람직하다. 폴리클로날 항체는 당업자에 알려진 종래방법에 따라 면역원인 바이오 마커 단백질 또는 그 단편을 외부 숙주에 주사함으로서 제조될 수 있다. 외부 숙주는 마우스, 래트, 양, 토끼와 같은 포유동물을 포함한다. 면역원은 근내, 복강내 또는 피하 주사방법으로 주사되며, 일반적으로 항원성을 증가시키기 위한 보조제(adjuvant)와 함께 투여된다. 외부숙주로부터 정기적으로 혈액을 채취하여 향상된 역가 및 항원에 대한 특이성을 보이는 혈청을 수거하거나 이로부터 항체를 분리, 정제한다.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.
모노클로날 항체는 당업자에 알려진 융합에 의한 불멸화된 세포주 생성기술(Koeher and Milstein 1975, Nature, 265:495)에 의해 제조될 수 있다. 그 제조방법은 간단히 설명하면 다음과 같다. 먼저 순수한 단백질을 20μg을 얻어서 Balb/C 쥐에 면역화를 시키거나 펩타이드를 합성하여 소혈청 알부민과 결합시켜 쥐에 면역화 시킨다. 그 후에 쥐에서 분리된 항원-생산 임파구를 인간 또는 마우스의 미엘로마와 융합하여 불멸화된 하이브리도마를 생성하며, 엘라이져(ELISA) 방법을 사용하여 원하는 모노클로날 항체를 생성하는 하이브리도마 세포만을 선택하여 증식한 후 배양물로부터 모노클로날 항체를 분리, 정제한다.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.
본 발명의 다른 양태에 따르면, 본 발명은 한우의 등심조직에서 상기 바이오마커 단백질의 존재를 검출하는 단계를 포함하는 한우의 고급육 선별방법을 제공한다.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.
본 발명의 선별방법에서, 상기 검출 단계는 한우의 등심조직으로부터 2차원 전기영동(2-DE)으로 바이오 마커 단백질의 존재를 직접 검출하거나, 등심조직을 본 발명의 항체와 접촉시켜 항원항체반응을 통해 바이오 마커 단백질의 존재를 간접적으로 확인하는 것을 포함한다. 항원항체반응으로서 현재 널리 알려진 면역분석(immunoassay)법은 효소면역측정법(ELISA, Coated tube), 항체결합 magnetic particle을 이용한 magnetic particle법, 항체결합 latex particle을 이용한 latex particle법 등이 있다.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.
본 발명의 다른 양태에 따르면, 본 발명은 상기 바이오마커 단백질 또는 그 면역원성 단편에 특이적으로 결합하는 항체를 유효성분으로 포함하는 한우 고급육 선별용 진단키트를 제공한다.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.
본 발명의 진단키트는 당업자에 알려진 종래의 제조방법에 의해 제조되며, 전형적으로 동결건조형태의 항체와 버퍼, 안정화제, 불활성 단백질 등을 포함한다. 상기 항체는 방사종(radionuclides), 형광원(fluorescors), 효소(enzymes) 등에 의해 표지화될 수 있다.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.
본 발명의 단일클론항체는 immunoassay 키트(ELISA, antibody coated tube test, lateral-flow test, potable biosensor)에 다양하게 이용될 수 있을 뿐만 아니라, 보다 높은 특이도와 민감도를 나타내는 항체의 개발을 통한 다양한 경제형질 스펙트럼을 갖는 단백질칩 개발에도 이용될 수 있다.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.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.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.
실시예 1. 한우 등심내 단백질 추출Example 1 Protein Extraction in Korean Beef Loin
등급판정부위인 13번째 늑골부위를 Spring-loaded biopsy instrument (Biotech Nitra, Slovak)를 이용하여 biopsy 한 뒤 액체질소에 즉시 냉동시키고 분석 전까지 -80℃에 보관하였다. 도 1에서와 같이 총 146두의 후대검정우의 등심샘플에서 도체중(CW)의 육종가주정치(EBV)의 순위에 따라 총 146개의 한우 등심조직 중에서 상위 20개와 하위 20개를 각기 상위그룹과 하위그룹으로 선정하여 2차원 전기영동을 실시하였다. 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.
육종가(breeding value, BV)란 다음 세대에 유전적으로 공여 될 수 있는 유전자의 가치 즉 부모로서 어느 개체가 갖고 있는 유전적 가치이다. 또한 이런 육종가의 예측값을 추정육종가(estimated breeding value, EBV)라 한다. 본 발명에서는 아래의 가축개체모형을 이용하여 MTDFREML Package로 육종가를 분석하였다.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.
여기서, Y: 관측치, μ: 전체 평균, YS: 연도 - 계절효과, L: 출생지역의 효과, D: 도축일령에 대한 co-variate, A: Animal Effects, e: Random Error 이다.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.
이렇게 분석된 도체중 관련 EBV 수치를 기준으로 총 40두의 후대검정우의 등심샘플에서 도체중(CW) 등 육종가주정치(EBV)의 순위에 따라 상위 20개(26.639~13.866)와 하위 20개(-7.364~-41.865)를 각기 고급육과 저급육 그룹으로 선정하여 2차원 전기영동을 실시하였다. 상위그룹 EBV의 평균은 (18.233)이고, 하위그룹 EBV의 평균은 (-14.361)이다 (도 5 참조).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).
실시예 2. IEF(1차원 전기영동)과 2-D 전기영동Example 2. IEF (1-D electrophoresis) and 2-D electrophoresis
(1) 1차원 전기영동(First-dimension isoelectric focusing, IEF)(1) First-dimension isoelectric focusing (IEF)
조직에서 추출한 단백질 100㎍에 80mM DTT, 0.5% IPG buffer를 넣고, rehydration solution으로 조직과 혈청의 전체 부피가 각각 340㎕, 250㎕ 되도록 혼합물을 준비하였다. 사용한 rehydration soluion의 조성은 다음과 같다: Urea 7M, Thiourea 2.8M, CHAPS 4%, Bromophenol blue 0.002%(w/v). Pharmalyte 3~10은 IPG buffer로 대신 사용하며 solution을 만들 시에는 첨가하지 않았다. 단, IPG buffer는 각 pH에 맞게 샘플을 rehydration solution에 첨가하여 loading시에 바로 첨가하였다. 준비된 mixture를 깨끗한 holder에 loading한 후 IPG strip(조직: pH3~10 and 18cm, serum: pH3~10 and 13cm)을 사용하여 isoelectrofocusing을 다음과 같은 조건으로 실시하였다. 12시간 동안 Rehydration 시킨 후 500V gradient 조건으로 1시간, 이후 8000V gradient로 1시간 전개 후 마지막으로 13시간동안 8000V steady-n-state level을 실시하였다.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,
(2) 2차원 전기영동(Second-dimension SDS-PAGE)(2) Second-dimension SDS-PAGE
Isoelectrofocusing을 마친 후 각 strip을 1% DTT(Dithiotheitol)와 2.5% IAA(iodoacetamide)로 각각 15분 동안 equilibration buffer(50mM Tris-HCl, pH8.8, 6M Urea, 30% glycerol, 2% SDS, 0.001% Bromophenol blue)를 사용하여 잘 혼합하면서 반응시켰다. Equilibration 시킨 각 strip을 12.5% SDS-PAGE에 올린 후 agrose sealing solution으로 채운 다음 SDS-PAGE 한 장당 2 Watt로 전개시켰다. 전개시킨 SDS-PAGE를 fixing solution (acetate acid 10%, ethanol 40%)에서 약 30분 정도 고정화시켰다.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 (
실시예 3. 은(silver) 염색 및 이미지 스캐닝, 분석Example 3 Silver Staining and Image Scanning, Analysis
고정화된 gel을 sensitizing solution(30% ethanol, 0.125%(w/v glutardialdehyde, 0.2%(w/v) sodium thiosulphate, 6.8% sodium acetate)과 30분간 반응시키고 증류수로 세척하였다. 0.25%(w.v) silver nitrate solution으로 다시 20분간 반응시키고 증류수로 세척하였다. 고정화된 gel을 developing solution (2.5%(w/v) sodium carbonate)과 8분간 반응시키고, 마지막으로 stopping solution 1.46%(w/v) EDTA?Na?2H2O와 반응시켰다. 염색된 gel을 ImageScanner flatbed scanner를 이용하여 scan한 후 각 protein spot을 ImageMaster 2-D melanie software(Amersham Bioscience Inc.)를 사용하여 분석하였다.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.).
도 3은 도체중(CW) 관련 육종가 추정치(EBV)가 변화된 스팟의 발현강 도(intensity)를 비교한 그래프이다.3 is a graph comparing the intensity of expression of spots in which carcass weight (CW) related breeding estimate (EBV) is changed.
실시예 4. 표현형 및 유전능력과 표지단백질간의 연관성 검토Example 4 Examination of Association Between Phenotype and Genetic Ability and Labeled Protein
(후보 표지단백질의 발현양과 경제형질간의 상관 분석 및 유의성 검정)(Correlation analysis and significance test between expression level of candidate marker protein and economic trait)
도체중(CW) 육종가추정치(EBV)의 순위에 따라 선발한 상위 20두 한우와 하위 20두 한우의 등심을 2차원 전기영동 등 화상의 spot 밀도에 대한 유의성 검증(T-test)을 실시하였고, 또한 SAS 8.12 package를 이용하여 spot의 상대밀도와 각 경제형질간의 상관분석을 실시하였다. 하기 표 1은 등심 단백질에 대한 도체중 관련 유의성 검정 결과 및 상관계수를 나타낸다.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.
[표 1]. TABLE 1
실시예 5. 질량분석기를 이용한 단백질 동정Example 5 Protein Identification Using Mass Spectrometry
이미지 분석을 통하여 단백질 발현 패턴에 차이가 보이는 spot을 선별한 다음, gel에서 해당 spot부분을 blue tip을 이용하여 분리하였다. 분리된 spot들은 30mM potassium ferricyanide와 100mM sodium thiosulfat를 1:1로 섞은 용액에 담궈 destaining 시켰다. destaining 된 spots을 100mM ammomium bicarbonate, pH 7.8 용액과 acetonitrile 용액을 사용하여 전처리 하고 trypsin과 반응시켰다.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.
In gel trypic digestion방법으로 처리한 후, 그 추출액을 nano-ESI-Q-TOF mass spectrometer (MS/MS)을 이용하여 분석하였다. 분석된 펩타이드 정보를 가지고 MASCOT search program에서 오차범위를 최소한으로 설정하여 일치되는 단백질을 검색하여 최종적으로 동정하였다. 하기 표 2는 ESI-Q-TOF를 이용해 확인한 등심 후보 표지단백질 목록이다.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.
[표 2]. TABLE 2
상기와 같이 동정된 단백질의 아미노산 서열은 NCBInr 데이터베이스(http;//www.ncbi.nlm.nih.gov/)를 서치하여 클리세르알데히드-3-포스 페이트 탈수소효소(Glyceraldehyde-3-phosphate dehydrogenase)는 서열번호 4의 아미노산 서열을 갖는 것을 확인하였다.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.
이상 설명한 바와 같이, 본 발명에 따르면, 단백질수준에서 한우의 도체중 등을 포함한 경제형질과 관련된 바이오마커 단백질을 프로테옴분석 기술을 이용하여 선발한 후 이를 활용하여 새로운 개념의 고급육 생산기술을 정착시킬 수 있다. 또한, 고비용의 표현형 위주의 능력검정작업을 최소화함과 동시에 성장초기나 배아상태에서의 유전전 잠재력과 조기예측이 가능할 수 있는 저비용의 검정방법으로 대치할 수 있다.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.
도 1은 도체중 관련 육종가추정치가 높은 등심과 낮은 한우 등심에서 발현하는 근육단백질을 나타내는 2-D 전기영동이미지이다.1 is a 2-D electrophoresis image showing muscle proteins expressed in the sirloin and low Hanwoo sirloin with high estimated carcass weight.
도 2는 도 1의 화살표 영역의 도체중 (CW) 관련 육종가 추정치(EBV)가 높은 등심조직(high CW group)과 낮은 등심조직(low CW group)에서의 스팟을 확대한 것이다.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.
도 3은 도 2에서 확인한 스팟들의 발현강도 (intensity)를 비교한 그래프이다.3 is a graph comparing the expression intensity (intensity) of the spots identified in FIG.
도 4a는 스팟 1758의 ESI-Q-TOF 질량분석 결과를 MASCOT 서치 프로그램을 이용하여 단백질 동정한 결과이고, 도 4b, 4c, 4d는 각각 서열번호 1, 2, 3의 펩타이드의 MS/MS fragmentation 스펙트럼이다.Figure 4a is a result of protein identification using the MASCOT search program ESI-Q-TOF mass spectrometry of the
도 5는 도체중 관련 EBV 수치를 기준으로 총 40두의 후대검정우의 각각 고급육과 저급육 그룹으로 선정한 결과이다.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 <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
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