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최 종 연구보고서 닭 전염성 낭병(Infectious bursal disease)의 진단법 및 백신개발 Development of Diagnostic Methods and Vaccine for Avian Infectious Bursal Disease 연 구 기 관 서 울 대 학 교 농 림 부

제 출 문 농림부 장관 귀하 본 보고서를 닭 전염성 낭병(Infectious bursal disease)의 진단법 및 백신개발 (제1 세부과제 제2세부과제 급성 전염성 낭 병의 사망 기전 규명과 대증 치료법 개발 협동과제 전염성 낭병의 유전자 재조합 백신 개발 )의 최종보고서로 제출합니다. 2004년 8 월 일 주관연구기관명: 총괄연구책임자: 제1세부연구책임자: 제2세부연구책임자: 협동연구기관명: 협동연구책임자: 서울대학교 김 선 중 김 선 중 류 판 동 강원대학교 한 태 욱

SUMMARY (영문요약문)

CONTENTS (영 문 목 차) Chapter I. Outline of Research Project ------------------------------- 21 Chapter II. Present Situation of Research --------------------------- 26 Chapter III. Materials and Methods --------------------------------- 29 1. The 1st Subtitle -------------------------------------------- 29 2. The 2nd Subtitle --------------------------------------------- 40 3. The Co-operative Project --------------------------------- 44 Chapter IV. Results ---------------------------------------------- 53 1. The 1st Subtitle -------------------------------------------- 53 2. The 2nd Subtitle -------------------------------------------- 71 3. The Co-operative Project --------------------------------- 90 Chapter V. Plan of Practical Use ------------------------------------111 Chapter VI. Data Collection during Project --------------------------- 112 Chapter VII. References--------------------------------------------- 123

목 차 제 1 장 연구개발과제의 개요 -------------------------------------- 21 제 2 장 국내외 기술개발 현황 ------------------------------------- 26 제 3 장 연구개발수행방법 ------------------------------------- 29 1. 제 1 세부과제 ----------------------------------------------- 29 2. 제 2 세부과제 ----------------------------------------------- 40 3. 협동과제 ----------------------------------------------- 44 제 4 장 연구수행내용 및 결과 ------------------------------------- 53 1. 제 1 세부과제 ----------------------------------------------- 53 2. 제 2 세부과제 ----------------------------------------------- 71 3. 협동과제 ----------------------------------------------- 90 제 5 장 연구개발결과의 활용계획 ------------------------------------111 제 6 장 연구개발과정에서 수집한 해외과학기술정보 --------------------- 112 제 7 장 참 고 문 헌 --------------------------------------------- 123

* Locations are based on the sequence of IBDV STC strain (Kibenge 등, 1990)

1.

Table 3-1. Korean isolates and vaccine strains used in this study Strain Virulence Description and origin SH92 VV Korean isolate (국립수의과학검역원) 1030 VV Korean isolate (서울대학교 수의과 대학) Blen A Merial Select Inc. USA Bur706 A Merial, France Bursine plus I plus Fort Dodge Animal Health, USA Bursine 2 I Fort Dodge Animal Health, USA Cevac I plus Sanofi VV, very virulent; A, attenuated; I, intermediate

Table 3-2. Oligonucleotide sequences used in RT-PCR Primer Oligonucleotide sequences Description IBDVP2-F1 IBDVP2-F2 IBDVP2-R1 IBDVP2-R2 IBDSegA-R 1 IBDSegA-R 2 5'-AGCTGGTACCATGACAAACCTG-3' 5'-ATATGGTACCAAACGATCGCAGCGATGACAAA C-3' 5'-GACTGAATTCTCAGCTTTTCCTGA-3' 5'-GACTGAATTCTCATGCTCTTGCTTTTCCTGAC G-3' 5'-TAAGCGGCCGCTTTGGGATGTTGT-3' 5'-ATTAGCGGCCGCCAATTTGGGATGTTGTAAGG -3' VP2와 sega의 증폭을 위한 forwarding sequence (KpnI site) VP2와 sega의 증폭을 위한 forwarding sequence (KpnI site) VP2의 증폭을 위한 reverse sequence (EcoRI site) VP2의 증폭을 위한 reverse sequence (EcoRI site) SegA의 증폭을 위한 reverse sequence (NotI site) SegA의 증폭을 위한 reverse sequence (NotI site) 나. Reverse transcriptase-polymerase chain reaction(rt- PCR) 및 PCR 생성물 확인

Amp R Clone the VP2 & sega into a baculovirus transfer vector Cotranfect the linearized baculovirus DNA and the recombinant vector in to Sf21 cells pbacpak8 baculovirus polyhedrin fusion transfer vector VP2 gene & sega gene Linearized AcMNPV Baculovirus DNA Homologous recombinant Recombinant Vector Budding recombinant virus Amplify recombinant virus by infecting Sf21 cells to obtain high titer viral stock high titer viral stock Infect Sf21 cells to produce recombinant protein Budding recombinant virus Fig. 3-1. Flow-diagram of generation of recombinant baculovirus containing VP2 & sega gene.

M 1 2 3 4 5 6 7 8 9 10 11 500 bp 491 bp

분리주 91108 9414 9423 9593 9596 9750 9818 9858 9912 9914 유래 L B B B BB B B L B B 치사율 (%) 71.4 0.0 13.3 80.0 58.8 88.7 27.0 93.3 53.3 78.6 분리주 9919 9927 9946 9960 9977 9993 99103 99107 99122 1030 유래 B B B B B B BB B B B 치사율 (%) 60.0 80.0 66.7 85.7 53.3 80.0 20.0 80.0 60.7 52.9

Incubation Bursal cell viability at PMA concentrations (μg/ml) (days) 0 0.1 0.25 0.5 1.0 2.5 5.0 10.0 1 2 2 2 3 4 4 4 3 2 0 0 0 1 3 3 3 2 3 0 0 0 0 3 3 3 1 4 0 0 0 0 2 3 3 0 5 0 0 0 0 1 2 2 0 IBDV isolates and infectivity titer (TCID 50 /25 μl) SNU 91108 SNU 9414 SNU 9423 SNU 9596 SNU 9993 SNU 1030 3.6 2.7 2.8 3.5 4.3 3.6

Virus neutralizing antibody titer (log 2 ) of monoclonal antibody 1) virus 33/25 23/10 33/29 35/11 23/14 32/12 24/11 23/11 23/12 24/17 35/24 24/19 35/21 SNU 91108 122) 12 12 11 11 12 12 8 11 12 12 12 12 SNU 9423 SNU 9415 8 12 12 9 12 10 12 8 12 12 11 12 10 10 12 12 9 12 12 12 9 12 12 12 12 12 Monoclonal antibody and VN titers (log 2 ) Virus 33/29 24/19 35/24 23/12 SNU9423 6 12 8 12 Lukert 2 6 3 6 SAL 4 9 5 9 D78 6 10 8 11 S706 9 12 9 12

. Antigenic relatedness among IBDV local isolates and vaccine viruses Virus Lukert SAL 10 * 7 Lukert (100) ** (71) 5 SAL 9 (100) SNU91108 3 1 SNU 91108 7 (9) 5 (12) 7 (100) SNU9325 4 1 8 Antiserum SNU 9325 9 (18) 8 (25) 8 (141) 8 (100) SNU9414 5 2 11 9 SNU 9414 4 (12) 4 (25) 4 (283) 6 (200) 5 (100) SNU9423 4 1 7 9 6 SNU 9423 9 (9) 8 (12) 10 (100) 10 (141) 10 (141) 10 (100) SNU9415 6 2 NT NT 6 10 * VN (virus neutralization) titer (log 2) ** R value(%) NT: not tested SNU 9415 9 (18) 9 (25) NT SNU 9428 7 8 10 NT 11 11 (200) 9 (70) 10 (100) 11

(Nucleotide sequence comparison) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 1 96.9 95.0 95.0 97.8 94.2 94.7 93.6 92.8 92.7 90.3 95.0 95.8 92.2 92.2 91.4 93.1 92.8 92.8 91.9 97.5 93.1 97.5 93.1 92.2 93.6 1 2 96.7 94.7 94.4 96.9 94.7 96.1 94.4 93.9 94.0 89.7 94.7 95.6 93.3 93.6 92.5 94.2 93.9 93.9 93.3 96.7 94.2 96.7 94.2 93.3 94.2 2 3 94.2 94.2 98.3 95.3 93.1 94.2 94.2 91.1 90.9 88.3 98.6 99.2 90.3 90.6 90.0 91.4 91.1 91.1 90.0 95.3 91.4 95.0 91.4 90.3 92.2 3 4 92.5 92.5 95.8 95.3 92.8 94.2 94.2 91.1 90.9 88.3 98.6 99.2 90.6 90.6 90.3 91.4 91.1 91.1 90.3 95.3 91.4 95.0 91.4 90.6 92.5 4 5 97.5 97.5 95.0 93.3 94.7 95.3 93.9 92.8 92.7 90.8 95.3 96.1 92.2 92.2 91.4 93.1 928 92.8 91.9 99.2 93.1 99.7 93.1 92.2 94.2 5 6 90.8 92.5 91.7 90.8 91.7 93.6 92.2 90.6 90.3 88.9 93.1 93.9 90.3 89.7 90.0 90.8 90.6 90.6 91.1 94.4 90.8 94.4 90.8 90.3 99.2 6 7 91.7 93.3 90.8 90.0 92.5 89.2 95.6 91.9 91.8 91.1 94.2 95.0 91.4 91.4 90.6 92.2 91.9 91.9 91.4 95.0 92.2 95.0 92.2 91.4 94.2 7 8 90.8 92.5 93.3 92.5 91.7 91.7 95.0 90.6 90.9 88.9 94.4 95.0 90.3 90.3 89.4 90.8 90.8 90.3 90.3 93.6 90.8 93.6 90.8 90.3 92.5 8 9 93.3 96.7 90.8 89.2 95.8 90.8 90.8 90.0 99.7 86.7 90.8 91.9 98.9 98.3 98.1 99.7 99.4 98.9 98.3 93.1 99.7 92.5 99.7 98.9 90.0 9 10 93.3 96.7 90.8 89.2 95.8 90.8 90.8 90.0 100 86.1 90.6 91.8 99.4 98.8 98.5 100 100 99.1 98.8 93.1 100 92.4 100 99.4 89.7 10 11 89.2 89.2 88.3 86.7 90.0 84.2 88.3 86.7 88.3 88.3 88.6 89.4 86.9 86.4 86.4 86.9 86.7 86.7 86.7 90.3 86.9 90.6 86.9 86.9 88.3 11 12 94.2 94.2 96.7 95.8 95.0 90.8 90.8 92.5 90.8 90.8 88.3 99.4 90.3 90.3 90.0 91.1 90.8 91.1 90.3 95.0 91.1 95.0 91.1 90.3 92.2 12 13 95.0 95.0 98.3 97.5 95.8 92.5 90.7 94.2 91.7 91.7 89.2 98.3 91.4 91.4 91.1 92.2 91.9 91.9 91.1 96.1 92.2 95.8 92.2 91.4 93.1 13 14 93.3 96.7 90.8 89.2 95.8 90.8 90.8 90.0 100 100 88.3 90.8 91.7 98.3 99.2 99.2 99.2 98.3 99.2 92.5 99.2 91.9 99.2 100 89.7 14 15 93.3 96.7 90.8 89.2 95.8 90.8 90.8 90.0 100 100 88.3 90.8 91.7 100 97.5 98.6 98.6 97.8 97.8 98.5 98.6 91.9 98.6 98.3 89.2 15 16 93.3 96.7 90.8 89.2 95.8 90.8 90.8 90.0 100 100 88.3 90.8 91.7 100 100 98.3 98.3 97.5 98.3 91.7 98.3 91.1 98.3 99.2 89.4 16 17 93.3 96.7 90.8 89.2 95.8 90.8 90.8 90.0 100 100 88.3 90.8 91.7 100 100 100 99.7 99.2 98.6 93.3 100 92.8 100 99.2 90.3 17 18 93.3 96.7 90.8 89.2 95.8 90.8 90.8 90.0 100 100 88.3 90.8 91.7 100 100 100 100 98.9 98.6 93.1 99.7 92.5 99.7 99.2 90.0 18 19 92.5 95.8 90.0 88.3 95.0 90.0 90.0 89.2 99.2 99.2 87.5 90.0 90.7 99.2 99.2 99.2 99.2 99.2 98.3 92.8 99.2 92.5 99.2 98.3 90.0 19 20 93.3 96.7 90.8 89.2 95.8 90.8 90.8 90.0 100 100 88.3 90.8 91.7 100 100 100 100 100 99.2 91.9 98.6 91.7 98.6 99.2 90.6 20 21 97.5 97.5 95.0 93.3 100 91.7 92.5 91.7 95.8 95.8 90.0 95.0 95.8 95.8 95.8 95.8 95.8 95.8 95.0 95.8 93.3 98.9 93.3 92.5 93.9 21 22 93.3 96.7 90.8 89.2 95.8 90.8 90.8 90.0 100 100 88.3 90.8 91.7 100 100 100 100 100 99.2 100 95.8 92.8 100 99.2 90.3 22 23 96.7 96.7 94.2 93.3 99.2 90.8 91.7 90.8 95.0 95.0 89.2 94.2 95.0 95.0 95.0 95.0 95.0 95.0 94.2 95.0 99.2 95.0 92.8 91.9 93.9 23 24 93.3 96.7 90.8 89.2 95.8 90.8 90.8 90.0 100 100 88.3 90.8 91.7 100 100 100 100 100 99.2 100 95.8 100 95.0 99.2 90.3 24 25 93.3 96.7 90.8 89.2 95.8 90.8 90.8 90.0 100 100 88.3 90.8 91.7 100 100 100 100 100 99.2 100 95.8 100 95.0 100 89.7 25 26 91.7 93.3 90.8 90.0 92.5 97.5 90.0 90.8 91.7 91.7 85.0 90.8 91.7 91.7 91.7 91.7 91.7 91.7 90.8 91.7 92.5 91.7 91.7 91.7 91.7 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 (Deduced amino acid sequence comparison) 1: STC 2: 5270 3: CU1 4: PBG98 5: 2512 6: Bursine-2 7: DELE 8: Variant A 9: 90-11 10: SNU91108 11: SNU9414 12: SNU9423 13: SNU9539 14: SNU9750 15: SNU9593 16: SNU9596 17: SNU9912 18:SNU9914 19: SNU9946 20: SNU9960 21: SNU9991 22: SNU9993 23: SNU9993-2 24: SNU99103 25: SNU99122 26: SNU99125

SNU91-108 ꠐ ꠉ SNU99-103 16ꠐ ꠏꠊ SNU99-12 ꠐ ꠐ 31ꠐ ꠉ SNU99-14 29 ꠏ ꠐ ꠏ ꠌ SNU99-93 ꠐ ꠉꠏꠏ 9011 ꠐ ꠐ ꠐ 10ꠐ ꠉꠏꠏ SNU85-7 14 ꠏ ꠐ ꠐ ꠌꠏꠏ SNU99-130 35ꠐꠐ ꠐ ꠐ 100 ꠐ ꠌꠏꠏꠏꠏ SNU99-46 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ ꠐꠐ ꠐ ꠐꠐꠐ ꠐ ꠏꠏꠏꠏꠏ SNU99-60 ꠐꠐꠐ ꠐꠐ ꠐꠐꠐ ꠐꠐ 51 SNU98-18 ꠐꠐꠐ ꠐꠐ ꠏ ꠐꠐꠐ ꠌ ꠐ ꠌ SNU99-83 ꠐꠐꠐ20ꠌꠏꠏ ꠐꠐꠐ 51ꠐ SNU97-50 ꠐꠐꠐ ꠌꠏ ꠐꠐꠐ 49ꠌ SNU99-122 ꠐꠐꠐ ꠐꠐꠌꠏꠏꠏꠏꠏꠏꠏꠏ SNU86-15 ꠐꠐ ꠐꠐ ꠏꠏ SNU99-136 ꠐꠌꠏꠏꠏꠏꠏꠏ 77ꠌꠏꠏ SNU99-141 ꠐ ꠐ ꠌꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ SNU95-96 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ 70 ꠏꠏ LUKERT 100 ꠏꠏ ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ ꠌꠏꠏ SNU99-125 ꠐ ꠌꠏꠏꠏꠏꠏ Bursine-2 100 ꠏꠏ DELE 40 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ ꠏꠏꠏꠏꠏꠏ ꠌꠏꠏ VariantE ꠐ ꠌꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ VariantA 83 2512 79 ꠏꠏꠏ 90 ꠏꠏ ꠌ SNU99-93-2 82 ꠏꠏꠏꠏꠏ ꠐ ꠐ ꠐ 52 ꠏꠏꠏꠏꠏꠏ ꠌꠏꠏꠏꠏ SNU99-177 ꠌꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ ꠐ ꠏꠏꠏꠏꠏ ꠐ ꠐ 74ꠐ ꠌꠏꠏꠏꠏꠏꠏꠏ SNU99-91 ꠐ ꠐ ꠐ ꠌꠏꠏꠏ ꠌꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ STC 51ꠐ ꠐ ꠐ ꠐ ꠌꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ 5270 ꠌꠏꠏꠏꠏꠏꠏ 30ꠐ ꠏꠏꠏꠏꠏꠏ SNU94-23 ꠐ ꠐ ꠏꠏꠏꠏꠏ PBG98 ꠐꠐ ꠐ ꠐꠐ ꠏꠏꠏꠏ CU1 ꠌꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ ꠐꠐ 100ꠌ ꠐ ꠏꠏ D78 42ꠌ ꠐ 36ꠌꠏ SNU91-108F15 41ꠌꠏ 41ꠌ SNU95-39 ꠐ ꠌꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ SNU94-14 Scale: each ꠏ is approximately equal to the distance of 0.000917

100 Competition ELIS A % competitio 80 60 40 20 33/25 24/17 0-8 -7-6 -5-4 -3-2 Dilution of anti-snu91108 serum

A B a A B

RBC Dilution of bursal homogenates RBC Conc(%) 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 Control 0.50 - - - - - - - - - - 0.25 + - - - - - - - - - 0.12 + + - - - - - - - - 0.06 + + + - - - - - - - 0.03 + + + + - - - - - - 0.015 + + + + + - - - - - 0.007 + + + + + + - - - - 0.003 + + + + + + + + - -

Treatment Hemolytic titer (log 2) Uninfected BH 0 IBDV-infected BH (IBH) 5 IBH, heated (56, 30 min.) 0 Purified IBDV(20 concentrated) 0 IBH, supernatant (100,000 g, 1 hr.) 5 IBH + IBDV immune serum (1/10, VN titer:1/128) 1 IBH + Fresh normal chicken serum (1/10) 1 IBH + Immune serum + Fresh chicken serum 1 IBH, heated + Immune serum + Fresh chicken serum 0 IBDV-infected chorio-allantoic membrane 0 RBC Control 0

PRIMER 90-11 SNU91-108 SNU95-93 야외주 SNU97-50 SNU98-18 SNU99-12 SNU99-103 D78 백신주 Lukert Bursine-2

A M 1 2 3 4 5 6 7 8 9 10 400 300 200 175 125 100 50 25 B M 1 2 3 4 5 6 7 8 9 10 400 300 200 175 125 100 50 25

품종 일령 페사율 (%) 7 1/10 (10.0) 14 3/10 (30.0) SPF 21 4/10 (40.0) 28 8/10 (80.0) 35 10/10 (100) 42 10/10 (100) A 35 4/12 (33.3) 갈색 산란계 42 14/29 (48.3) 브로일러 A 31 0/12 (0.0)

Fig. 2-1. Temperature and mortality of SPF chickens inoculated with IBDV Isolate 1030 (Trial 1) 45 45 44 40 43 35 Temp. (C) 42 41 40 39 30 25 20 15 Moatality (%) 38 10 37 5 36 N AM AM PM AM PM AM N PM AM PM AM AN AM AM PM 0 0 1 2 3 4 5 7 8 9 10 Days post-inoculation Temp. (C) Mortality (%)

Fig. 2-2. Bursa (B/B), spleen (S/B) and liver (L/B) weight (mg) to body weight (g) ratios in commercial brown layer chickens inoculated with IBDV SNU 9818 (Trial 2) B/B, S/B ratio 6.0 5.0 4.0 3.0 2.0 1.0 0.0 60 50 40 30 20 10 0 0 1 2 3 4 5 6 7 10 12 15 20 28 Days after inoculation L/B ratio B/B ratio S/B ratio L/B ratio Fig. 2-3. Changes of erythrocyte sedimentation rate (ESR) and packed cell volume (PCV) in 39 days old commercial brown layer chickens inoculated with IBDV SNU 9818 (Trial 2) % 40 35 30 25 20 15 10 5 0 1 2 3 4 5 6 7 10 12 15 20 Days of post inoculation PCV ESR

32 Low virulence High virulence 28 PCV 24 20 16 * * * * * * C T L D 2 D 3 D 4 D 5 D 6 D 7 D 8 D 9 D 10 Days postinoculation

50 * Low virulence High virulence ESR 40 30 20 * * * 10 0-10 CTL D2 D3 D4 D5 D6 D7 D8 D9 D10 Days postinoculation

43.0 Body temperature 42.5 42.0 41.5 * * * Low virulence High virulence 41.0 CTL DPI-1 DPI-2 DPI-3 DPI-4 DPI-5 DPI-6 DPI-7 DPI-8 DPI-9 DPI-10 Days postinoculation 43 42 Body temperature 41 40 39 38 D0 D1 D2-1 D2-2 D3-1 D3-2 Days postinoculation

03.06.02-03.06.12 04.06.01-04.06.08 04.07.26-04.07.31 Age (week) virulence of IBDV Days postinoculation (DPI) 0 1 2 3 4 5 6 7 8 Mortalit y (%) 7 Low 0 0 0 0 2 0 0 0 0 2/20(10) 7 High 0 0 0 0 0 3 2 1 0 6/20(30) 8 High 0 0 1 2 1 1 0 0 0 5/10(50) 8 High 0 0 0 2 4 0 0 0 0 6/17(35)

Body Temperature 44 43 42 41 CTL Virus Vit.E SMT Dexa Asp 40 0 1 2 3 4 5 6 Day postinoculaiton

50 DPI-2 DPI-3 40 Body temperature 30 20 10 0 Live (n=33) Moribund (n=8) Live (n=30) Moribund (n=11)

NO concentratin (µm) 160 CTL Virus Vit. E 120 SMT Dexa Asp 80 40 0 0 1 2 3 4 5 6 Day postinoculation

160 * NO concentration (µm) 120 80 40 0 Live (n=11) Moribund (n=30) plasma NO concentration( µm ) 120 100 80 60 40 20 DPI-2 DPI-3 DPI-4 DPI-5 0 live moribund (n=9) (n=1) live (n=7) moribund (n=2) live moribund (n=5) (n=2) live moribund (n=4) (n=1)

2500 Corticosterone (ng/dl) 2000 1500 1000 500 * * * CTL Virus Dexa 0 0 1 2 3 4 5 6 Day postinoculation

3.5 3.5 3.0 3.0 Bursa/Body Ratio 2.5 2.0 1.5 1.0 0.5 * Bursa/Body Ratio 2.5 2.0 1.5 1.0 0.5 * * * * 0.0 CTL Virus Vit. E SMT DEXA ASP 0.0 CTL Virus Vit. E SMT DEXA ASP Fig. 2-14. Bursa/Body weight ratio of survived (left) and dead chickens (right) of each treatment groups. (*, p<0.05 by t-test).

M 1 2 3 4 5 M 6 7 3,054 1,636 1,018 Fig. 3-2. Amplification of VP2 from IBDV vaccines and isolates strains. M : 1kbp DNA marker, lane 1. Blen, lane 2. Bur-706, lane 3. Bursine plus, lane 4. Bursine 2, lane 5. Cevac, lane 6. SH-92, lane 7. 1030 M 1 2 3 4 5 6 7 3,054 1,636 1,018 Fig. 3-3. Amplification of IBDV sega DNA fragment from IBDV vaccines and isolates strains. M : 1kbp DNA marker, lane 1. SH-92, lane 2. 1030, lane 3. Blen, lane 4. Bur-706, lane 5. Bursine plus, lnae 6. Bursine 2, lane 7. Cevac 나. IBDV SH/92 strain VP2 와 SegA 유전자 및 아미노산의 염기서열 분석 증폭된 각각의 유전자를 1% agarose gel에서 전기영동한 후 겔에서 각각의 DNA를 gel extraction kit (Qiagen)를 사용하여 추출한 하였다. 증폭된 VP2유전자의 경우 제한효소로

처리한 후 pbluescripii vector에 클로닝하였고 증폭된 SegA유전자의 경우 pcr2.1-topo vector (TOPO TA cloning kit)에 제공된 방법에 따라 클로닝하였다. VP2 및 sega의 삽 입유무는 제한효소인 EcoRI digestion에 의해 검사하였고 선발된 각각의 클론에서 plasmid DNA를 miniprep kit (Qiagen)로 추출한 후 염기서열을 분석하였다. 얻어진 염기서열 과 아 미노산 서열을 DNASIS로 분석하였다. VP2의 경우 Fig. 3-4에 SegA의 경우 Fig. 3-5에 나타내었다.

Table 3-4. ELISA titer of anti-ibdv antibody obtained from field chicken No. ELISA titier 1 6910 2 6482 3 7710 4 5622 5 7362 6 7160 7 5382 8 6764 9 6379 10 5226 11 9570 12 9093

(A) (B)

1 2 3 4 5 6 M KDa 1 2 3 4 5 6 M KDa 116 97.4 66 97.4 66 45 45 31 31 21.5

KDa M 1 2 3 4 5 6 97.4 66 45 31 21.5

Table 3-5. Amtigenicity of baculovirus-expressed proteins against positive antisera to field IBDV No. ELISA titier 92VP2 92SegA 1 6910 0.043±0.023 0.048±0.012 2 7710 0.051±0.014 0.067±0.022 3 5622 0.038±0.043 0.057±0.021 4 7362 0.067±0.055 0.061±0.039 5 5382 0.032±0.026 0.037±0.021 6 6764 0.047±0.027 0.056±0.011 7 6379 0.032±0.026 0.049±0.016 8 9093 0.097±0.045 0.193±0.101 9 9570 0.177±0.082 0.227±0.078 Negative control - 0.012±0.011 0.019±0.006

Fig. 3-10. Analysis of recombinant VP2 and SegA protein expression rate by culture time. Same amount of cell lysates were analysed by dot blot hybridization using IBDV positive antisera (A) and IBDV monoclonal antibody (B). Lanes 1-4, recombinant SegA grown for 1, 2, 3, and 4 days, respectively; 5-8, recombinant VP2 grown for 1, 2, 3, and 4 days, respectively.

(A) (a) (b) (c) (B)

주 의 1. 이 보고서는 농림부에서 시행한 농림기술개발사업의 연구보고서입니다. 2. 이 보고서 내용을 발표할 때에는 반드시 농림부에서 시행한 농림기술개발사업의 연구결과임을 밝혀야 합니다. 3. 국가과학기술 기밀유지에 필요한 내용은 대외적으로 발표 또는 공개하여서는 아니됩니다.