초록 ▼

Ⅳ. 연구개발결과
1. 젖소발굽관리를 통한 생산성 향상 연구
가. 발굽질환 관리방안 연구 및 원인 분석
- 발굽삭제만을 실시한 처리 1구의 경우 처지전 보행지수 3이상인 개체가 62.5%였으며 처리 후 28일 기준 12.5%로 감소하였다. 발굽삭제 및 Oxytetracycline 붕대를 한 처리 2구의 경우 처리전 보행지수 4이상인 개체가 58.3%이였으며 처치 7일후 8.3%로 감소하였다. 발굽삭제 및 Oxytetracycline 분무인 처리 3구는 처리전 보행지수 3이상인 개체가 40%였으며 처치 14일후 20%

Ⅳ. 연구개발결과
1. 젖소발굽관리를 통한 생산성 향상 연구
가. 발굽질환 관리방안 연구 및 원인 분석
- 발굽삭제만을 실시한 처리 1구의 경우 처지전 보행지수 3이상인 개체가 62.5%였으며 처리 후 28일 기준 12.5%로 감소하였다. 발굽삭제 및 Oxytetracycline 붕대를 한 처리 2구의 경우 처리전 보행지수 4이상인 개체가 58.3%이였으며 처치 7일후 8.3%로 감소하였다. 발굽삭제 및 Oxytetracycline 분무인 처리 3구는 처리전 보행지수 3이상인 개체가 40%였으며 처치 14일후 20%로 감소하였다. Oxytetracycline 분무군인 처리 4구는 처리전 보행지수 4이상인 개체가 42.9%였으며 처치 14일후 14.3%로 감소하였다.
- 기립 및 보행시 자세를 기준으로 5단계로 구분되는 보행지수를 조사한 결과 삭제구와 우상피부염 발생축 모두 개선되는 것으로 조사되었으며 특히 처치 7일후에 보행이 개선되는 결과를 보였다.
- 발굽삭제 및 우상성피부염 치료후 유량은 전반적으로 증가하는 경향을 보였다. 우상피부염이 발생되지 않은 처리 1구(삭제)보다 우상피부염이 발생된 처리 2, 3, 구의 유량이 감소되어 있는 것으로 조사되었으며 우상피부염이 치료되면서 유량은 약 2% 가량 증가하는 추세를 보였다.
나. 젖소 발굽질환 발생 조사
- 전국의 총 46개 젖소농가를 조사하였으며 젖소 2,990두를 대상으로 발굽질환 발생률을 조사한 결과 발굽질환이 있는 개체는 467두로 15.6%의 발생률을 나타내었다.
- 세부 발굽질환중 파행증을 나타내는 우상피부염의 발생이 52.5%로 가장 높았고 제저궤양(24.2%), 백선병(12.8%), 제저출혈(4.3%) 순으로 조사되었다.
다. 삭제방법에 따른 젖소 생리학적 모니터링
- 횡와식 발굽처치는 발굽삭제만 실시하였을 경우 15.1±4.2분이었으며 삭제 및 우상피부염 치료를 위한 항생제 붕대 치료군과 삭제 및 제저궤양 치료를 위한 블럭장착군은 각각 17.8±4.6분, 24.8±3.4분으로 조사되었다.
- 발굽처치 전후 스트레스지표는 총 25두를 대상으로 분석하였으며 삭제 및 치료군 모두 처치 후에 코티솔 농도는 상승하였다. 삭제전후 혈청내 코티솔 평균 농도는 각각
1.4±1.4ug/dL, 5.3±1.7 ug/dL으로 분석되었다.
2. 소 락토페린 단백질 발현 사료용 형질전환 미세조류 개발 및 항균/항바이러스 활성 규명
가. Bovine lactoferrin cloning and transient expression
- 담배에서 젖소유래 락토페린을 발현하기 위해서, 락토페린 유전자는 유선조직에서 RT-PCR로 분리하였고, pCAMBIA1304 과 pRI101에 클로닝 되었다.
- 담배 형질전환에 사용된 발현벡터는 pCAMBIA1304-bLf1, pCAMBIA1304-bLf2, pRI-bLf12 및 pRI-bLf23이다. 이 벡터들은 agroinfiltration으로 담배에 접종되었고, 5일 동안 발현시켰다.
- RT-PCR로 락토페린 유전자가 삽입된 형질전환를 선별하였다.
- pCAMBIA1304-bLf1, pCAMBIA1304-bLf2, pRI-bLf12 및 pRI-bLf23이 감염된 잎에서 total RNA를 추출하여 확인였고, 약 2.0kb 크기의 예상된 밴드를 확인하엿다. 또한 이 잎들에서 총단백질을 추출하여 western blot으로 락토페린 발현 유무를 확인하엿다.
- pCAMBIA1304-bLf2 및 pRI-bLf23이 감염된 잎에서 약 70kDa의 예상햇던 단백질 밴드가 확인되었다.
나. Expression of recombinant lactoferrin in transformed microalgae
- 미세조류 클로렐라에서 락토페린 N-lobe를 발현하기 위해서 클로렐라 핵 유전자와 유사하게 코돈 최적화를 하여 유전자의 크기는 1014bp이고 GC 함량은 61%로 최적화하였다. 이 유전자는 pRTL2 vector에 클로닝하였고, pRTL2-Lfb-N으로 명명하였으며, 발현카세트로 하였다. 이 발현카세트는 EcoRI and Hind III로 절단하여 pCAMBIA1304의 MCS에 삽입하였고, 이 발현벡터를 pGG12로 명명하였다.
- pGG12로 형질전환된 Agrobacterium tumefaciens를 Agrobacterium-mediated transformation 방법으로 클로렐라를 형질전환시켰다. 형질전환체는 선택배지(BG-11 plates containing 20 mg/L hygromycin and 150 mg/L cefoxaime)에서 선별되었다. 선택배지에서 7일 동안 배양된 단일콜로니를 임의적으로 선별하여 액상배지에 배양하였다. 배양된 형질전환체에서 DNA를 추출하여 PCR로 최종 후보 형질전환체를 선별하였
다. 형질전환체 CT7와 CT9에서 예상했던 크기의 1.0kb fragment가 확인되었다. 형진 전환된 클로렐에서 락토페린 발현 유무를 western blot으로 확인하였다. 형질전환체 CT7와 CT9에서 예상했던 크기의 35kDa fragment가 확인되었다. CT7의 발현정도는 0.17%(TSP)였고, CT9의 발현정도는 0.13%(TSP)로 확인되었다. 또환 재조합 락토페린의 항균 활성은 5개 병원균(E.coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus bovis, Salmonella typhimurium)에서 확인되었다.
- 대량 생산 조건은 CT7을 이용하여 확립되었으며, BG-11을 기본으로 0.1% 마늘추출물과 0.5% 포도당이 첨가된 배지에서 4000 lux와 16:8(light:dark)의 조건으로 배양하였고, 락토페린 발현량은 0.3%(TSP)로 확인되었다.
다. Safety and feeding test
- 6주령된 SD rats(male and female)에 재조합 락토페린이 함유된 클로렐라 추출물을 단회경구투여하여 안전성 테스트를 수해하였다. 투여량을 체중1kg당 추출물 20ml을 경구 투여하여 14일 동안 치사율, 임상적 변화 및 체중을 관찰하였다. 관찰기간 동안 대조구와 처리구 사이에 특별환 소견이 보이지 않은 관계로 재조합 락토페린 함유된 클로렐라 추출물은 안전한 것으로 확인되었다.
- 이 추출물을 포도당과 전분을 혼합하여 분만화 하였고, 이 분말을 말분과 혼합하여 시제품을 제조하였다. 시제품을 젖소에 30g/head/day의 양으로 TMR과 같이 급이하였고, 시험기간은 3개월으로 하였다. 대조구는 급이 전이며 처리구는 급이 후로 하였고, 15일 간격으로 체세포수를 관찰하였다. 테스트 종료 후 대조구와 처리구 사이에 유의차가 크지 않았다.
3. 젖소 이등유 발생 저감을 위한 대사산물 탐색 및 활용기술 개발
가. 젖소 이등유 발생실태 조사 및 이등유 관련 대사산물 탐색
(1) 국내 젖소 농가의 시기별 이등유 발생실태 비교 조사
- 우리 나라 중남부지역 9개 젖소 목장을 대상을 5월에서 9월 사이 이등유 발생 실태를 조사한 결과 시점 유병률은 평균 15.4%를 나타내었으며, 농가별 가장 높은 유병률은 34.4%였다.
- 40여두의 착유규모의 농가를 대상으로 12개월 동안 월별 이등유 발생률은 조사한 결과
월별 발생률은 2월(32.6%)과 11월(33.3%)에 가장 높은 발생률을 나타내었고, 늦겨울인 2월부터 초여름인 6월까지 높은 발생률을 보였다. 평균기온이 25℃ 이상을 나타낸 7월에 감소하였으나 일평균기온이 30℃이상을 나타낸 8월에 증가하여 하절기 고온 스트레스가 이등유 발생에 영향을 미쳤을 것으로 판단되며, 환절기인 2월과 3월 그리고 6월에 비교적 높게 발생되고 있어 환절기 역시 이등유의 발생에 영향을 미치고 있을 것으로 판단된다.
- 5농가 785두를 대상으로 비유시기별 이등유 발생실태를 조사한 결과 이등유 발생률은 분만후 높게 나타내어 비유개시 1개월 이내에 34.1%로 매우 높았으며, 비유개시 60-90일 까지 발생률이 3.5%로 감소하였다. 이후 비유중기 및 말기로 진행됨에 따라 이등유의 발생률이 유의적으로 증가하는 경향을 나타내었다(p< 0.05). 한편 비유개시 270-300일째에 발생률이 36.1% 가장 높게 나타내었다.
(2) 이등유우유와 정상우유의 산도분포 및 산도에 따른 이등유 발현 위험도
- 이등유와 정상유의 평균산도를 비교한 결과 평균산도는 각각 0.159±0.026%와 0.158± 0.027%로 차이가 없었다.
- 이등유와 정상우유에 있어서 산도에 따른 위험도 분석결과 기여위험도가 1.06, 비교위험도는 1.04를 나타내었으나 통계적 유의차가 인정되지 않아(p=0.7659), 본 연구에서 알콜검사에서 양성반응을 나타낸 우유는 산도의 영향을 거의 받지 않은 이등유 이었다.
(3) 이등유 발생우군과 비발생우군의 혈액 대사산물 측정 비교 연구
- 이등유를 나타내는 젖소의 조기진단을 위한 영양대사산물을 설정하기 위하여 이등유를 보인 젖소와 정상유를 나타낸 젖소에 대한 혈액 성분을 비교한 결과 이등유를 나타낸 젖소에서 간 손상시 증가하는 효소인 AST(GOT) 값이 유의적으로 높았으며(p< 0.001), 흡수단백질의 부족 또는 간기능장애가 있을 때 낮은 값을 보이는 Albumin 역시 유의적으로 낮았다(p< 0.001). 역시 간기능 감퇴 시 낮은 값을 보이는 NEFA·cholesterol 비는 통계적인 유의차는 없었으나 이등유를 나타내지 않은 젖소에서 낮은 경향을 나타내었다. 이러한 결과에서 이등유를 나타내는 착유소는 미약하나마 간기능의 감퇴가 있음을 알 수 있다. 또한 이등유 발현된 젖소에서 Total protein 및 Triglyceride가 유의적으로 낮은 값을 나타내고 있으며, 유의적인 차이를 없으나 glucose, NEFA 및 BUN 값이 낮은 경향을 보여 영양결핍을 보인 소에서 이등유의 발현이 높은 것으로 생각된다.
(4) 대사산물 측정을 통한 이등유 발생 예측도 설정 연구
- 이등유 발현젖소와 정상유 젖소에서 유의적인 차이를 보인 혈액성분의 민감도, 특이도, 양성예측도 및 음성예측도를 계산한 결과 착유중인 젖소를 대상으로 본 혈액대사물질을 조사하여 높은 AST값을 보이며, Albumin, Total protein 그리고 Triglyceride 값이 낮았을 경우 이등유의 발현이 상대적으로 높을 것으로 예측할 수 있는 지표로 활용이 가능할 것으로 생각된다.
나. 제형화 천연물질(민들레 및 레스베라트롤)의 효과검증
(1) 실험동물(랫트)에서 민들레추출물에 대한 간기능 개선 및 항산화효과 규명
- 민들레추출물의 단기 및 장기 급여시험 결과 비슷한 경향의 결과를 나타내었다.
- 간 독성을 유발하기 위하여 TAA 투여 시 증체율의 감소, 간 중량의 증가, 중심정맥주위의 염증세포 침윤과 간세포 괴사가 나타났고, 백혈구 증가 및 AST, ALT 및 GGT의 증가, TG 합성이 감소되었으며, TAA 단독 투여군에 비해 민들레 추출물 고용량 투여 시 증체율 증가, 중심정맥 주위의 경미한 염증세포 침윤, 백혈구 감소를 보였으며, AST, ALT 및 GGT의 감소, TG 합성 증가를 확인하였다. 이를 통해 민들레 추출물이 TAA로 유도된 간세포 괴사 및 염증반응을 억제하는 사실을 확인하였다.
- TAA 단독 투여군에서 MDA의 증가와 GSH, GR, GST, Catalse, SOD와 같은 항산화 작용 요소들의 유의적으로 감소하고, 민들레 추출물 투여군에서 MDA가 감소하고 GSH, GR, GST, Catalse, SOD의 활성이 증가한 것으로 보아 TAA에 의해 유도되는 간 손상이 산화적 스트레스와 관련되어 있으며, 민들레 추출물이 항산화 기전과 관련하여 간을 보호하는 효과가 있음을 확인하였다.
- 이러한 결과는 민들레추출물을 단기 및 장기급여기 급여기간에 따른 차이를 나타내지 않았다. 이상의 결과를 통해, 민들레 추출물 급여가 TAA로 유도된 간 손상에서 간을 보호하는 효과를 확인하였다.
(2) 실험동물(랫트)에서 레스베라트롤 대한 간기능 개선 및 항산화효과 규명
- TAA 투여 시 사료섭취량의 감소, 체중 및 간 중량, 증체율의 감소, 간의 창백한 변화등이 나타났으며, RES와 TAA를 병용 투여한 군에서 TAA 단독투여군보다 AST, ALT, GGT, TB 값이 감소하고 TG 값이 증가하는 양상을 보여 통계적으로 유의한 수준의 차이가 아니지만, RES가 간 세포의 손상을 예방, 치료하는 작용이 어느 정도 효과가 있는 것으로 판단된다.
- TAA가 간에 일으키는 산화적 손상과 이에 대한 RES의 효과를 실험하기위해 실시한 항산화 측정 실험에서는 예방효과실험에서 GSH와 CAT에서 RES와 TAA 병용 투여군이 TAA 단독투여군에 비해 유의적인 차이를 나타내었으나 나머지 항목에서는 RES의 항산화 효과에 대한 유의적인 결과를 얻지 못했다. 치료효과 실험에서는 RES와 TAA의 병용 투여군이 TAA 단독투여군에 비해 어떠한 항목도 유의적인 값의 차이를 나타내지 않았으나 예방효과 실험군과 비슷하게 RES 투여군에서 증가하거나 감소되는 경향을 나타내어 RES가 간 세포를 산화적 손상으로부터 보호하는 경향을 보이기는 하지만 본 연구에서 설정한 농도 수준에서는 효과를 기대하기 어려웠으며, 치료효과 실험에서 RES의 항산화효과를 확인하기는 어려웠다.
다. 예방물질활용에 의한 이등유발생 저감 및 간기능개선효과 구명
(1) 간기능 개선 및 항산화제 첨가사료 대량 생산공정 표준화
- 간기능개선제(민들레 추출물) : 뿌리, 줄기 및 잎의 열수 추출 방법 정립
∙ 추출공정 : 건조 (수분 5%이하)→분쇄(100메쉬 이하) → 100℃에서 3시간 교반(분말 : 물 = 1:3) → 여과(100수 면포) → 농축(40%)
- 항산화제(레스베라트롤) : 에탄올 추출법 정립
∙ 추출공정 : 건조 호장근 1kg + 에탄올 1000ml → 실온의 어두운 곳에서 72시간 방치
→ 2회 반복 → 40℃ 갑압 농축 → 영하 50℃ 동결건조 (21.15g 고형분 수거, 레스베라트롤 함량 57%) → 10% 레스베라트롤 용액제조(용매 : 에탄올)
(2) 천연물질의 사료 제품 제형화 및 대량생산 공정 확보
- 간기능개선제(민들레 추출물)
∙ 젖소 간기능 개선을 위한 민들레 추출물 대량생산 시스템 구축
· 제형화 : 주정박을 흡착제로하여 농축액 10%를 함유하여 추출 고형분 4%를 함유하는 제형
∙ 장기간 보관시 천연물질의 산화, 중량에 의한 제품의 응집현상 등의 변성 방지를 위한 제형의 표면처리 기술개발 및 적용
·분체표면 코팅 : 1-3㎛ 제올라이트 3% 비율
- 항산화제(레스베라트롤)
· 제형화 : 주정박을 흡착제로하여 레스베라트롤 0.5%을 함유 하는 제형
· 분체표면 코팅 : 1-3㎛ 제올라이트 3% 비율
(3) 제형화 천연물질(민들레 및 레스베라트롤)의 간기능개선 및 이등유 발생 예방효과 규명
- 민들레추출물 급여후 간조직 손상이 진행되었을 경우 증가하여 나타나는 AST의 평균 혈청효소치는 급여전 105.4±23.0 U/L에서 85.5±14.4 U/L로 유의적인 감소를 나타내었으며(p< 0.001), 만성 간 손상시에 증가하는 GGT의 평균은 급여전 36.7±9.4 U/L에서 32.1±7.7 U/L로 유의적으로 감소하여(p< 0.001), 민들레추출물급여가 착유 젖소의 간 기능의 저하를 억제하는 효과가 있는 것으로 확인하였다.
- 항산화제인 레스베라트롤 급여후 AST의 평균값이 급여전 93.2±27.7 U/L에서 급여후 84.1±19.1 U/L로 유의적인 감소를 나타내었으며(p< 0.01), GGT 역시 평균 36.8±12.0 U/L에서 31.7±11.4 U/L로 유의적으로 감소하여(p< 0.01), 레스베라트롤 역시 항산화작용을 통하여 간 기능 저하를 예방하는 효과가 있는 것을 확인하였다.
- 민들레추출물 급여시 에너지대사와 관련된 Total cholesterol (p< 0.01)과 유리지방산(NEFA) (p< 0.001)은 유의적으로 감소하였으며, 단백질 대사와 관련된 Albumin, Total protein 및 BUN 등이 유의적으로 증가하여(P< 0.001) 간기능 개선과 함께 영양대사가 개선되고 있는 것으로 나타났다.
- 항산화제인 레스베라트롤 급여시 NEFA는 유의적으로 감소하였으며(p< 0.001), Albumin, Total protein 및 BUN이 유의적으로 증가하고 있어, 항산화제인 레스베라트롤 급여 역시 간기능 개선에 의한 에너지 및 단백질 대사에 영향을 미친 것으로 확인되었다.
- 민들레추출물 급여전∙후 유의적인 차이는 없었으나 MDA는 민들레추출물 급여 후 감소였고, GSH, CAT 및 SOD는 증가하는 경향을 나타내어 제형화된 민들레추출물을 젖소에 사료첨가제로 급여하였을 경우 항산화효과가 있음을 확인 하였다.
- 레스베라트롤 급여후 혈중 농도 및 활성은 유의적인 변화를 나타내지 않았으나, MDA는 감소하는 경향을 나타내었으며, GSH와 CAT는 증가하는 경향을 나타내었으며, 평균 SOD 역시 증가하는 경향을 나타내어 레스베라트롤을 사료첨가제로 착유중인 젖소에 급여시 항산화작용이 있음을 확인하였다.

Abstract ▼

Ⅱ. Results of Research and Development
1. Development of control for dairy production by hoof care
Hoof diseases cause severe economic losses due to premature culling of affected animals, decreased milk production, weight loss, reduced fertility and the high costs of treatment. It is also a pr

Ⅱ. Results of Research and Development
1. Development of control for dairy production by hoof care
Hoof diseases cause severe economic losses due to premature culling of affected animals, decreased milk production, weight loss, reduced fertility and the high costs of treatment. It is also a problem regarding animal welfare. Digital dermatitis(DD) is a contagious disease of cattle affecting the skin of the distal extremities. The inflammation causes varying degrees of irritation and pain and may cause severe lameness. It is thought that the infection is complex and caused by more than one single species of bacteria contributing to the initial onset and continual progression of this disease.
Treponemes are a bacterial family thought to be involved with the onset of the disease but little in known of their specific biology at this time. Is known to effective treatments based on oxytetracycline. The objective of this study was to evaluate the efficacy of the oxytetracycline application method for treatment of DD in dairy cows.
A total of 32 Holstein cows were usead at National Livestock Research Institute in Korea. Cows were divided into four groups(Groups A-D), designated only hoof trimming(A, 8cows), hoof trimming and oxytetracycline bandages for 3days(B, 8), hoof trimming and 10% oxytetracycline aerosol for 3days(C, 8) and only 10% oxytetracycline aerosol for 3days(D, 8). All groups except Group A cows with DD lesions on the skin of interdigital space and on other location. We judged locomotion score divided into 1 through 5. Locomotion scoring is based on the observation of cows standing and walking with special emphasis on their back posture. Milk yield was investigated at intervals of a week from before treatment to day 28. Blood samples were collected once a week to determine metabolic parameters and cortisol concentration of serum. On day 0 before treatment, 37.5%, 0%, 60%, and 42.9% of the group A-D were not lame at walk(Locomotion score 1-2) the other cows showed a locomotion score 3-5.On day 14 all groups had Improved 50%, 58.3%, 80%, and 71.4%, respectively. The R2 value of changed of milk yield from day 0 to day 21 in Group A, B, C and D were 0.009, 0.997, 0.2859 and 0.3835, respectively. The Mean milk yield of DD lesions groups(Group B, C and D) were significantly increased higher than Group A. The serum chemistry items showed not significantly changed for study period. The cortisol levels of all groups at day 7 were significantly lower compared to day 0. The Hoof care and use of oxytetracycline appeared significantly effective that less of lameness, increased of milk yield and decreased of cortisol levels among cows with DD lesions. And the oxytetracycline application methods may be a valuable to treatment for DD in dairy cows.
The influence of DD on leukocyte subpopulation(ACT, Bcell, CD2, CD4, CD8, CD25, CD26, MHC1, MHC2, WCI), serum chemistry and cortisol concentration to evaluate the effectiveness of a DD treatment using hoof care.
For prevalence of foot lesions in a cross-sectional study of 2,990 Korea Holstein cows in 46 herds using loose-housing systems. There were 52.5%, 24.2%, 12.8%, 4.3% cows with hoof lesion of digital dermatitis, sole ulcer, white line disease, sole hemorrhage, respectively. And study efficacy of the sugar and iodine therapy for topical treatment of digital dermatitis in dairy cows. Change of locomotion score were 3.0±0.8 and 2.3±0.5 Before and after sugar therapy, respectively.
2. Developement of mictroalgae producing bovine lactoferrin as feed-additives and identification of antibacterial/antiviral activity
The final objective of this study is to develop feed-additives using recombinat bovine lactoferrin in microalgae and to industrialize this product through the development of mass extract protocol, in vivo and in vitro efficacy test, and bovine feeding test. Lactoferrin, a member of the transferrin family, functions as an iron-binding glycoprotein and is primarily found in mammal’s milk. Lactoferrin has been shown to have various physiological effects, including antimicrobial, antiviral, anti-inflammatory, anticancer, proliferative, and antioxidant activities. There are many studies for using broad physiological effect of lactoferrin, and livestock and medical industry have accomplished purpose about cursing disease and antibiotic effect. People can get lactoferrin only from colostrum, so it is very expensive about 300$ per kg. Many country including south Korea have tried to make large scale production of recombinant lactoferrin using genetic
engineering. But there is no example about effective commercialization of lactoferrin in our country. We should totally depend on importation of lactoferrin if we need it. In our research, lactoferrin is chosen as feed additives instead of antibiotics. The final goal of this research is the large scale production of recombinant human lactoferrin and making animal feed.
(1) Bovine lactoferrin cloning and transient expression
To express bovine lactoferrin in tobacco plant (Nicotiana benthamiana), a bovine lactoferrin(bLf) gene was amplified by RT-PCR from bovine mammary gland library and engineered to be expressed by a plant expression binary vector, pCAMBIA1304 and pRI101. Tobacco transformation were carried out with pCAMBIA1304-bLf1, pCAMBIA1304-bLf2, pRI-bLf12 and pRI-bLf23. The constructs were tested for its ability to express bLf gene after transient expression for 5day by agroinfiltration into tobacco.
The presence and integration of bLf gene in plant were confirmed by RT-PCR. In plant pCAMBIA1304-bLf1, pCAMBIA1304-bLf2, pRI-bLf12 and pRI-bLf23, the expected 2.0kb fragment were amplified from total RNA extracted from agroinfiltrated leaves. The expression of bLf gene in plant were confirmed by western blot. In plant pCAMBIA1304-bLf2 and pRI-bLf23, the expected 70kDa fragment were amplified from total protein extracted from agroinfiltrated leaves.
(2) Expression of recombinant lactoferrin in transformed microalgae
We optimized the Lfb-N gene to resemble Chlorella nuclear gene codons, i.e., to have a GC content of 61% and a gene size of 1014 bp. The digested Lfb-N was cloned in the pRTL2 vector. The constructed plasmid was named pRTL2-Lfb-N and used expression cassette. The expression cassette was cut out from pRTL2-Lfb-N with EcoRI and Hind III and ligated into the MCS of pCAMBIA1304, a binary vector, to produce pGG12.
Agrobacterium tumefaciens harboring pGG12 was used to transform C. vulgaris by Agrobacterium-mediated transformation. Transformed cells were selected on BG-11 plates containing 20 mg/L hygromycin and 150 mg/L cefoxaime. A putative hygromycin-resistant single colonies, confirmed within 7 days on selection media, were randomly selected, grown in liquid media before the DNA was extracted, and used in PCR analysis. In CT7 and CT9, the expected 1.0kb fragment were amplified from total DNA extracted from transformants. The expression of bLf-N gene in transformed C. vularis were confirmed by western blot. In CT7 and CT9, the expected 35kDa fragment were amplified from total protein extracted from transformed C. vulgaris. In CT7, the expression level of rbLf-N was 0.17% of the total soluble protien from transformed C. vulgaris. In CT9, the expression level of rbLf-N was 0.13% of the total soluble protein from transformed C. vulgaris. We also confirmed antibacterial activity of by 5 pathogens(E.coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus bovis, Salmonella typhimurium). For mass production of rbLf-N from transformed C. vulgaris, CT7 which is an excellent transformant having strong expression was used and the mass production condition such as optimum of rbLf-N production was established. The optimal condition for rbLf-N production was that BG-11 media contating 0.1% garlic extract and 0.5% glucose, 4000 lux and 16:8(light:dark) were best for transformant growth and the expression level of rbLf was about 0.3% of the total soluble protein.
(3) Safety and feeding test
Six weeks old SD rats(male and female) were treated with transformed C. vulgaris extract containing rbLf-N protein once orally at 20ml/kg and observed for 14 days. At the doses used, no mortality or abnormal clinical signs in animals were shown during at the observation period. In addition, no differences were found between control and treated groups in body weight. In view of the physical characteristics of the formulation for the transformed C. vulgaris extract containing rbLf-N protein, the best one was the formulation consisted of glucose and starch. The experimental feed-additive was produced by a mix of powdery Chlorella extract and wheat sort.
In feeding test, daily cows were fed TMR supplemented with 30g/head/day of the experimental feed-additive for 3 months. The control was pre-feeding time and the treatment was post-feeding time. In this test, change of somatic cell counts before and after test were observed at 15 days intervals. After 3 months, change of somatic cell counts were slightly recognized.
3. M etabolic products exploration and application technology development for the reduction of second grade milk in dairy cow
Recently, economic losses in dairy cow has been raised because of the increase of metabolic disorder incidence rate. Also, Liver function had been decreased of using concentrate feed to dairy cow. Therefore, development of liver function enhancing and antioxidant effect feed additive agent is needed which can substitute chemical additive agent and also, eco-friendly. The 2nd grade milk shows a positive reaction to the alcohol test and has a normal acidity range. In order to produce a first class milk, heat stability of milk is very important. However, 2nd grade milk is less stable to heat treatments than fresh milk so that affects the quality of condensed milk and it makes economic losses.
When the seasons change, the production of second class milk is increased. Changing feeds, nutritional deficiencies, feed the fungus on silage, excess calcium supplements, mastitis, and reproduction disorder are cause of producing 2nd grade milk. But theres no early diagnosis method yet. In this study, we examined incidence of low quality milk in farms and suggest standard of early parameter of second grade milk production. The genus Taraxacum, known by the common name Dandelion, is a medicinal herb in the family Asteraceae. It has been traditionally used as a folk medicine for the treatment or prevention of various diseases due to its anti-inflammatory, diuretic, choleretic, and anti-oxidative, anti-carcinogenic properties. Although the medical uses of Dandelion are legion, scientific information about medicinal herb therapy is limited and most of its effects are proven only empirically. This study was aimed to investigate the hepatoprotective effect of Dandelion water extract on TAA-induced Oxidative liver damage and elucidate its potent mechanisms through in vivo experiments.
This research was performed the following; (1) the seasonal occurrence rate of the second grade milk in dairy farms to investigate and the correlation between the second grade milk and normal grade milk production cow' s serum composition were determined; and (2) the hepatoprotective and antioxitant effects of the dandelion(Taraxacum officinale) extract and resveratrol against thioacetamide-induced hepatotoxicity in rats; and (3) Enhance liver function and antioxidant effects of the dandelion extract and resveratrol supplementation in lactating dairy cows.
(1) Investigation of the seasonal occurrence rate of second grade milk in dairy farms and analysis of the correlation between the second grade milk and normal grade milk production cow' s serum composition.
As a baseline study for the establishment to prevention protocol of second grade milk in dairy farm, the seasonal incidence rate of second grade milk and the co-relationship was determined in the present study. The incidence rate of second grade milk in nine dairy farm on the central-southern region of our country from May to September was investigated. it was found that the overall incidence rate showed average 15.4%, and the highest one was 34.4%.
Seasonal survey revealed that the morbidity was higher between late winter (February) and early summer (June), and highest in February (32.6%) and November (33.3%) from the monthly investigation in a herd with around 40 cows. It was higher in August in which the temperature was over 30℃ despite of low incidence in July (over 25℃). This can be interpreted as the stress from the temperature change may affect the incidence of the second grade milk. Changes of seasons also can result in high morbidity in February, March and June.
Categorical analysis in lactating period showed that the second grade milk was most frequently found in postpartum; 34.1% within one month of postpartum, and 3.5% in 60-90 days of lactating period (n=785, 5 herds). The morbidity tends to increase thereafter, on the other hand, the morbidity was highest (36.1%) between 270-300 days of lactation.
The acidity was not significantly different between second grade milk (0.159±0.026%) and normal milk (0.158±0.027%). Hazard analysis did not show significant difference (P=0.7659) between second grade milk and normal milk (1.06 of attributable risk and 1.04 of relative risk), which suggests positive results in alcohol precipitation test was not affected by acidity.
Finally, blood test was performed in order to compare metabolites for early diagnosis of second grade milk. Among the cows producing second grade milk, liver enzyme (AST=GOT) was significantly higher (p< 0.001) and albumin was significantly lower(p< 0.001) than cows producing normal milk. NEFA, cholesterol which indicates decreased liver function was also found to be low but statistic difference was not found.
This results suggest cows producing second grade milk might have decreased liver function. Furthermore, total protein and triglyceride was significantly low, and glucose, NEFA and BUN was also low in cows producing second grade milk. Nutritional deficiency probably affected this results. Statistic analysis including sensitivity, specificity, positive prediction index, negative prediction index showed high AST, low albumin, total protein and triglyceride can predict second grade milk. It was concluded that serological parameters especially live function & metabolic related ones( AST, Albumin, Total protein and Triglyceride) were significantly influenced. These results indicating that the liver function of cows with second grade milk can be seen that the decay is.
(2) The hepatoprotective and antioxitant effects of the dandelion(Taraxacum officinale) extract and resveratrol against thioacetamide-induced hepatotoxicity in rats
(A) Hepatoprotective effects of the aqueous extract from Taraxacum officinale (Dandelion) against thioacetamide-induced hepatotoxicity in rats
In this study, we attempted to evaluate protective effects of Dandelion related with anti-oxidative activity to Thioacetamide(TAA)-indeced liver damage. 36 rats were randomly assigned to six experimental groups (n＝6 per group): Control, Dandelion water extract(DWE), TAA, TAA&DWE 300, TAA&DWE 600, TAA&DWE 1200 groups. Rats in DWE and TAA&DWE groups were pretreated with DWE (300,600 or 1200 mg/kg BW) by gavage for 7 days, while the rats in the control and TAA groups received equal volume of distilled water. All rats were treated intraperitoneally with TAA (200mg/kg BW) or normal saline at 12 hours after last oral administration and sacrificed at 12 hours after last treatment. Final body weight were checked before sacrifice. Blood was collected by caudal vena cava and liver tissue was excised, weighed.
Weight gain (%) in TAA&DWE groups was increased compared with that in TAA group (P< 0.001). On the basis of Complete blood cell count results, levels of WBC and Neutrophil count were significantly decresed in TAA&DWE 1200 group compared with that in TAA group (p< 0.05). To examine the protective effects of DWE on serum parameter, serum AST, ALT, GGT, Triglycerides, total bilirubin were determined using an auto chemistry analyzer. In TAA&DWE 600 and TAA&DWE 1200 groups, serum AST, ALT, GGT levels were lower than TAA group (p< 0.05). The serum TG level was significantly elevated in TAA&DWE groups compared with those in TAA group. The results of the histopathological examination, the control and DWE groups presented livers with normal appearances. Liver tissues from TAA group showed extensive histopathological changes, characterized by moderate or severe hepat ocytes degeneration, inflammatory cell infiltration, and congestion. In the TAA&DWE group, The severity of histopathological lesions were decreased in TAA&DWE groups compared to those in the TAA group. The concentration of MDA, an end product of LPO, was significantly increased, and GSH content was significantly decreased in the TAA group compared to those in the control group. But the MDA concentration was significantly decreased and GSH content was significantly increased in the TAA&DWE 1200 group compared to those in the TAA group. The activities of GR, CAT, GST in the TAA group significantly decreased compared to those in the control group. However, GR, CAT and GST activities in the TAA&DWE 1200 group were significantly increased compared to those in the TAA group.
To determine the preventive effect of dandelion extract on TAA induced liver damage, rats were treated with dandelion extract for 20 days prior to TAA administration. when Dandelion extract was administrated for a long time, It did not induce hepatotoxicity and oxydative stress. WBC, AST, ALT, GGT and TB in TAA alone treated group was increased. TAA treated with DWE groups prevented the elevation of serum ALT, GGT, TB and increased TG synthesis, but not significantly. The concentration of MDA were decreased in the TAA & DWE groups comapared to TAA groups. The level of activity were increased for GSH, CAT, SOD in TAA & DWE group, DWE was certificated that it has prophylactic effect on oxydative damage. To investigate the curative effects on TAA induced liver damage, Rats were treated with dandelion extract for 20 days and with TAA every 3 days. In TAA & DWE groups, AST and ALT were decreased and TG synthesis was increased compatred to TAA group. GSH concentration and SOD activity were significantly increased compared to those in the TAA group.
(B) Hepatoprotective effects of the resveratrol from R eynoutria japonica (Reynoutria) against thioacetamide-induced hepatotoxicity in rats Resveratrol is a polyphenol phytoalexin derived from a variety of plant species. It has been many researches about biological actions of resveratrol include anti-inflammatory, antioxidation, and anticancer effects. In this study, we investigated whether resveratrol was capable of preventing TAA-induced oxidative liver damage.
Treatment with TAA caused inappetence, decreased weight gain and liver weight, pale liver color. In RES & TAA group, AST, ALT, GGT, TB levels were lower and TG level was higher than TAA group. But the results were not significant, it was ambiguous to said that resveratrol have hepato-protective effect from hepatic injury. In the antioxydative study, GSH content and CAT activity of the preventive effect test were significantly different from RES & TAA group to TAA group. But, MDA, GST, SOD were not show significant difference between RES & TAA group and TAA group. Moreover, in the treatment effect test, RES & TAA group and TAA group did not show any significant difference. This study showed that RES has some antioxydative and hepatoprotective effects, but at the concentration used in this study, RES did not show significant effect.
Especially in the treatment effect test, it is difficult to identify the antioxydative effect of RES. So, to verify the antioxydative effect of RES, further studies are required. And based on this study, additional study is needed to determine the most effective concentration of RES.
(3) Enhance liver function and antioxidant effects of the dandelion extract and resveratrol feed additive supplementation in lactating dairy cows
Two dairy farms trials were conducted to test dandelion extract and resveratrol supplement as a additive to lactating dairy diet. Trial 1 was conducted with 2 dairy farms (no of lactating cows are 41 and 43, respectively), 100g dandelion extract additive feed (/day/head) was supplemented for 3 month. Trial 2 was conducted with 2 dairy farms (no of lactating cows are 37 and 30, respectively), 50g resveratrol additive feed (/head/day) was supplemented for 3 month.
Mean serum AST and GGT level were significantly(p< 0.01) decreased after fed diets supplemented with dandaelion exatract (trial 1; AST 105.4±23.0 U/L vs 85.5±14.4 U/L, GGT 36.7±9.4 U/L vs 32.1±7.7 U/L) and resveratrol (trial 2; AST 93.2±27.7 U/L vs 84.1±19.1 U/L, GGT 36.8±12.0 U/L vs 31.7±11.4 U/L). Accordingly, those additive feeds were effective in preventing the decrease in liver function cause by several reason, such as oxidative stress etc, has been known.
Among the metabolic profile blood parameters, In trial 1, Total cholesterol (p< 0.01) and NEFA (p< 0.001) level were significantly increased in after fed diets supplemented with dandelion extract compared before supplementation. Albumin, Total protein and BUN levels were significantly decreased in after supplementation. Cows fed resveratrol diert was significantly decreased in serum NEFA(p< 0.001) and was significantly increased in Albumin, Total protein and BUN(p< 0.05) than before fed resveratrol additive feed in trial 2.
The concentration of MDA and GSH, and the activities of SOD and CAT were no significant differences between before and after fed the dandelion exact supplement.
However, The concentration of MDA was decreased, and GSH concentration, CAT and SOD activities were tended to increase after fed the dandelion extract supplement. Such a result was the same that when resveratrol feed additive was fed to the lactating cows.
In conclusion, feeding dadalieon extract and resveratrol supplement in lactating cows showed enhance liver function and antioxidant activity. In addition, lactating cows fed those supplements to improve the metabolism of nutrients were compared before fed.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 15
• 목차 ... 23
• 제 1 장 서 론 ... 24
• 제 1 절 연구개발의 필요성 ... 24
• 제 2 절 연구개발의 최종 목표 ... 26
• 제 2 장 국내외 기술개발 현황 ... 27
• 제 1 절 국내 기술 개발 현황 ... 27
• 제 2 절 국외 기술 개발 현황 ... 28
• 제 3 장 연구개발수행 내용 및 결과 ... 30
• 제1절 젖소발굽관리를 통한 생산성 향상 연구 ... 30
• 1. 서 론 ... 30
• 2. 연구개발 수행내용 및 결과 ... 31
• 제2절 소 락토페린 (bovine lactoferrin; bLF) 단백질 발현 사료용 형질전환 미세조류 개발 및 항균.항바이러스 활성 규명 ... 43
• 1. 재조합 락토페린 클로닝 및 transient expression ... 43
• 2. 형질전환된 미세조류에서 재조합 락토페린 발현 ... 57
• 3. 재조합 락토페린 안정성 테스트 및 현장 적용 테스트 ... 76
• 제3절 젖소 이등유 발생저감을 위한 대사산물탐색 및 활용기술개발 ... 84
• 1. 젖소 이등유 발생실태 조사 및 이등유 관련 대사산물 탐색 ... 84
• 2. 제형화 천연물질(민들레 및 레스베라트롤)의 안전성 검사 및 이등유 발생 예방효과 구명 ... 91
• 제 4 장 연구개발목표 달성도 및 대외기여도 ... 156
• 제1절 : 연구개발 목표 달성도 ... 156
• 제2절 : 정량적 성과 ... 157
• 제 5 장 연구개발결과의 활용계획 ... 160
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 161
• 제 7 장 기타 중요 변동사항 ... 163
• 제1절 연구과제(과제번호 PJ907096) 계획서 변경내역 ... 163
• 제 8 장 국가과학기술종합정보시스템에 등록한 연구장비 현황 ... 166
• 제 9 장 참고문헌 ... 167
• 끝페이지 ... 172