KR960023066A - 전에스 (s) 2 펩티드 함유 비 (b) 형 간염 표면항원의 정제 방법 - Google Patents
전에스 (s) 2 펩티드 함유 비 (b) 형 간염 표면항원의 정제 방법 Download PDFInfo
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- KR960023066A KR960023066A KR1019950044795A KR19950044795A KR960023066A KR 960023066 A KR960023066 A KR 960023066A KR 1019950044795 A KR1019950044795 A KR 1019950044795A KR 19950044795 A KR19950044795 A KR 19950044795A KR 960023066 A KR960023066 A KR 960023066A
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- 102000036639 antigens Human genes 0.000 title claims abstract description 16
- 108091007433 antigens Proteins 0.000 title claims abstract description 16
- 239000000427 antigen Substances 0.000 title claims abstract description 15
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 5
- 238000000034 method Methods 0.000 title claims abstract 21
- 208000006454 hepatitis Diseases 0.000 title 2
- 231100000283 hepatitis Toxicity 0.000 title 2
- 150000003839 salts Chemical class 0.000 claims abstract description 8
- 230000003196 chaotropic effect Effects 0.000 claims abstract description 6
- 208000002672 hepatitis B Diseases 0.000 claims abstract description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract 10
- 210000004027 cell Anatomy 0.000 claims abstract 10
- 239000000377 silicon dioxide Substances 0.000 claims abstract 5
- 238000004587 chromatography analysis Methods 0.000 claims abstract 3
- 230000002209 hydrophobic effect Effects 0.000 claims abstract 3
- 238000004440 column chromatography Methods 0.000 claims abstract 2
- 238000001641 gel filtration chromatography Methods 0.000 claims abstract 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims 6
- 239000004094 surface-active agent Substances 0.000 claims 5
- 239000007853 buffer solution Substances 0.000 claims 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims 3
- 239000004202 carbamide Substances 0.000 claims 3
- 239000006167 equilibration buffer Substances 0.000 claims 3
- 239000000243 solution Substances 0.000 claims 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 2
- 239000000356 contaminant Substances 0.000 claims 2
- 229960003964 deoxycholic acid Drugs 0.000 claims 2
- 230000007717 exclusion Effects 0.000 claims 2
- 238000001914 filtration Methods 0.000 claims 2
- 239000000499 gel Substances 0.000 claims 2
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 claims 2
- 238000005406 washing Methods 0.000 claims 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims 1
- 229920001213 Polysorbate 20 Polymers 0.000 claims 1
- 239000007983 Tris buffer Substances 0.000 claims 1
- 239000013504 Triton X-100 Substances 0.000 claims 1
- 229920004890 Triton X-100 Polymers 0.000 claims 1
- 239000011543 agarose gel Substances 0.000 claims 1
- SOIFLUNRINLCBN-UHFFFAOYSA-N ammonium thiocyanate Chemical compound [NH4+].[S-]C#N SOIFLUNRINLCBN-UHFFFAOYSA-N 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 239000000872 buffer Substances 0.000 claims 1
- 210000000170 cell membrane Anatomy 0.000 claims 1
- 238000003795 desorption Methods 0.000 claims 1
- SPSXSWRZQFPVTJ-ZQQKUFEYSA-N hepatitis b vaccine Chemical compound C([C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC1N=CN=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)OC(=O)CNC(=O)CNC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)C1=CC=CC=C1 SPSXSWRZQFPVTJ-ZQQKUFEYSA-N 0.000 claims 1
- 229940124736 hepatitis-B vaccine Drugs 0.000 claims 1
- 239000002245 particle Substances 0.000 claims 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims 1
- 239000008363 phosphate buffer Substances 0.000 claims 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims 1
- 229920000053 polysorbate 80 Polymers 0.000 claims 1
- ZNNZYHKDIALBAK-UHFFFAOYSA-M potassium thiocyanate Chemical compound [K+].[S-]C#N ZNNZYHKDIALBAK-UHFFFAOYSA-M 0.000 claims 1
- 229940116357 potassium thiocyanate Drugs 0.000 claims 1
- 230000001376 precipitating effect Effects 0.000 claims 1
- 230000001737 promoting effect Effects 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 claims 1
- 238000010298 pulverizing process Methods 0.000 claims 1
- 239000011347 resin Substances 0.000 claims 1
- 229920005989 resin Polymers 0.000 claims 1
- 239000011780 sodium chloride Substances 0.000 claims 1
- VGTPCRGMBIAPIM-UHFFFAOYSA-M sodium thiocyanate Chemical group [Na+].[S-]C#N VGTPCRGMBIAPIM-UHFFFAOYSA-M 0.000 claims 1
- 239000006228 supernatant Substances 0.000 claims 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 238000001556 precipitation Methods 0.000 abstract 1
- 238000001179 sorption measurement Methods 0.000 abstract 1
- 210000005253 yeast cell Anatomy 0.000 abstract 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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Abstract
본 발명은 세포 파쇄시 카오트로픽 염(chaotropic salt)을 이용하여 전S2 펩티드의 유실을 억제하는 것을 특징으로 하여, 그 이후의 추출범, 침전법, 실리카 흡착법, 소수성 컬럼 크로마토그래피법 및 젤 여과 크로마토그래피법 등의 과정을 포함하는, 재조합 효모 세포로부터 전S2 펩티드 함유 B형 간염 표면항원을 정제하는 방법에 관한 것이다.
Description
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제1도는 전S2 펩티드 함유 B형 간염 표면항원의 정제시 카오트로픽 염의 존재여부에 따른 B형 간염 표면항원중 전S2 펩티드의 함량을 15% 소디움도데실설페이트 폴리아크릴아미드 겔 전기영동(SDS-PAGE)으로 확인한 결과를 나타낸다.
Claims (17)
- 전S2 펩티드 함유 B형 간염 표면항원 단백질이 발현된 재조합 생물체 세포를 파쇄하여 이를 정제하는 방법에 있어서, 상기 재조합 생물체 세포를 카오트로픽 염(chaotropic salt)이 포함된 완충용액으로 파쇄하는 것을 특징으로 하는 방법.
- 제1항에 있어서, 상기 카오트로픽 염이 티오시안산 나트륨, 티오시안산 칼륨, 티오시안산 암모늄, 염산 구아니듐 또는 요소인 방법.
- 제1항에 있어서, 상기 완충용액의 카오트로픽 염의 농도가 1∼8M인 방법.
- 제1항에 있어서, (1) 상기 세포 파쇄액에 계면활성제를 첨가하여 표면항원을 세포막에서 추출하는 단계 ; (2) (1)단계에서 얻은 세포 추출액에 포함되어 있는 표면항원의 용해성을 높이고 입자 형성을 촉진시키는 단계; (3) (2)단계에서 얻은 세포 추출액으로부터 세포 찌꺼기와 오염물질을 침전 제거하는 단계; (4) (3)단계에서 얻은 표면항원이 포함된 용액을 실리카와 접촉시켜 표면항원을 실리카에 흡착시킨 후, 오염 단백질을 세척하고 완충용액으로 표면항원을 탈착시키는 단계; (5) (4)단계에서 얻은 표면항원이 포함된 분획을 소수성 컬럼 크로마토그래피로 정제하는 단계; 및 (6) (5)단계에서 얻은 표면항원이 포함된 용출 분획을 분자량 배제 겔 여과 크로마토그래피로 여과 정제하는 단계를 더욱 포함하는 방법.
- 제4항에 있어서, 상기 (1)단계에서 얻은 세포 추출액에 염을 첨가한 후 세포 찌거기와 오염 물질을 제거하고 상충액을 다이아 여과하는 단계를 더욱 포함하는 방법.
- 제4항에 있어서, 상기 (1)단계의 계면활성제가 트윈(tween) 20, 트윈 80, 트리톤 X-100 또는 소디움 데옥시콜레이트인 방법.
- 제4항에 있어서, 상기 (1)단계의 계면활성제가 0.1∼0.5%(W/V)의 농도인 방법.
- 제4항에 있어서, 상기 (2)단계가 세포 추출액의 pH를 11.0∼13.5로 상승시키는 방법.
- 제4항에 있어서, 상기 (3)단계가 세포 추출액의 pH를 4.5∼6.0으로 낮추는 방법.
- 제4항에 있어서, 상기 (4)단계의 실리카가 100m2/g∼500m2/g 범위의 표면적을 갖는 것인 방법.
- 제4항에 있어서, 상기 (4)단계에서 실리카로부터 표면항원의 탈착이 1∼8M의 요소 또는 0.1∼0.3%의 계면활성제를 포함하는 pH 8.8∼11.0의 완충용액으로 되는 방법.
- 제11항에 있어서, 상기 계면활성제가 소디움 데옥시콜레이트인 방법.
- 제4항에 있어서, 상기 (5)단계에서 소수성 수지가 페닐기가 붙은 아가로즈 겔인 방법.
- 제4항에 있어서, 상기 (5)단계의 크로마토그래피가 1∼4M 요소를 포함하는 pH 8.8∼11.0의 평형 완충 용액으로 평형화시킨 컬럼에 표면 항원이 포함된 분획을 통과시키고, 10∼40%의 에틸렌 글리콜이 포함된 평형 완충용액으로 컬럼을 세척한 후, 60∼80%의 에틸렌 글리콜이 포함된 평형 완충용액으로 표면항원을 용출시킴으로써 수행되는 방법.
- 제4항에 있어서, 상기 (6)단계가 적어도 1,000.000의 분자량 배제 한도를 갖는 텍스트란 겔 또는 폴리아크릴이미드 겔에서 수행되는 방법.
- 제4항에 있어서, 상기 (6)단계의 크로마토그래피가 0.1∼0.2M 염화 나트륨이 포함된 PH 6∼8의 트리스 또는 인산염 완충용액으로 평형화된 컬럼에 표면항원 함유 분획을 통과시킨 후 동일 완충액으로 용출시켜 수행하는 방법.
- 제1항의 방법에 따라 제조된 B형 간염 백신.※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR19940033594 | 1994-12-10 | ||
| KR94-33594 | 1994-12-10 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR960023066A true KR960023066A (ko) | 1996-07-18 |
| KR0177254B1 KR0177254B1 (ko) | 1999-04-01 |
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| Application Number | Title | Priority Date | Filing Date |
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| KR1019950044795A KR960023066A (ko) | 1994-12-10 | 1995-11-29 | 전에스 (s) 2 펩티드 함유 비 (b) 형 간염 표면항원의 정제 방법 |
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| Country | Link |
|---|---|
| US (1) | US6362320B1 (ko) |
| EP (1) | EP0716094B1 (ko) |
| KR (1) | KR960023066A (ko) |
| CN (1) | CN1057532C (ko) |
| AR (1) | AR002006A1 (ko) |
| AT (1) | ATE264341T1 (ko) |
| BG (1) | BG63699B1 (ko) |
| BR (1) | BR9505739A (ko) |
| CA (1) | CA2164672C (ko) |
| CO (1) | CO4480040A1 (ko) |
| CZ (1) | CZ291024B6 (ko) |
| DE (1) | DE69532878T2 (ko) |
| DK (1) | DK0716094T3 (ko) |
| ES (1) | ES2217272T3 (ko) |
| JO (1) | JO1885B1 (ko) |
| PE (1) | PE56396A1 (ko) |
| PL (1) | PL182103B1 (ko) |
| PT (1) | PT716094E (ko) |
| RO (1) | RO113308B1 (ko) |
| RU (1) | RU2122430C1 (ko) |
| TR (1) | TR199501557A2 (ko) |
| UY (1) | UY24112A1 (ko) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100405174B1 (ko) * | 2001-02-02 | 2003-11-12 | 씨제이 주식회사 | 재조합 β형 간염바이러스 표면항원의 정제방법 |
| KR100948127B1 (ko) * | 2007-06-27 | 2010-03-18 | 주식회사 중앙백신연구소 | 복합호흡기 질환 돼지의 조직유제를 함유하는 백신 조성물 |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| UA79735C2 (uk) * | 2000-08-10 | 2007-07-25 | Глаксосмітклайн Байолоджікалз С.А. | Очищення антигенів вірусу гепатиту b (hbv) для використання у вакцинах |
| JPWO2005111620A1 (ja) * | 2004-05-19 | 2008-03-27 | 株式会社先端生命科学研究所 | Ｂ型肝炎ウイルスの検出方法 |
| GB0522765D0 (en) * | 2005-11-08 | 2005-12-14 | Chiron Srl | Combination vaccine manufacture |
| CA2885263C (en) | 2012-09-17 | 2021-11-16 | W. R. Grace & Co.-Conn. | Chromatography media and devices |
| FR2997222B1 (fr) * | 2012-10-19 | 2015-01-16 | Alstom Technology Ltd | Dispositif d'etablissement et/ou de coupure de courant a contacts permanents a usure reduite |
| EP3137209B1 (en) | 2014-05-02 | 2022-10-05 | W.R. Grace & CO. - CONN. | Functionalized support material and methods of making and using functionalized support material |
| BR112017026193B1 (pt) | 2015-06-05 | 2021-09-14 | W.R. Grace & Co-Conn | Adsorventes, método de produção dos adsorventes e uso dos adsorventes |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5340926A (en) * | 1983-03-25 | 1994-08-23 | Celltech, Limited | Process for the recovery of recombinantly produced protein from insoluble aggregate |
| US4649192A (en) * | 1985-05-30 | 1987-03-10 | Smith Kline-Rit | Method for the isolation and purification of hepatitis B surface antigen using polysorbate |
| ES2043808T3 (es) * | 1987-03-09 | 1994-01-01 | Merck & Co Inc | Purificacion de antigeno superficial de hepatitis b recombinante. |
| EP0310178A1 (en) * | 1987-09-30 | 1989-04-05 | Merck & Co. Inc. | Recovery of pres2+S antigen |
| EP0314240A3 (en) * | 1987-10-26 | 1990-03-28 | Merck & Co. Inc. | Process for purifying recombinant hepatitis antigens |
| US5004688A (en) * | 1988-04-15 | 1991-04-02 | Phillips Petroleum Company | Purification of hepatitis proteins |
| US5030720A (en) * | 1988-09-01 | 1991-07-09 | Merck & Co., Inc. | Pres2+S hepatitis B vaccine derived from plasma |
| EP0384058A1 (en) * | 1989-02-09 | 1990-08-29 | Development Center For Biotechnology | Isolation of hepatitis B surface antigen from transformed yeast cells |
| CU22290A1 (es) * | 1990-10-08 | 1995-01-31 | Cigb | Procedimiento para la obtencion de antigeno de superficie del virus de la hepatitis b de superior capacidad inmunoganica y su uso en un preparado vacunal |
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1995
- 1995-11-29 KR KR1019950044795A patent/KR960023066A/ko not_active IP Right Cessation
- 1995-12-07 PE PE1995286655A patent/PE56396A1/es not_active Application Discontinuation
- 1995-12-07 CO CO95057860A patent/CO4480040A1/es unknown
- 1995-12-07 JO JO19951885A patent/JO1885B1/en active
- 1995-12-07 CA CA002164672A patent/CA2164672C/en not_active Expired - Fee Related
- 1995-12-08 PT PT95119383T patent/PT716094E/pt unknown
- 1995-12-08 CZ CZ19953247A patent/CZ291024B6/cs not_active IP Right Cessation
- 1995-12-08 DK DK95119383T patent/DK0716094T3/da active
- 1995-12-08 BG BG100212A patent/BG63699B1/bg unknown
- 1995-12-08 DE DE69532878T patent/DE69532878T2/de not_active Expired - Fee Related
- 1995-12-08 AT AT95119383T patent/ATE264341T1/de not_active IP Right Cessation
- 1995-12-08 RU RU95120587A patent/RU2122430C1/ru not_active IP Right Cessation
- 1995-12-08 RO RO95-02141A patent/RO113308B1/ro unknown
- 1995-12-08 EP EP95119383A patent/EP0716094B1/en not_active Expired - Lifetime
- 1995-12-08 PL PL95311735A patent/PL182103B1/pl not_active IP Right Cessation
- 1995-12-08 ES ES95119383T patent/ES2217272T3/es not_active Expired - Lifetime
- 1995-12-11 UY UY24112A patent/UY24112A1/es not_active IP Right Cessation
- 1995-12-11 AR ARP950100498A patent/AR002006A1/es active IP Right Grant
- 1995-12-11 CN CN95120408A patent/CN1057532C/zh not_active Expired - Fee Related
- 1995-12-11 TR TR95/01557A patent/TR199501557A2/xx unknown
- 1995-12-11 BR BR9505739A patent/BR9505739A/pt not_active Application Discontinuation
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1999
- 1999-11-02 US US09/432,300 patent/US6362320B1/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100405174B1 (ko) * | 2001-02-02 | 2003-11-12 | 씨제이 주식회사 | 재조합 β형 간염바이러스 표면항원의 정제방법 |
| KR100948127B1 (ko) * | 2007-06-27 | 2010-03-18 | 주식회사 중앙백신연구소 | 복합호흡기 질환 돼지의 조직유제를 함유하는 백신 조성물 |
Also Published As
| Publication number | Publication date |
|---|---|
| BG100212A (bg) | 1996-12-31 |
| CO4480040A1 (es) | 1997-07-09 |
| CZ324795A3 (en) | 1996-06-12 |
| DE69532878T2 (de) | 2005-06-09 |
| BR9505739A (pt) | 1997-12-23 |
| EP0716094A1 (en) | 1996-06-12 |
| RO113308B1 (ro) | 1998-06-30 |
| BG63699B1 (bg) | 2002-09-30 |
| JO1885B1 (en) | 1997-12-15 |
| CN1057532C (zh) | 2000-10-18 |
| ATE264341T1 (de) | 2004-04-15 |
| EP0716094B1 (en) | 2004-04-14 |
| CA2164672A1 (en) | 1996-06-11 |
| US6362320B1 (en) | 2002-03-26 |
| UY24112A1 (es) | 1996-05-10 |
| CZ291024B6 (cs) | 2002-12-11 |
| KR0177254B1 (ko) | 1999-04-01 |
| ES2217272T3 (es) | 2004-11-01 |
| PL182103B1 (pl) | 2001-11-30 |
| RU2122430C1 (ru) | 1998-11-27 |
| CN1132211A (zh) | 1996-10-02 |
| PE56396A1 (es) | 1996-12-14 |
| AR002006A1 (es) | 1998-01-07 |
| DK0716094T3 (da) | 2004-07-26 |
| TR199501557A2 (tr) | 1997-03-21 |
| PL311735A1 (en) | 1996-06-24 |
| DE69532878D1 (de) | 2004-05-19 |
| PT716094E (pt) | 2004-08-31 |
| CA2164672C (en) | 2004-06-22 |
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