JPH01196293A - Extraction of hepatitis b virus surface antigen - Google Patents
Extraction of hepatitis b virus surface antigenInfo
- Publication number
- JPH01196293A JPH01196293A JP63017422A JP1742288A JPH01196293A JP H01196293 A JPH01196293 A JP H01196293A JP 63017422 A JP63017422 A JP 63017422A JP 1742288 A JP1742288 A JP 1742288A JP H01196293 A JPH01196293 A JP H01196293A
- Authority
- JP
- Japan
- Prior art keywords
- hbsag
- polyethylene oxide
- hepatitis
- oxide condensate
- sorbitan ester
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108091007433 antigens Proteins 0.000 title claims abstract description 14
- 239000000427 antigen Substances 0.000 title claims abstract description 13
- 102000036639 antigens Human genes 0.000 title claims abstract description 13
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- 241000700721 Hepatitis B virus Species 0.000 title claims abstract description 7
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- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 10
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- -1 fatty acid sorbitan ester Chemical class 0.000 claims description 3
- 230000000813 microbial effect Effects 0.000 claims description 2
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Landscapes
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Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は遺伝子操作により得られるＢ型肝炎ウィルス表
面抗原（以下、ＨＢＳＡｇという。）の抽出方法に関す
る。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for extracting hepatitis B virus surface antigen (hereinafter referred to as HBSAg) obtained by genetic manipulation.
ＨＢｓＡｇはＢ型肝炎ワクチンの主成分である。 HBsAg is the main component of the hepatitis B vaccine.
Ｂ型肝炎ワクチンは、ＨＢｓＡｇ陽性のキャリアー血液
からＨＢｓＡｇ粒子を分離精製し、ホルマリン処理など
の不活化操作を行った後、ＨＢｓＡｇの免疫原性を高め
るために、アジュバントとしてアラム・ゲルを加えたも
のとして開発、実用化された。Hepatitis B vaccine is made by separating and purifying HBsAg particles from HBsAg-positive carrier blood, performing inactivation procedures such as formalin treatment, and then adding alum gel as an adjuvant to increase the immunogenicity of HBsAg. It was developed and put into practical use.
このようにして作られたＢ型肝炎ワクチンは感染発症の
予防だけでなく、感染源となるキャリアーの発生阻止に
も有効であることから、Ｂ型肝炎の絶滅も可能である。The hepatitis B vaccine produced in this way is effective not only in preventing the onset of infection but also in preventing the generation of carriers, which are the source of infection, so it is possible to eradicate hepatitis B.
しかし、Ｂ型肝炎ワクチンの生産についてはいくつかの
問題点がある。第１は、ＨＢｓＡｇの原材料をキャリア
ーの血液に依存していることから、ワクチンの供給が制
約されるということである。However, there are several problems with the production of hepatitis B vaccines. The first is that the supply of vaccines is restricted because the raw material for HBsAg relies on the blood of carriers.
第２は、キャリアーの血液中には少量ながら感染性のＢ
型肝炎ウィルス（ＨＢＶ）が含まれることから、唯一の
感染しえる動物であるチンパンジーを用いての安全試験
が必要になるということである。Second, there is a small amount of infectious B in the carrier's blood.
Because it contains hepatitis virus (HBV), safety testing using chimpanzees, the only animals that can be infected, is required.
このようなワクチンの生産上の難点を克服する方法とし
て遺伝子操作の技術が導入された。Genetic engineering technology has been introduced as a way to overcome these difficulties in vaccine production.
組換えＤＮＡ技術は異種のＤＮＡをファージ又はプラス
ミドＤＮＡにつなぎ、大腸菌で増やすという方法で開始
された。そして、ＨＢＶ−ＤＮＡの全塩基配列の決定、
ワクチンとして使われるべきＨＢｓＡｇ遺伝子の同定、
大腸菌での形質発現を経て酵母でのＨＢｓＡｇの産生へ
と発展していった。Recombinant DNA technology began by ligating heterologous DNA to phage or plasmid DNA and propagating it in E. coli. Then, determination of the entire base sequence of HBV-DNA,
Identification of the HBsAg gene to be used as a vaccine,
After expression in Escherichia coli, the process progressed to production of HBsAg in yeast.
たとえば、酵母由来ＨＢｓＡｇ （ｙ−ＨＢｓＡｇ）は
、５ＤＳ−ポリアクリルアミドゲル電気泳動によると、
分子量２３． ＣＯＯ〜２５．０００のポリペプチドの
みからなり、これはヒト由来ＨＢｓＡｇ （ｈ−ＨＢｓ
Ａｇ）のグリコジル化されていないポリペプチドと一致
するものである。また、ｈ−ＨＢｓＡｇと同じ２２ｎｍ
の直径をもち、塩化セシウム溶液中での浮上密度は、ｈ
−ＨＢｓＡｇと同じである。For example, according to 5DS-polyacrylamide gel electrophoresis, yeast-derived HBsAg (y-HBsAg)
Molecular weight 23. It consists of only COO ~ 25,000 polypeptides, which are derived from human HBsAg (h-HBs
This corresponds to a non-glycosylated polypeptide of Ag). In addition, the same 22 nm as h-HBsAg
The floating density in a cesium chloride solution is h
- Same as HBsAg.
前述したＢ型肝炎ワクチンの生産上の難点を克服するた
めに、遺伝子操作の技術によりＨＢＳＡｇを大量に得、
それを高度精製することによりワクチンとする方法が確
立されつつある。In order to overcome the above-mentioned difficulties in producing hepatitis B vaccines, we obtained large amounts of HBSAg using genetic engineering technology.
A method to use it as a vaccine by highly purifying it is being established.
遺伝子操作により微生物菌体から産生されたＨＢｓＡｇ
を回収するには、菌体を適当な溶媒に懸濁後、菌体を粉
砕することによって、ＨＢｓＡｇを菌体内外から抽出す
る方法が用いられる。HBsAg produced from microbial cells through genetic manipulation
In order to recover the HBsAg, a method is used in which the bacterial cells are suspended in an appropriate solvent and then pulverized to extract HBsAg from inside and outside the bacterial cells.
しかし、遺伝子操作により得られたＨＢｓＡｇは水溶性
に乏しいため、抽出操作が極めて困難であった。ＨＢｓ
Ａｇを可溶化するために抽出操作の時に界面活性剤を添
加するのが常法であるが、界面活性剤の種類においては
ＨＢｓＡｇを失活させるものもあることが判明した。However, HBsAg obtained by genetic manipulation has poor water solubility, making extraction operations extremely difficult. HBs
Although it is a conventional method to add a surfactant during the extraction operation in order to solubilize Ag, it has been found that some types of surfactants can deactivate HBsAg.
そこで、本発明者らはこれらの事情に鑑み鋭意研究を重
ねた結果、遺伝子操作を経た菌体内または菌体外に産生
されたＨＢｓＡｇを効率よく抽出し、しかも抗原性を低
下させない方法を見出し、本発明を完成した。In view of these circumstances, the present inventors have conducted intensive research and have found a method for efficiently extracting HBsAg produced inside or outside of the bacterial cell through genetic manipulation, without reducing its antigenicity. The invention has been completed.
すなわち、本発明の目的は、遺伝子操作により得られる
ＨＢｓＡｇを高級脂肪酸ソルビタンエステル−ポリエチ
レンオキサイド縮合物の存在下に抽出する方法を提供す
ることにある。That is, an object of the present invention is to provide a method for extracting HBsAg obtained by genetic manipulation in the presence of a higher fatty acid sorbitan ester-polyethylene oxide condensate.
〔課題を解決するための手段及び作用〕■ 出発原料
本発明において用いられるＨＢｓＡｇは遺伝子操作に由
来して調製されたものであれば、特に限定されない。従
って遺伝子操作を経てＨＢｓＡｇを発現する菌体（例え
ば、大腸菌、酵母、枯草菌など）を用いる。[Means and effects for solving the problem] ■ Starting material The HBsAg used in the present invention is not particularly limited as long as it is prepared by genetic manipulation. Therefore, bacterial cells (eg, Escherichia coli, yeast, Bacillus subtilis, etc.) that express HBsAg through genetic manipulation are used.
遺伝子操作によりＨＢｓＡｇを発現させる方法は公知の
手段に基づく。すなわち、ＨＢｓＡｇ遺伝子（ＨＢｓＡ
ｇをコードするｃＤＮＡ）を調製して適当なベクターに
組込んで組換えベクターを調製し、これを微生物菌体に
導入して形質転換させた後、この形質転換株を培養して
菌体にＨＢｓＡｇを発現・産生せしめる。The method for expressing HBsAg by genetic manipulation is based on known means. That is, the HBsAg gene (HBsA
A recombinant vector is prepared by preparing a cDNA encoding G. HBsAg is expressed and produced.
具体的には、例えば、大腸菌を用いる方法として特開昭
５５−１０４８８７など、酵母を用いる方法として特開
昭５９−４８０８２などが例示される。Specifically, examples of methods using E. coli include JP-A-55-104887, and examples of methods using yeast include JP-A-59-48082.
■ 抽出
菌体を高級脂肪酸ソルビタンエステル−ポリエチレンオ
キサイド縮合物の存在下に、ｐＨ７〜９の緩衝液（例え
ば、１０ｍＭリン酸緩衝液（ｐ Ｈ７，５）１ｍＭエチ
レンジアミン四酢酸＋１ｍＭフッ化フェニルメチルスル
ホニル含有１０ｍＭ ） ’Ｊス緩Ｉ液（ｐＨ７，５）
など）に懸濁後、菌体を粉砕することによって、ＨＢｓ
Ａｇを菌体内外から抽出する。■ The extracted bacterial cells were placed in a buffer solution of pH 7 to 9 (e.g., 10 mM phosphate buffer (pH 7,5) containing 1 mM ethylenediaminetetraacetic acid + 1 mM phenylmethylsulfonyl fluoride in the presence of a higher fatty acid sorbitan ester-polyethylene oxide condensate). 10mM) 'Jsu mild I solution (pH 7,5)
HBs
Ag is extracted from inside and outside the bacterial body.
高級脂肪酸ソルビタンエステル−ポリエチレンオキサイ
ド縮合物における高級脂肪酸としてはラウリン酸、パル
ミチン酸、ステアリン酸、オレイン酸などが挙げられる
。Examples of higher fatty acids in the higher fatty acid sorbitan ester-polyethylene oxide condensate include lauric acid, palmitic acid, stearic acid, and oleic acid.
具体的には、
モノラウリン酸ソルビタンエステル−ポリエチレンオキ
サイド縮合物（Ｔｗｅｅｎ−２０、同一２１）、モノパ
ルミチン酸ソルビタンエステル−ポリエチレンオキサイ
ド縮合物（Ｔｗｅｅｎ−４０）、モノステアリン酸ソル
ビタンエステルーポＪＪエチレンオキサイド縮合物（Ｔ
ｗｅｅｎ−６０、同一６１）、トリステアリン酸ソルビ
タンエステル−ポリエチレンオキサイド縮合物（Ｔｗｅ
ｅｎ−６５）、モノオレイン酸ソルビタンエステル−ポ
リエチレンオキサイト縮合物（Ｔｗｅｅｎ−８０、同８
１）、トリオレイン酸ソルビタンエステル−ポリエチレ
ンオキサイト縮合物（Ｔｗｅｅｎ −８５）などが例示
される。Specifically, sorbitan monolaurate ester-polyethylene oxide condensate (Tween-20, same 21), sorbitan monopalmitate ester-polyethylene oxide condensate (Tween-40), sorbitan monostearate ester-polyethylene oxide condensate (T
ween-60, same 61), tristearate sorbitan ester-polyethylene oxide condensate (Twe
en-65), monooleate sorbitan ester-polyethylene oxide condensate (Tween-80,
1), trioleic acid sorbitan ester-polyethylene oxide condensate (Tween-85), and the like.
高級脂肪酸ソルビタンエステル−ポリエチレンオキサイ
ド縮合物の添加濃度としては０．１〜２．０ｗ／ｖ％程
度であり、好ましくは、０．１〜０．５１１１／ｖ％程
度である。The concentration of the higher fatty acid sorbitan ester-polyethylene oxide condensate is about 0.1 to 2.0 w/v%, preferably about 0.1 to 0.5111/v%.
なお、菌体の粉砕に際しては、既知の方法、例えば、凍
結融解法、ガラスピーズ法、高圧法、超音波処理法、酵
素処理法などが用いられる。In addition, when pulverizing the bacterial cells, known methods such as a freeze-thaw method, a glass bead method, a high pressure method, an ultrasonic treatment method, an enzyme treatment method, etc. are used.
■ 精製・製剤化
得られたＨＢｓＡｇ抽出画分は、公知の方法、例えば溶
解度差に基づく分画（硫安、ＰＥＧ、アクリノールなど
）、コロイド珪酸塩による吸着クロマト、疎水性基を有
する担体による吸着クロマト、ＨＢｓ抗体を用いたアフ
ィニティクロマト、ゲル濾過、超遠心分離、透析、限外
濾過、不活化処理（加熱、ホルマリン処理）、などによ
り精製される。■ Purification and formulation The obtained HBsAg extraction fraction is processed using known methods such as fractionation based on solubility difference (ammonium sulfate, PEG, acrinol, etc.), adsorption chromatography using colloidal silicate, adsorption chromatography using a carrier having a hydrophobic group. , affinity chromatography using HBs antibody, gel filtration, ultracentrifugation, dialysis, ultrafiltration, inactivation treatment (heating, formalin treatment), etc.
さらに、ＨＢｓＡｇ精製画分は既知の方法、例えば、免
疫補助剤、賦形剤、安定化剤、防腐剤の添加、小分は分
注、除菌濾過、凍結乾燥により製剤化される。Further, the purified HBsAg fraction is formulated by known methods, such as addition of immunoadjuvants, excipients, stabilizers, and preservatives, dispensing into small portions, sterile filtration, and lyophilization.
具体的には、特開昭５９−１０１４２６ 、同５９−１
１５１８９、同６０−２５８１２７ 、同６１−１０３
８９５ 、同６２−２２７２８に記載された方法に準じ
ればよい。Specifically, JP-A-59-101426, JP-A-59-1
15189, 60-258127, 61-103
895, 62-22728.
本発明の方法により、ＨＢｓＡｇを効率的に抽出でき、
しかも、ＨＢｓＡｇを失活させないという特徴を有する
。By the method of the present invention, HBsAg can be efficiently extracted,
Moreover, it has the characteristic of not deactivating HBsAg.
従って、本発明の方法は遺伝子操作により得られたＨＢ
ｓＡｇを抽出する方法として極め、て有用である。Therefore, the method of the present invention is applicable to HB obtained by genetic manipulation.
This is an extremely useful method for extracting sAg.
この結果は、最酩精製品の回収率の向上、比活性の向上
をもたらしうる。従って、ＨＢｓＡｇの製造全体にとっ
ても有用である。This result can lead to an improvement in the recovery rate and specific activity of the most distilled product. Therefore, it is also useful for the overall production of HBsAg.
実施例Ｉ
ＨＢｓＡｇを発現した酵母〔サツ力ロマイセスセレビジ
ｘ （Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉ
ａｅ） Ｇ ＲＦＩＩＩ ｒＩｈ ｏ９０株） ｔ−集
菌し、この５ｋｇを５βの１ｍＭエチレンジアミン四酢
酸（ＥＤＴＡ） 、１ｍＭフッ化フェニルメチルスルホ
ニノヘ０．１％（Ｗ／Ｖ）モノラウリン酸ソルビタンエ
ステル−ポリエチレンオキサイド縮合物（Ｔｗｅｅｎ
２０）を含む１０ｍＭ ）リス緩衝液、ｐＨ７，５に懸
濁し、グラスビーズ法（Ｄｙｎ。Example I Yeast expressing HBsAg [Saccharomyces cerevisi x]
ae) G RFIII rIh o90 strain) t- Harvest and add 5 kg of this to 5β, 1mM ethylenediaminetetraacetic acid (EDTA), 1mM fluorinated phenylmethylsulfonino, and 0.1% (W/V) monolauric acid sorbitan ester-polyethylene oxide. Condensate (Tween
Glass bead method (Dyn.
−Ｍｉｌｌ使用）により酵母からＨＢｓＡｇを抽出した
。抽出液にアクリノールを終濃度で０．３％（Ｗ／Ｖ’
）加え、室温で３０分撹拌後、生成した沈澱を遠心分離
（４０（ｆｏｒ、 ｐ、 ｍ、 、 ３０分間）により
除去し上清を得た。HBsAg was extracted from yeast using the following method. Acrinol was added to the extract at a final concentration of 0.3% (W/V'
), and after stirring at room temperature for 30 minutes, the generated precipitate was removed by centrifugation (40 p, m, , 30 min) to obtain a supernatant.
この上清にベントナイトを０．５％（Ｗ／Ｖ）加え、室
温で３０分撹拌後、遠心分離（４０００ｒ、 ｐ、 ｍ
、 、 ３０分間）し、得られた上清にケイ酸アルミン
酸マグネシウム〔エアロジルＲ３８０、Ｄｅｇｕｓａ社
製〕を２．５％（ｗ／ｖ）になるように添加し、３７℃
で３時間撹拌した。撹拌後、ｙ−ＨＢｓＡｇを吸着した
ケイ酸アルミン酸マグネシネウムを遠心分ｍ （３００
０ｒ、ｐｏｍ、。0.5% (W/V) bentonite was added to this supernatant, and after stirring at room temperature for 30 minutes, centrifugation (4000 r, p, m
, for 30 minutes), and magnesium aluminate silicate (Aerosil R380, manufactured by Degusa) was added to the resulting supernatant at a concentration of 2.5% (w/v), and the mixture was heated at 37°C.
The mixture was stirred for 3 hours. After stirring, the magnesium aluminate silicate adsorbed with y-HBsAg was centrifuged for 300 m
0r, pom,.
１５分間）により回収し、回収したｙ−ＨＢｓＡｇ吸着
ケイ酸アルミン酸マグネシウム３．１５Ｍ ＥＤＴＡと
０．１５Ｍ塩化ナトリウムより成るｐＨ７，５の緩衝液
で洗浄した。洗浄後、０．５％（Ｗ／Ｖ）デオキシコー
ル酸ナトリウムを含有するｐＨ９，０の緩衝液で吸着さ
れたｙ−ＨＢｓＡｇを溶出した。遠心分離（３０００ｒ
、ｐ、ｍ、、 １５分間）により回収した溶出液はｐＨ
を７．２に調整したのち、抽出液１！当り硫安を７０ｇ
添加し、別途調製されたフェニルアラニン−セファロー
ス４Ｂのカラムへ注入ＬＨＢｓ抗原を吸着させた。上記
と同じ濃度の硫安液で十分に洗浄し、不純物質を除去し
た後、ＩＯＶ／Ｖ％エタノール含有０．１ＭＩＪン酸緩
衝波緩衝液７．２）を用いてＨＢｓ抗原を溶出した。尚
フェニルアラニン−セファローズ４Ｂの調製法は臭化シ
アンで活性化したセファローズ４Ｂにフェニルアラニン
溶液を加え、室温で１６時間反応させることにより行っ
た。The collected y-HBsAg adsorbed magnesium aluminate silicate was washed with a pH 7.5 buffer consisting of 3.15M EDTA and 0.15M sodium chloride. After washing, the adsorbed y-HBsAg was eluted with a pH 9.0 buffer containing 0.5% (w/v) sodium deoxycholate. Centrifugation (3000r
, p, m, for 15 minutes), the eluate was collected at pH
After adjusting to 7.2, extract 1! 70g of ammonium sulfate per serving
The LHBs antigen was then injected onto a separately prepared phenylalanine-Sepharose 4B column to adsorb it. After thorough washing with ammonium sulfate at the same concentration as above to remove impurities, the HBs antigen was eluted using 0.1 MIJ acid buffer buffer 7.2) containing IOV/V% ethanol. Phenylalanine-Sepharose 4B was prepared by adding a phenylalanine solution to Sepharose 4B activated with cyanogen bromide and reacting at room temperature for 16 hours.
得られたＨＢｓＡｇ画分はショ糖直線密度勾配（２０〜
５５％）ゾーナル超遠心分離（４３，００Ｏｒ、 ｐ９
ｍ、。The obtained HBsAg fraction was analyzed using a sucrose linear density gradient (20~
55%) Zonal ultracentrifugation (43,00Or, p9
m.
４８時間）により、単一ピークとして回収され、このピ
ークと吸光度のピークは一致した。48 hours), a single peak was collected, and this peak and the absorbance peak coincided.
回収したｙ−ＨＢｓＡｇ画分は、ｐＨ７のリン酸緩衝液
で透析した後、アジュバントとして水酸化アルミニウム
を加えてワクチンを得た。The collected y-HBsAg fraction was dialyzed against a pH 7 phosphate buffer, and then aluminum hydroxide was added as an adjuvant to obtain a vaccine.
次に精製ｙ−ＨＢｓＡｇの性状分析を行った。Next, the properties of purified y-HBsAg were analyzed.
精製Ｖ−ＨＢｓＡｇ中の酵母成分の有無を、酵母成分に
対する抗体とＨＢｓＡｇを含まない酵母成分の感作ヒツ
ジ赤血球による血球凝集阻止法（ＨＡｌ法）、および精
製ｙ−ＨＢｓＡｇと酵母成分に対する抗体を用いてモル
モット皮膚被動性アナフィラキ−（ＰＣＡ）反応で検討
した結果、いずれの方法でも酵母成分は検出されなかっ
た。５ＤＳ−ポリアクリルアミドゲル電気泳動の分析に
よれば、精製ｙ−ＨＢｓＡｇはｈ−ＨＢｓＡｇの分子量
２５０００に相当するペプチドのみから成るものであっ
た。電子顕微鏡観察では、精製ｙ−ＨＢｓＡｇはｈ−Ｈ
ＢｓＡｇとほぼ同じ直径、形状を持った均一な球形粒子
として認められた。The presence or absence of yeast components in purified V-HBsAg was determined using an antibody against yeast components and a hemagglutination inhibition method (HAl method) using sensitized sheep red blood cells with yeast components that do not contain HBsAg, and purified y-HBsAg and antibodies against yeast components. As a result of an investigation using a guinea pig cutaneous anaphylaxis (PCA) reaction, yeast components were not detected by any of the methods. According to analysis by 5DS-polyacrylamide gel electrophoresis, purified y-HBsAg consisted only of peptides corresponding to the molecular weight of h-HBsAg of 25,000. In electron microscopy, purified y-HBsAg is h-H
It was recognized as uniform spherical particles with approximately the same diameter and shape as BsAg.
実施例２
抽出における各種添加剤の有効性を調べた。添加剤とし
て
アルキルフェノール−ポリエチレンオキサイド縮合物（
Ｔｒｉｔｏｎ Ｘ−１００）
モノラウリン酸ソルビタンエステル−ポリエチレンオキ
サイド縮合物（Ｔｗｅｅｎ ２０）モノオレイン酸ソル
ビタンエステル−ポリエチレンオキサイド縮合物（Ｔｗ
ｅｅｎ ８０）を選んだ。Example 2 The effectiveness of various additives in extraction was investigated. Alkylphenol-polyethylene oxide condensate (
Triton X-100) Sorbitan monolaurate-polyethylene oxide condensate (Tween 20) Sorbitan monooleate-polyethylene oxide condensate (Tw
I chose een 80).
実施例１に準じて抽出を行い、抽出液中のＨＢｓＡｇ活
性をＲＰＨＡ法により測定した（第１表）。Extraction was performed according to Example 1, and the HBsAg activity in the extract was measured by the RPHA method (Table 1).
実施例３ 精製ＨＢＳＡｇに対する添加剤の影響を調べた。Example 3 The effect of additives on purified HBSAg was investigated.
用いた添加剤は実施例２と同じである。The additives used were the same as in Example 2.
一定力価の精製ＨＢｓＡｇ溶液（２０ｐｇ／ｍｌ）に各
種濃度の添加剤を等量混合し２５℃で１・時間静置した
のちＲＰＨＡ法で抗原価を測定し抗原の失活の程度を測
定したく第２表）。Additives of various concentrations were mixed in equal amounts with a purified HBsAg solution (20 pg/ml) of a constant titer, left to stand at 25°C for 1 hour, and then the antigen titer was measured using the RPHA method to determine the degree of antigen inactivation. Table 2).
１、抽出における界面活性剤の有効性（第１表）２、精
製ＨＢｓ抗原に対する界面活性剤の影響（第２表）以上
の結果より酵母からＨＢｓ抗原を抽出する場合界面活性
剤を入れることによって効果的に抽出できる力や１表）
、界面活性剤の種類によっては、ＨＢｓ抗原を失活させ
る場合もある＠２林しかし’ｉ’ｗｅｅｎ ２０の場合
、抽出効率はＴｒｉｔｏｎ Ｘ−１００と変わらず、し
かもかなり高濃度までＨＢｓ抗原を失活させないという
特徴を持つ。1. Effectiveness of surfactants in extraction (Table 1) 2. Effect of surfactants on purified HBs antigen (Table 2) Based on the above results, when extracting HBs antigen from yeast, adding a surfactant Power that can be extracted effectively (Table 1)
, depending on the type of surfactant, HBs antigen may be inactivated @2 Hayashi However, in the case of 'i'ween 20, the extraction efficiency is the same as Triton It has the characteristic of not being activated.
（ほか３名） 手続補正占 昭和６３年３り／６日(3 others) Procedural correction divination 3/6, 1985
Claims (4)
子を組込んだ組換えベクターを用いて形質転換した微生
物菌体から産生されたＢ型肝炎ウィルス表面抗原を抗原
性を低下させずに抽出する方法において、高級脂肪酸ソ
ルビタンエステル−ポリエチレンオキサイド縮合物の存
在下に行うことを特徴とするＢ型肝炎ウィルス表面抗原
の抽出方法。(1) A method for extracting hepatitis B virus surface antigen produced from microbial cells transformed using a recombinant vector into which the hepatitis B virus surface antigen gene has been incorporated by genetic manipulation without reducing antigenicity. A method for extracting a hepatitis B virus surface antigen, which is carried out in the presence of a higher fatty acid sorbitan ester-polyethylene oxide condensate.
キサイド縮合物の添加量が０．１〜２．０ｗ／ｖ％であ
る請求項１記載の抽出方法。(2) The extraction method according to claim 1, wherein the amount of the higher fatty acid sorbitan ester-polyethylene oxide condensate added is 0.1 to 2.0 w/v%.
キサイド縮合物がモノラウリン酸ソルビタンエステル−
ポリエチレンオキサイド縮合物である請求項１記載の抽
出方法。(3) Higher fatty acid sorbitan ester - polyethylene oxide condensate is monolaurate sorbitan ester -
The extraction method according to claim 1, wherein the extract is a polyethylene oxide condensate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63017422A JPH01196293A (en) | 1988-01-29 | 1988-01-29 | Extraction of hepatitis b virus surface antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63017422A JPH01196293A (en) | 1988-01-29 | 1988-01-29 | Extraction of hepatitis b virus surface antigen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01196293A true JPH01196293A (en) | 1989-08-08 |
Family
ID=11943578
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63017422A Pending JPH01196293A (en) | 1988-01-29 | 1988-01-29 | Extraction of hepatitis b virus surface antigen |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01196293A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05252976A (en) * | 1991-03-15 | 1993-10-05 | Merck & Co Inc | Stabilization of recombinant hepatitis b virus surface protein obtained from recombinant host cell |
-
1988
- 1988-01-29 JP JP63017422A patent/JPH01196293A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05252976A (en) * | 1991-03-15 | 1993-10-05 | Merck & Co Inc | Stabilization of recombinant hepatitis b virus surface protein obtained from recombinant host cell |
| JPH0734753B2 (en) * | 1991-03-15 | 1995-04-19 | メルク エンド カムパニー インコーポレーテッド | Method for stabilizing recombinant hepatitis B virus surface protein obtained from recombinant host cell |
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