발간등록번호 효모표면발현및분비시스템을 활용한재조합백신생산기반 기술개발 (Recombinant vaccine production by yeast expression platform technology) 한국생명공학연구원 농림축산식품부
제출문 농림축산식품부장관귀하 이보고서를 효모표면발현및분비시스템을활용한재조합백신생산기반기술개 발 과제의보고서로제출합니다. 2014 년 10 월 22 일 주관연구기관명 : 한국생명공학연구원 주관연구책임자 : 최의성 세부연구책임자 : 안정오 연 구 원 : 박경민 연 구 원 : 서성화 연 구 원 : 박선주 협동연구기관명 : 중앙백신연구소 협동연구책임자 : 유성식 - 1 -
요약문 Ⅰ. 제목 Ⅱ. 연구개발의목적및필요성 Ⅲ. 연구개발내용및범위 Ⅳ. 연구개발결과 Ⅴ. 연구성과및성과활용계획 - 2 -
- 3 -
SUMMARY The vaccine candidate genes of Erysipelothrix rhusiopathiae were screened by signal trap methods. SpaA gene coding for a highly immunogenic surface protein of Erysipelothrix rhusiopathiae was expressed as a fusion protein with CBD (cellulose binding domain) in Escherichia coli and the protein was immobilized in cellulose containing matrix resulting in improved vaccine efficacy in an animal experiment. This technology was filed as a patent. The capsid protein of porcine circovirus type 2 encoded by ORF2 was expressed in Saccharomyces cerevisiae. The recombinant capsid protein was found to form virus-like particle, which is considered to be very important for proper immunogenicity. The recombinant capsid protein tended to aggregate during the isolation and purification procedures. Several different forms of the recombinant capsid protein including cell free extract and whole cell were scored for vaccine efficacy in animal experiments using ginea pigs and pigs. The recombinant whole cell expressing the capsid VLP was the most potent as a vaccine. International patent application was filed for this technology and the technology was licensed to the ChoongAng Vaccine Lab.. After further animal experiments with a larger scale, the technology is planned to be commercialized. - 4 -
CONTENTS ( 영문목차 ) - 5 -
목 차 제 1 장연구개발과제의개요 7 제 1 절연구개발의필요성 7 제 2 장국내외기술개발현황 11 제 1 절돈단독백신 11 제 2 절 PCV-2 백신 12 제 3 장 연구개발수행내용및결과 14 제 1 절 1차년도연구결과 14 제 2 절 2차년도연구결과 40 제 3 절 3차년도연구결과 70 제 4 장목표달성도및관련분야에의기여도 116 제 5 장연구개발성과및성과활용계획 119 제 1 절연구개발성과 119 제 2 절성과활용계획 121-6 -
제 1 장 연구개발과제의개요 * 연구개발의목적, 필요성및범위등을기술 동물의약품 시장 - 7 -
- 8 -
서브유닛백신개발의필요성 - 9 -
그림 재조합서브유닛백신기술 - 10 -
제 2 장 국내외기술개발현황 제품명 제조사 년판매액 백만원 기술수준 항원구성 포실리스 인터벳 리터가드화이자 기타 서브유닛백신으로필요항원만 발현정제한차세대백신 국내시장의 이상을점유 소계 - 11 -
- 12 -
- 13 -
제 3 장 연구개발수행내용및결과 Primer 290-Fr1-BamH1 290-Fr2-BamH1 290-Fr3-BamH1 290-R-SalI Sequence (5' 3') a agtcggatccgctgattggagctcttttagtatt cagtggatccagctgattggagctcttttagtatt tcagggatcccagctgattggagctcttttagtatt ttttgtcgacttactgggctttataaactcgtatccaatc - 14 -
- 15 -
ATGACATATCCGCGTAGACGTTACAGAAGACGTAGACACAGACCAAGGTCGCATCTGGGCCAAATCCTACGCCGAAGACCTTGGCTTGTACAC CCCAGACACAGATACAGATGGCGTAGGAAGAATGGAATATTCAACACTAGACTATCAAGGACATTCGGATACACAATAAAACGGACTACTGTG AAAACGCCCTCTTGGGCTGTTGATATGATGAGGTTTAATATAAATGACTTCCTACCCCCAGGTGGAGGTTCTAACCCAAGAAGTGTTCCATTT - 16 -
GAGTATTATAGAATTAGAAAAGTCAAGGTTGAGTTCTGGCCTTGTTCCCCAATAACGCAAGGAGATAGAGGTGTGGGTTCTTCTGCTGTAATT TTGGACGACAACTTTGTCACTAAGGCCACAGCTCTCACTTATGACCCTTATGTTAACTACTCCTCAAGACATACTATCACTCAGCCATTTTCT TACCATAGCAGATATTTCACCCCAAAGCCTGTCTTAGACTCCACTATTGATTACTTTCAGCCTAATAATAAAAGAAATCAACTTTGGCTGCGA TTGCAAACCACTGGTAATGTTGACCATGTTGGGTTGGGTACCGCATTTGAAAACTCTATATACGATCAAGAATACAACATTAGGGTTACAATG TATGTTCAGTTCAGAGAATTTAATTTAAAAGATCCACCTTTGAACCCTTAG EcoRI-PCV2-F SalI-His-ORF2-R AATT GAATTC AAAAATGACATATCCGCGTAGACGT TAT GTCGAC CTAGTGATGGTGATGGTGATG AGGGTTCAAA GGTGGATCT - 17 -
Ty1-F Ty1-R Ty1-PCV2 SalI-ORF2-R atta gaattc aaaa atggaatcccaacaattatctc tttggttgtattcgtatagcgc gcgctatacgaatacaaccaaa AGACTATCAAGGACATTCGGATAC aatt gtcgac CTAAGGGTTCAAAGGTGGATC - 18 -
sscbd-ecori-f CBD-EcoRI-F CBD-R CBD-BORF2 SalI-ORF2-R aatt GAATTCATGATGGTTGCATG aatt GAATTC ATGCAACAGACAGTCTGGGGTCA AGAAGAAGGAGGTGGAGTAGC GCTACTCCACCTCCTTCTTCTAGACTATCAAGGACATTCG GATAC aatt gtcgac CTAAGGGTTCAAAGGTGGATC α - 19 -
- 20 -
- 21 -
- 22 -
- 23 -
- 24 -
- 25 -
- 26 -
- 27 -
재조합 항원선별 결과 - 28 -
재조합 시험백신조성 재조합 시험백신역가측정 결과 - 29 -
돼지단독재조합단백질시험백신조성 - 30 -
단독재조합단백질시험백신 접종후 결과 돼지단독재조합단백질시험백신방어시험결과 - 31 -
[ 제 1 협동과제 ] 지적사항 2 추가자료 - 32 -
- 33 -
- 34 -
- 35 -
- 36 -
- 37 -
시험백신 항원함량및혼합비율 PCV-1 PCV2-BORF2 total protein 3mg/ ml adjuvant* 10% 면역증강제 200 μg / ml PCV-2 PCV2-CBD BORF2 total protein 3mg/ ml adjuvant 10% 면역증강제 200 μg / ml PCV-3 PCV2-Ty1 BORF2 total protein 3mg/ ml adjuvant 10% 면역증강제 200 μg / ml PCV-4 Control vector total protein 3mg/ ml adjuvant 10% 면역증강제 200 μg / ml Circo-one 양성대조군 1 ( 중앙백신연구소 ) Cricoflex 양성대조군 2 ( 베링거인겔하임 ) PBS 음성대조군 - 38 -
시험백신 항원함량및혼합비율 SpaA-1 Soluble SpaA 20 μg / ml adjuvant* 30% SpaA-2 Soluble SpaA CBD 20 μg / ml adjuvant 30% SpaA-3 Avicel-CBD SpaA 20 μg / ml adjuvant 30% ERT2T-1 양성대조군 ( 중앙백신연구소 ) PBS 음성대조군 - 39 -
α α 서열번호 1 ggttaccaatcattcgaagccgtcaacgaggaaattaactctatcgtctccgaattgaaaaatgagggaatgtctctacaaaat attcatcacatgtttaaacaatccatccagaacctggctactcgtatcggttataggtcttttatgcaggatgctatgtacttggag aatttcgaacgtttgacaattcctgaattagatgaagcctatgttgacttgttggttaattatgaagtcaagcacagaatcttagtt aagtatgaggacaaagtcaagggtagagcaccactagaagcattcatcgttccactgaggaacaggattagatctatgaacg aaattgctgcagaagttaattatctgcctgaagctcatgaagatttcctagtcagtgattcttcagagtacaacgacaaattgaat aacattaattttgccttgggtttaggcgtttccgaatttatcgactacaatagattagagaacatgatggagaaagagattcatcc actttatttggaattgtacgcaatgaggcgtaacagacagatccaagtagttcgtgacgtctatcctaatttagaaagagctaac gcagttgtcgaaagtcttaagacaattaaagatattaaacagcgtgagaagaaactacaagaattgttggagatttacattcaa agatctggtgacgttaggaaaccagatgtcttacagagattcatcggaaaatatcagtcagttgttgatgaagagaagaataag ctacaagactatcttgaaagtgatatctttgatagctattcagtcgatggcgaaaagattagaaataaagagattaccttgatca accgtgacgcttacttgtccatgatttacagagctcaatctatttcagagatcaagactatcagggccgacttagaatctttagta aagagttttcagaatgaggaatctgactctaaagttgaacctgaatcaccagtcaaggttgagaaacctgtcgataaagagaa gcctaaggattaa spaa opt-ecori spaa opt-sali XbaI-spaA opt atat gaattc atgggttaccaatcattcgaagc aatt gtcgac ttaatccttaggcttctctttatc atat tctagataagagaggttaccaa - 40 -
ClaI-spaA opt ClaI-cwp1 SalI-cwp1 aatt atcgat atccttaggcttctctttatc atat atcgat ttctactctgctctgtacgag aatt gtcgac ttacataagaagaacggccattc 그림 1 그림 2-41 -
μ - 42 -
그림 3 α α 표 1 Xba1-ORF2 XbaI-ORF2-NLS SalI-His-ORF2-R aatt tctaga taagagaatgacatatccgcgtagacgtt aaaa tctaga taagagaagactatcaaggacattcggatac atat gtcgac ctagtgatggtgatggtgatg agggttcaaaggt ggatct - 43 -
그림 4 α 그림 5 α α - 44 -
표 2 XbaI-Ty1 Ty1-R Ty1-R-His EcoRI-Amyss Amyss-R atat tctagataagaga atggaatcccaacaattatctc tttggttgtattcgtatagcgc aatt gtcgac ttagtgatggtgatggtgatg tttggttgtattcgtatagcgc aatt gaattc aaaa atgatggttgcatggtggtct gagataattgttgggattccat agctaatgctggagcagcta 그림 6-45 -
그림 7 α α - 46 -
ptef_f ptef_r ppdc_f ppdc_r ptef-borf2 ppdc-borf2 NotI-His-BORF2 NotI-BORF2 aaaa cccggg gggatttaaatataactgtcg attggcgaataactaaaatgtatgtag aaaa cccggg gggacaagcacacgattacc tgttgtgtactagctaattgattgatg ctacatacattttagttattcgccaat atgacttatccaagacgtcgttat catcaatcaattagctagtacacaaca atgacttatccaagacgtcgttat aatt gcggccgc ttagtgatggtgatggtgatggggttcaagggaggatcttt aatt gcggccgc ttacgggttcaagggaggatc 그림 8-47 -
그림 9 HpGAP-F HpGAP-R HpGAP-BORF Hind3-His-BORF2-R aatt ggatcc cctttgctcaatgccgtttt attgtttctatattatctttgtact agtacaaagataatatagaaacaat atgacttatccaagacgtcg atat aagctt ttagtgatggtgatggtgatg gggttcaagggaggatct - 48 -
그림 10 그림 11-49 -
clontech-f clontech-r GAATGGCGAATGGCGGGGATCCATCGCTTCGCTGATTAA ATACCGCATCAGGCGACAATGAGCCTTGCTGCAACATCC 그림 12-50 -
그림 13-51 -
그림 14 그림 15-52 -
그림 16-53 -
그림 17 표 3 A ORF2-HIS ORF2 세포파쇄액 Loading (µl) 0.5 1 1.5 2 2.5 3 0.025 0.05 0.1 0.25 0.5 Sample (µl) 0.375 0.750 1.125 1.500 1.875 2.250 0.01875 0.0375 0.075 0.1875 0.375 Protein concentration (mg/ml) 0.173 0.347 0.520 0.693 0.866 1.040 0.607 0.864 1.113 1.246 1.382 Density (vol. %) 0.838 1 1.005 1.407 1.787 2.071 1.212 1.742 2.257 2.53 2.811 B ORF2-HIS ORF2 세포파쇄액 Loading (µl) 0.5 1 1.5 2 2.5 3 0.025 0.05 0.1 0.25 0.5 Sample (µl) 0.375 0.750 1.125 1.500 1.875 2.250 0.01875 0.0375 0.075 0.1875 0.375 Protein concentration 0.173 0.347 0.520 0.693 0.866 1.040 0.862 1.032 1.286 1.492 1.915 (mg/ml) Density (vol. %) 0.833 1 0.99 1.27 1.599 1.947 1.604 1.918 2.387 2.767 3.548-54 -
그림 18-55 -
표 1 전배양배지 본배양배지 공급배지 성분 농도 (g / L) 성분 농도 (g / L) 성분 농도 (g / L) 포도당 20 포도당 15 포도당 600 효모추출액 10 MgSO 4 2 - - 펩톤 20 효모추출액 20 - - - - 펩톤 10 - - - - (NH 4 )SO 4 20 - - - - KH 2 PO 4 7 - - - 56 -
그림 1-57 -
그림 2 바인딩단백질이결합된항원단백질의고정화시스템 - 58 -
표 2 의생산을위한유가식배양에사용된배지조성 성분 초기배지 첨가배지 - 59 -
그림 재조합대장균 의유가식배양결과 - 60 -
표 발현된 의정량 수용성단백질중 의발현량 배양말기 배양말기 총단백질농도 발현양 표 4 의아비셀결합능력 구분용해성 정제 순도 - 61 -
표 1 시험백신 1 시험백신 2 시험백신 3 시험백신 4 시험백신 5 시험백신 6 시험백신 7 ORF2 ORF2 효모효모 ( 열-사멸 ( 포르말린- 화 ) 불활성화 ) 2x10 9 2x10 9 조성 cells/ml cells/ml ORF2 효모 ( 세포파쇄물 ) ORF2 (MF ORF2 (-NLS) ORF2 (+His) 양성대조군 2 mg/ml 0.6mg/ml 2 mg/ml 2 mg/ml - 보조제 수산화알루미늄젤 - 비고 NLS- 히스티딘포르말린- 열-사멸된 MF-처리결핍표지된사멸된세포효모세포 OFR2 ORF2 효모파쇄물전세포파쇄물세포세포전세포파쇄물파쇄물 Circoflex - 62 -
표 2 시험 시험 시험 시험 시험 시험 시험 음성 백신 1 백신 2 백신 3 백신 4 백신 5 백신 6 백신 7 대조군 ORF2효모 ( 열-사멸 ) ORF2 효모 ( 포르말린불활성화 ) ORF2 효모 ( 세포파쇄물 ) ORF2 (MF) ORF2 (-NLS) ORF2 +His Circoflecx ( 양성대조군 ) - 시료 1 2.36 2.37 2.09 1.58 0.44 1.72 1.96 0.34 시료 2 2.23 2.22 2.33 2.53 0.89 1.02 2.31 0.48 시료 3 1.96 2.32 2.39 2.37 0.66 1.59 2.24 시료 4 1.94 2.59 1.84 1.46 0.73 2.63 2.34 시료 5 2.01 2.09 2.40 2.55 0.87 1.15 2.47 시료 6 1.96 2.56 2.53 1.22 0.86 2.45 2.52 시료 7 2.32 2.23 2.02 0.61 2.16 2.10 시료 8 2.18 1.19 0.56 1.28 2.36 평균 2.11 2.36 2.25 1.87 0.70 1.75 2.29 0.41 T / C 5.15 5.76 5.49 4.55 1.71 4.28 5.58-63 -
그림 1-64 -
표 3 조성백신 1 백신 2 백신 3 백신 4 효모발현 PCV ORF2 효모균체 세포파쇄액 MF 처리군 효모균체 전체단백질 전체단백질 흡광도기준 기준 기준 양성대조군 (Circoflex) 접종량 2x10 9 cells/ ml 2.2 mg / ml 1.0 mg / ml 항원보조제 수산화알루미늄젤 표 4 그룹개체번호접종 1 주전접종 2 주후접종 4 주후접종 6 주후 시험백신 1 전세포 시험백신 2 세포파쇄물 시험백신 3 MF 처리된 세포파쇄물 양성대조군 (Circoflex) S/P S/P S/P S/P 1 0.94 0.55 0.59 0.41 2 0.88 1.01 0.5 0.32 3 0.82 0.75 0.66 0.52 평균 0.88 0.77 0.58 0.42 1 1.05 0.84 0.84 0.67 2 0.96 0.91 0.8 0.5 3 1.03 0.8 0.34 0.33 평균 1.01 0.85 0.66 0.5 1 0.65 0.71 0.81 0.7 2 0.96 0.92 0.73 0.54 3 0.91 0.87 0.95 0.95 평균 0.84 0.83 0.83 0.73 1 1.04 0.76 0.7 0.5 2 1 0.75 0.67 0.48 3 0.86 0.72 0.71 0.54 평균 0.97 0.74 0.69 0.51 음성대조군 1 0.95 0.98 0.96 0.97-65 -
그림 2 표 5 조성 vac1 vac2 vac3 vac4 vac5 vac6 vac7 vac8 vac9 vac10 항원 효모발현PCV-ORF2 전세포 PCV- 전세포 PCV-ORF2 squalene OFR2 동시발현전세포 +Ty1 항원량 2x10 7 / ml 2x10 8 / ml 2x10 9 / ml 2x10 9 / ml 2x10 7 / ml 2x10 8 / ml 2x10 9 / ml 2x10 9 / ml circoflex 무접종 군 백신 보조제 유유유무무무무무 비고 항원량결정시험 cell wall의면역증강효과 항원의효능확인항원량결정시험 항원의백신효능확인 양성 대조군 음성 대조군 - 66 -
표 6 채혈 3 주차 ELSIA 결과 혈청 1 혈청 2 혈청 3 혈청 4 혈청 5 평균OD T/C값 판정 vac 1 3.437 3.354 3.250 3.173 3.437 3.330 7.00 양성 vac 2 3.241 3.212 2.924 3.172 3.206 3.151 6.63 양성 vac 3 2.964 3.183 3.041 3.260 3.255 3.141 6.60 양성 vac 4 3.310 3.194 3.215 3.222 3.283 3.245 6.82 양성 vac 5 0.956 0.485 1.184 0.906 0.587 0.824 1.73 음성 vac 6 3.432 2.371 2.777 3.201 3.355 3.027 6.37 양성 vac 7 3.425 3.297 3.030 3.194 3.271 3.243 6.82 양성 vac 8 3.390 3.468 3.407 2.907 3.440 3.322 6.99 양성 양성대조군 2.886 3.305 3.001 2.846 2.976 3.003 6.32 양성 음성대조군 0.449 0.502 0.476 * 양성기준은 T/C값이 2.0 이상 표 7 채혈 5 주차 ELISA 결과 혈청 1 혈청 2 혈청 3 혈청 4 혈청 5 평균OD T/C값 판정 vac 1 3.444 3.445 3.394 3.346 3.374 3.401 8.49 양성 vac 2 3.416 3.220 3.430 3.421 3.078 3.313 8.27 양성 vac 3 3.331 3.202 3.410 3.333 3.318 3.319 8.29 양성 vac 4 3.419 3.501 3.462 3.469 3.212 3.413 8.52 양성 - 67 -
vac 5 0.932 0.759 0.594 2.186 0.489 0.992 2.48 양성 vac 6 3.442 3.442 3.461 3.488 3.458 8.63 양성 vac 7 2.921 2.369 2.990 2.793 2.618 2.738 6.84 양성 vac 8 2.763 2.462 2.760 2.413 2.600 6.49 양성 양성대조군 3.259 3.131 3.027 3.206 2.967 3.118 7.79 양성 음성대조군 0.354 0.447 0.401 그림 3-68 -
- 69 -
그림 1-70 -
1. general glycosylation pathway protein (624bp) locus_tag= HMPREF0357_10927 gtgaataaattatataggaaatttctaaagagagtattagattttgttctttctttagttgggattatattactatcaccagtattagtaattgttagtgta gttgggggaatatttctaaaaggtaacccattttttctacaagaaagacctggtaagaatggtaaaatattcaaattaataaaatttagaacgatga gcaatgaaaaagatgataacggtaatttactacctgcggatgaaagacttattccttatggaaagtttttaagatcaacatctatcgatgaattaccc gaactttttaacatactaaaaggagatatggcaattgttggtccacgtccattactaccaaagtatcttccgttatacaatagtgaacaaaaaagaag acatgatattttacctggattgacaggatatgctcaagttcatggacgaaatgatattgattggagtaaacgattagcattggatgtttggtatgttg ataattgttcttttgtactagattgtaaaatttttattaaaacggtacttgtaatttttaaacgcgatggaattacatcaacaaacgcgggacaaacattt atgacggagtttaaaggagacgaaaattag translation : 207aa, 23.6 kda, pi : 9.47 MNKLYRKFLKRVLDFVLSLVGIILLSPVLVIVSVVGGIFLKGNPFFLQERPGKNGKIFKLIKFRTMSNEKDDNGNLLPADERLIPYGKFLR STSIDELPELFNILKGDMAIVGPRPLLPKYLPLYNSEQKRRHDILPGLTGYAQVHGRNDIDWSKRLALD VWYVDNCSFVLDCKIFIKTVLVIFKRDGITSTNAGQTFMTEFKGDEN" 2. sialidase A : neuraminidase : locus_tag= ERH_0299 atgagaggggtatttaaattgaagaaaataagtaacattttcgtcgtgcttcttacacttgtattgggtattagtctttacccaagttctatctttgctat gaatgatctaaatcttaatgacctacacttaaagggagaaggtgctgatatttccgaccatttatcacacatcaaagaatttaaccaaggttccctaa ccttacgtttccgttctgaatcactatccagtgatcttactgcactgttttcaatatcaaacgcaaatttggaaaacaactatttcgcgctttactatcaaa acagtaccggtaagcttggcattgaattcagagatgaaaccggcaaacatattatcaatacttcgggcacaagtacccagcttgccaacgcaaact ggcacaccgttaccgttttaaatcatggaaatcgtgttgaaatatatgcagatggaatcttacttgcatcccaagataatattgatttcacgaaaatcc aatctttaccttggaatcgtatgaaactgggtggcgttgcacgtttaaaaggtgaaaacttatggccttttagtgggaaaatttcttccctaaacctaa cgcatgacatcctcgatgtcgataccattaaaacactgcataacgacactgcttcaactgttgatccatcgacttcagaagcaattgagctctatgca ccgggacaagatggcagtaaaaactaccgcattccaagtcttcttacaacgcaagatgggaccgtcttagctgcgattgataaacgtattacccat caagcagactggggaaatatcgatatcgcaatccgtagaagtcaagacagcggtaaaacatggtctgataccgaagtgattattgatctaaagtc agatccaaatgtatctcaggaaaatgtaaatacgaactatcaaagttccgcattcctcattgatgcggcaatggcacaagataaccgtaatggacg tatctatcttttagtcgatatgttcccagaatcacgtggattctttagtgtccaaaatgaagaccaagactttggtcaaccttatgtaaccaaagaagat ggtacccatattgttctgaaaggtactgatggcaaaaaatattacgcaaaagctgatggaaccgtatttgatctaaatactgatatcatcacgaatta ccgtgttgtcttagaatctgagaaaggcatgcctttcaatgatcttggtgacctttatgaaggtgatacgtatgtgggaaatatctaccttaagaaaa gtcctctacgcattcgtcaaaccgcttatttatggttaacccattcagatgatgacggtcaaacatggtcatcaccccatgatattacaccacaagtca aagcggattggatgcagttcattggaacgggtcccggtgttgggattcaacttaaaaatggcgaacttgccttcccgatttaccatactaaccgtca tggtggtaattcacaatctacatcaatgatcaaaagtgccgatggtattacatggcataaaacggaatcactcaatgacaatcgtaactttaacggtc aagttttaaattctaaaaccttaaataacggcggtgcattggtaactgaatctcaagtggtccaacttaataatggaaccctgaaacaatttgcgcga aatgtatccggaaaagttttggtaggtacttcttatgataatggagaaacctgggagaatgatcttgaaacacttccaattaccgatgtttactcaca aatgtccgccctacattataaccatgaaggtaaagagtatatcttacttgcaaatccaaatggtcctaaacgtaccaatggggctgtgaaactgcttc aagttttagaaggtgaacgtctcgtacttctttctaatacgcatatgcaaagtggaaattacgagtacaatgtggttcaaccacttcctgatggacatt tcggcttaatgtatgaacataaaaaacctgaaaacggggataatatgtcgatgatgtttaagcgcttctcctttgaagatttaggtgtcaaagatacc - 71 -
tcaagtctaagtcttgatattcaggatacctatcttgaaatcaaatcggataaggctttaattcctagttcaaacttaacgttaacgtataatacaactca aacaacaaaaccgaaacgtgttgatacgaacacattacgctttaacttagcagaacccgtcaccattctcaatggatttactgaagggtatcttgaa acacttgatggaaaagcactcaatattgcgaataaacaaggtcttgatgaatcgcttgaaacacttaaagcccttgattttgaaaacttagcaccaa gtgtgaaggatgcagcggatcaactgattaccaaagcagaactcttaagtgataatccctttaccatcggtgatttcatccaacaaattcatgcaatg attggtgcgattgatccaacttacattccaaactcaatgtttgatgtaacggcatcaagtcaagaagatgaaaaagggaatacacaagtagaaggt cccatcggtttagcctttgatggagacccaagtaccttctggcactccaaatggggtgataagaacccgccatttgatgtaacggtttcgttccatca tgacatggttgtgaatgcgttgacctatctaccacgtcaagacggaacccttaatggtgtcgtcacagaatatcaaatcttaggtaaattgggcgat gcaccacttcatgttctcaatgaaggaacccttgatacaaaccttacaaagaaaataatcacgtttgaggatgctcatgtggatacactgatctttaa agttgtgaaaggtcttgaaaactatggaaatgctgcggaaattaccctgcatggaaaaacgtatcgtgcacctcttgattattcaagcttaaatcaa gtacttaaaacacttgaaaactacaaccaaagtctttataccgaagcaagttttaaaccattacttgaattaaaagaaaaagcctatgagcttctcgat aactcagaaacaacacaggaaatgattgatgaaatggttataaacctcaatgaattcatcaataatctcgagttaagcaactatactttagattcatta cgtctgaaaattcaagaggttgaagattatatgaatacacttgatttggctctctacacacagacaagtgtacaggctgtttatgatgctcttgattca gcgaaagcactgcttcaaacgccacgcttcagttttagaagtctcttcaaagcttctacaccaaacgaagctcagattaaagaagccattgaatcctt ggactcagccgttagaggtttaagcaaacgtgaggttgaaacagaggaaccatcaaatcctgatgaaaacatcacacctccaatcgtagatccaa aaccggatacggattctcaaaaacctgaaactccagatagcaatactgaagacagtgctcaaatttcacctaccgacaaaaccgatgtaacacttc ctaaaacaggacaagctgtttctggatttatcggacttggtatgcttgtatccctatgtggttttgtgatttttaaacgaaaacaataa translation : 1,204 aa, 133.8 kda, pi: 4.86 MRGVFKLKKISNIFVVLLTLVLGISLYPSSIFAMNDLNLNDLHLKGEGADISDHLSHIKEFNQGSLTLRFRSESLSSDLT ALFSISNANLENNYFALYYQNSTGKLGIEFRDETGKHIINTSGTSTQLANANWHTVTVLNHGNRVEIYADGILLASQDNIDFTKIQSL PWNRMKLGGVARLKGENLWPFSGKISSLNLTHDILDVDTIKTLHNDTASTVDPSTSEAIELYAPGQDGSKNYRIPSLLTTQDGTVLA AIDKRITHQADWGNIDIAIRRSQDSGKTWSDTEVIIDLKSDPNVSQENVNTNYQSSAFLIDAAMAQDNRNGRIYLLVDMFPESRG FFSVQNEDQDFGQPYVTKEDGTHIVLKGTDGKKYYAKADGTVFDLNTDIITNYRVVLESEKGMPFNDLGDLYEGDTYVGNIYLKK SPLRIRQTAYLWLTHSDDDGQTWSSPHDITPQVKADWMQFIGTGPGVGIQLKNGELAFPIYHTNRHGGNSQSTSMIKSADGIT WHKTESLNDNRNFNGQVLNSKTLNNGGALVTESQVVQLNNGTLKQFARNVSGKVLVGTSYDNGETWENDLETLPITDVYSQ MSALHYNHEGKEYILLANPNGPKRTNGAVKLLQVLEGERLVLLSNTHMQSGNYEYNVVQPLPDGHFGLMYEHKKPENGDNMS MMFKRFSFEDLGVKDTSSLSLDIQDTYLEIKSDKALIPSSNLTLTYNTTQTTKPKRVDTNTLRFNLAEPVTILNGFTEGYLETLDGKALNIA NKQGLDESLETLKALDFENLAPSVKDAADQLITKAELLSDNPFTIGDFIQQIHAMIGAIDPTYIPNSMFDVTASSQEDEKGNTQVEGP IGLAFDGDPSTFWHSKWGDKNPPFDVTVSFHHDMVVNALTYLPRQDGTLNGVVTEYQILGKLGDAPLHVLNEGTLDTNLTKKIIT FEDAHVDTLIFKVVKGLENYGNAAEITLHGKTYRAPLDYSSLNQVLKTLENYNQSLYTEASFKPLLELKEKAYELLDNSETTQEMIDEMV INLNEFINNLELSNYTLDSLRLKIQEVEDYMNTLDLALYTQTSVQAVYDALDSAKALLQTPRFSFRSLFKASTPNEAQIKEAIESLDSAVR GLSKREVETEEPSNPDENITPPIVDPKPDTDSQKPETPDSNTEDSAQISPTDKTDVTLPKTGQAVSGFIGLG ml VSLCGFVIFKRKQ. 3. hypothetical cytosolic protein : locus_tag= ERH_0130 atgcttagtactgtttgggaggagatgtccatgaaagaaactaaaaaaattaaaagaagaattattcttagtattttcgtattaattgttgcatgcgcatgtctata ttacttagttacatttgatgcgggtgagagtcctcaagaatttccatatgagagtggaaactatattgtgggaacagatttagatccaggagaatatatgcttctta cggatcaagaaggctattttgaaataagaaatcaaaattcatcacagattaagagcaacgacaattttaaagggaattcaattatcactttaaatgagaatg aagtaattgaggtttcaagagcacttatatatccttatatagcttcgccagaactcaagcctcaagcagatggcatgtataagattggggaacatttagatgctg gaacgtataaattaaaaactgaattaagtggttattatgaaatcacaacatcgtccgatcataatgtcgacgatattcttgagaataataattttacggatacagt tacgatttatgttaaggacggggagtatctaaa acttaattcagccttgattcttgaacgaccatcatcataa translation : 199 aa, 22.6 kda, pi: 4.51 ml STVWEEMSMKETKKIKRRIILSIFVLIVACACLYYLVTFDAGESPQEFPYESGNYIVGTDLDPGEY ml LTDQEGYFEI RNQNSSQIKSNDNFKGNSIITLNENEVIEVSRALIYPYIASPELKPQADGMYKIGEHLDAGTYKLKTELSGYYEITTSSD - 72 -
HNVDDILENNNFTDTVTIYVKDGEYLKLNSALILERPSS. 4. small conductance mechanosensitive ion channel (MscS) family transporter : NC_015601, locus_tag= ERH_0358 atggaaaatatcatattattttttgaaaaaccgattgttttacgtttgataagcattgcattaacgctgcttattgtgtttgtgattgttcgagtcatacgtagaagtttaa ttaaaattgaaaataaagtaccacttgggaaacaacaacgaactgtatacccaatcatttcaagaacactggtgtatgttgtttatttcattgcgtttatcataatttt agaaacaattggattggatacaaaaccgattcttgcggtcacaagtattggaagtgttgcggtagggtttggtgcgcaacaacttgtaaaggatgtcattaatg gattctttattctatctgagaatcaatttaatgttggtgatgtggtttcgttgtcagatgtgacaggaacggttgaagacatcacattacgtacgacgcgtttacgcaa tggcactgatgggaaggtttatatcatacccaatggtgagattaagatggttacaaatatgtcgaaagagtatatgtttgcggttgtcaatgtacccgttccttacg ataaagatctggatactattttagaagttttaaatgataccgtgaaacactaccaaaatccgggttcaatcatgcaggcaccagttgtacagggtgttacagaat atgaggaaagctcattaaatatacggattacatgtaaaacgtatgtgggtaagaattgggcggtggaacgcgatctgcgacgtgttattatctatgcacttgag gcacatgatatttcactaccatttccaacacgtactgtttatgttgaacaagatttatccaactag translation : 271 aa, 30.5 kda, pi: 6.18 MENIILFFEKPIVLRLISIALTLLIVFVIVRVIRRSLIKIENKVPLGKQQRTVYPIISRTLVYVVYFIAFIIILETIGLDTKPILAVTSIGSVAVGFGA QQLVKDVINGFFILSENQFNVGDVVSLSDVTGTVEDITLRTTRLRNGTDGKVYIIPNGEIKMVTNMSKEYMFAVVNVPVPYDKDLD TILEVLNDTVKHYQNPGSIMQAPVVQGVTEYEESSLNIRITCKTYVGKNWAVERDLRRVIIYALEAHDISLPFPTRTVYVEQDLSN. 5. hypothetical protein HMPREF0357_1207 : locus_tag= ERH_1503 atgaagcaacacagtaaaggtcttaccatttttatattaattctttcacttacaactttgagtgtaggtggtttttttgcgtaccgtgcataccaatcaaaaacaagtatt gatggtggtactacaagcgagaacagtttttattcacttcgcaaaaatgcaacggaatatcaaaaagaactctataaggaacttacctcgaaacttaaagaa gatccaagggatgataaagtaatctcagagctgatcgctcaaaactttgtcgctgatttttatacatggaccaataagttgcgttttaacgatgtaggtggaatgc agtatattcataaggacctcgactgggtttatggacaagcgctggatactttctataatgatatgcgctactacaaagaaaaagggaagttagaccaaacactt gaagttacttcatcatcagcaagtgctaaaaaagataagttagttttaatagaacaagaagatgagttggtgacgcttgaagatggtacggttaatacggtta ccaacgatgtggaacgcaccattccagtatatcgtgtctcgattacatggaaatacaaagactcagatgttttaaatgtttctgagtttcaacagaaagcagatatt tatgtaacgaaagatgaagatggattgtattcaatcatggaggtagacgatggccaagtcaaagaaacaacaaactaa translation : 236 aa, 27.2 kda, pi: 4.83 MKQHSKGLTIFILILSLTTLSVGGFFAYRAYQSKTSIDGGTTSENSFYSLRKNATEYQKELYKELTSKLKEDPRDDKVIS ELIAQNFVADFYTWTNKLRFNDVGGMQYIHKDLDWVYGQALDTFYNDMRYYKEKGKLDQTLEVTSSSASAKKDKLVLIEQEDEL VTLEDGTVNTVTNDVERTIPVYRVSITWKYKDSDVLNVSEFQQKADIYVTKDEDGLYSIMEVDDGQVKETTN. 6. hypothetical protein HMPREF0357 : locus_tag= ERH_1567 atgaaacgtctaagttctgttttagttttattgcttattttaacagggtgtgcaaagactccagaagaacctgaagtcattaaaccacccgtgaatacctttgaaacc attgtgtttaaattaggaggtaaagattatcaacttcctgtatcttatcaaacgttatttgaagatggttgggagccaactacagatattgattccataacacttgcttc caatagttatacagacggatatgctttacgtaaagatcgtaatattatttggattgcatttttcaatgccgcagatgaggaaaaacttttagaagaaacgcttgttg catcaataagtgcagagaatcgcgagtcctattacgatgatccggttgatattgttgtgcatgaagggatttcatttgatacacctaaagaacaaatcattgagc aattaggtgaatttaaggaagatgagaatgcccgatttaaaaacataacatttgagcacaataaaaaagctaagagtactttcaagtttgatgttgattcagg acgcatggaatatattgaaataactaattatcgaaaaccgattcagtaa translation : 193 aa, 22.2 kda, pi: 4.64 MKRLSSVLVLLLILTGCAKTPEEPEVIKPPVNTFETIVFKLGGKDYQLPVSYQTLFEDGWEPTTDIDSITLASNSYTDGY ALRKDRNIIWIAFFNAADEEKLLEETLVASISAENRESYYDDPVDIVVHEGISFDTPKEQIIEQLGEFKEDENARFKNIT FEHNKKAKSTFKFDVDSGRMEYIEITNYRKPIQ. 7. pyridine nucleotide-disulphide oxidoreductase family protein : locus_tag= ERH_0304 atgagtaaaagaatattaattgttggaggggtagctggaggtgcttcagttgcagctcgtgttcgtcgtcaagatgaacatgcggaagtcattatgtttgaacgc ggaccgaatgtgtccttctcaaactgtgccttgccttaccacttaagtggcattgtcgcatcaagtgatgacttagtcttaatgacaccagaacaatttaaaaaaca - 73 -
atataacatcattgcacgcacaagcactgaagttctaaaaattaatcgtgatgcgaaaactgtaacggttaaaaatcttgaaaccggtgaagaaatggatga agcatatgacaagcttgtcttatcaccaggagcaagtgcaattttaccacgcagtatcgaaggcgttgacggtgataatgtctttaccgtacgtaatgttgtcgaca ttaaaaaattaaacggacatatcgtcaacaacaatattgaaaacattaccgttgttggtggtggatttatcggtgttgaagttgcggaaaacttgcgtcatgcag gtaaaaacgtaacacttgtggaagcaattgatcaaatcatgagtccattcgactatgatatggcacaagtactacataaagaaatgatggatcacggcattaa cctgattctgggtgatggcgttaagaagattacagaaacaagtgttgaactgggttcaggaacaatcttagaatccggtgcagttgtaatggcaatcggtgttg cacctgaaacaagtcttgcgaaagctgcagatcttgaaatcggtgttacaggtggaattaaagttgatcacaattaccttacaaatgataaaaacatttatgca gtgggtgatgcgattgaagtcttccatcaaatcacacataaaccaacacgtcttgcacttgcaggtcctgctcaacgtcaagcacgtgcagccgcagatcatatgt atggaattcctcataacaataaaggtgttgttggatcatcatgtgttcaagtatttgacttaaatgctgcttcaaccggtcttaatgaaaaaatggcaaaggctaa cggtttcgattatgactttgtttacattatccctggtgataaagttggattaatgccagatgcaaatccaatgcactttaaactcttatttgaatatccaacaggtaaatt actgggtgcacaagcaatcggtaagggaaatgttgataaacgtattgatgtgattgcgacaatgattcaaatgggtggaactcttgaagatcttaaagaagtt gaattatgctatgcaccaccatttggtacggctaaagatgtcgtgaatcatgcagcgcttgttggtttaaaccttctttacggtcgtttcaaccaagttcctgtaacgca agttcgtgaattggtagagtccaatgcatgtattatagatgttcgtgaagaacatgaatatgcattaagtcaccttatcaatgcagtaaatattccattaagccaa attcgtgatcgcttggatgaaattcctacagataaacctgtatatttacactgtcgttcaagccaacgttcatacaatgctgtaatggcacttcaaaaccttggttttga taatgtgattaatatttcaggatcatatcttggaatctgtgtttatgaatactttaatgatcaagtcacaggccgtgataaaatcgttacagaatacaactttaacta a translation : 565 aa, 61.5 kda, pi: 5.37 MSKRILIVGGVAGGASVAARVRRQDEHAEVIMFERGPNVSFSNCALPYHLSGIVASSDDLVLMTPEQFKKQYNIIARTSTEVLKINR DAKTVTVKNLETGEEMDEAYDKLVLSPGASAILPRSIEGVDGDNVFTVRNVVDIKKLNGHIVNNNIENITVVGGGFIGVEVAENLR HAGKNVTLVEAIDQIMSPFDYDMAQVLHKEMMDHGINLILGDGVKKITETSVELGSGTILESGAVVMAIGVAPETSLAKAADLEIG VTGGIKVDHNYLTNDKNIYAVGDAIEVFHQITHKPTRLALAGPAQRQARAAADHMYGIPHNNKGVVGSSCVQVFDLNAASTGL NEKMAKANGFDYDFVYIIPGDKVGLMPDANPMHFKLLFEYPTGKLLGAQAIGKGNVDKRIDVIATMIQMGGTLEDLKEVELCYAP PFGTAKDVVNHAALVGLNLLYGRFNQVPVTQVRELVESNACIIDVREEHEYALSHLINAVNIPLSQIRDRLDEIPTDKPVYLHCRSSQR SYNAVMALQNLGFDNVINISGSYLGICVYEYFNDQVTGRDKIVTEYNFN. ( 밑줄 : KEX2 cleavage site, 빨간색 : N-glycosylation site) 표 1-74 -
그림 2-75 -
그림 3 그림 4-76 -
- 77 -
표 2-78 -
표 3-79 -
표 4 GeneSymbol Systematic Name Mutant type 1 KAR1 YNL188W DAmP Description Protein involved in karyogamy and spindle pole body duplication 2 YPT52 YKR015C Knock out Putative protein of unknown function 3 MTR4 YJL049W Knock out Putative protein of unknown function 4 VPS51 YKR023W Knock out Putative protein of unknown function 5 IFM1 YOL023W Knock out Mitochondrial translation initiation factor 2 6 IPL1 YPL209C DAmP Aurora kinase subunit of the conserved chromosomal passenger complex 7 KEL2 YGR238C Knock out Protein that negatively regulates mitotic exit 8 KAR3 YPR141C Knock out Minus-end-directed microtubule motor 9 ZIP1 YDR285W Knock out Transverse filament protein of the synaptonemal complex 10 VIK1 YPL253C Knock out Protein that forms a complex with Kar3p at the spindle pole body 11 RBS1 YDL189W Knock out Protein of unknown function 12 TOM40 YMR204C Knock out Peripheral membrane protein of peroxisomes involved in peroxisomal inheritance 13 UBP16 YPL072W Knock out Deubiquitinating enzyme anchored to the outer mitochondrial membrane 14 CIN8 YEL061C Knock out Kinesin motor protein 15 SHE3 YBR130C Knock out Protein adaptor between Myo4p and the She2p-mRNA complex 16 TID3 YIL144W DAmP Component of the kinetochore-associated Ndc80 complex 17 PZF1 YPR186C DAmP Transcription factor IIIA (TFIIIA) 18 KAR5 YMR065W Knock out 19 BUB1 YGR188C Knock out Protein required for nuclear membrane fusion during karyogamy Protein kinase involved in the cell cycle checkpoint into anaphase - 80 -
- 81 -
그림 5 α α 표 5 VIK1-EcoRI-F VIK1-SalI-F KAR3-MunI-F KAR3-SalI-R pgal10-saci-f pgal10-sphi-r ZIP1-SphI-F ZIP1-SalI-R TATA GAATTC AAAA ATGGCGTCACAGCAAAAC TATA GTCGAC TTA AGTGAGCTTTTTCTTTCTTTTAC TATA CAATTG AAAA ATGGAATCACTTCCACGTAC TATA GTCGAC TCA TTTTCTACTAACCAATCTGGT TATC GAGCTC GGTACCCGGG AATA GCATGC TTGAATTTTCAAAAATTCTTACT ATAT GCATGC AAAA ATGTCAAACTTTTTTAGAGATAG TTAA GTCGAC CTA TTTCCTTCTCCTTTTCTTG 유 - 82 -
그림 6-83 -
그림 7-84 -
그림 8-85 -
그림 9 [ 서열 1] - 86 -
GGTTACCAATCATTCGAAGCCGTCAACGAGGAAATTAACTCTATCGTCTCCGAATTGA AAAATGAGGGAATGTCTCTACAAAATATTCATCACATGTTTAAACAATCCATCCAGAA CCTGGCTACTCGTATCGGTTATAGGTCTTTTATGCAGGATGCTATGTACTTGGAGAA TTTCGAACGTTTGACAATTCCTGAATTAGATGAAGCCTATGTTGACTTGTTGGTTAA TTATGAAGTCAAGCACAGAATCTTAGTTAAGTATGAGGACAAAGTCAAGGGTAGAGC ACCACTAGAAGCATTCATCGTTCCACTGAGGAACAGGATTAGATCTATGAACGAAATT GCTGCAGAAGTTAATTATCTGCCTGAAGCTCATGAAGATTTCCTAGTCAGTGATTCT TCAGAGTACAACGACAAATTGAATAACATTAATTTTGCCTTGGGTTTAGGCGTTTCC GAATTTATCGACTACAATAGATTAGAGAACATGATGGAGAAAGAGATTCATCCACTT TATTTGGAATTGTACGCAATGAGGCGTAACAGACAGATCCAAGTAGTTCGTGACGTC TATCCTAATTTAGAAAGAGCTAACGCAGTTGTCGAAAGTCTTAAGACAATTAAAGAT ATTAAACAGCGTGAGAAGAAACTACAAGAATTGTTGGAGATTTACATTCAAAGATCT GGTGACGTTAGGAAACCAGATGTCTTACAGAGATTCATCGGAAAATATCAGTCAGTT GTTGATGAAGAGAAGAATAAGCTACAAGACTATCTTGAAAGTGATATCTTTGATAGC TATTCAGTCGATGGCGAAAAGATTAGAAATAAAGAGATTACCTTGATCAACCGTGAC GCTTACTTGTCCATGATTTACAGAGCTCAATCTATTTCAGAGATCAAGACTATCAGG GCCGACTTAGAATCTTTAGTAAAGAGTTTTCAGAATGAGGAATCTGACTCTAAAGTT GAACCTGAATCACCAGTCAAGGTTGAGAAACCTGTCGATAAAGAGAAGCCTAAGGATT AATAG spaa opt-ecori : ATATGAATTCATGGGTTACCAATCATTCGAAGC spaa opt-sali : AATTGTCGACTTAATCCTTAGGCTTCTCTTTATC spaa opt-xbai : ATATTCTAGATAAGAGAGGTTACCAA spaa opt-clai : AATTATCGATATCCTTAGGCTTCTCTTTATC α α α - 87 -
그림 10 그림 11-88 -
나. E2 의클로닝및발현확인 [ 서열 2] ATGAGATTGGCATGTAAAGAAGATTATCGTTACGCATTGAGTTCAACTAATGAAATT GGTTTGTTGGGAGCAGGTGGTCTTACAACTACCTGGGAAGAGTATAATCACGATTTG CAACTAAATGATGGTACAGTTAAGGCTATTTGTGTAGCTGGTTCCTTTAAGATAACT GCCCTAAATGTGGTGTCTAGGAGGTACTTAGCTAGCTTGCATAAAGAAGCCTTACCA ACTTCCGTTACTTTCGAACTTTTGTTTGATGGCACTAATCCATCAACTGAAGAGATG GGTGATGACTTTGGCTTTGGCTTGTGTCCATTCGACACAAGTCCAGTTGTAAAAGGT AAATATAATACAACTCTATTAAATGGATCAGCTTTCTACTTAGTTTGTCCAATAGGA TGGACTGGTGTTATTGAGTGTACCGCGGTTTCTCCAACTACTTTGAGAACCGAAGTC GTTAAGACTTTCAGAAGAGACAAACCGTTTCCACATAGGATGGATTGCGTCACAACTA CAGTAGAGAACGAAGATTTATTTTATTGTAACTGGGGAGGTAACTGGACTTGTGTTA AAGGAGAGCCAGTGGTTTATGCTGGTGGCCTTGTTAAACAATGCAGATGGTGCGGTT TCGATTTTAACGAACCTGATGGTTTGCCACACTATCCAATCGGAAAGTGCATTTTGG CTAACGAGACTGGATACAGAATAGTTGACTCTACAGATTGTAATAGAGATGGTGTTG TAATTAGTACTGAAGGCTCACATGAATGTTTAATCGGTAATACCACTGTCAAAGTAC ATGCTTCCGACGAACGTTTAGGACCAATGCCATGTAGACCAAAAGAGACTGTCTCGTC TGAAGGTCCAGTTAGGAAGACGTCTTGCACATTTAACTATGCAAAGACTTTGAAGAA TAAATACTATGAGCCACGTGATAGTTATTTTCAGCAATACATGTTGAAAGGTGAATA TCAGTACTGGTTCGACTTGGATGTTACAGATCGACATTCTGACTATTTTGCCGAATA ATAG E2 opt-ecori:atatgaattcatgagattggcatgtaaagaag E2 opt-sali:aattgtcgacttattcggcaaaatagtcagaatg E2 opt-xbai:atattctagataagagaatgagattgg - 89 -
E2 opt-clai:aattatcgatttcggcaaaatagtcagaatg α α 그림 12 그림 13-90 -
α α α 그림 14-91 -
α F: gaatggcgaatggcg gggatccatcgcttcgctgattaa R: ataccgcatcaggcg acaatgagccttgctgcaacatcc 그림 15-92 -
황산암모늄 % 0 10 15 20 25 30 40 50 60 황산암모늄mg 0 56 84 114 144 176 214 313 390 그림 1-93 -
그림 2 PEG6000 % 0 3 6 9 NaCl M 0 0.5 0 0.5 0 0.5 0 0.5-94 -
그림 3-95 -
그림 4 그림 5-96 -
그림 6-97 -
표 1 그림 7-98 -
표 2-99 -
그림 8-100 -
그림 9-101 -
그림 10 그림 10. MBP:TEV:ORF2 와 TEV 반응기작 그림 11-102 -
그림 12 다. - 103 -
그림 13 μ - 104 -
- 105 -
- 106 -
시험백신 1 시험백신 2 시험백신 3 시험백신 4 시험백신 5 조성 spaa 효모전세포 2x10 8 cell/ ml spaa 효모 spaa 효모양성대조군음성대조군세포파쇄물세포파쇄물 20μg / ml 100μg / ml ERT2T - 보조제수산화알루미늄겔 - - 시험백신 1 시험백신 2 시험백신 3 시험백신 4 시험백신 5 조성 spaa 효모 spaa 효모 spaa 효모양성대조군음성대조군 - 107 -
전세포세포파쇄물세포파쇄물 접종방법피하피하피하피하 - 마리수 15 15 15 15 5 시험결과 0/15 8/15 12/15 15/15 0/5 ( 생존율 ) (0%) (53%) (80%) (100%) (0%) - 108 -
시험백신 1 시험백신 2 시험백신 3 시험백신 4 시험백신 5 조성 spaa-e2 효모전세포 spaa-e2 세포파쇄액 양성대조군 1 양성대조군 2 음성대조군 2x10 9 / ml 1 mg / ml CSFV E2 ERT2T - 보조제수산화알루미늄겔 - - - 시험백신 1 시험백신 2 시험백신 3 시험백신 4 시험백신 5 조성 spaa-e2 효모전세포 spaa-e2 세포파쇄액 양성대조군 1 (CSFV E2) 양성대조군 2 (ERT2T) 음성대조군 접종방법피하피하 - 피하 - 마리수 10 10-10 5 시험결과 ( 생존율 ) 1/10 (10%) 5/10 (50%) - 10/10 (100%) 0/5 (0%) - 109 -
시험백신 1 시험백신 2 시험백신 3 시험백신 4 시험백신 5 조성 spaa-e2 효모전세포 spaa-e2 세포파쇄액 양성대조군 1 (CSFV E2) 양성대조군 2 (ERT2T) 음성대조군 시료 1 0.961 0.878 0.713-0.983 시료 2 0.905 0.916 0.566-0.935 시료 3 0.887 1.008 0.815 - 시료 4 0.918 0.921 0.571 - 시료 5 0.923 0.893 0.827 - 평균 0.918 0.923 0.6984-0.94 Blocking rate 2.0% 1.5% 25.5% - 0% 시험백신 314TMX 01 항원함량및혼합비율 Yeast PCV ORF2 2x10 8 CFU/ ml Adjuvant 30% 분병수량 2 ml / 두분 500 병 - 110 -
시험백신 22 37 NA NB Thio NA NB Thio 비고 314TMX 01 -* - - - - - 시험백신 시험재료 배양방법 시험결과 비고 314TMX 01 Chocolate agar 37, 24~48시간 집락형성 (-) 대조군 Chocolate agar 37, 24~48시간 집락형성 (-) - 111 -
시험백신 314TMX 01 추세선수식 (Linear) y=0.0112x + 0.0006 R 2 =0.9952 OD 520 (x 값 ) 희석배수포르말린판정기준판정농도 (%) 0.100 100 배 0.17 0.2% 적합 Group 314TMX 01 동물 접종부위및접종량 관찰기간 생존수 / 전체수 임상관찰결과 마우스 복강, 0.5ml 7일 8/8 이상없이생존 기니픽 피하, 2.0ml 7일 2/2 이상없이생존피내, 0.1ml 7일 2/2 이상없이생존 - 112 -
TMX 01 대조군 Group O.D.1 O.D.2 평균 OD 값 1 0.994 0.964 2 0.720 0.785 3 0.971 0.999 4 0.730 0.596 5 1.335 1.215 6 0.973 0.875 C13 0.195 0.207 C14 0.231 0.228 C15 0.329 0.300 C16 0.184 0.168 T/C (Test OD/Control OD) 0.930 4.04 0.230 Group 구분 접종수 접종부위및접종량 TMX 01 vaccination 2 근육, 4ml PBS Control 1 근육, 4ml 관찰기간 21 days 임상관찰결과이상없이생존이상없이생존 - 113 -
Group 314TMX 01 양성대조군음성대조군 PCV-2 ELISA S/P ratio (JENOBIOTECH) 접종전 1차접종 2주후 1차접종 3주후 51 0.41 1.10 1.17 52 0.59 1.54 1.40 53 0.91 1.05 0.89 54 0.45 1.41 1.34 55 0.68 0.97 1.00 61 0.51 0.32 0.35 62 1.36 0.74 0.56 63 0.52 0.68 0.72 64 0.72 0.57 0.59 65 0.99 0.63 0.69 66 0.60 0.49 0.60 67 0.78 1.08 1.35 68 0.78 0.44 0.42 Group Average S/P ratio (JENOBIOTECH) 접종전 1 차접종 2 주후 1 차접종 3 주후 314TMX 01 0.61 1.21 1.16 양성대조군 0.78 0.58 0.56 음성대조군 0.79 0.66 0.77-114 -
- 115 -
- 116 -
- 117 -
제 4 장 목표달성도및관련분야기여도 제 세부과제 유효항원스크리닝및효모발현시스템구축 제 세부과제 항원생산공정개발 협동과제 백신효능검정 돈단독백신후보유전자스크리닝 백신후보유전자 발현카세트제조 실험실규모에서의재조합항원후보단백질생산공정개발 실험실규모에서의재조합항원후보단백질분리정제공정개발 동물실험을통한유효항원선발시험 돈단독원인균 균 주의 를 벡터에구축하여유효항원을스크리닝하고 서열분석을통하여돈단독백신후보유전 자를선별 백신후보유전자 를효모최적 코돈으로합성하고 등과의 융합발현을시도하였으며단백질발현과전 자현미경을통해 형성을확인 융합발현 시스템개발 유가식배양공정을통한항원단백질생산 공정개발 를 으로사용한항원단백 질 분리정제공정개발 항원단백질을셀룰로우즈담체에고정화 하는기법을돈단독질병예방을위한백신 에적용마우스 기니픽을통한재조합항원의면역 원성및안정성확인 백신보조제비교시험및선발 항원과백신보조제의제제화실험 - 118 -
제 세부과제 유효항원스크리닝및효모발현시스템구축 제 세부과제 항원생산공정개발 협동과제 백신효능검정 돈단독항원유전자효모발현시스템구축 백신유전자발현시스템구축 실험실규모재조합항원단백질의생산공정확립 동물실험을위한재조합항원단백질의생산 시험백신제제화 백신의안전성및유효 성평가 돈단독원인균 균주의표면항원 를효모세포표면부착매개단백질 과융합하여사 카로마이세스세레비지에에서발현하고세포부착을확인함 백신유전자 를효모최적코돈으로합성하고발현 최적화를위한숙주세포및프로모터를확정하였고분비발현 가능성을조사하였으며 등과융합발현 뿐만아니라 및 등비전통효모에서 유전자발현율을조사 역할을하는스쿠알렌을 와동시에 생산하는사카로마이세스세레비지에균주를구축 를순수정제하여재조합효모에서생산되는 의 정량법확립 유가식배양공정을통한재조합효모및재조합대장균항원 단백질생산공정개발 재조합효모를통한항원단백질생산및분리정제공정개발 발효 원심분리 세포회수 세포파쇄 원 심분리 실험실규모 리터 발효조를사용하여재조합효모균체를 수득하고 후 를제조하여 과함께동물시험을위해협동기관에공급 실험실규모 리터 발효조를이용하여 가부착된 돈단독항원단백질 를생산하여 셀룰로우즈담체에결 합된항원단백질을협동기관에공급 과 처리를통한백신의불활성화 시험수행 백신제제화를위하여백신보조제및면역증강제를포함하는 시험백신을제조 정제수준 에따른백신의안전성및유효성시험실시 실험동물 기니픽 에서항원및항원량결정시험수행 목적동물 돼지 에서면역원성지속기간확인을위해 주및 주차채혈 실시 - 119 -
제 세부과제 유전자발현유효항원최적화스크리닝및효모발현시스템구축 돈단독원인균의 를구축하고 종의 을스크리닝하여발현후항혈청과의반응성을조사 를과발현하는효모숙주세포에서발현량이증감하는유전자를 를통하여도출하고이들의결손시와과발현시 의발현량에미치는영향을조사 유전자발현의최적효모숙주세포를선별하고최적프로모터를선정 표면발현최적화 와 를각각분비발현및표면발현하고형광현미경등을통하여표면발현을확인함 동시발현도수행 제 세부과제 항원생산공정개발 협동과제 백신효능검정 파일럿스케일에서의생산공정개발파일럿스케일에서의분리정제공정개발 시험백신대량생산 시험백신의목적동물안전성및효능성시험 리터발효조를사용하여 를발효생산한후대장균에서순수분리정제한표준품을사용하여정량적생산성을분석 효모에서생산된 의분리및안정화를수행하였으며대장균에서 을통한가용발현및순수분리공정을개발하고표준품을제조 돼지 두분의시험백신을제조하고특성시험 무균시험 불활화학인시험 방부제정량시험등을수행 및 의효모표면발현시면역원성을마우스를사용하여조사 시험백신의실험동물에대한안전성시험및면역원성시험 그리고목적동물에대한안전성시험및효력시험을수행 - 120 -
제 5 장 연구개발성과및성과활용계획 - 121 -
항원성단백질 고정화를통한돈단독백신 제조방법 - 122 -
- 123 -
- 124 -
년도 사업목표 사업화과제 * 야외농장시험후 2016년 효능평가품목허가 ( 항체가확인시험취득및목적동물 공격접종시험 ) * 제품의대량생산 * 최초생산 Lot. 의자가검정제품생산합격및 * 국가검정합격국가검정합격, * 국내판매개시국내판매, 2017년 * 국내마케팅기획 / 해외등록진행실행 * 해외등록서류 ( 영문 ) 진행 사업 화 품목 투자계획 판매계획 재료비인경상생산및매출 ( 수출 ( 건운영계계획설비투억원 ) 억원 ) 비비자비 계 1.3 3.0 0.3 4.6 _ 1.1 2.7 0.3 4.1 30 _ 30 국내판매 / 해외등록준비 2018 년국내판매 / 해외판매 * 해외 ( 동남아 ) 등록 ( 베트남, 태국, 필리핀 ) 국내판매및동남아판매 - - - 35-35 - 125 -
- 126 -
보고서겉표지뒷면하단에다음문구삽입 주 의 이보고서는농림축산식품부에서시행한생명산업기술개발사업의연구보고 서입니다 이보고서내용을발표할때에는반드시농림축산식품부에서시행한생명 산업기술개발사업의연구결과임을밝혀야합니다 국가과학기술기밀유지에필요한내용은대외적으로발표또는공개하여서는 아니됩니다 - 127 -