초록 ▼

○ 연구결과 : 젖소를 각각 임신소 및 비임신소로 구분하여 임신기간별로 오줌시료를 채취하여, 50% 범위의 ammonium sulfate로 오줌단백질을 분리 및 농축하였다. 농축된 오줌단백질을 IEF 방법과 2-D gel 분석을 실시하여 반복성 높은 단백질 분리방법을 성공적으로 확립하였다. 2-D gel 분석결과를 이용하여 PDQUest program으로 image analysis를 수행하여 proteomic map을 완성하였으며 약 140종류의 protein spots을 확인할 수 있었다. 또한 MALDI-TOF mass spec

○ 연구결과 : 젖소를 각각 임신소 및 비임신소로 구분하여 임신기간별로 오줌시료를 채취하여, 50% 범위의 ammonium sulfate로 오줌단백질을 분리 및 농축하였다. 농축된 오줌단백질을 IEF 방법과 2-D gel 분석을 실시하여 반복성 높은 단백질 분리방법을 성공적으로 확립하였다. 2-D gel 분석결과를 이용하여 PDQUest program으로 image analysis를 수행하여 proteomic map을 완성하였으며 약 140종류의 protein spots을 확인할 수 있었다. 또한 MALDI-TOF mass spectrometry 사용조건을 최적화하였으며, 임신특이적으로 증가하는 10종의 단백질과 감소하는 2종의 단백질에 대한 분석을 이미 완료하였으며, 각각의 단백질들을 성공적으로 확인하였다. 임신특이 단백질 중 특이성이 가장 높은 #2 spot에 대하여 N-termial 및 internal peptides의 microsequencing을 수행하였다. 이 결과를 BLAST search로 비교했을 때 일치하는 단백질이 발견되지 않았음으로 #2는 임신특이적으로 발현되는 새로운 단백질로 판단된다. 본 연구팀은 polyclonal antibody 및 monoclonal antibody 제조 및 진단 Kit의 성능검정에 대한 연구를 수행하여 성공적인 연구결과를 얻었다. 이 항체를 이용하여 Western blot을 수행한 결과 임신특이 단백질에 대해서 비임신소의 오줌에 서는 감지하지 않았고 임신소의 오줌에서 특이하게 감지하였다. 또한 이 항체는 전체적으로 90% 이상의 정확도를 나타내었다. 이 같은 성적은 지금까지 보고된 다른 소임신진단 Kit에 의한 결과보다 20% 이상 우수한 결과이다.

Abstract ▼

In this research, we developed a method for the localization of pregnancy-specific protein from cow urine on 2-D gel. The proteins were transferred to membrane and microsequenced. One of the proteins, bovine pregnancy-associated protein 2 (bPAP 2), was identified, characterized by N-terminal and int

In this research, we developed a method for the localization of pregnancy-specific protein from cow urine on 2-D gel. The proteins were transferred to membrane and microsequenced. One of the proteins, bovine pregnancy-associated protein 2 (bPAP 2), was identified, characterized by N-terminal and internal sequencing, and mass spectrometric analysis, and the pregnancy-association was confirmed by Western blot analysis. It will be a good candidate for developing a cow pregnancy detection assay.
Total proteins from pregnant and non-pregnant cow urine were obtained with addition of trichloroacetic acid. Approximately 0.35 mg protein of each sample was loaded onto each first dimension isoelectric focusing gel. Generally, the protein concentration of 60 ∼ 90 mg/L in pregnant urine was about three-folds higher than that of non-pregnant urine. Interestingly though, the total proteins in the four urine samples from the different cows in terms of age and status of pregnancy showed similar migration patterns on 2-D gels. For better separation and improved spot intensity on the gel, urine samples were fractionated with ammonium sulfate. When the proteins were separated on 2-D gels after the 50% ammonium sulfate fraction, several pregnancy-associated and non-pregnancy specific spots were found on the gels. In particular, spot 2 was isolated very well at 20 kDa and PI 6.2, and expressed at a significant level in pregnancy-associated urine. Considering the spot intensity after silver staining, the amount of the protein in the spot was believed to be in the tens of nanograms range. Throughout this experiment, we focused on proteins whose expression was pregnancy-associated.
To determine a correlation between the expression level of the proteins and duration of pregnancy, the relative expression levels of spots 1 and 2 in the urine of cow #46 was compared at 2, 4 and 5 months of pregnancy. In this period, the expression of the spot 2 protein increased significantly while that of the spot 1 protein remained constant as the pregnant state proceeded. The possibility of the influence of feeding conditions or other genetic differences on the expression of the pregnancy-associated proteins localized on the 2-D gel was ruled out by analyzing pregnant and non-pregnant cow urine from another institute housing a different lineage of cows and receiving different feed. Interestingly, the profiles of the total urine protein of the cows from two different institutes were highly similar on 2-D gels. As the pregnant cow was artificially inseminated four months before the urine sampling and the protein was expressed at a significant level, it is possible to speculate that this protein is expressed much earlier than the four-months state, and perhaps even earlier than two months after artificial insemination.
Proteomic analysis of the urine proteins separatated on 2-D gels was not very informative. Among over 200 spot analyzed, only <5% of the proteins could be annotated by PMF (peptide mass fingerprinting) due to the incomplete nature of existing bovine genomic and protein databases. Some of the protein spots were identified with low MOWSE scores. The spot 2, bPAP 2, showed a similarity to human (or bovine) collagen by PMF. The collagen-like sequence in the N-terminus of bPAP 2 could be digested by trypsin to form some responsible peptides matched to collagen, which is a huge molecule with repetitive Gly-X-X sequence. The pregnancyassociation of bPAP 2 was analyzed and other additional pregnancy-associated proteins were sought on the 2-D gel. Due to the variations of urine protein components among individual cows, isolation of pregnancy-associated protein by comparing 2-D gels required many urine samples from pregnant and non-pregnant cows. From the analysis of the spots on the gel stained with Coomassie Brilliant Blue R-250, we set the cutoff for pregnancy-specific proteins as repeatedly positive in pregnant urines and negative in non-pregnant urines from at least three cows in different gestation periods and feeding conditions. Proteins from 30 pregnant and 20 non-pregnant urines were prepared, separated on 2-D gels, and analyzed using the image analysis software, PDQuest. The bPAP 2 protein spot was the only pregnancy-associated detected protein on 2-D gels stained with Coomassie Brilliant Blue. There were two false positive and on false negative urines among 20 non-pregnat and 30 pregnant urine samples, respectively, in terms of bPAP 2 expression. Considering the high correlation of bPAP expression to bovine pregnancy (over 97%) and low background (10%), the bPAP 2 protein can be utilized for the diagnosis of bovine pregnancy by an immunological format.
The expression of bPAP 2 in the non-pregnant urine was compared with that of the pregnant urine using a silver nitrate staining. Non-pregnant or pregnant urine proteins in gels were first stained with Coomassie Brilliant Blue. The gel images were scanned and stained with silver nitrate. In the Coomassie Brilliant Blue stained gels, the bPAP 2 protein was clearly apparent in the pregnant urine but not in the non-pregnant urine. The bPAP 2 protein not shown by the Coomassie Brilliant Blue staining could be detected as a faint smear on the silver nitrate stained gel. Urine (50 ∼ 100 mL) was fractionated with 50% ammonium sulfate to run one 2-D gel, suggesting that the basal expression of bPAP 2 is lower than the detection limit of silver staining, at the nmol level. The concentration of bPAP 2 protein in urine increases with the onset of pregnancy and reached a concentration higher than the nmol level, as detected by Coomassie Brilliant Blue staining.
The identification of bPAP 2 was pursued by N-terminal and internal sequencing. Several spots of bPAP 2 on PVDF membranes were investigated for Edman degradation and 18 amino acid sequences from the N-terminus of bPAP 2 were identified. Database search by BLAST using the amino acid sequence suggested that the N-terminus of the protein has a high similarity to collagen alpha. The similarity residues in the four Gly-X-X repeats in the sequence, which is the typical repeat of collagen proteins. Further sequence analysis by internal sequencing provided an extension of the collagen-like Gly-X-X repeats up to 37 amino acids with some ambiguities. However, the other internal sequence of bPAP 2 did not show similarities to collagen but to several different protein within short sequence ranges without any common features. Given that the molecular mass of the protein is about 20 kDa and the average molecular mass of an amino acid is 110 Da, the protein should have about 190 amino acids and at least 20% of the protein should form a collagen-like structure. The rest of the sequence forms a different kind of structure. This structural feature of bPAP 2 shows similarity to human and bovine lung surfactant protein D (SP-D). SP-D forms a quaternary structure with the oligomers of trimeric subunits. Likewise, bPAP 2 protein could form a quaternary structure via the collagen-like structure.
The amino acid sequence data obtained by a MS/MS spectrometric analysis of the tryptic peptides of bPAP 2 are summarized in Table 2. The peptide sequences do not identify any bovine protein but show limited similarities to various proteins from ifferent species. The early stage of bovine protein and genomic databases limit the proteomic approaches to identify proteins from bovine tissues. Although it might be an inefficient way to identify bovine proteins by PMF or peptide sequencing by a MS/MS spectrometry until the full-length bovine genome sequencing is completed, in the current study, the results from the mass spectrometric analyses provided an ample amount of sequence and structural information of bPAP 2.
Polyclonal antibodies and monoclonal antibodies against the peptide sequences obtained by N-terminal sequencing (DSELAGPRGARG) and MS/MS analysis (DGQELELVSSG) of bPAP 2 were generated rabbits and mouses, respectively. The polyclonal antibodies showed such high nonspecific binding that the antisera could not show the specific bPAP 2 band until the anti-bPAP 2 antibodies were purified using the affinity column. The specificity of the antibodies was analyzed by Western blot nalysis on 2-D gel separated pregnant and non-pregnant urines. Both antibodies indicated the bPAP 2 protein to have a pH of 6.2 and mass of 20 kDa. However, the affinity purified antibody against the peptide of DGQELELVSSG still showed high nonspecificity in Western blotting. Thus, the antibody to the peptide of DSELAGPRGARG was used for Western blot analysis. Approximately 5 ∼ 15 mL of the bovine urines at different gestation periods were fractionated with 50% ammonium sulfate, separated by SDS-PAGE, and transferred to the PVDF membrane. The blot was blocked with blocking buffer and incubated with the affinity purified polyclonal antibody in blocking buffer. Result shows that the purified antibody selectively recognized the 20 kDa band and that the intensity of the band was not correlated with the gestation period. The antibody recognized nonspecific bands with molecular mass lower than 20 kDa in some non-pregnant urines. The specific recognition of the bPAP protein by the antibody was not changed although the urines were collected at different times and places. In other words, the bPAP 2 protein in the pregnant urine is expressed in a pregnancy-associated manner. This result suggests that bPAP 2 is a bovine pregnancy-associated protein and that the expression of the protein in the urine varies in the individual cows. From our screening experiments, we saw <10% of false positives and <3% of false negatives. These results suggest that there could be veterinary reasons for this variation of the fluctuation of the expression level among individua l cows. If the bPAP 2 protein expression level during pregnancy can be quantitated, the correlation between the embryonic mortality which accounts for major reproductive wastage in farm animals, and the fluctuation of bPAP 2 can possibly be investigated. We have successfully produced a more specific monoclonal antibodies, Mono-1 and Mon-2, to develop a diagnostic Kit for bovine pregnancy test.
In conclusion, from the proteomic analysis of urine proteins from Holstein cows, a bovine pregnancy-associated protein was characterized by 2-D gel and image analysis, PMF (proteomic mass fingerprinting), N-terminal sequencing and MS/MS analysis. Due to the incomplete nature of current bovine genomic and protein databases, the identification of the protein was not quite successful. However, using the peptide sequence information, antibodies against the bPAP 2 protein were generated. Western blot analysis using the purified antibody to bPAP 2 showed the protein is indeed a pregnancy-associated protein and that the bPAP 2 concentration in the urine of pregnant cows fluctuates among individuals. The correlation between the fluctuation of bPAP 2 expression and veterinary symptoms need to be analyzed. This antibodies can be utilized to develop a reagent for the demonstration of bovine pregnancy and should be a valuable aid to help understand embryonic mortality in bovine pregnancy.

목차 Contents

• 제출문 ... 1
• 요약문 ... 2
• SUMMARY ... 10
• CONTENTS ... 15
• 목차 ... 17
• 제1장 연구개발과제의 개요 ... 19
• 제2장 국내외 기술개발 현황 ... 20
• 제3장 연구개발수행 내용 및 결과 ... 22
• 제1절 연구개발 수행 내용 ... 22
• 제2절 연구재료 및 방법 ... 23
• 제3절 연구개발 결과 ... 29
• 1. 소의 인공수정 및 실험동물 사육 ... 29
• 2. 정상소 및 임신소의 오줌 채취 ... 29
• 3. 정상소, 임신소의 오줌단백질 분리 ... 32
• 4. 임신기간별 임신특이 단백질의 발현량 변화조사 ... 39
• 5. 선발된 특이단백질의 amino acid microsequencing 분석을 통한 단백질 정성분석 ... 41
• 6. 정상소, 임신소의 오줌단백질의 protein fingerprint database 구축 ... 44
• 7. 오줌단백질의 proteomic map 작성 및 특이단백질 선발 ... 56
• 8. 선발된 특이 단백질의 mass spectrometry 분석 ... 60
• 9. 소임신특이 단백질에 대한 polyclonal antibody 제조 및 Western blotting ... 79
• 10. 소임신특이 단백질에 대한 monoclonal antibody 제조 및 Western blotting ... 94
• 11. 소의 임신진단용 Kit의 성능검정 ... 109
• 제4장 목표달성도 및 관련분야에의 기여도 ... 111
• 제1절 연구개발의 최종목표 ... 111
• 제2절 연구평가의 착안점 ... 111
• 제3절 연구개발 목표의 달성도 ... 112
• 1. 제 1차년도 ... 112
• 2. 제 2차년도 ... 113
• 3. 제 3차년도 ... 115
• 제5장 연구개발결과의 활용계획 ... 118
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 119
• 제7장 참고문헌 ... 120
• 끝페이지 ... 123