보고서 정보
주관연구기관 |
충북대학교 Chungbuk National University |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2015-03 |
과제시작연도 |
2011 |
주관부처 |
농촌진흥청 Rural Development Administration(RDA) |
등록번호 |
TRKO201500010206 |
과제고유번호 |
1395021572 |
사업명 |
차세대바이오그린21 |
DB 구축일자 |
2015-07-11
|
DOI |
https://doi.org/10.23000/TRKO201500010206 |
초록
▼
Ⅳ. 연구개발결과
○ 돼지 게놈에서 항미생물 펩타이드 및 선천성 면역 바이오마커 유전자의 발굴, 발현 조절 및 분석 (PMAP-23, Defensin, LAMPs, Ubiquilin 등)
○ Short & Medium Chain Fatty Acids에 의한 면역바이오마커 유전자들의 발현 유도 구명
○ 아미노산 결핍반응에 의한 염증 질환 및 장관 병원균의 세포내 면역 반응분석 (한약재 상산에서 유래한 Halofuginone의 항염증 작용기전 구명)
○ 할로퓨지논(HF)에 의한 세포외 기질(ECM) 단백질 발현수준
Ⅳ. 연구개발결과
○ 돼지 게놈에서 항미생물 펩타이드 및 선천성 면역 바이오마커 유전자의 발굴, 발현 조절 및 분석 (PMAP-23, Defensin, LAMPs, Ubiquilin 등)
○ Short & Medium Chain Fatty Acids에 의한 면역바이오마커 유전자들의 발현 유도 구명
○ 아미노산 결핍반응에 의한 염증 질환 및 장관 병원균의 세포내 면역 반응분석 (한약재 상산에서 유래한 Halofuginone의 항염증 작용기전 구명)
○ 할로퓨지논(HF)에 의한 세포외 기질(ECM) 단백질 발현수준 조절 분석 (2014년 Cell 논문 발표)
⊙ TLR2 리간드에 의한 돼지 장상피세포의 타잇정션 단백질 발현 증가 및 디옥시니발레놀에 의한 타잇정션 단백질 감소 억제 (그림 1)
- LR2 리간드에 의한 타잇정션 단백질(CLDN3, occludin, ZO-1) 발현이 증가하였으며, 또한 디옥시니발레놀에 의한 타잇정션 단백질의 감소가 완화되었음을 확인(그림 1A) 하였으며, confocal 현미경으로도 같은 결과 확인 (그림 1B)
⊙ Bacillus subtilis 에 의한 타잇정션 단백질 발현 증가 (그림 2)
- 시제품 가능성을 알아보기 위해 Bacillus subtilis를 이용하여 타잇정션 단백질 변화 확인
- Bacillus subtilis 처리하면 LTA(Bacillus subtilis 유래) 와 비슷한 수준으로 타잇정션 단백질 발현 증가
- 디옥시니발레놀에 의한 타잇정션 단백질 발현 감소 효과 완화
○ 기능성 유산균 선발 및 유전체 분석을 통한 장관흡착 유전자군 발굴
- 선발된 유산균 중에서 Lactobacillus johnsonii PF01과 L. mucosae LM1 균주의 유전체를 확보하고 분석하였으며, 장관흡착과 관련된 다수의 유전자군을 발굴하였음
○ 유용미생물 도입에 의한 장관계질환 억제효과 검증
- 유전자 전달 host로 사용될 L. johnsonii C1-10 균주를 자돈에 급여한 후에 설사유발 살모넬라균에 대한 방어능 조사 및 장내균총을 비교 분석한 결과, 돼지분변에서 분리한 L. johnsonii C1-10 균주는 자돈에 있어 살모넬라균 감염에 방어효과가 있는 것으로 판단되었다.
⊙ 돼지의 면역기능 평가를 위한 혈액 중 면역세포의 표적 바이오마커활용으로 새로운 항생 제대체제의 개발 및 평가에 활용 가능
⊙ 돼지 예방백신 모델 확립을 통해 발굴된 바이오마커를 이용하여 질병 저항능력 향상 여부를 예측하여 종돈선발과 면역능력 향상을 위한 기술 개발에 기여
* 개발기술(적용기술): 표적 바이오마커를 이용하여 질병 저항능력 향상 여부를 예측, 종돈 선발, 면역능력 향상 기술에 적용
- 돼지의 비침습적 방법을 통한 질병저항성 바이오마커 평가 및 검증기법 확립
Abstract
▼
1세부:
The innate immune system in animal is the first line of defense that constitutes an important part of the general immune system comprising the cells and mechanisms that defend the host from infection by other organisms. The cells of the innate system recognize and respond to pathogens in a g
1세부:
The innate immune system in animal is the first line of defense that constitutes an important part of the general immune system comprising the cells and mechanisms that defend the host from infection by other organisms. The cells of the innate system recognize and respond to pathogens in a generic way, but, unlike the adaptive immune system, it does not provide long-lasting immunity to the animal. Innate immune systems provide immediate defense to prevent intestinal pathogens from triggering intestinal diseases including diarrhea. Although the genomes of major domestic animals including Pig and Cow have been elucidated, there remains a on-going effort for identifying genes of economical values. Despite its significance in the animal industry, there has been a lack of through investigation for the identification of biomarkers and their specific roles for the health of animal. In addition, recent evidences have demonstrated a critical interplay between nutrient sensing pathways and innate immunity to bacterial pathogens, viruses and parasites. Based on the significance of amino acid response (AAR) in the animal immunity, there has been a need to screen a therpautic tool to control the AAR and immunity.
Through this project, we attempted to identify candidate biomarkers for innate immunity and/or economically important genes and also to elucidate their action mechanisms to set the basis for the development of: (1) therapeutic tool against the pathogenes, (2) therapeutic tool to control the immune response of the animal.
Here, we report several biomarkers that are involved in the innate immunity against Salmonella. Moreover, we present experimental evidences that support the role of Halofuginon and its derivative in the regulation of AAR and inflammatory response. Using CRISPR/Cas9 and Zebrafish model animal, we established a basis to modify novel genes at the animal level for the identification of their functions. The results in this report will set the basis for identification of many economically important biomarkers for domestic animals potentially as diagnostic or selection marker for disease resistant animals.
1협동:
Intestinal epithelial cells play a major role in innate immune responses and protect host from external hazards including pathogenic microorganisms and toxins. Intestinal epithelium is the first line of defense against food and feed contaminants. Deoxynivalneol is a secondary toxic metabolite from fusarium mycotoxin that is harmful to humans and animals, and pigs being the most sensitive. Deoxynivalenol is known to disrupt intestinal structure and interrupt with the intestinal barrier function. Toll-like receptor (TLR) 2 in intestinal epithelial cells are selectively expressed on the basolateral membrane fraction in response to its ligands and plays an important role in protection by inducing inflammatory activity at the local area.
In the present study, we investigated the effect of TLR2 ligands on the expression of tight junction proteins, induction of barrier integrity and anti-apoptotic effect in IPEC-J2, swine intestinal epithelial cells treated with deoxynivalenol. Interestingly, pre-treatment with TLR2 ligands significantly suppressed the level of deoxynivalenol-mediated apoptosis in IPEC-J2 cells when compared to the cells without pre-treatment. IPEC-J2 cells treated with TLR2 ligands and Bacillus subtilis, contrary to the cells without treatment, showed the increased expression of tight junction proteins, claudin-3, zonula occluden-1 (ZO-1), and occludin and their integrity. The results using confluent IPEC-J2 cells have shown resistant effect of PCSK and LTA, Bacillus subtilis on the reduction of TEER induced by deoxynivalenol suggesting that TLR2 ligands induced increase of tight junction proteins in intestinal epithelial cells, which enhanced the paracellular resistance. It was noting that the blockade of TLR2 signaling leads to failure of PCSK- and LTA-mediated barrier protection against the damage induced by deoxynivalenol.
We examined the importance of PI3K-Akt signaling in IPEC-J2 cells treated with TLR2 ligand. The results indicated that PSCK and LTA induced the expression of tight junction proteins in IPEC-J2 cells, which was dependent on the activation of phosphatidylinositil-3-kinase/Akt (PI3K-Akt) and TLR2 signaling pathway. This was further validated that IPEC-J2 cells pre-treated with PCSK or LTA induced barrier protection was significantly suppressed by PI3K inhibitor and anti-TLR2 antibody.
In conclusion, the present study showed that pre-treatment of TLR2 ligands to swine intestinal epithelial barrier could have protective role against the damage induced by deoxynivalenol through PI3K-Akt pathway. It is likely that probiotics which express TLR2 ligands could cause a health gut in pigs.
2협동:
Probiotics, including lactic acid bacteria, play an important role in the regulation of gene expression of pigs, which affects the health and growth performance of pigs.
Initially, we isolated putative probiotics from the feces of pigs based on their abilities to withstand bile stress and to adhere to mucin well, which are important characteristics for gut colonization and subsequently provide their probiotic benefits.
Among them, Lactobacillus johnsonii PF01 and L. mucosae LM1 were selected and their genome was sequenced for further studies. Both LM1 and PF01 showed both high adherence into mucin and inhibition of the colonization of pathogens such as E. coli K88 and Salmonella Typhimurium KCCM 40253.
Genomic analysis showed that PF01 strain had three different types of bile salt enzymes and two types of bile transporter genes that could explain why this strain has excellent bile resistance. Proteomic analysis of PF01 under bile stress was done using the iTRAQ method, showing that PF01 may respond to the presence of these bile acids in a protective manner, choosing several metabolic pathways that will conserve energy and protect itself from bile toxicity in intestine.
Likewise, the genomic analysis of LM1 strain was performed to understand how this strain shows excellent adherence to pig mucin. Several genes which are involved in the adherence into mucin were found in the genome of LM1 strain. Adhesion ability of LM1 into mucin was characterized for its ability for non-specific and specific adhesion manner both. In addition, quantitative proteomic analysis of LM1 strain co-cultured with intestinal pig cells (IPEC-J2 cells) was performed using Nano-HPLC and LC-MS/MS. It showed the up-regulation of several metabolic pathways which help cell survival and proliferation.
To understand the relationship between intestinal microbiota and pig health, pig's fecal DNAs were obtained and pyrosequencing using the Roche 454 GS FLX Titanium platform was done. First, the fecal bacterial diversity of 15-week-old pigs from three purebred lines (Duroc, Landrace, and Yorkshire) was compared. The microbiomes of Yorkshire and Landrace were more closely related to each other than to that of Duroc pigs, which may be due to gene pool similarities between the two breeds. Among the core microbiome of Duroc, Prevotella genus was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. it will be of great importance in the future to assess the correlation of O. valericigenes in the gut of Duroc pigs. In addition, analysis of fecal microbial shifts of healthy piglets during the weaning transition was performed. Firmicutes (54.0%) and Bacteroidetes (59.6%) were the most dominant phyla during pre-weaning and post-weaning, respectively. During the weaning transition a distinct shift from Bacteroides to Prevotella as the most abundant genus was observed. On the other hand, Salmonella challenge experiment in pigs showed that probiotic Lactobacillus spp. changed the intestinal microbiota of pigs and inhibited the growth of Salmonella in vivo. Finally, a porcine-targeted gene delivery system using L. johnsonii C1-10 and pBCEG29 plasmid was constructed. L. johnsonii C1-10 transformed with pBCEG29 vector showed successful expression of a foreign gene under bile stress.
3협동: There are not many reliable immunologic biomarkers to assess the domestic animal's disease resistance. Through this study, we attempted to identify biomarkers from swine blood by non-invasive manner to accurately predict immune reaction. We report here that several biomarkers including MYB, RAG-1, NOR-1, HBEGF, LYZ are differentially regulated at the transcription level in PED vaccinated pigs compared to those of controls.
This information is expected to be valuable in the prediction of disease resistance of pigs and also in the screening of antibiotic candidate substances.
목차 Contents
- 표지 ... 1
- 제출문 ... 2
- 요 약 문 ... 3
- SUMMARY ... 10
- 목차 ... 13
- 제 1 장 서 론 ... 14
- 1세부 ... 14
- 1협동 ... 16
- 2협동 ... 17
- 3협동 ... 18
- 제 2 장 국내외 기술개발 현황 ... 20
- 1세부 ... 20
- 1협동 ... 21
- 2협동 ... 23
- 3협동 ... 24
- 제 3 장 연구개발수행 내용 및 결과 ... 27
- 1세부 ... 27
- 1협동 ... 46
- 2협동 ... 64
- 3협동 ... 103
- 제 4 장 연구개발목표 달성도 및 대외기여도 ... 131
- 1절. 세부과제별 정성적 연구목표대비 달성도 ... 131
- 2절. 정량적 목표대비 달성도 ... 135
- 제 5 장 연구개발결과의 활용계획 ... 13
- 1세부 ... 136
- 1협동 ... 137
- 2협동 ... 137
- 3협동 ... 137
- 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 138
- 1세부 ... 138
- 1협동 ... 138
- 2협동 ... 138
- 3협동 ... 138
- 제 7 장 기타 중요 변동사항 ... 139
- 1세부 ... 139
- 1협동 ... 139
- 2협동 ... 139
- 3협동 ... 139
- 제 8 장 국가과학기술종합정보시스템에 등록한 연구장비현황 ... 140
- 1세부 ... 140
- 1협동 ... 140
- 2협동 ... 140
- 3협동 ... 140
- 제 9 장 참고문헌 ... 141
- 끝페이지 ... 148
※ AI-Helper는 부적절한 답변을 할 수 있습니다.