초록

○ 연구결과
돼지 위축성 비염예방을 위하여 점막면역유도를 위하여 점막점착성 키토산 나노입자를 만들어 여기에 BBD를 담지시켜 in vitro에서의 방출조건을 확립하였고 in vitro의 면역세포에서의 면역능력을 물론 in vivo의 실험동물인 쥐나 목적동물인 돼지에서의 면역능력이 대조군보다 월등하게 유도되는 것을 확인하였다.

Abstract ▼

Ⅲ. Results and Discussion
1. Confirmation of infectious disease of swine and preparation of antigen
1) Extraction of Bordetella bronchiseptica (BB) and Pasteurella multocida type D(PMTD)
From the extraction and confirmation of infectious disease of swine, dermonecrosis toxin (DNT) gene dete

Ⅲ. Results and Discussion
1. Confirmation of infectious disease of swine and preparation of antigen
1) Extraction of Bordetella bronchiseptica (BB) and Pasteurella multocida type D(PMTD)
From the extraction and confirmation of infectious disease of swine, dermonecrosis toxin (DNT) gene detected by PCR amplification showed that BB had 643bp and PMTD had 1080bp.
2) Confirmation of protein
From analysis by SDS-PAGE and Western blot, BB showed about 40KDa protein and PMTD showed about 140KDa one.
2. 1n vitro study of the immune stimulation activity of BBD-loaded chitosan micro spheres
1) Preparation of chitosan microspheres (CMs)
CMs were prepared based on the ionotropic gelation of chitosan dissolved in acetic acid with sodium tripolyphosphate (TPP).
2) Loading of BBD into CMs
BBD dispersed in PBS containing CMs were kept at 37℃ for 24h under shaking.
The loading content of BBD determined by BCA method was 65-75 wt-%.
3) Observation of CMs
The morphology of CMs by SEM was observed as spherical shapes although BBD-loaded CMs became bigger.
4) Measurement of particle sizes of CMs
Particle size distribution of CMs and BBD-loaded CMs measured by DLS was 1.94±0.72 and 4.39±0.68 ㎛, respectively.
5) Release of BBD from BBD-loaded CMs in vitro
More BBD was released with a decrease of molecular weight of chitosan and with an increase of medium pH.
6) Immune stimulating activity of RAW264.7 cells production of tumor necrosis factor-α (TNFα) and nitric oxide (NO) from RAW 264.7 cells was increased in a time-dependent manner similar to LPS as a control.
3. In vitro study of the immune stimulation activity of BBD-loaded pegylated chitosan microspheres
1) Pegylation of chitosan
Chitosan was reacted with polytethylene glycol) (PEG) using EDC/NHS as activating agents at 4℃ for 48h.
2) Preparation of pegylated micro spheres (PCMs)
PCMs were prepared based on the ionotropic gelation of pegylated chitosan dissolved in acetic acid with TPP.
3) Loading of BBD into PCMs
BBD dispersed in PBS containing PCMs were kept at 37℃ for 24h under shaking. The loading content of BBD determioed by BCA method was 65-75 wt-%.
4) Observation of PCMs
The morphology of PCMs by SEM was observed as spherical shapes although BBD-loaded PCMs became bigger.
5) Measurement of particle sizes of PCMs
Particle size distribution of PCMs and BBD-loaded PCMs measured by DLS was 2.71±O.75 and 5.47±2.35 ㎛, respectively.
6) Release of BBD from BBD-loaded PCMs in vitro
More BBD was released from PCMs than CMs due to hydrophilic property of PEG.
7) Immune stimulating activity of RAW264.7 cells production of tumor necrosis factor-α (TNFα) and nitric oxide (NO) from RAW 264.7 cells was increased in a time-dependent manner similar to LPS as a control.
4. In vitro study of the inunune stimulation activity of BBD-loaded chitosan microspheres prepared in the presence of F127 as a stabilizer
1) CMs were prepared in the presence of F127 as a stabilizer, based on the ionotropic gelation of chitosan dissolved in acetic acid with TPP.
2) Loading of BBD into CMs
BBD dispersed in PBS containing F127-CMs were kept at 37℃ for 24h under shaking. The loading content of BBD determined by BCA method was 65-75 wt-%.
3) Observation of F127-CMs
The morphology of F127-CMs by SEM was observed as spherical shapes although BBD-loaded F127-CMs became bigger.
4) Measurement of particle sizes of F127-CMs
Particle size distribution of F127-CMs and BBD-loaded F127-CMs measured by DLS was 3.42±O.62 and 5.21±1.04 ㎛, respectively.
5) Release of BBD from BBD-loaded F127-CMs in vitro
More BBD was released from F127-CMs than CMs owing to hydrophilic property of F127.
6) Immune stimulating activity of RAW264.7 cells production of tumor necrosis factor-α (TNFα) and nitric oxide (NO) from RAW 264.7 cells was increased in a time-dependent manner similar to LPS as a control.
5. In vivo study of the immune stimulation activity of BBD-loaded chitosan rnicrospheres
1) Immune stimulating activity of mouse associated to BBD-loaded CMs
IgA from the mouse associated to BBD-loaded CMs after nasal washing was increased with an increase of time and concentration whereas IgA and IgG were only detected in the mouse blood.
6. Comparison of inunune stimulating activity of mouse between CMs prepared in the presence of F127 and mannosylated CMs (MCMs)
I) Mannosylation of chiotsan
Chitosan was reacted with mannose having isothiocyanate.
2) Preparation of MCMs
MCMs prepared based on the ionotropic gelation of mannosylated chitosan dissolved in acetic acid with TPP.
3) Immune stimulating activity of mouse associated to BBD-loaded CMs prepared in the presence of F127 and BBD-loaded MCMs
The highest immune stimulating activity of mouse associated to BBD-Ioaded CMs prepared in the presence of F127 was obtained owing to synergistic effect of F127 to immune sitmulating activity.
7. In vivo study of the immune stimulation activity of swine associated with BBD-Ioaded chitosan microspheres
1) Immune stimulating activity of swine associated to BBD-Ioaded CMs
IgA from the swine associated to BBD-Ioaded eMs after nasal washing was increased after 10 dyas and especially 5 times after 40 days.

목차 Contents

• 제출문 ... 1
• 요약문 ... 2
• SUMMARY ... 7
• CONTENTS ... 11
• 목차 ... 13
• 제1장 연구개발과제의 개요 ... 15
• 1절 연구개발의 목적 ... 15
• 2절 연구개발의 필요성 ... 15
• 3절 연구개발 내용 및 범위 ... 16
• 제2장 국내외 기술개발 현황 ... 18
• 1절 국내 ... 18
• 2절 국외 ... 18
• 3절 앞으로의 전망 ... 18
• 4절 기술도입의 타당성 ... 19
• 제3장 연구개발수행 내용 및 결과 ... 21
• 1절 돼지 위축성비염 주요병원성인자 분석 및 항원 준비 ... 21
• 제2절 BBD가 담지된 키토산미립자에서의 in vitro에서의 면역활성연구 ... 32
• 제3절 BBD가 담지된 폐길화된키토산미립자에서의 in vitro에서의 면역활성연구 ... 43
• 제4절 안정화제로서 프르로닉 F 127를 사용하여 키토산미립자를 제조하고 이들의 미립자에 BBD를 담지한후 in vitro에서의 면역활성연구 ... 57
• 제5절 마우스에서 키토산 미립자에 담지시킨 Bordetella bronchiseptica DNT 의 면역반응 ... 67
• 제6절 F127 존재하 또는 만노스와 결합시킨 BBDNT/키토산미립자의 면역반응 비교 ... 80
• 제7절 돼지에서 키토산 미립자에 담지시킨 Bordetella bronchiseptica DNT의 면역반응 ... 92
• 제4장 목표달성도 및 관련분야에의 기여도 ... 112
• 제1절 제1세부과제 ... 112
• 제2절 제2세부과제 ... 113
• 제5장 연구개발결과의 활용계획 ... 114
• 제1절 연구종료 2년후 예상연구성과 ... 114
• 제2절 연구종료 2년후 예상연구성과 ... 114
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 115
• 제1절 키토산미립자의 점막점착성의 응용 ... 115
• 제2절 효율적인 항원전달에 관한 정보 ... 115
• 제3절 DNA 항원전달에 관한 정보 ... 115
• 제7장 참고문헌 ... 116
• 끝페이지 ... 124