초록 ▼

Ⅳ. 연구개발결과
PMWS 발병 방지를 위한 면역조절 기전 구명 및 이를 이용한 질병저항성 활용 기술 개발을 위해 PCV2 ORF와 상호작용하는 면역 숙주세포 내 표적 분자를 동정하고 하부신호를 탐색하여 발병메커니즘을 확인하였다. 또한, PCV2 ORF 발현에 의해 변화되는 숙주세포의 마이크로 RNA와 그 표적 유전자를 동정하여 발현조절에 따른 PCV2 감염저항성조절 기반기술을 개발하였다. PCV2 증식관련 측정형질을 발굴하여 PCV2증식억제관련 후보유전자를 선정하였고, 후보유전자 내 신규 염기다형성을 발굴하였다. 또한, 연관

Ⅳ. 연구개발결과
PMWS 발병 방지를 위한 면역조절 기전 구명 및 이를 이용한 질병저항성 활용 기술 개발을 위해 PCV2 ORF와 상호작용하는 면역 숙주세포 내 표적 분자를 동정하고 하부신호를 탐색하여 발병메커니즘을 확인하였다. 또한, PCV2 ORF 발현에 의해 변화되는 숙주세포의 마이크로 RNA와 그 표적 유전자를 동정하여 발현조절에 따른 PCV2 감염저항성조절 기반기술을 개발하였다. PCV2 증식관련 측정형질을 발굴하여 PCV2증식억제관련 후보유전자를 선정하였고, 후보유전자 내 신규 염기다형성을 발굴하였다. 또한, 연관성분석을 통해 PCV2증식수준 최적 유전자형을 선정하였고, 돼지 SNP chip 분석을 통해 PCV2증식관련 유용 SNP도 발굴하였다. 뿐만 아니라, 자돈 생존폐사와 관련된 SNP를 대량 발굴하여 200개의 기능성 후보유전자를 검색하고, 자돈소모성 질환과 관련된 원인유전자 및 기능을 규명하는데 정보를 제공하였다. 돼지 MHC(SLA) 타이핑을 실시하여 면역관련 유전자의 유전자형 분석을 실시하였다. 이에 따라 viral epitope 결합력 분석 시스템을 개발하여 질병저항성 품종개발에 필요한 SLA class I 및 II의 대립 유전자 데이터베이스를 확장하였다.

Abstract ▼

< Development of genetic markers related to the resistance to PMWS by dissecting the molecular mechanism of PCV2 and host cell interactions >
Porcinecircovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which leads to serious economic losses

< Development of genetic markers related to the resistance to PMWS by dissecting the molecular mechanism of PCV2 and host cell interactions >
Porcinecircovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which leads to serious economic losses in the pig industry worldwide. The mechanism of pathogenicity associated with PCV2 infection is still not fully understood. Nevertheless, the fact that large amounts of proinflammatory cytokines within lymphoid tissues are released during the early stage of PCV2 infection may induce chronic inflammatory responses followed by the destruction of lymphoid tissues. However, how PCV2 infection causes an excessive inflammatory response in the host immune system during the early stage of PCV2 infection is still not elucidated. In this study, we show that direct interaction between the PCV2 ORF3 and regulator of G protein signaling 16 (RGS16) within the cytoplasm of host cells leads to ubiquitin-mediated proteasomal degradation of RGS16. Facilitated degradation of the RGS16 by PCV2 ORF3 further enhances NFkB translocation into the nucleus through the ERK 1/2 signaling pathway and increased IL-6 and IL-8 mRNA transcripts. Consequently, more severe inflammatory responses and leukocyte infiltration occur around host cells. Also, we analyzed the expression of C1qBP that other target protein of PCV2 ORF. The results show that the expression of C1qBP is increased in PCV2 ORF2 overexpressed host cells and phagocytosis activity is also increased. And, we employed Solexa deep sequencing technology to determine which cellular miRNAs were differentially regulated after expression of each of three PCV2-encoded ORFs in host cells. We identified 51 ORF1-regulated miRNAs, 74 ORF2-regulated miRNAs, and 32 ORF3-regulated miRNAs that differed in abundance compared to the control. Among the potential target genes of ORF-regulated miRNAs, two genes encoding proteins that are known to interact with PCV2-encoded proteins, zinc finger protein 265 (ZNF265) and RGS16, were selected for further analysis. We provide evidence that ZNF265 and RGS16 are direct targets of miR-139-5p and let-7e, respectively, which are both down-regulated by ORF2.
< Identification of PCV2 proliferation resistance DNA marker in pig >
The goal of this project is identification of PCV2 proliferation resistance DNA marker in pig. Therefore, we decided a major trait of PCV2 proliferation measurement on a PCV2 genomic DNA quantity in porcine peripheral blood. PCV2 genomic DNA quantity (Viral load) in porcine peripheral blood from 200 pigs were quantified using qPCR. We decided two candidate genes, Porcine Regulator of G protein signaling 16 (RGS16) and MYC, which are interact with PCV2 through the information about PCV2 related genes. 22 single nucleotide polymorphisms (SNPs) were founded in RGS16 and 1 SNP and 1 InDel were founded in MYC gene using pooling DNA sequencing. However, the SNP and InDel in MYC were identified in Berkshire breed. So, we could not type the genotype of SNP in MYC because, we had Yorkshire breed samples. Genotypes of each SNPs from 142 Yorkshire pigs were typed using PCR-RFLP and direct sequencing methods. Three SNPs (SNP2, 8, 11) showed significant correlation between genotypes and PCV2 viral load (each P = 0.022, P = 0.033, P = 0.027). Additionally, we discovered another candidate gene, DNAJC6, using Genome wide association study (GWAS) and identified a new SNP and genotyped about 142 Yorkshire pigs using PCR-RFLP. The association study between genotypes in DNAJC6 SNP and PCV2 viral load was shown significant correlation (P < 0.040). For effectiveness verification of DNA markers, we quantified PCV2 viral load, meat quality traits, and lean meat production traits from 417 Yorkshire pigs in normal farms. Association study showed significant correlation between the genotype of SNP2 in RGS16 and lymphocyte count (P < 0.042). Also, the SNP2 in RGS16 showed significant correlation with Lightness (L*) (P < 0.013). The SNP11 in RGS16 significantly correlated with backfat thickness (P < 0.026), pH_45min (P < 0.031), and Lightness traits (L^*) (P < 0.002).
< Detection of SNPs related to blood immune components and survival/death of piglets >
The importance of the genes related to SLA, immune response and resistance to PMWS disease in pigs is increasing due to the big loss of swine production caused by infectuous disease. A set of 40 significant SNPs were detected by genome-wide association analysis using high throughput porcine SNP chips in an F2 cross population between Korean native pigs and Yorkshire. About 40 functional candidate genes were found which were located near the significant SNPs for the blood immune components. A set of 449 SNPs were detected for survival & death of piglets using the Illumina porcine 64K SNP chip using the samples of 72 live and dead piglets, and about 200 functional candidate genes were found near the significant SNPs. The SNPs would be provided as SNP resources to find causal genes for resistance to PMWS.
< Characterization and excavating of the immune response related genes for improving the tolerance on wasting disease in the piglet >
Unlike the long-term information has been accumulated human and mouse, pig Major histocompatibility complex (MHC) and the study of the association between disease-resistant case can not be made a lot of research because of the technical limitations debilitating disease and swine MHC allele-specific reactivity differences associated the need for research to identify significant. To this end, the case of swine SLA information stored on the allele is very necessary and it is possible to develop epitope prediction program for swine MHC and TCR a future based on it. Research on swine MHC (SLA) is a restriction fragment length polymorphism (RFLP) haplotype constructed by using the method compared with the HLA (. Chardon et al, 1985) class I and class II haplotype analysis for the occasion and these genes a recombinant haplotype for the analysis (Chardon et al., 1985; Lie et al., 1987; Singer et al., 1987; Sachs et al., 1988; Martens et al., 2003) are primarily been used came. DNA molecular marker-based selection through the pig genome using DNA markers by combining genetic techniques (MAS, marker assisted selection) in situations that are less precise molecular transition is known about the cause of the excessive growth inhibition and immune-deficient PCV2 far and to develop and provide a fundamental solution of PMWS. Piglets through an analysis using the half of the money intended for commercial analysis and the main body of the SLA genes immunomodulatory genes are expected to be the most important gene in relation to the development of useful DNA markers with wasting disease resistance, and resistance to common diseases associated with existing reported to the usefulness gene (FUT1, Mx1, Nramp1) and if there is a relationship between rates and livestock wasting disease requires a further study of the patent avoidance technologies. For the analysis of SLA genotype and haplotype, two groups of 300 early died and 300 normal piglets were typed using genomic DNA sequence based typing (GSBT; developed by our previous research). Based on the GSBT results, the haplotype of both groups were analyzed through the pedigree analysis. To analyze the genotype of non-SLA immunomodulatory genes, between early died group and normal group, piglets diarrhea E. coli associated resistance and the virus-related genes such as FUT1, TNFA, TNFB, Mx1, Nramp1, TLR, MUC4, SLC12A8, TFRC, MYD88, pBD, play an important role in the regulation of immune response and defense of foreign antigens. For the excavation of SNPs, SNPs was confirmed by DNA sequencing after amplification by PCR using gene specific primer sets. The minor allele frequencies of SNP alleles among the excavated using a PCR-RFLP, direct sequencing or Snapshot way to target the SNP should only be carried out at least 10% of SNP typing. Analysis of statistical significance for the SLA haplotype between early died and normal piglets performed using POPGENE 1.31 software for the observed and expected allele frequency measurement. Statistical analysis to estimate the SNP genotype and allele effects on piglet survival significance tests to be conducted by chi-square test (chi-square test). For multiple test correction, a Bonferroni correction applied. Newly excavated SLA alleles were added to the largest international SLA database. Developed database characterized on a specific pathogen pig MHC epitope (epitope) and related vaccines against swine MHC alleles with disease allele. SLA alleles can be used as a useful data for the development of information-based disease-resistant varieties.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 5
• 목차 ... 8
• 제 1 장 서 론 ... 9
• 제 1 절 연구개발대상 기술의 경제적․산업적 중요성 및 연구개발의 필요성 ... 9
• 제 2 장 국내외 기술개발 현황 ... 15
• 제 1 절 국내 연구 현황 ... 15
• 제 2 절 국외 연구 현황 ... 16
• 제 3 절 국내외 연구현황 비교 및 필요 연구 분야 ... 20
• 제 3 장 연구개발수행 내용 및 결과 ... 26
• <제1세부과제 : 이유후전신소모성증후군(PMWS) 발병 방지를 위한 면역 조절 기전 구명 및 이를 이용한 질병저항성 활용 기술 개발> ... 26
• <제2세부과제 : 돼지 유전체분석을 통한 PCV2증식억제관련 DNA마커 개발> ... 84
• <제1협동과제: 통계·분자유전학적 기법을 적용한 자돈소모성질환 및 면역형질관련 SNP 분석> ... 132
• <제2협동과제: 돼지자돈의 소모성질환 내성(tolerance) 향상을 위한 면역조절 유전자 발굴> ... 146
• 제 4 장 연구개발목표 달성도 및 대외기여도 ... 173
• 제 1 절 목표대비 달성도 ... 173
• 제 2 절 정량적 성과 ... 178
• 제 5 장 연구개발결과의 활용계획 ... 179
• <1세부과제 : 이유후전신소모성증후군(PMWS) 발병 방지를 위한 면역 조절 기전 구명 및 이를 이용한 질병저항성 활용 기술 개발> ... 179
• <제2세부과제 : 돼지 유전체분석을 통한 PCV2증식억제관련 DNA마커 개발> ... 179
• <1협동과제 : 통계·분자유전학적 기법을 적용한 자돈소모성질환 및 면역형질관련 SNP 분석> ... 180
• <2협동과제 : 돼지자돈의 소모성질환 내성(tolerance) 향상을 위한 면역조절 유전자 발굴> ... 181
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 182
• <1세부과제 : 이유후전신소모성증후군(PMWS) 발병 방지를 위한 면역 조절 기전 구명 및 이를 이용한 질병저항성 활용 기술 개발> ... 182
• <제2세부과제 : 돼지 유전체분석을 통한 PCV2증식억제관련 DNA마커 개발> ... 182
• 제 7 장 기타 중요 변동사항 ... 184
• 제 8 장 국가과학기술종합정보시스템에 등록한 연구장비 현황 ... 184
• 제 9 장 참고문헌 ... 185
• 【붙임】 연구성과 근거자료-총 연구기간 성과 ... 191
• 끝페이지 ... 206