초록 ▼

○ 연구결과
ND, ILT 및 IB에 대한 in ovo vaccination용 항원과 DNA 백신 개발에 대한 연구를 수행하여 시험 제조된 약독 및 불활화 백신 과 DNA 백신의 부화계란에 대한 병원성과 안전성을 규명하였다. 또한 시험 제조된 in ovo 백신 접종에 따른 닭에 대한 면역원성과 강독바이러스 공격에 대한 방어효과를 시험하였다. 일련의 시험을 통하여 약독화된 NDV, ILTV 및 IBV의 in ovo 백신용 항원 개발 기초 정보를 확립하였고, DNA 백신의 분자생물학적 특성과 부화계란에 대한 높은 안전성과 해당 바

○ 연구결과
ND, ILT 및 IB에 대한 in ovo vaccination용 항원과 DNA 백신 개발에 대한 연구를 수행하여 시험 제조된 약독 및 불활화 백신 과 DNA 백신의 부화계란에 대한 병원성과 안전성을 규명하였다. 또한 시험 제조된 in ovo 백신 접종에 따른 닭에 대한 면역원성과 강독바이러스 공격에 대한 방어효과를 시험하였다. 일련의 시험을 통하여 약독화된 NDV, ILTV 및 IBV의 in ovo 백신용 항원 개발 기초 정보를 확립하였고, DNA 백신의 분자생물학적 특성과 부화계란에 대한 높은 안전성과 해당 바이러스 대한 면역원성 및 방어효과의 유도 가능성을 확인할 수 있었다.

Abstract ▼

II. The objectives and necessity of study
Vaccination in poultry industries is a laborious - and high cost job, therefore, we will establish antigen production technologies for in ovo vaccination, which has been known to be simple and cost-saving method, to enhance productivity and international

II. The objectives and necessity of study
Vaccination in poultry industries is a laborious - and high cost job, therefore, we will establish antigen production technologies for in ovo vaccination, which has been known to be simple and cost-saving method, to enhance productivity and international competitiveness by saving labor and time for vaccination.
In this study, we will conduct experiments to produce antigens, which can confer higher safety and effective protectivity when it is applied into egg embryos, by serial passage of antigen on tissue culture or egg embryo, modifying replication conditions, specific antibody treatment, and by chemical treatment to reduce or remove virulence of antigens. The vaccine antigens include Newcastle disease(ND) virus, Infectious laryngotracheitis (ILT) virus and Infectious broncheitis(IB) virus those have been big economic losses in poultry industries.
We will also conduct study on production of recombinant DNA vaccine on DNV, ILTV, and IB.
III. Contents and field of study
The research project consists of 3 subjects including " Study on the development of ND virus antigen for inovo vaccination", " Study on the development of ILT virus antigen for inovo vaccination", and " Study on the development of IB virus antigen for inovo vaccination", The detail research contents in each research subject are production of inactivated antigens, safety and efficacies test on inactivated antigen, vaccine virus, and attenuated field virus isolates in egg embryos and chicken. The immunogenecity studies using different inoculation methods were also conducted in egg embryos. Characterization of antibody inducing genes of NDV, ILTV and IB virus, production of DNA vaccines and test of their characteristics, safety, immunogenecity and efficacy were also carried out.
IV. Results and suggestion for application
The summary of this study is as follows;
- Study on the development of ND virus antigen for in ovo vaccination ; A live attenuated NDV CBP-1 strain was produced by 250 times serial passage or cold adaption in egg embryos. The immunogenecity of the NDV B1 and CBP-1 strain inactivated by formalin or EMS treatment were determined by the inovo vaccination into egg embryos. B1 strain, CBP-1 strain treated with polyvalent antisera, monoclonal antibody and IgY showed high safety and high titers in HI test and ELISA. Sequence of F gene, HN gene of NDV CBP-1 and its amino acid sequence was determined and were compared with B1 strain, Lasota strain, Texas G.B. strain, Beaudette C stran, and Ulster strain. CBP-1 strain share an homology more than 89% in nucleotide sequence and 91.3% in amino acid sequence with reference strains. The recombinant plasmid DNAs pSLIA-F and pSLIA-HN were constructed using F and HN genes obtained from CPB-1 strain. Expression of F and HN protein in COS-7 cell transfected with pSLIA-F or pSLIA-HN was confirmed by FA test, SDS-PAGE and Western blot.
Antigenicity of pSLIA-F and pSLIA-HN in mouse was confirmed by ELISA.
Hatchebility and safety of DNA vaccines pSLIA-tsF, pSLIA-tsHN, pSLIA-F+HN, pSLIA, and attenuated NDV CBP-1(250th) reacted with IgY were up to 80 to 100%, when they were inoculated into egg embryo. High booster effects by adminstration of B1 oral vaccine were demonstrated in groups inoculated with the DNA vaccine and attenuated NDV CBP-1(250th) reacted with IgY. When the challenge injection with wild type CBP-1 virus was made into chicken administered with B1 oral vaccine , immunigenicity and protectivity were significantly high.
- Study on the development of ILT virus antigen for in ovo vaccination ; The field isolate ILTV-N91B01 was adapted well to LMH cells and was passaged serially up to 181 passages. The LD50 value of the ILT virus against 18 days old egg embryos was 1x10^-2.3/0.1ml, indicating a big loss of virulence. Treatment of ILT virus with EMS, polyclonal antibody, or monoclonal antibody resulted in loss of virulence, but antigenicity of ILT virus antigen was low. Hatchability, survivability for 10day after hatching of egg embryos inoculated with attenuated ILT virus(1x10^1.25TCID50/0.1ml) were similar to those of egg embryos injected with PBS. Serum neutralization antibody titers at 4-.5-. 6-. and 7 weeks post-inoculation were 1:2∼1:64, 1:2 ∼1:64,1:2∼1:32, and 1:2∼1:32 , respectively.
Immune response in chickens less than 2 weeks of age do not respond as well to vaccination as do older birds. It has been known that humoral immune responses to ILTV are not the primary mechanism for protection and a poor correlation generally has been found between serum antibody titer and immune status of flocks. The protection mechanism rely on a local cell mediated immune response, therefore, relative lower immune response may not be problems in protection of birds from ILT infection. Recombinant plasmid DNA vaccine pCI-neo-gB was constructed by inserting gB gene, which was amplified by PCR technique, into pC-neo-vector DNA. Safety of recombinant plasmid DNA vaccine pCI-neo-gB against 18 days old egg embryos was hight. No perceivable SN antibody titer was demonstrated, but 50% plaque reduction effects was found in sera collected from chickens inoculated with recombinant plasmid DNA vaccine pCI-neo-gB at 18 days old SPF egg embryos.
- Study on the development of IB virus antigen for inovo vaccination : IBV-V and IBV-KM91 strain is highly pathogenic and cause abnormal egg production, dwarfing or curling when they were inoculated in egg embryos.
They were subjected 10 to 20 egg embryos passage. Virulence was reduced but not lost completely. IBV inactivated with β- propiolactone or polyclonal antibody or monoclonal antibody showed high safety and significant immune response in egg embryos, The S1 gene of IBV-KM91p6 strain was cloned into pCRII vector and the neucleotide sequence was analyzed. The S1 gene was expressed in Sf 9 cell using Baculovirus transfer vector and the expression S1 gene protein was confirmed. The recombinant protein of rAc-KMp6S1 showed lower protectivity than that of inactivated KM91p6 virus antigen, but it induced protective immune response against high virulent IBV when it applied repeatedly. We constructed recombinat plasmid DNA pCI-KMp6S1 and pTriEx-KMp6S1 and examined the expression of their S1 gene protein in euaaryotic cells. The pCI-KMp6S1and pTriEx-KMp6S1 had more than 70% hatchablity on 18 days old egg embryos, The booster effects of DNA vaccine was significantly high in chicken flocks inoculated with IB vaccine.
The results obtained in this study may be useful data in developing antigens for inovo vaccination which is an emerging new technology. Especially, the results obtained from the in ovo vaccination experiments using virus-antibody complex antigen and recombinant DNA vaccine will be used in production of antigens and DNA vaccine for in ovo vaccination to ND, ILT and IB.
This study is only first step in developing antigen for in ovo vaccination, therefore, it must be continued and be commercialized through a cooperative study with industry.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 7
• CONTENTS ... 11
• 목 차 ... 13
• 제 1 장 연구개발과제의 개요 ... 15
• 제 2 장 국내외 기술개발 현황 ... 16
• 제 3 장 연구개발수행 내용 및 결과 ... 17
• 제 1 절 닭 뉴켓슬병에 대한 in ovo vaccination용 항원 개발에 관한 연구 ... 17
• 1-1. 재료 및 방법 ... 17
• 1-2. 결과 ... 23
• 1-3. 결론 ... 56
• 제 2 절 전염성후두기관지염에 대한 in ovo vaccination용 항원 개발에 관한 연구 ... 58
• 2-1. 재료 및 방법 ... 58
• 2-2. 결과 ... 68
• 2-3. 결론 ... 84
• 제 3 절 전염성기관지염에 대한 in ovo vaccination용 항원 개발에 관한 연구 ... 86
• 3-1. 재료 및 방법 ... 86
• 3-2. 결과 ... 96
• 3-3. 결론 ... 113
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 115
• 제 5 장 연구개발결과의 활용계획 ... 115
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 116
• 제 7 장 참고문헌 ... 117
• 끝페이지 ... 121