발간등록번호 - PDF Free Download

발간등록번호 11-1541000-000475-01

농림수산식품부장관귀하 이보고서를 이종항원발현생균백신 system 을이용한 PRRSV 백신및감별진단법 개발 과제의보고서로제출합니다. 2010 년 08 월 11 일 주관연구기관명 : ( 주 ) 바이오리쏘스주관연구책임자 : 정승기세부연구책임자 : 정승기협동연구기관명 : 전남대학교협동연구책임자 : 이재일협동연구기관명 : 전남대학교협동연구책임자 : 김태중 - 1 -

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SUMMARY Porcine reproductive and respiratory disease(prrs) is an economicay important disease around the gobe. It is we estabished that PRRS is caused by an enveoped, singe-stranded positive-sense RNA virus known as procine reproductive and respiratory syndrome virus(prrsv). GP5 and GP6 is the major enveope poypeptide of PRRSV, and it is associated with the deveopment of neutraizing antibodies and protection the GP5 and GP6 protein. In this study, a heteroogous PRRS gycoprotein 5(GP5) and used as a ive vaccine vehice. To study the effect of BBS-GP5. we estabished a antigen-specifec antibody(igg & IgA) inductivity induced by BBS-GP5 using mouse and pig. Mice were primed with recombinant GP5(rGP5) foowed by boosting with ive BBS-GP5 via the subcutaneous and intranasa route. Pig experiment is the same that. As a resut, anti-gp5 IgG was significanty induced. our data is beieved to be a good candidate for the deveopment of a ive-attenuated vaccine against PRRSV infections. furthermore antigen-specificity and sensitivity is more usefu than other kit. - 6 -

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Carbon (%) Nitrogen (%) Commercia sugar Gray Backish White coor coor coor Starch Dextrose Saccharose Lactose Gycero Gaactose Soy 3:10 Z) (68.6) 3:2(75.6) 3:15(70.8) 3:0.5(34.4) 3:10(64.1) 3:10(57.3) 3:3(56.5) 3:10(32.8) 3:1.5(23.2) Soy bean mik 1:10(6.9) 1:2(8.9) 3:10(26.8) 1:0.5(5.4) 3:10(12.4) 1:5(9.2) 2:8(12.7) 1:6(14.5) 1:5(12.9) (NH 4) 2SO 4 1:10(17.9) 1:2(23.2) 1:10(53.3) 3:0.5(8.2) 1:2.5(15.6) 1:2.5(18.4) 1:2.5(21.4) 2:5(12.9) 2:4.5(19.8) (NH 4) 2CO 0.5:1(1.8) 1:2(7.5) 1:3(27.3) 1:0.5(8.4) 1:2(1.6) 0.5:1(7.5) 1.2:2(14.8) 1.6:3(25.2) 1.2:4(11.5) NH 4C 1:5(19.6) 1:2(25.4) 1:5(44.8) 3:0.5(2.4) 1:5(18.4) 1:5(18.6) 1:4.5(19.3) 1:4.2(12.3) 1:4(15.2) Poypeptone 1:10(89.4) 1:2(87.0) 1:10(65.7) 3:0.5(76.2) 1:5(97.0) 1:10(85.9) 1:8(45.2) 1:5(65.3) 1:8(81.6) Peptone 1:3(20.2) 2:5(36.2) 7:5(62.8) 5:1.9(25.6) 7:1.5(70.5) 7:1.5(100) 6:3(96.2) 5:1.5(30.2) 6:5(83.1) - 15 -

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Name Amino acid sequence Encompassing amino acid ocation GP5-VR* #1 CLLDTKGGLYRWRSPV 145-159 th GP5-VR* #2 CVIIEKRGKVEVEGHLID 159-175 th GP5-LV** #1 CIVDDRGRVHRWKSPI 147-161 th GP5-LV** #2 CIVVEKLGKAEVDGNLV 161-176 th M-VR* CANDNHAFVVRRPGSTT 127-142 th M-LV** CSGNRAYAVRKPGLTSV 127-142 th - 25 -

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antigen Reacted antibodies raised against GP5-VR* #1 GP5-VR #1 (3.17±0.17) GP5-VR #2 (0.34±0.20) GP5-LV#1 (0.18±0.02) GP5-LV #2 (0.52±0.31) GP5-VR #2 GP5-VR #2 (3.05±0.50) GP5-VR #1 (0.18±0.11) GP5-LV #1 (0.09±0.07) GP5-LV #2 (0.28±0.17) GP5-LV** #1 GP5-LV #1 (2.28±0.24) GP5-VR #1 (0.64±0.18) GP5-VR #2 (0.27±0.03) GP5-LV #2 (0.16±0.01) GP5-LV #2 GP5-LV #2 (3.01±0.60) GP5-VR #1 (0.50±0.23) GP5-VR #2 (0.25±0.01) GP5-LV #1 (0.14±0.02) M-VR M-VR (3.43±0.36) M-LV (0.24±0.01) M-LV M-LV (3.64±0.61) M-VR (0.20±0.07) - 27 -

항원명 양성대조군 음성대조군 시료 양성개체수 (%) GP5-VR* #1 0.394±0.081 0.278±0.091 0.344±0.295 28 (10) GP5-VR #2 0.452±0.101 0.299±0.088 0.353±0.319 32 (12.3) GP5-LV** #1 0.352±0.111 0.191±0.078 0.298±0.233 0 GP5-LV #2 0.414±0.091 0.222±0.104 0.335±0.312 0 M-VR 0.372±0.127 0.279±0.083 0.310±0.239 23 (8.8) M-LV 0.466±0.077 0.245±0.101 0.361±0.308 0-28 -

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성분명 함량 (g/l) Caf brain Infusion from 200g 7.7 Beef heart Infusion from 250g/L 9.8 Proteose peptone 10 Dextrose 2 Sodium choride 5 Disodium phosphate 2.5 ph 7.4± 0.2 성분 대두박 (%) African ocuse beans(%) 단백질 47.73 30.69 수분 4.83 3.4 지방 17.72 23.03-42 -

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Beef extract(g/l) 미네랄혼합물 (g/l) 비타민혼합물 (g/l) O.D 600nm 2 0.72 5 10 15 5 10 15 5 10 15 5 10 15 5 0.82 10 0.86 2 0.69 5 0.76 10 0.77 2 0.84 5 0.65 10 0.77 2 0.51 5 0.63 10 0.55 2 0.79 5 0.82 10 0.97 2 0.88 5 0.81 10 0.77 2 0.62 5 0.66 10 0.75 2 0.64 5 0.63 10 0.79 2 0.86 5 0.83 10 0.89-44 -

대두에서조제한펩톤 (g/l) African ocust bean 에서조제한 펩톤 (g/l) 5 10 15 5 10 15 PRRSV GP5 2.3*10 10 5.9*10 10 3.5*10 10 3.4*10 10 2.5*10 10 1.5*10 10 Wid type 3.7*10 12 5.2*10 12 5.1*10 12 2.4*10 12 4.3*10 12 3.2*10 12-45 -

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일시 Group 1 Group 2 Group 3 rgp5 200ug in PBS + rgp5 200ug in PBS + rgp5 200ug in PBS + Day 0 Freund's compete Freund's compete Freund's compete adjuvant, SC* adjuvant, SC adjuvant, SC Day 14 rgp5 200ug in PBS + Freund's incompete adjuvant, SC 1 10 8 BBS-PRRSV GP5**/kg, nasa 1 10 8 BBS-aroA*** /kg, nasa Day 21 모든자돈에서채혈및항체가측정 Day 28 rgp5 200ug in PBS + Freund's incompete adjuvant, SC 1 10 8 BBS-PRRSV GP5/kg, nasa 1 10 8 BBS-aroA/kg, nasa Day 35 모든자돈에서채혈및항체가측정 Day 42 PRRSV chaenge Day 47 채혈및부검실시 - 49 -

Group 평균 OD 값 (±SD) 통계적의의 1 0.47 (0.08) 없음 2 0.36 (0.07) 없음 3 0.45 (0.09) 없음 Group 평균 OD 값 (±SD) 통계적의의 1 2.02 (0.30) 있음 (1-2: P<0.05, 1-3; P<0.01) 2 1.27 (0.37) 있음 (2-3: P<0.1) 3 0.67 (0.13) 없음 - 50 -

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A: supeficia LN B: submandibuar LN C: mediastina LN D: media iiac LN - 52 -

Group 1 Group 2 Group 3 비고 Superficia LN 21.74 (1.83) 23.49 (1.69) 30.71 (3.26) P<0.01 Submandibuar LN 22.01(2.45) 23.04 (2.49) 22.90 (1.45) Mediastina LN 22.42 (1.23) 23.84 (2.78) 25.15 (3.29) Media iiac LN 21.65 (1.20) 23.02 (2.33) 24.24 (3.90) - 53 -

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Antigens N* VR #1** VR #2** VR #1+2 rgp5 Positive 209 28 32 208 253 Negative 49 230 226 50 5 True positive 23 28 181 208 Fase positive 5 4 27 45 Fase negative 186 181 28 1 True negative 44 45 22 4 Sensitivity 11.00 13.40 86.60 99.52 Specificity 89.80 91.84 44.90 8.16-55 -

Antigens N* LV #1** LV #2** Positive 209 2 4 Negative 49 256 252 True positive 0 0 Fase positive 2 4 Fase negative 256 252 True negative 0 0-56 -

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Group 항원 formuae boosting 마리수 1 PBS PBS PBS 3 2 rgp5 50ug PBS rgp5+pbs 3 3 rgp5 50ug 4 rgp5 50ug 5 rgp5 50ug 6 rgp5 50ug 7 rgp5 50ug 8 rgp5 50ug Freund's compete 0.25% chitosan so. 0.25% chitosan 과립 0.5% chitosan 과립 1% chitosan 과립 2% chitosan 과립 rgp5+freund's incompete rgp5+0.25% chitosan so. rgp5+0.25% chitosan 과립 rgp5+0.5% chitosan 과립 rgp5+1% chitosan 과립 rgp5+2% chitosan 과립 3 3 3 3 3 3-58 -

Group 항원항체가 (SD) 통계적의의 (P) 1 PBS 0.29 (0.21) 의의없음 2 rgp5 50ug 0.32 (0.16) 의의없음 3 rgp5 50ug 1.83 (0.65) 의의없음 4 rgp5 50ug 0.05 (0.00) 의의없음 5 rgp5 50ug 0.04 (0.00) 의의없음 6 rgp5 50ug 0.72 (0.45) 의의없음 7 rgp5 50ug 1.08 (0.61) 의의있음 8 rgp5 50ug 1.16 (0.39) 의의있음 - 59 -

Antigens OD 값 (SD) 음성값 (SD) 음성대비 P 값 rgp5 2.871 (0.04) 0.6 (0.01) P < 0.01 rgp6 2.14 (0.01) 0.6 (0.06) P < 0.01 Antigens OD값 (SD) 음성값 (SD) 음성대비 P값 rgp5 1.6 (0.3) P < 0.01 0.5 (0.1) rgp6 1.2 (0.2) P < 0.05-60 -

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Group Experimenta condition Tota Pigs 백신접종 group BBS PRRS GP5 비강분무 10 백신비접종 group PBS 비강분무 10-67 -

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10 9 Cinica scores for experimenta pigs treated nontreated 8 7 Cumuative cinica score 6 5 4 3 2 1 0 1 2 3 4 5 6 7 Days post chaenge 15 Cinica score for experimenta pigs treated nontreated Cumuative cinica score 10 5 0 1 2 3 4 5 6 7 8 9 10 Days post chaenge - 70 -

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일시 Group 1 Group 2 Group 3 Day 0 rgp5 200ug in PBS + Quiaza saponin, SC* rgp5 200ug in PBS + Quiaza saponin, SC rgp5 200ug in PBS + Quiaza saponin, SC Day 14 rgp5 200ug in PBS + Quiaza saponin, SC 1 10 8 BBS-PRRSV GP5**/kg, Quiaza saponin, nasa 1 10 8 BBS-aroA*** /kg,quiaza saponin, nasa Day 21 모든자돈에서채혈및항체가측정 Day 28 rgp5 200ug in PBS + Quiaza saponin, SC 1 10 8 BBS-PRRSV GP5/kg, Quiaza saponin, nasa 1 10 8 BBS-aroA/kg, Quiaza saponin, nasa Day 35 Day 42 Day 47 모든자돈에서채혈및항체가측정 PRRSV chaenge 채혈및부검실시 - 73 -

Group 평균 OD 값 (±SD) 통계적의의 1 0.23 (0.07) 없음 2 0.27 (0.12) 없음 3 0.46 (0.53) 없음 Group 평균 OD 값 (±SD) 통계적의의 1 0.42 (0.62) 없음 2 0.37 (0.37) 없음 3 0.67 (0.13) 없음 - 74 -

Test group 1(n=12) 2(n=10) 3(n=9) Days post-inocuation Test item 0 3 5 7 9 11 15 21 28-98 105 발병 0 0 0 0 0 0 2 1 5 1 병원체분리 0 0 0 0 0 0 2 1 5 1 폐사 0 0 0 0 0 0 0 1 2 1 발병 0 0 0 0 0 0 1 3 3 1 병원체분리 0 0 0 0 0 0 1 3 3 1 폐사 0 0 0 0 0 0 1 1 1 0 발병 0 0 0 0 0 0 4 1 2 1 병원체분리 0 0 0 0 0 0 4 1 2 1 폐사 0 0 0 0 0 0 0 1 1 1-75 -

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1 0.1 0.01 0.001 1 10 100 1 0.1 0.01 0.001 1 10 100 1 0.1 0.01 0.001 1 10 100 rgp5 Ag coating ysate Ag coating (-)* (-) (-) (-) (-) (-) (-) (+)** (+) (+) (+) (+) (+) (+) 1 2 3 4 5 6 7 시료 1 2.227 1.740 0.503 0.636 0.810 1.315 1.097 시료 2 1.860 1.619 0.492 0.516 0.878 1.019 1.134 시료 3 1.769 1.265 0.547 0.494 0.945 1.152 1.208 mean 1.952 1.541 0.514 0.548 0.877 1.162 1.146 SD 0.242 0.246 0.029 0.076 0.067 0.148 0.056 (-) 0.367 0.137 0.101 0.103 0.128 0.108 0.106 (+) 1.585 1.519 0.415 0.357 0.671 0.957 0.933-77 -

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pate 1 차 2 차 3 차 4 차 5 차 6 차 평균 197 199.5 170 132 125 185 stock CFU/m 1.97 10 9 1.99 10 9 1.7 10 9 1.32 10 9 1.25 10 9 1.85 10 9-79 -

pate 1 차 2 차 3 차 4 차 5 차 6 차 평균 25 28 22.5 17 15 29 stock CFU/m 2.5 10 9 2.8 10 9 2.2 10 9 1.7 10 9 1.5 10 9 2.9 10 9-80 -

pate 1 차 (10/21) 2 차 (12/21) 3 차 (2/21) 4 차 (4/21) 평균 184 213 197 194 stock CFU/m 1.84 10 9 2.13 10 9 1.97 10 9 1.94 10 9 pate 1 차 (10/21) 2 차 (12/21) 3 차 (2/21) 4 차 (4/21) 평균 24.7 21 21 22 stock CFU/m 2.4 10 9 2.1 10 9 2.1 10 9 2.2 10 9-81 -

name sequence(5' 3') size T7po-F T7pro-po-R T7po-pro-F GP5-T7pro-R T7pro-GP5-F GP5-BamH-R AATTGGTACCATGAACACGATTAACATCGC TAGTGAGTCGTATTAATTTTACGCGAACGCGAAGT GCGTTCGCGTAAAATTAATACGACTCACTATAGGG GCTGCTGTCGTTGCTCATATCCTTATCGTCATCGTCGT GATGACGATAAGGATATGAGCAACGACAGCAGCTCCCA AATTGGATCCCTAAGGACGACCCCATTGTT 2.6kb 180bp 0.5kb - 82 -

name sequence(5' 3') size kan-kpn-6his-r ATAGGTACCATGATGATGATGATGATGGAAGAACTCGTC AAG 0.8kb - 83 -

name T7po-kpnI-R Kan-KpnI-F Kan-BamHI-R T7po-6h-Bg-F sequence(5' 3') AATGGTACCCGCGAACGCGAAGTCCGACT CTGGGTACCATGATTGAACAAGATGGATT TCGGGATCCTTAGAAGAACTCGTCAAGAAGGC TCGAGATCTCATCATCATCATCATCATATGAACACGATTAACATCGC - 84 -

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출원연도 특허명 출원인 출원국 출원번호 2008 바이오리쏘스, 약독화 Bordetea bronchiseptica에서발현된이종항원을전남대학교이용한 PRRSV 백신산학협력단 한국 바이오리쏘스, 2008 펩타이드항원을이용한 PRRSV wses법 전남대학교산학협력단 한국 - 90 -

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