KR970073604A - Purification method of recombinant hepatitis B surface antigen - Google Patents

Purification method of recombinant hepatitis B surface antigen Download PDF

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KR970073604A
KR970073604A KR1019960017426A KR19960017426A KR970073604A KR 970073604 A KR970073604 A KR 970073604A KR 1019960017426 A KR1019960017426 A KR 1019960017426A KR 19960017426 A KR19960017426 A KR 19960017426A KR 970073604 A KR970073604 A KR 970073604A
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South Korea
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hbs antigen
antigen
molecular weight
hbs
weight exclusion
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KR1019960017426A
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Korean (ko)
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KR100194247B1 (en
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이영미
윤경희
임국진
박순재
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성재갑
주식회사 Lg 화학
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Abstract

본 발명은 재조합 B형 간염 표면 항원(HBs항원)의 정제 방법에 관한 것으로, (a) B형 간염 표면 항원(HBs항원)이 발현된 효모 세포를 계면 활성제의 존재하에 용해시킨 후 HBs항원을 포함하는 상등액을 분리하는 단계；(b)단계 (a)에서 얻어진 HBs항원을 함유하는 상등액을 실리카와 접촉시킨 후, HBs항원이 흡착된 실리카를 pH 6-8의 완충용액으로 세척하고, pH8.8-11.0의 완충용액으로 HBs항원을 탈착시키는 단계；(c)단계 (b)에사 얻어진 HBs항원 함유약을 pH 7 내지 9로 조정하고 0.5M 이하의 염을 첨가하여 음이온 교환수지에 통과시킨 다음, HBs항원 함유 분획을 투석여과(diafilitration) 및 농축시키는 단계；(d) 농축된 HBs항원 함유 분획을 분자량 배제 제한을 갖는 물질을 사용하여 겔 여과 크로마토그래피 한 후, 투석여과하는 단계；(e)단계 (d)에서 얻어진 HBs항원 함유 분획을 단백질 분해 효소로 처리한 후, 투석여과 및 농축하는 단계；및 (f)단계 (e)에서 얻어진 HBs항원 함유액을 분자량 배제 제한을 갖는 물질을 사용하여 겔 여과 크로마토그래피하는 단계를 포함하는, 본 발명의 HBs항원 정제방법에 의하면 HBs항원을 불순물 없이 고순도로 정제할 수 있다.The present invention relates to a method of purifying recombinant hepatitis B surface antigen (HBs antigen), comprising the steps of (a) dissolving yeast cells expressing hepatitis B surface antigen (HBs antigen) in the presence of a surfactant, (B) contacting the supernatant containing the HBs antigen obtained in step (a) with silica, washing the HBs antigen-adsorbed silica with a buffer solution having a pH of 6-8, (C) adjusting the pH of the HBs antigen-containing drug obtained in step (b) to a pH of 7 to 9, adding a salt of 0.5 M or less, passing the solution through an anion exchange resin, (D) subjecting the concentrated HBsAg-containing fraction to gel filtration chromatography using a substance having a molecular weight exclusion limit, followed by dialyzing filtration; (e) (d) < RTI ID = 0.0 > (F) subjecting the HBs antigen-containing solution obtained in step (e) to gel filtration chromatography using a substance having a molecular weight exclusion limit, and , According to the HBsAg purification method of the present invention, HBsAg can be purified with high purity without impurities.

Description

재조합 B형 간염 표면 항원의 정제방법Purification method of recombinant hepatitis B surface antigen

본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음Since this is a trivial issue, I did not include the contents of the text.

Claims (12)

(a) B형 간염 표면 항원(HBs항원)이 발현된 효모 세포를 계면활성제의 존재하에 용해시킨 후 HBs항원을 포함하는 상등액을 분리하는 단계；(b)단계 (a)에서 얻어진 HBs항원을 함유하는 상등액을 실리카와 접촉시킨 후, HBs항원이 흡착된 실리카를 pH6-8의 완충용액으로 세척하고, pH8.8-11.0의 완충용액으로 HBs항원을 탈착시키는 단계；(c)단계 (b)에사 얻어진 HBs항원 함유약을 pH 7 내지 9로 조정하고 0.5M 이하의 염을 첨가하여 음이온 교환수지에 통과시킨 다음, HBs항원 함유 분획을 투석여과(diafilitration) 및 농축시키는 단계；(d) 농축된 HBs항원 함유 분획을 분자량 배제 제한을 갖는 물질을 사용하여 겔 여과 크로마토그래피 한 후, 투석여과하는 단계；(e)단계 (d)에서 얻어진 HBs항원 함유 분획을 단백질 분해 효소로 처리한 후, 투석여과 및 농축하는 단계；및 (f)단계 (e)에서 얻어진 HBs항원 함유액을 분자량 배제 제한을 갖는 물질을 사용하여 겔 여과 크로마토그래피하는 단계를 포함하는, 재조합 효모로부터 발현된 B형 간염 표면 항원의 정제 방법.(a) dissolving yeast cells expressing hepatitis B surface antigen (HBs antigen) in the presence of a surfactant, and then separating the supernatant containing HBs antigen; (b) (B) washing the silica adsorbed with the HBs antigen with a buffer solution having a pH of 6-8, desorbing the HBs antigen with a buffer solution having a pH of 8.8-11.0, (c) contacting the supernatant with silica, Adjusting the obtained HBs antigen-containing drug to a pH of 7 to 9, adding a salt of 0.5 M or less, passing through an anion exchange resin, and then diafiltration and concentrating the HBs antigen-containing fraction; (d) (E) fractionating the HBs antigen-containing fraction obtained in step (d) with proteolytic enzyme, followed by dialyzing and filtration; and (e) subjecting the antigen-containing fraction to a gel filtration chromatography using a substance having a restriction of molecular weight exclusion, And (f) comprising the step of subjecting the HBs antigen-containing solution obtained in step (e) to gel filtration chromatography using a substance having a molecular weight exclusion limit, thereby to purify the hepatitis B surface antigen expressed from the recombinant yeast. 제1항에 있어서, 상기 단계(a)에서 상기 계면활성제가 트윈 20, 트윈 80, 트리톤 X-100 또는 나트륨 데옥시콜레이트인 것을 특징으로 하는 방법.The method of claim 1, wherein in step (a), the surfactant is Tween 20, Tween 80, Triton X-100, or sodium deoxycholate. 제2항에 있어서, 상기 계면활성제의 농도가 0.05 내지 1.5%인 것을 특징으로 하는 방법.The method of claim 2, wherein the concentration of the surfactant is 0.05 to 1.5%. 제1항에 있어서, 상기 단계(b)에서 상기 실리카가 100 내지 500㎡/g의 표면적을 갖는 것을 특징으로 하는 방법.The method of claim 1, wherein the silica in step (b) has a surface area of from 100 to 500 m < 2 > / g. 제1항에 있어서, 상기 단계(b)에서 1 내지 8M의 요소 또는 0.1 내지 0.3%의 나트륨 데옥시콜레이트를 포함하는 pH 8.8내지 11.0의 완충용액으로 HBs항원을 탈착시키는 것을 특징으로 하는 방법.The method according to claim 1, wherein the step (b) desorbs the HBs antigen with a buffer solution having a pH of 8.8 to 11.0 containing 1 to 8 M of element or 0.1 to 0.3% of sodium deoxycholate. 제1항에 있어서, 상기 단계(b)에서 상기 음이온 교환 수지가 DEAE 또는 Q-세파로즈인 것을 특징으로 하는 방법.The method of claim 1, wherein in step (b), the anion exchange resin is DEAE or Q-Sepharose. 제1항에 있어서, 상기 투석여과가 5,000 내지 500,000의 분자량 배제 제한을 갖는 막을 사용하여 수행되는 것을 특징으로 하는 방법.The method of claim 1, wherein the dialysis filtration is performed using a membrane having a molecular weight exclusion limit of 5,000 to 500,000. 제1항에 있어서, 상기 겔 여과 크로마토그래피가 100만 이상의 분자량 배제 제한을 갖는 아가로스, 덱스트란 또는 폴리아크릴아미드 겔로 충전된 컬럼에서 수행되는 것을 특징으로 하는 방법.The method of claim 1, wherein the gel filtration chromatography is performed in a column packed with agarose, dextran, or polyacrylamide gel having a molecular weight exclusion limit of at least 1 million. 제1항에 있어서, 상기 단계(e)에서 상기 단백질 분해 효소가 트립신 또는 펩신인 것을 특징으로 하는 방법.The method according to claim 1, wherein in step (e), the protease is trypsin or pepsin. 제1항에 있어서, 상기 단계(e)에서 HBs항원 함유 분획의 HBs항원 농도가 전체 단백질의 5% 이상인 것을 특징으로 하는 방법.The method of claim 1, wherein the HBs antigen concentration of the HBs antigen-containing fraction in step (e) is at least 5% of the total protein. 제1항에 있어서, 단백질 분해 효소를 HBs항원 함유 분획의 전체 단백질 양의 1/2,000 내지 1/20의 비율로 첨가하는 것을 특징으로 하는 방법.The method according to claim 1, wherein the protease is added at a ratio of 1 / 2,000 to 1/20 of the total protein amount of the HBs antigen-containing fraction. 제9항에 있어서, 단백질 분해 효소 처리를 25 내지 45℃의 온도에서 2 내지 30 시간 동안 실시하는 것을 특징으로 하는 방법.10. The method according to claim 9, wherein the proteolytic enzyme treatment is carried out at a temperature of 25 to 45 DEG C for 2 to 30 hours. ※ 참고사항：최초출원 내용에 의하여 공개하는 것임.※ Note: It is disclosed by the contents of the first application.
KR1019960017426A 1996-05-22 1996-05-22 Method for Purifying Recombinant Hepatitis B Surface Antigen KR100194247B1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100405174B1 (en) * 2001-02-02 2003-11-12 씨제이 주식회사 Purification method of surface antigen of recombinant B-type hepatitis virus
KR100436655B1 (en) * 2001-07-25 2004-06-22 (주)엘피스바이오텍 Method of concentration and purification of protein applicable to wide range of protein

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117042805A (en) 2021-04-05 2023-11-10 株式会社Lg化学 Vaccine composition against coronavirus

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100405174B1 (en) * 2001-02-02 2003-11-12 씨제이 주식회사 Purification method of surface antigen of recombinant B-type hepatitis virus
KR100436655B1 (en) * 2001-07-25 2004-06-22 (주)엘피스바이오텍 Method of concentration and purification of protein applicable to wide range of protein

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