KR101463124B1 - Protein marker for predicting fertility of Hanwoo and predicting method thereof - Google Patents
Protein marker for predicting fertility of Hanwoo and predicting method thereof Download PDFInfo
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- KR101463124B1 KR101463124B1 KR1020130000124A KR20130000124A KR101463124B1 KR 101463124 B1 KR101463124 B1 KR 101463124B1 KR 1020130000124 A KR1020130000124 A KR 1020130000124A KR 20130000124 A KR20130000124 A KR 20130000124A KR 101463124 B1 KR101463124 B1 KR 101463124B1
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Abstract
본 발명은 한우의 수정능력 예측을 위한 한우 특이적 단백질 마커 및 이를 이용한 한우 수정능력 예측방법에 관한 것이다. 더욱 상세하게는, 본 발명은 수정능력 향상과 관련된 정장 단백질 Spermadhesin Z13 precursor및 Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module의 발현위치 및 발현량을 조사하여 한우의 수정능력을 예측하는 방법에 관한 것으로, 본 발명의 정장 단백질 마커를 이용하면 한우의 수정능력을 간편하게 예측할 수 있으며, 이를 통해 수정률이 높은 종모우를 선발하여 개량하는데 유용하게 사용될 수 있다.The present invention relates to a Hanwoo-specific protein marker for predicting the fertilization ability of Hanwoo and a method for predicting the ability of Hanwoo fertilization using the same. More particularly, the present invention relates to a method for predicting the fertilization ability of Hanwoo by investigating the expression position and expression level of the Spermadhesin Z13 precursor and Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module, The use of the suitably protein marker of the present invention makes it possible to easily predict the fertilizing ability of Hanwoo and it can be usefully used for selecting and repairing sows with high fertilization rate.
Description
본 발명은 한우의 수정능력 예측을 위한 한우 특이적 단백질 마커 및 이를 이용한 한우의 수정능력 예측 방법에 관한 것으로, 한우의 수정능력과 관련하여 개체의 정장에서 특이적으로 발현이 증가하는 정장 단백질을 확인하여 이를 통해 한우의 수정능력을 예측할 수 있는 기능성 유전자 특이적 단백질 마커를 개발하는 방법에 관한 것이다.The present invention relates to a Hanwoo-specific protein marker for predicting the fertilization ability of Hanwoo and a method for predicting the fertility of Hanwoo using the same. And a method for developing a functional gene-specific protein marker capable of predicting the fertilizing ability of Korean beef cattle.
본 발명은 한우의 수정능력 예측을 위한 한우 특이적 단백질 마커 및 이를 이용한 한우의 수정능력 예측 방법에 관한 것이다. 인공수정이 보편화 되고 수정란이식의 기술이 상용화에 이르면서 수정에 영향을 주는 단백질에 대한 연구가 활발히 진행되고 있다. 국 내외로 활발히 이루어지고 있는 연구 중의 하나는 단백질체학(Proteomics)을 통한 정액 중의 정자와 정장 단백질의 분석이다. The present invention relates to a Hanwoo-specific protein marker for predicting the fertilization ability of Hanwoo and a method for predicting the fertilization ability of Hanwoo using the same. As artificial insemination becomes commonplace and fertilization embryo transfer technology is commercialized, researches on proteins affecting fertilization are actively being carried out. One of the active researches in Korea and abroad is the analysis of sperm and sperm proteins in semen through proteomics.
정장은 정자의 생존을 위한 최적의 삼투압을 유지시켜주고 완충요소 역할을 수행, 정액 pH 변화를 막아주며 정장에 포함되어 있는 단백질들은 정자가 자성 생식기관으로 이동한 후에 정자의 운동성 및 수명을 연장시켜주는 역할을 하게 된다. 정장에서 정자의 수정능을 획득하는데 도움이 되는 단백질이 무엇인지, 또한 수정능 획득에 대한 명확한 분자기전을 밝히려는 연구들이 아직도 활발히 진행중에 있다. The suicide maintains the optimal osmotic pressure for sperm survival, acts as a buffer factor, prevents the change of the sperm pH, and the protein contained in the sperm prolongs the sperm motility and lifespan after the sperm has moved to the magnetic reproductive tract The state becomes a role. Studies are still underway to identify what proteins are helpful in acquiring fertility of spermatozoa and how to clarify the molecular mechanisms of obtaining fertility.
단백질체학은 세포 내에서 발현하는 단백질체(Proteome)의 확인과 동정을 대량으로 처리하는 연구 분야이다. 단백질체학에서는 SDS-PAGE 방법 등으로 단백질의 분자량(Molecular weight)의 차이에 따라 단백질을 분리하는 1차원 전기영동과 등전점(Isoelectric weight=pI ; net charge가 0일 때의 pH값)과 분자량(Molecular weight)의 차이로 발현되는 단백질을 분리하는 2차원 전기영동(Two dimensional electrophoresis, 2DE)을 이용해 발현되는 단백질의 비율을 확인한다. 또한, 이런 방법을 이용해 분리된 단백질의 발현을 시각화 한 다음 SDS-PAGE 상의 band와 2DE gel상의 spot 밀도값을 분석 소프트웨어를 이용하여 비교 및 분석하게 된다. Proteomics is a field of research that massively identifies and identifies proteomes expressed in cells. In protein biology, one-dimensional electrophoresis, which separates proteins according to the difference in molecular weight (molecular weight) of proteins by SDS-PAGE method, isoelectric point (pH value when the net charge is 0) and molecular weight (2DE), which separates the protein expressed by the difference in the weight of the protein (2E). In addition, this method is used to visualize the expression of isolated proteins and then to compare and analyze the spot density values of 2DE gels on bands on SDS-PAGE using analysis software.
이와 같은 단백질체학을 이용한 정액연구에 대한 분석은 외국에서 이미 활발히 이루어지고 있지만, 국내에서는 미흡한 것이 현실정이다.Analysis of the semen study using this proteomics has already been actively conducted in foreign countries, but it is not realistic in Korea.
본 발명은, 한우의 수정능력과 관련하여 특이적으로 발현이 증가하는 정장 내 단백질 확인을 통해 한우의 수정능력을 예측할 수 있으며, 이를 토대로 수정률이 높은 종모우를 선발하여 개량할 수 있는 방법을 제공하는 것을 목적으로 둔다.The present invention provides a method for predicting the fertilization ability of Korean beef cattle through the confirmation of the protein in the specimen whose expression is specifically increased with respect to the fertilization ability of the Korean beef cattle, For the purpose of.
상기 목적을 달성하기 위해 본 발명은 단백질마커로서 Spermadhesin Z13 precursor 및 Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는 한우 수정능력 예측용 마커를 제공한다.In order to accomplish the above object, the present invention provides a marker for predicting the Korean cattle breeding ability, wherein the protein marker is at least one selected from the group consisting of Spermadhesin Z13 precursor and Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type II Module .
또한 본 발명은 상기 Spermadhesin Z13 precursor는 서열번호 1의 아미노산 서열을 가지고 15,000-16,000 Da의 분자량을 갖는 것을 특징으로 하는 한우 수정능력 예측용 단백질 마커를 제공한다.Also, the present invention provides a protein marker for predicting Korean Native Cattle modification ability, wherein the Spermadhesin Z13 precursor has an amino acid sequence of SEQ ID NO: 1 and a molecular weight of 15,000-16,000 Da.
또한 본 발명은 상기 Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module은 서열번호 2의 아미노산 서열을 가지고 13,000-14,000 Da의 분자량을 갖는 것을 특징으로 하는 한우 수정능력 예측용 단백질 마커를 제공한다.Also, the present invention provides a protein marker for predicting the ability of Korean Native Cattle, wherein the Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type II Module has the amino acid sequence of SEQ ID NO: 2 and a molecular weight of 13,000-14,000 Da.
또한 본 발명은 Spermadhesin Z13 precursor의 발현량이 증가할수록 한우의 수정능력이 향상되는 것을 특징으로 하는 한우 수정능력을 예측하는 방법을 제공한다.In addition, the present invention provides a method for predicting the ability of the Korean Native Cattle to improve the fertilization ability of Hanwoo as the expression amount of Spermadhesin Z13 precursor increases.
또한 본 발명은 Chain A, Bull Seminal Plasma PDC-109 Fibronecin Type Ⅱ Module의 발현량이 증가할수록 한우의 수정능력이 향상되는 것을 특징으로 하는 한우 수정능력을 예측하는 방법을 제공한다.Also, the present invention provides a method for predicting the ability of Korean beef cattle to improve the fertilization ability of Hanwoo as the expression level of Chain A, Bull Seminal Plasma PDC-109 Fibronecin Type Ⅱ Module increases.
상기에서 살펴본 바와 같이, 본 발명에 따른 한우의 수정능력 예측을 위한 한우 특이적 단백질 마커 및 이를 이용한 한우의 수정능력 예측방법은, 한우의 수정능력과 관련하여 특이적으로 발현이 증가하는 정장 내 단백질 마커를 확인하는 방법으로 이를 통해 한우의 수정능력을 간편하게 예측할 수 있으며, 이를 토대로 수정률이 높은 종모우를 선발하여 개량하는데 유용하게 사용될 수 있다.As described above, the Hanwoo-specific protein marker for predicting the fertility of Hanwoo according to the present invention and the method for predicting the fertility of Hanwoo using the same, This method can easily predict the ability of Hanwoo to modify the markers, and it can be used to select and improve breeding sows with high fertilization rate.
도 1은 한우의 정장샘플 중 HW1 개체와, HW1 개체보다 수정능력이 우수한 HW3 개체를 이용하여 특이 정장 단백질 발현을 확인하기 위해 실시한 이차원 전기영동 결과이다.
도 2는 Spermadhesin Z13 precursor로 동정된 한우의 이차원 전기영동 결과이다.
도 3은 이차원 전기영동 결과로 Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module로 동정된 한우의 이차원 전기영동 결과이다.FIG. 1 shows the results of two-dimensional electrophoresis carried out in order to confirm the expression of a specific protein by using HW1 and HW3 individuals, which are superior to HW1, in the suit samples of Hanwoo.
Fig. 2 shows the results of two-dimensional electrophoresis of Hanwoo which was identified as Spermadhesin Z13 precursor.
Fig. 3 shows the result of two-dimensional electrophoresis of Hanwoo identified as Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module by two-dimensional electrophoresis.
이하, 본 발명의 구체적인 방법을 실시예를 들어 상세히 설명하고자 한다. Hereinafter, specific methods of the present invention will be described in detail with reference to examples.
단, 하기 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 의해 한정되는 것은 아니다.
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.
실시예 1 : 공시축Example 1: Disclosed axis
전라북도 김제의 전라북도축산위생연구소 축산시험장과 강원도 대관령 한우시험장에서 종모우 한우(Hanwoo, HW)4두의 정액을 채정하여 본 발명의 공시재료로 사용하였으며, 상기 사용된 공시재료의 특성치는 표 1에 나타내었다. 채정한 정액은 35℃를 유지시켜 실험실로 운반한 후, 사출된 정액의 정자를 제외한 액상부분의 정장만을 취하기 위해 1.5㎖ 튜브에 500㎕씩 분주하고 4℃, 12,000rpm에서 10분간 원심분리하여 정자와 정장을 분리하여 사용하였으며, 사용 전까지 -80℃에 보관하였다. The semen of Hanwoo (HW) 4 was sampled from the Livestock Experiment Station of Jeonbuk National Livestock Research Institute, Gyeongbuk Province, Jeonbuk Province and the Hanwoo Experiment Station of Gangwon Province, and used as the disclosure material of the present invention. . The semen was kept at 35 ° C and transferred to the laboratory. To obtain only the liquid portion of the sperm except the sperm injected, 500 μl of each was dispensed into a 1.5 ml tube and centrifuged at 12,000 rpm for 10 minutes at 4 ° C. And suits were separated and stored at -80 ℃ until use.
(%)PMOT 1
(%)
(%)MOT 2
(%)
Volume (ml)Ejaculate
Volume (ml)
Volume (ml)BSP3
Volume (ml)
PMOT : 정자의 전진 운동성(Proceed motility)PMOT: Progressive motility of sperm
MOT : 운동성(Motiility)MOT: Motility
BSP Vol. : 정장 샘플 량(Bovine seminal plasma volume)BSP Vol. : Bovine seminal plasma volume
위 표에서 보는 바와 같이, 정자의 운동성과 관련된 PMOT 및 MOT의 값은 HW3과 HW4에서 우수한 특성치를 확인할 수 있었으며, 그 외, HW1과 HW2의 경우 HW3 및 HW4에 비해 낮은 특성치로 확인되었다.
As shown in the above table, PMOT and MOT values related to sperm motility were found to be excellent in HW3 and HW4, and HW1 and HW2 were found to be lower than HW3 and HW4.
실시예 2 : 단백질 정량법 (Bradford assay)Example 2: Protein quantitation method (Bradford assay)
단백질 정량은 분광학적 방법으로 실시하였고, Spectrophotometer로 750nm에서 흡광도를 측정하였다. 상기 실시예 1에서 분리한 각 정장샘플은 RIPA 버퍼 pH 7.5로 10배 희석하여 분비하였고, 정량에 필요한 대조구는 BSA(1㎎/㎖)를 사용하였다. 모든 정장샘플과 대조구는 3반복 실험으로 단백질 정량을 실시하였으며 그 결과를 표 2에 나타내었다. Protein quantification was performed spectroscopically and absorbance was measured at 750 nm with a spectrophotometer. Each of the formal samples isolated in Example 1 was diluted 10 times with RIPA buffer pH 7.5 and secreted. BSA (1 mg / ml) was used as a control for quantification. Protein quantification was carried out in all replicate samples and control in 3 replicates. The results are shown in Table 2.
AverageStandard (BSA)
Average
실시예 3 : 이차원 전기영동(Two-dimensional electrophoresis)Example 3: Two-dimensional electrophoresis
3-1. 이차원 전기영동(Two-dimensional electrophoresis)3-1. Two-dimensional electrophoresis (two-dimensional electrophoresis)
이차원 전기영동을 하기 위해 실시예 1을 통해 분리된 4개체의 정장 샘플을 표 2를 참조하여 스트립에 로딩하여 전기영동을 실시하였다. 1단계에서는 50mA/Strip, 30분간 250V를 유지시켜주고, 2단계에서는 8,000V까지 60분간 가동시켜 주었다. 3단계에서는 8,000V로 35kV hrs가 될 때까지 유지하였다. 상기 로딩이 끝난 스트립은 1차 평형화 용액 [75mM Tris-HCI, pH 8.8, 6M Urea, Glycerol 29.3%, SDS 2%, Bromophenol blue stock solution 0.001%, Double-distilled water and DTT 1%] 을 넣고 15분간 반응시킨 후 버리고, 다시 이차 평형화 용액 [75mM Tris-HCI, pH8.8, 6M Urea, Glycerol 29.3%, SDS 2%, Bromophenol blue stock solution 0.002%, 2차 증류수, iodoacetamide 4.8%] 을 넣고 15분간 재반응 시켰다. In order to perform two-dimensional electrophoresis, the four sets of the formal samples separated through Example 1 were loaded on a strip with reference to Table 2, and electrophoresis was performed. In the first stage, 50mA / Strip was maintained at 250V for 30 minutes. In the second stage, the system was operated at 8,000V for 60 minutes. In the third step, the voltage was maintained at 8,000 V until 35 kV hrs. The loaded strips were loaded with primary equilibration solution [75 mM Tris-HCI, pH 8.8, 6M Urea, Glycerol 29.3%, SDS 2%, Bromophenol blue stock solution 0.001%, Double-distilled water and DTT 1% After the reaction, discard the solution and add again the secondary equilibration solution [75 mM Tris-HCl, pH8.8, 6M Urea, Glycerol 29.3%, SDS 2%, Bromophenol blue stock solution 0.002%, secondary distilled water, iodoacetamide 4.8% Lt; / RTI >
15%로 만들어진 SDS-acrylamide gel [2차 증류수, Acrylamide/Bis (30%T, 2.67%C), 10%(w/v) SDS, 1.5M Tris-HCI pH 8.8, 10% APS, TEMED(Bio-Rad, USA), Agarose)위에 상기 반응이 끝난 스트립을 장착하고, 또한 분자량의 크기도 식별하기 위하여 거름종이를 4mm/8mm로 잘라 분자량 표준(Precision Plus ProteinTM Standards - Dual color, Bio-Rad, USA)을 13㎕ 첨가하여 0.5% 아가로즈로 고정시켜 넣어주었다. SDS-acrylamide gel [secondary distilled water, 30% T, 2.67% C, 10% SDS, 1.5M Tris-HCI pH 8.8, 10% APS, -Rad, USA), Agarose) mounted over the strip end of the reaction, and also the filter paper to identify the size of the molecular weight cut to 4mm / 8mm molecular weight standard (Precision Plus Protein TM Standards - Dual color, Bio-Rad, USA) were added and fixed with 0.5% agarose.
3-2. 염색(Staining)3-2. Staining
이차원 전기영동이 끝난 뒤 gel을 가시화 하기 위해 10% Acetic acid, 40% Ethanol로 고정 후, Coomassie Brilliant Blue G-250를 사용하여 24시간 염색하고 2차 증류수로 세척하였다. After two-dimensional electrophoresis, gel was fixed with 10% acetic acid and 40% ethanol, and stained with Coomassie Brilliant Blue G-250 for 24 hours and washed with secondary distilled water.
3-3. 이미지 분석(Image Analysis)3-3. Image Analysis
실험한 gel들을 분석하기 위해 먼저 VersaDoc MP 5000(Bio-Rad laboratories, USA)로 이미지화 하였으며(도 1), 분리된 단백질 점들에 대한 영상 및 통계 분석은 이차원 전기영동 이미지 분석 프로그램(PDQuest, Bio-Rad lab, USA)으로 실시하였다.
To analyze the gels, images were first imaged with a VersaDoc MP 5000 (Bio-Rad Laboratories, USA) (Fig. 1) and image and statistical analyzes of the separated protein points were performed using a two-dimensional electrophoresis image analysis program (PDQuest, Bio-Rad lab, USA).
실시예 4 : 단백질 동정Example 4: Identification of Protein
실시예 3의 이차원 전기영동으로 정성분석을 끝낸 gel의 조각을 떼어내어 50% MeoH와 증류수의 혼합물 100㎕을 첨가하여 진탕배양기에서 5분간 방치시켜 세척하였다. 이 후, 다시 200mM 탄화수소암모늄(ammonium bicarbonate)으로 20분간 배양기에 방치시켜 반응한 후, 아세토나이트릴(aceronitrile) 100㎕을 첨가하여 젤이 하얗게 변할 때까지 탈수시켰다. 이 gel을 진공 원심분리기에 넣어 젤로부터 용액을 완전히 없앴다. 건조된 gel 조각은 0.2㎍의 트립신(Promega)이 들어있는 50mM 탄화수소암모늄 20㎕로 얼음 상에서 45분간 함수시켰다. 반응용액을 제거한 후 50mM 탄화수소암모늄 30㎕을 넣은 후 37℃로 밤새 반응시켰다. 그 펩티드 용액은 C18 나노 칼럼(nano column)을 이용하여 염분을 제거하였다.The piece of gel which had been subjected to qualitative analysis by two-dimensional electrophoresis of Example 3 was removed, and 100 μl of a mixture of 50% MeoH and distilled water was added, followed by washing in a shaking incubator for 5 minutes. After incubation for 20 minutes with 200 mM ammonium bicarbonate in an incubator, 100 μl of acononitrile was added to dehydrate the gel until the gel turned white. The gel was placed in a vacuum centrifuge to completely remove the solution from the gel. The dried gel pieces were incubated for 45 minutes on ice with 20 [mu] l of 50 mM ammonium hydrogencarbonate containing 0.2 [mu] g of trypsin (Promega). After removing the reaction solution, 30 50 of 50 mM ammonium hydrogencarbonate was added, and the mixture was reacted at 37 캜 overnight. The peptide solution was desalted using a C18 nano column.
크로마토그래피 칼럼(chromatographic columns)은 질량분석 이전에 펩티드 염분제거와 농축용으로 사용되었다. Poros reverse phase R2 material(20-30㎛ bead size perSeptive Biosystems)로 구성된 칼럼은 단단히 조여진 GELoader tip(Effendorf, Hamburg, Germany)에 팩킹하였다. 상기 염분이 제거된 용액을 100mL 주사기를 사용하여 천천히 압력을 가해 주입시켜 주었고, 펩티드 용액 30㎕은 5% 포름산(formic acid)용액 내에서 희석되어 칼럼 안으로 이동되었다. 그리고 30㎕은 5% 포름산 용액으로 세척되었다. MS/MS 분석을 위해 펩티드는 borosilicate nanoelectrospray needle(Econo TipTM, New Objective, USA)에 50% methanol/49% H2O/1% formic acid와 섞여 용출시켰다. Chromatographic columns were used for peptide salt removal and concentration prior to mass spectrometry. Columns made of Poros reverse phase R2 material (20-30 μm bead size perSeptive Biosystems) were packed in a tightly packed GELoader tip (Effendorf, Hamburg, Germany). The saline-free solution was slowly poured into the solution using a 100 mL syringe and 30 μl of the peptide solution was diluted in 5% formic acid solution and transferred into the column. And 30 μl was washed with 5% formic acid solution. Peptides were eluted with 50% methanol / 49% H 2 O / 1% formic acid on a borosilicate nanoelectrospray needle (Econo Tip TM , New Objective, USA) for MS / MS analysis.
MS/MS 펩티드는 nano-ESI Q-TOF2 mass spectrometer(AB Sciex instruments, Foster City, CA94404 USA)로 수행하였다. 실온에서 실험이 실시되었으며, 여과시키는 borosilicate nanoelectrospray needle(Econo Tip TM, New Objective, USA)은 1kV로 맞춰주고 이온 소스(ion source)와 함께 일정하게 결합시켜 주고 0-5psi의 배압을 가해주면서 질소를 10-30nL/min로 주입하였다. 그 원뿔(Cone) 전류는 40V였으며, 충돌가스(collision gas)는 아르곤(Ar)에 6-7x10-5mbar의 압력을 가해주었고 충돌 에너지(collision energy)는 25-40V였다. 생성 이온은 반사기(reflector)와 작은 통로판 검사기(micro-channel plate) 그리고 time to digital 변화기에 부착되어있는 orthogonal TOF analyzer를 사용하여 분석하였다. 그리고 분석하여 얻은 자료는 Peptide sequence system을 이용하여 아미노산 서열을 분석하였다.
MS / MS peptides were run on a nano-ESI Q-TOF2 mass spectrometer (AB Sciex instruments, Foster City, CA 94404 USA). The experiment was carried out at room temperature. The filtered borosilicate nanoelectrospray needle (Econo Tip TM, New Objective, USA) was set at 1 kV and fixed with an ion source. A pressure of 0-5 psi was applied, -30 nL / min. The cone current was 40V and the collision gas applied argon (Ar) at a pressure of 6-7x10-5 mbar and the collision energy was 25-40V. The product ions were analyzed using a reflector, a micro-channel plate, and an orthogonal TOF analyzer attached to a time-to-digital converter. The amino acid sequence was analyzed using the peptide sequence system.
실시예 5 : 통계분석(Statistical Analysis)Example 5: Statistical Analysis < RTI ID = 0.0 >
이차원 전기영동 분석 후 단백질 동정을 위해 선택된 점(spot)의 농도의 차이는 SAS program GLM procedure로 분석하였다. 종모우에서 동정된 정장 단백질의 평균 농도를 Duncan's Multiple Range Test를 사용하여 유의성 p<0.05 수준에서 비교 분석 하였다.
Differences in the concentration of selected spots for protein identification after two-dimensional electrophoresis analysis were analyzed by the SAS program GLM procedure. The average concentration of formalin proteins identified in sow moths was analyzed by Duncan 's Multiple Range Test at a significance level of p <0.05.
실시예 6 : 이차원 전기영동으로부터 얻은 단백질 점(Protein spot) 동정 결과Example 6: Identification of protein spot obtained from two-dimensional electrophoresis
실시예 3의 이차원 전기영동에서 확인된 단백질 점(spot)은 PDQuest software에 의해 이미지 분석을 실시하였다. 먼저 유의적으로 차이를 보이는 점(spot)의 밀도(density)를 PDQuest software를 이용하여 측정하여 밀도 값(density value)를 Statistical Analysis System(SAS)로 분석, 그래프로 표시하였다(도 2,3). 이 후, 분리된 단백질 점(spot)은 동정하여 아미노산 시퀀스(amino acid sequence)을 확인하고 해당 점(spot)의 단백질을 확인하였다(표3).Protein spots identified in two-dimensional electrophoresis of Example 3 were analyzed by PDQuest software. First, density of spots showing significant differences was measured using PDQuest software, and the density value was analyzed by a statistical analysis system (SAS) and displayed in a graph (FIGS. 2 and 3) . Thereafter, the isolated protein spot was identified to identify the amino acid sequence and identify the protein at the spot (Table 3).
동정 결과protein
Identification result
시퀀스Peptides
sequence
Z13 precursorSpermadhesin
Z13 precursor
taurusBos
taurus
EIIEGPPESSNSR.K 9471 R.DVHLNCNKESL
EIIEGPPESSNSR.K 94
taurusBos
taurus
QDGPAELPEDEEC
VFPFVYR.N 311 -.DQDEGVSTEPT
QDGPAELPEDEEC
VFPFVYR.N 31
Spot No. 1 단백질은 분자량 15,496 Da인 소의 단백질 Spermadhesin Z13 precursor로 동정이 되었고 펩티드 시퀀스는 71-94에서 일치하였다(도 1). Spot No. 2 단백질은 분자량 13,244 Da인 소의 단백질 Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module로 동정되었고 펩티드 시퀀스는 1-31에서 일치하였다(도 1).
Spot No. 1 protein was identified as the bovine protein Spermadhesin Z13 precursor with a molecular weight of 15,496 Da and the peptide sequence was consistent at 71-94 (Figure 1). Spot No. 2 protein was identified as a protein of Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module with a molecular weight of 13,244 Da, and the peptide sequence was consistent at 1-31 (FIG. 1).
실시예 7 : 동정된 정장 단백질과 수정능력과의 상관관계Example 7: Correlation between Identified Suit Protein and Correction Ability
본 발명에서는 이차원 전기영동 실험을 통해 등전점과 분자량을 이용하여 단백질 점(spot)을 분리하고 Spermadhesin Z13 precursor와 Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module 단백질을 동정하였다. 이 후 그 동정 결과로 나타난 각 단백질 점(spot)의 발현량(density) 값을 개체별 그래프로 표시하여 도 2, 도3에 나타내었다.In the present invention, protein spots were separated using isoelectric point and molecular weight through two-dimensional electrophoresis, and Spermadhesin Z13 precursor and Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type II Module proteins were identified. The density values of the respective protein spots as a result of the identification are shown in individual graphs and shown in FIG. 2 and FIG. 3. FIG.
상기 도면에서 확인되는 바와 같이, 정자의 운동성이 높아 수정능력이 비교적 높은 HW3, HW4에서는 정자의 운동성이 낮아 수정능력이 낮은 HW1, HW2에 비해 spot 1에 해당하는 Spermadhesin Z13 precursor 단백질이 확연히 높게 발현되는 것을 확인하였다.As can be seen from the figure, Spermadhesin Z13 precursor protein corresponding to spot 1 is significantly higher than HW1 and HW2 having low fertility due to low mobility of sperm in HW3 and HW4, which have high sperm motility and relatively high fertility Respectively.
또한 수정능력이 비교적 높은 HW3과 HW4에서 수정능력이 낮은 HW1, HW2에 비해 spot 2에 해당하는 Chain A, Bull Seminal Plasma PDC-103 Fibronectin Type Ⅱ Module 단백질의 발현량이 비교적 높게 나타나는 것을 확인하였다. In addition, the expression levels of Chain A and Bull Seminal Plasma PDC-103 Fibronectin Type Ⅱ Module proteins, which correspond to spot 2, were relatively higher than those of HW1 and HW2, which have relatively low ability of cleavage in HW3 and HW4.
이러한 결과로 보아, Spermadhesin Z13 precursor와 Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module 단백질은 수정능력이 높은 개체에서 유의적으로 증가함을 알 수 있었다.These results suggest that Spermadhesin Z13 precursor and Chain A and Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module proteins are significantly increased in individuals with high fertility.
따라서, Spermadhesin Z13 precursor와 Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module이 발현되는 단백질 점(spot)의 위치 및 발현량을 확인함으로 한우의 수정능력을 예측할 수 있는 마커로서 유용할 것으로 생각되며, 수정능력이 우수한 종모우를 선발하는 도구로 활용할 수 있다.Therefore, it may be useful as a marker for predicting the fertilization ability of Hanwoo by confirming the location and expression level of protein spot where Spermadhesin Z13 precursor and Chain A, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module are expressed , And can be used as a tool to select a breeder with excellent fertility.
Spermadhesin Z13 precursor
Chaine, Bull Seminal Plasma PDC-109 Fibronectin Type Ⅱ Module
<110> ChonBuk National University Foundation of University-Industry Cooperation <120> Protein marker for predicting fertility of Hanwoo and predicting method thereof <130> P12-0042 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 134 <212> PRT <213> Spermadhesin Z13 precursor <400> 1 Met Lys Leu Ser Ser Val Ile Pro Trp Ala Leu Leu Leu Ser Thr Ala 1 5 10 15 Ser Ser Asp Ser Thr Asp Gly Leu Leu Val Lys Asp Lys His Leu Cys 20 25 30 Gly Asp Leu Tyr Gly Glu Glu Tyr Gly Val Ile Phe Pro Tyr Leu Gly 35 40 45 Leu His Thr Glu Cys Leu Trp Ile Ile Lys Met Asp Pro Gly Tyr Arg 50 55 60 Ile Leu Leu Glu Val Arg Asp Val His Leu Asn Cys Asn Lys Glu Ser 65 70 75 80 Leu Glu Ile Ile Glu Gly Pro Pro Glu Ser Ser Asn Ser Arg Lys Ile 85 90 95 Cys Asp Thr Ser His Ala Glu Tyr Thr Ser Cys Thr Asn Thr Met Thr 100 105 110 Val Lys Tyr Thr Arg Lys Pro Asn His Pro Ala Pro Asp Phe Phe Leu 115 120 125 Ile Phe Arg Arg Val Leu 130 <210> 2 <211> 109 <212> PRT <213> Chain A Bull Seminal Plasma PDC-109 Fibronectin Type II Module <400> 2 Asp Gln Asp Glu Gly Val Ser Thr Glu Pro Thr Gln Asp Gly Pro Ala 1 5 10 15 Glu Leu Pro Glu Asp Glu Glu Cys Val Phe Pro Phe Val Tyr Arg Asn 20 25 30 Arg Lys His Phe Asp Cys Thr Val His Gly Ser Leu Phe Pro Trp Cys 35 40 45 Ser Leu Asp Ala Asp Tyr Val Gly Arg Trp Lys Tyr Cys Ala Gln Arg 50 55 60 Asp Tyr Ala Lys Cys Val Phe Pro Phe Ile Tyr Gly Gly Lys Lys Tyr 65 70 75 80 Glu Thr Cys Thr Lys Ile Gly Ser Met Trp Met Ser Trp Cys Ser Leu 85 90 95 Ser Pro Asn Tyr Asp Lys Asp Arg Ala Trp Lys Tyr Cys 100 105 <110> ChonBuk National University Foundation of University-Industry Cooperation <120> Protein marker for predicting fertility of Hanwoo and predicting method thereof <130> P12-0042 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 134 <212> PRT <213> Spermadhesin Z13 precursor <400> 1 Met Lys Leu Ser Ser Val Ile Pro Trp Ala Leu Leu Leu Ser Thr Ala 1 5 10 15 Ser Ser Asp Ser Thr Asp Gly Leu Leu Val Lys Asp Lys His Leu Cys 20 25 30 Gly Asp Leu Tyr Gly Glu Glu Tyr Gly Val Ile Phe Pro Tyr Leu Gly 35 40 45 Leu His Thr Glu Cys Leu Trp Ile Ile Lys Met Asp Pro Gly Tyr Arg 50 55 60 Ile Leu Leu Glu Val Arg Asp Val His Leu Asn Cys Asn Lys Glu Ser 65 70 75 80 Leu Glu Ile Ile Glu Gly Pro Pro Glu Ser Ser Asn Ser Arg Lys Ile 85 90 95 Cys Asp Thr Ser Ala Glu Tyr Thr Ser Cys Thr Asn Thr Met Thr 100 105 110 Val Lys Tyr Thr Arg Lys Pro Asn His Pro Ala Pro Asp Phe Phe Leu 115 120 125 Ile Phe Arg Arg Val Leu 130 <210> 2 <211> 109 <212> PRT <213> Chain A Bull Seminal Plasma PDC-109 Fibronectin Type II Module <400> 2 Asp Gln Asp Glu Gly Val Ser Thr Glu Pro Thr Gln Asp Gly Pro Ala 1 5 10 15 Glu Leu Pro Glu Asp Glu Glu Cys Val Phe Pro Phe Val Tyr Arg Asn 20 25 30 Arg Lys His Phe Asp Cys Thr Val His Gly Ser Leu Phe Pro Trp Cys 35 40 45 Ser Leu Asp Ala Asp Tyr Val Gly Arg Trp Lys Tyr Cys Ala Gln Arg 50 55 60 Asp Tyr Ala Lys Cys Val Phe Pro Phe Ile Tyr Gly Gly Lys Lys Tyr 65 70 75 80 Glu Thr Cys Thr Lys Ile Gly Ser Met Trp Met Ser Trp Cys Ser Leu 85 90 95 Ser Pro Asn Tyr Asp Lys Asp Arg Ala Trp Lys Tyr Cys 100 105
Claims (6)
Spermadhesin Z13 precursor 및 Chain A, Bull Seminal plasma PDC-109 Fibronectin Type Ⅱ Module로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는 한우 수정능력 예측용 단백질 마커 조성물.As a protein marker composition,
Spermadhesin Z13 precursor and Chain A, Bull Seminal plasma PDC-109 Fibronectin Type Ⅱ Module.
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