JP3733152B2 - Method for culturing pathogenic non-acid-fast bacteria - Google Patents
Method for culturing pathogenic non-acid-fast bacteria Download PDFInfo
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- JP3733152B2 JP3733152B2 JP31175194A JP31175194A JP3733152B2 JP 3733152 B2 JP3733152 B2 JP 3733152B2 JP 31175194 A JP31175194 A JP 31175194A JP 31175194 A JP31175194 A JP 31175194A JP 3733152 B2 JP3733152 B2 JP 3733152B2
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Description
【０００１】
【産業上の利用分野】
本発明は病原性非抗酸菌の培養方法に関する。
【０００２】
【従来の技術】
伝染性の疾患の予防のためワクチンが広く用いられている。百日咳やジフテリアなどの病原性非抗酸菌に対するワクチンの製造においてはこれら病原性細菌を培養し、得られた全菌体を不活性化して利用（不活性化ワクチン）したり、さらに菌体成分中から有効成分を取り出してワクチン原料として用いられている。 百日咳においては、接種後の発熱防止のため百日咳菌体から百日咳繊維状赤血球凝集素（ＦＨＡ）、百日咳毒素（ＰＴ）、百日咳外膜蛋白（６９Ｋ−ＯＭＰ）、百日咳線毛（Ｆimb）などの感染防禦抗原を取り出し、内毒素（ＥＴ）を除去した精製百日咳コンポーネントワクチンも実用化されつつある。
ジフテリアにおいては、菌体から遊離したジフテリア毒素を精製してジフテリアトキソイドとしてジフテリア症の予防に用いられている。
【０００３】
好気性非抗酸菌である百日咳菌やジフテリア菌は、培養時に菌の集落が培地表面に層状に形成して成長する。また、菌体自体が物理的刺激に弱い上にワクチンとしての有効成分の産生量が生育条件に深く関係する。
従って、これまで百日咳菌やジフテリア菌のワクチン原料の取得を目的とする培養は、静置培養を行い、表層に生育するこれら病原菌の菌体を利用して来た。
百日咳菌の培養用の担体としては、通常ガラス製のルー瓶が用いられている。この培養法は、例えば１５００mlの容量のルー瓶に４５０mlの培地を充填し、３５℃で５日間静置して培養するものであり、百日咳毒素、百日咳繊維状赤血球凝集素、百日咳外膜蛋白および百日咳線毛を良く産生するが、ワクチンを生産する際は大量のルー瓶と大きな培養室が必要となり、生産効率上問題がある。
一方、大量に百日咳菌を培養するためにガラス瓶による振盪培養〔インフェクション アンド イミュニティー（Infection and Immunity、 第２５巻、７８４頁、１９７９年）〕や大型のタンクによる撹拌培養が行われている〔アプライト ミクロバイオロジー バイオテクノロジー（Applied Microbiology Biotechnology、第２８巻、３５６頁、１９８８年）〕が、これらの培養法では、百日咳毒素はある程度産生されるものの、百日咳繊維状赤血球凝集素の産生量はルー瓶による静置培養法に比べ極端に低い。
また、ジフテリア菌の培養用の担体としては、通常ガラス製のコルベンが用いられている。この培養方法は、例えば３０００mlの容量のコルベンに３００mlの培地を充填し、３３℃で５日間静置して培養するものであり、ジフテリア毒素を良く産生するが、ワクチンを生産する際は大量のコルベンと大きな培養室が必要となり、大量生産に適した培養方法とはいえない。
【０００４】
【発明が解決しようとする課題】
このように、上記した病原菌の性状および菌体中のワクチン成分の含有量の維持を計る必要があるため、これらの細菌を大量培養する際には強制通気や撹拌が必須となり、細菌の大量培養で一般に行われるようなタンク培養を行うことはできなかった。
【０００５】
【課題を解決するための手段】
本発明は、培地に非イオン性界面活性剤を添加して、撹拌下培養することを特徴とする好気性の病原性非抗酸菌の培養方法を提供するものである。
上記好気性の病原性非抗酸菌とは、ワクチンや改良ワクチンの製造のためのコンポーネントを分離、取得するために培養する細菌であり、百日咳菌やジフテリア菌などが挙げられる。
本発明の培地に添加する非イオン性界面活性剤は、複数の親水性基と親油性基を有し、水中でイオン化しないものであり、例えばポリプロピレングリコールを疎水基としてエチレンオキサイドを附化したポリマーの非イオン性界面活性剤であるプルロニック系界面活性剤やポリオキシエチレンソルビタン脂肪酸エステルであるソルビタン系界面活性剤（ツィーン２０、ツィーン８０など、いずれも和光純薬株式会社製など）などが挙げられる。
とりわけ毒性が低く、低起泡性であるプルロニック系界面活性剤が好ましく、平均分子量が１，０００〜５，０００程度、平均エチレンオキサイド含量が３０重量％を超えるプルロニック系界面活性剤、好ましくは平均エチレンオキサイド含量が５０〜８０重量％程度のもの、例えばプルロニックＦ−６１、同Ｆ−６８，同Ｆ−８８，同Ｆ−１０８（いずれも、旭電化工業株式会社製など）などが挙げられ、とりわけプルロニックＦ−６８またはＦ−８８がより好ましく用いられる。
非イオン性界面活性剤として、例えばプルロニック系界面活性剤の場合、最終濃度０.００５〜５.０重量％（培地容量に対する添加する界面活性剤の重量％）、とりわけ０.０２〜３.０重量％、さらに好ましくは０．０５〜２.０重量％で培地に添加する。非イオン性界面活性剤は通常、培地に添加後高圧加熱などにより滅菌する。
培養は、細菌の生育に悪影響を与えない範囲内で撹拌して行われる。撹拌としては、例えば回転撹拌する場合は、培養槽内で回転体を５０〜１５０回転／分程度で回転させて行われる。ここで言う回転体とは、通常、培養の撹拌に用いられているものであれば特に限定はなく、例えば回転翼、回転羽根および回転子などが挙げられる。撹拌培養は、培地を入れたコルベンをロータリー式の振盪機に設置し回転撹拌してもよい。例えば５００mlの容量のコルベンに５０〜３００ml程度の培地を充填して培養する場合は、８０〜１５０回転／分程度、好ましくは１２０〜１４０回転／分程度でロータリー回転させて行われる。
本発明の培養方法においては、培養槽内に通気管を設置し、培養液中に空気，酵素ガス，酸素ガスと炭酸ガス（０〜４５％）の混合気体など放出する通気条件下に行うことができる。これらの通気は内圧０〜１kg／cm2G程度で行う。
【０００６】
百日咳菌の培養に関し、百日咳菌〔ボルデテラ パターシス（Bordetella pertussis）〕としては、例えば百日咳Ｉ相菌東浜株、百日咳Ｉ相菌山口株、百日咳Ｉ相菌１８−３２３株などの公知株が挙げられるが、なかでも百日咳Ｉ相菌東浜株〔インフェクション アンド イミュニティー（Infection and Immunity）、第６巻、８９９−９０４頁（１９７２年）〕が百日咳ワクチン製造株として好都合に用いられる。 培地としては公知の基本培地、例えばコーエンウイラー培地（Cohen-Wheeler Medium、アメリカン・ジャーナル・オブ・パブリック・ヘルス(American Journal of Public Health)、第36巻、371-376頁、(1946年)）、 ステナー ショルト培地（Stainer-Scholte Medium、ジャーナル・オブ・ジェネラル・マイクロバイオロジー（Journal of General Microbiology、第63巻、211-220頁、(1971年)）等の液体培地を用いることができるが、なかでもステナーショルト培地が好ましい。ステナーショルト培地に対し、最終濃度が約１〜５重量％になるようグルタミン酸ソーダを増量した改良培地を使用することもできる。
培養槽としては、ガラス製などの小型培養タンクのみならず、大型（３０〜６００リットル程度）の培養タンクを用いることができ、３０〜３７℃で２４〜１２０時間培養する。
【０００７】
本発明の培養方法により製造される百日咳菌は、これまで用いられた培養方法により得られた菌体と同様に、培養上清あるいは菌体を精製し、不活性化すること（特開昭６４−９２８号公報参照）により百日咳不活性化ワクチンとして用いることができる。また、さらに本培養方法により得られる百日咳菌またはその培養液から百日咳繊維状赤血球凝集素（ＦＨＡ）、百日咳毒素（ＰＴ）、百日咳外膜蛋白（６９Ｋ−ＯＭＰ）、百日咳線毛（Ｆimb）などの感染防禦成分を取り出し、百日咳コンポーネントワクチンの製造にも用いることができる。
ジフテリア菌の培養に関して、ジフテリア菌〔コリネバクテリウム ジフテリア（Coeynebacterium diphtheria）〕としては、例えばＰＷ−Ｎo.８、トロント株などがジフテリアトキソイド製造株として好都合に使用される。培養に用いられている公知の培地としては、例えばマーチン培地（Martin Medium）、ポープスミス培地（Pope-Smith Medium）、ポープの変法培地（Modified Pope Medium）、ミューラー培地（Mueller Medium）等の液体培地を用いることができるが、なかでもミューラー培地が好ましい。培養は３０〜３７℃で、１日〜１０日間行う。 ジフテリア菌培養中に産生されるジフテリア毒素は培養物を培養上清と菌体とに分離し、ついで培養上清を従来の公知の方法、例えば塩析による沈殿法、イオン交換ゲルや各種ゲル等への吸脱着法等、汎用されている常套手段を組み合わせて精製することにより得ることができる。
本発明の培養方法によると、好気性の病原性非抗酸菌の大量培養が可能である。さらに、本発明培養法では菌体量が増加するだけでなく有用な培養生成物の産生が促進され、特に百日咳菌の培養においては、従来促進方法が知られていなかった百日咳外膜蛋白および百日咳線毛が良く産生され、百日咳繊維状赤血球凝集素の生産量も増大する。
【０００８】
【作用および実施例】
以下に本発明を実施例により具体的に説明するが、本発明の範囲がこれらに限定されるものでないことは言うまでもない。
百日咳菌の培養に用いたステナーショルト培地の調製は培地１０リットル当たり下記の組成を用いた。基礎組成の部分は高圧加熱滅菌し、補液部分は濾過滅菌した。使用時に両者を混合して最終濃度１０リットル当たり下記の組成になるよう蒸留水を加えて調製した。
基礎組成： 補液組成：
塩化ナトリウム 25.0g Ｌ−シスチン 0.4g
リン酸二水素カリウム 5.0g 硫酸鉄五水塩 0.1g
塩化カリウム 2.0g ナイアシン 0.04g
塩化マグネシュウム六水塩 1.0g アスコルビン酸 0.2g
塩化カルシュウム二水塩 0.26g グルタチオン 1.0g
Ｌ−プロリン 2.4g
グルタミン酸ソーダ 107.0g
トリスバッファー 15.3g
【０００９】
実施例１
回転撹拌器および通気管を有する培養タンク（２リットル容量）にステナーショルト培地１.３リットルおよびプルロニックＦ−６８（ポリプロピレングリコールの分子量，１７５０；エチレンオキシド８０％）を０.１重量％（w／v）を加えて高圧加熱滅菌した。
これに百日咳菌Ｉ相東浜株を最終濃度２０億個／mlになるように接種し、９０回転／分で撹拌しながら、約３５℃で、２４〜１２０時間培養を行った（プルロニック添加法）。なお、培養中は通気量１リットル／分（酸素ガス１００％）で通気（バブリング）した。
対照としてそれぞれプルロニックを加えずに、１.５リットル容量のルー瓶を用いる静置培養、および培養槽に設置した通気管から酸素ガスを培養液中に放出する通気撹拌培養（バブリング法）を、上記と同様の培養条件下に行った。
各々の培養開始後、２４時間毎に培養液のサンプリングを行い、各検体の菌数、赤血球凝集活性（ＨＡ）、百日咳毒素（ＰＴ）、百日咳繊維状赤血球凝集素（ＦＨＡ）、百日咳外膜蛋白（６９Ｋ−ＯＭＰ）および百日咳線毛（Ｆimb）を下記の方法に従って測定した。その結果を〔表１〕に示す。
菌数の測定：比濁法により測定した。比色計（Coleman製）で、標準菌液から検体の菌数を算出した。単位は１０億個／ml。
赤血球凝集活性の測定：検体を０.０１Ｍリン酸緩衝液で２倍段階希釈後、０.６ v/v％のヒヨコ固定化血球を添加混合し、赤血球凝集反応を行った。凝集をおこした検体の最高希釈倍数を赤血球凝集素価（Haemagglutinin Titer：ＨＡ価） とした。
百日咳毒素、百日咳繊維状赤血球凝集素、百日咳外膜蛋白および百日咳線毛の測定：ＥＬＩＳＡ法により、精製した百日咳毒素、百日咳繊維状赤血球凝集素、百日咳外膜蛋白および百日咳線毛を標準として算出した。単位はμｇ蛋白／ml。
【００１０】
【表１】
【００１１】
実施例２
実施例１と同様にステナーショルト培地にプルロニックＦ−６８を濃度を変えて添加した培養の結果を〔表２〕に示す。
【００１２】
【表２】
実施例３
回転撹拌器および通気管を有する培養タンク（２リットル容量）に、ミューラー培地１.３リットルおよびプルロニックＦ−６８を０.３重量％（W/V ％）加え、高圧加熱滅菌した。これにジフテリア菌ＰＷ−No.８、トロント株を接種した。約３５℃で２４〜１２０時間、９０回転／分で撹拌培養した。培養中は、通気量０.３リットル／分（酸素ガス１００％）で通気（バブリング）した。対照としてプルロニックＦ−６８を加えずに培養槽に設置した通気管から酸素ガスを培養液中に放出する通気撹拌培養（バブリング法）を行った。各々の培養開始後、２４時間毎に培養液のサンプリングを行い、各検体の菌数、ジフテリア毒素量としてＬf およびＥＬＩＳＡ抗原価を下記の方法に従って測定した。その結果を〔表３〕に示す。
菌数の測定：培養液を遠心（約１０００Ｇ，３０min）し再浮遊した菌数を比色計（Coleman 製）で比濁法（６５０nm）により測定した。単位はＯＤ（６５０nm）値で表した。
ジフテリア毒素の測定（Ｉ）：標準抗毒素を用いるフロキュレーション法により測定した。単位はＬf／ml。
ジフテリア毒素の測定（II）：ＥＬＩＳＡ法により測定し、精製ジフテリアトキソイドを標準として算出した。単位はＥu／ml。
【表３】
【発明の効果】
本発明の培養法により、病原性非抗酸菌のタンク大量培養が可能となり、ワクチンの効率的な生産ができる。[0001]
[Industrial application fields]
The present invention relates to a method for culturing pathogenic non-acid-fast bacteria.
[0002]
[Prior art]
Vaccines are widely used to prevent infectious diseases. In the production of vaccines against pathogenic non-acid-fast bacteria such as whooping cough and diphtheria, these pathogenic bacteria are cultured, and the entire cells obtained are inactivated and used (inactivated vaccine), and further, cell components Active ingredients are extracted from the inside and used as vaccine raw materials. In pertussis, infection from pertussis cells to pertussis fibrous hemagglutinin (FHA), pertussis toxin (PT), pertussis outer membrane protein (69K-OMP), pertussis fimbria (Fimb), etc. to prevent fever after inoculation. A purified whooping cough component vaccine from which an anti-antigen is removed and endotoxin (ET) is removed is also being put into practical use.
In diphtheria, diphtheria toxin released from bacterial cells is purified and used as diphtheria toxoid for the prevention of diphtheria.
[0003]
Bordetella pertussis and diphtheria, which are aerobic non-acid-fast bacteria, grow when the colonies of bacteria form a layer on the surface of the medium during culture. In addition, the bacterial cells themselves are vulnerable to physical stimulation, and the production amount of active ingredients as a vaccine is closely related to the growth conditions.
Therefore, until now, culture aimed at obtaining vaccine raw materials for Bordetella pertussis and Diphtheria has been carried out by static culture and using cells of these pathogenic bacteria that grow on the surface layer.
As a carrier for the cultivation of Bordetella pertussis, a glass roux bottle is usually used. In this culture method, for example, a 1500 ml capacity bottle is filled with 450 ml of medium and left to stand at 35 ° C. for 5 days for cultivation. Pertussis toxin, pertussis filamentous hemagglutinin, pertussis outer membrane protein and Pertussis cilia are produced well, but when producing vaccine, a large number of roux bottles and a large culture room are required, which is problematic in terms of production efficiency.
On the other hand, in order to cultivate Bordetella pertussis in large quantities, shaking culture using a glass bottle (Infection and Immunity (Vol. 25, p. 784, 1979)) and agitation culture using a large tank are performed [Aplite Micro Biologic Biotechnology (Applied Microbiology Biotechnology, Vol. 28, 356, 1988)), but in these culture methods, pertussis toxin is produced to some extent, but the amount of pertussis filamentous hemagglutinin produced depends on the roux bottle. Extremely low compared to static culture.
Further, glass Kolben is usually used as a carrier for culturing diphtheria. In this culture method, for example, 300 ml of Kolben in a capacity of 3000 ml is filled and left to stand at 33 ° C. for 5 days for culturing, and diphtheria toxin is produced well. Kolben and a large culture room are required, which is not a culture method suitable for mass production.
[0004]
[Problems to be solved by the invention]
Thus, since it is necessary to measure the properties of the above-mentioned pathogenic bacteria and the content of vaccine components in the bacterial cells, forced aeration and agitation are indispensable when culturing these bacteria in large quantities. In general, tank culture was not possible.
[0005]
[Means for Solving the Problems]
The present invention provides a method for culturing an aerobic pathogenic non-acid-fast bacterium characterized by adding a nonionic surfactant to a medium and culturing with stirring.
The aerobic pathogenic non-acid-fast bacterium is a bacterium that is cultured to isolate and obtain components for the production of vaccines and improved vaccines, and examples include Bordetella pertussis and Diphtheria.
The nonionic surfactant to be added to the culture medium of the present invention has a plurality of hydrophilic groups and lipophilic groups and does not ionize in water. For example, a polymer in which ethylene glycol is added with polypropylene glycol as a hydrophobic group Pluronic surfactants that are nonionic surfactants of the above, and sorbitan surfactants that are polyoxyethylene sorbitan fatty acid esters (Tween 20, Tween 80, etc., both of which are manufactured by Wako Pure Chemical Industries, Ltd.) and the like. .
Pluronic surfactants having particularly low toxicity and low foaming properties are preferred, pluronic surfactants having an average molecular weight of about 1,000 to 5,000 and an average ethylene oxide content exceeding 30% by weight, preferably an average Examples having an ethylene oxide content of about 50 to 80% by weight, such as Pluronic F-61, F-68, F-88, F-108 (all manufactured by Asahi Denka Kogyo Co., Ltd.), etc. In particular, Pluronic F-68 or F-88 is more preferably used.
As a nonionic surfactant, for example, in the case of a pluronic surfactant, the final concentration is 0.005 to 5.0% by weight (% by weight of the surfactant to be added to the medium volume), especially 0.02 to 3.0. It is added to the culture medium at a weight percentage of 0.05 to 2.0 weight%. Nonionic surfactants are usually sterilized by high-pressure heating after addition to the medium.
Culturing is performed with stirring within a range that does not adversely affect the growth of bacteria. As stirring, for example, when rotating and stirring, the rotating body is rotated at a rate of 50 to 150 rotations / minute in the culture tank. The rotator as used herein is not particularly limited as long as it is usually used for culture agitation, and examples thereof include a rotor blade, a rotor blade, and a rotor. In the stirring culture, Kolben containing the medium may be placed on a rotary shaker and rotated and stirred. For example, when culturing a 500 ml Kolben filled with about 50 to 300 ml of medium, it is carried out by rotating at about 80 to 150 rotations / minute, preferably about 120 to 140 rotations / minute.
In the culturing method of the present invention, an aeration tube is installed in the culture tank, and the culturing method is carried out under aeration conditions for releasing air, enzyme gas, mixed gas of oxygen gas and carbon dioxide gas (0 to 45%) into the culture solution. Can do. These aerations are performed at an internal pressure of about 0 to 1 kg / cm 2 G.
[0006]
Regarding the cultivation of Bordetella pertussis, examples of Bordetella pertussis include known strains such as Bordetella I phase Higashihama strain, Bordetella pertussis I Yamaguchi strain, Bordetella pertussis 18-323 strain. Among them, the pertussis I phase Higashihama strain (Infection and Immunity, Vol. 6, 899-904 (1972)) is advantageously used as a pertussis vaccine production strain. As the medium, a known basic medium, such as Cohen-Wheeler Medium (American Journal of Public Health, Vol. 36, pages 371-376, (1946)), Liquid media such as Stainer-Scholte Medium, Journal of General Microbiology, 63, 211-220, (1971) can be used. However, a stearic salt medium is preferable, and an improved medium in which sodium glutamate is increased to a final concentration of about 1 to 5% by weight can be used.
As a culture tank, not only a small culture tank made of glass or the like but also a large culture tank (about 30 to 600 liters) can be used, and the culture is performed at 30 to 37 ° C. for 24 to 120 hours.
[0007]
The pertussis produced by the culturing method of the present invention is purified and inactivated by purifying the culture supernatant or the microbial cells in the same manner as the microbial cells obtained by the culturing methods used so far (JP-A-64). Can be used as a pertussis inactivating vaccine. Further, Bordetella pertussis obtained by the present culturing method or a culture solution thereof such as pertussis fibrous hemagglutinin (FHA), pertussis toxin (PT), pertussis outer membrane protein (69K-OMP), pertussis fimbria (Fimb), etc. Infectious antifungal ingredients can be removed and used for the production of pertussis component vaccines.
Regarding the culture of diphtheria, as diphtheria (Coeynebacterium diphtheria), for example, PW-No. 8, a Toronto strain, etc. are advantageously used as a diphtheria toxoid producing strain. Examples of known culture media used for the culture include liquid media such as Martin Medium, Pope-Smith Medium, Modified Pope Medium, Mueller Medium, and the like. Among them, Mueller medium is preferable. The culture is performed at 30 to 37 ° C. for 1 to 10 days. Diphtheria toxin produced during diphtheria culture is separated into culture supernatant and bacterial cells, and then the culture supernatant is separated from conventional culture methods such as precipitation by salting out, ion exchange gels and various gels, etc. It can be obtained by purification by combining conventional means such as adsorption / desorption method.
According to the culture method of the present invention, large-scale culture of aerobic pathogenic non-acid-fast bacteria is possible. Furthermore, the culture method of the present invention not only increases the amount of bacterial cells but also promotes the production of useful culture products. In particular, in the culture of Bordetella pertussis, pertussis outer membrane protein and pertussis, which had not been known in the past, have been known. Pili are well produced, and pertussis fibrous hemagglutinin production is also increased.
[0008]
[Operation and Examples]
EXAMPLES The present invention will be specifically described below with reference to examples, but it goes without saying that the scope of the present invention is not limited to these examples.
The following composition was used per 10 liters of the medium for the preparation of Steiner-Scholt medium used for cultivation of Bordetella pertussis. The basic composition portion was sterilized by high-pressure heat, and the replacement fluid portion was sterilized by filtration. At the time of use, both were mixed and distilled water was added so as to have the following composition per final concentration of 10 liters.
Basic composition: Fluid replacement composition:
Sodium chloride 25.0g L-cystine 0.4g
Potassium dihydrogen phosphate 5.0 g Iron sulfate pentahydrate 0.1 g
Potassium chloride 2.0g Niacin 0.04g
Magnesium chloride hexahydrate 1.0 g Ascorbic acid 0.2 g
Calcium chloride dihydrate 0.26g glutathione 1.0g
L-proline 2.4g
Sodium glutamate 107.0g
Tris buffer 15.3g
[0009]
Example 1
In a culture tank (2 liter capacity) having a rotary stirrer and an aeration tube, 1.3 liters of stenerscholt medium and Pluronic F-68 (molecular weight of polypropylene glycol, 1750; ethylene oxide 80%) are 0.1% by weight (w / v). Was added and sterilized by high-pressure heat.
This was inoculated with Bordetella pertussis I phase Higashihama strain to a final concentration of 2 billion cells / ml, and cultured at about 35 ° C. for 24 to 120 hours with stirring at 90 rpm (pluronic addition method). . During culture, aeration (bubbling) was performed at an aeration rate of 1 liter / min (oxygen gas 100%).
As a control, static culture using a 1.5-liter roux bottle without adding pluronics, and aeration and agitation culture (bubbling method) for releasing oxygen gas from the aeration tube installed in the culture tank into the culture solution, The culture conditions were the same as described above.
The culture solution is sampled every 24 hours after the start of each culture, and the number of bacteria in each sample, hemagglutination activity (HA), pertussis toxin (PT), pertussis fibrous hemagglutinin (FHA), pertussis outer membrane protein (69K-OMP) and pertussis cilia (Fimb) were measured according to the following method. The results are shown in [Table 1].
Measurement of the number of bacteria: Measured by the turbidimetric method. The number of bacteria in the specimen was calculated from the standard bacterial solution using a colorimeter (Coleman). The unit is 1 billion / ml.
Measurement of hemagglutination activity: The sample was diluted 2-fold with 0.01M phosphate buffer, and 0.6 v / v% chick-immobilized blood cells were added and mixed to carry out hemagglutination reaction. The highest dilution factor of the agglutinated specimen was defined as the haemagglutinin titer (HA value).
Measurement of pertussis toxin, pertussis hemagglutinin, pertussis outer membrane protein and pertussis cilia: ELISA was used to calculate purified pertussis toxin, pertussis hemagglutinin, pertussis outer membrane protein and pertussis cilia as standards. . The unit is μg protein / ml.
[0010]
[Table 1]
[0011]
Example 2
Table 2 shows the results of the culture in which Pluronic F-68 was added at different concentrations to the Stainer-Short medium as in Example 1.
[0012]
[Table 2]
Example 3
To a culture tank (2 liter capacity) having a rotary stirrer and an aeration tube, 1.3 liters of Mueller medium and 0.3% by weight (W / V%) of Pluronic F-68 were added and sterilized by high-pressure heat. This was inoculated with Diphtheria PW-No. 8, Toronto strain. The culture was stirred at 90 rpm for 24 to 120 hours at about 35 ° C. During the culture, aeration (bubbling) was performed at an aeration rate of 0.3 l / min (oxygen gas 100%). As a control, aeration and agitation culture (bubbling method) in which oxygen gas was released into the culture solution from an aeration tube installed in the culture tank without adding Pluronic F-68 was performed. The culture medium was sampled every 24 hours after the start of each culture, and the number of bacteria and the amount of diphtheria toxin in each sample were measured according to the following method. The results are shown in [Table 3].
Measurement of the number of bacteria: The culture solution was centrifuged (about 1000 G, 30 min), and the number of resuspended bacteria was measured by a turbidimetric method (650 nm) with a colorimeter (manufactured by Coleman). The unit is represented by OD (650 nm) value.
Measurement of diphtheria toxin (I): Measured by flocculation method using standard antitoxin. The unit is Lf / ml.
Measurement of diphtheria toxin (II): Measured by ELISA and calculated with purified diphtheria toxoid as a standard. The unit is Eu / ml.
[Table 3]
【The invention's effect】
By the culture method of the present invention, large-scale culture of pathogenic non-acid-fast bacteria is possible, and vaccines can be produced efficiently.
Claims (7)
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| JP31175194A JP3733152B2 (en) | 1993-12-15 | 1994-12-15 | Method for culturing pathogenic non-acid-fast bacteria |
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| JP5-314722 | 1993-12-15 | ||
| JP31472293 | 1993-12-15 | ||
| JP31175194A JP3733152B2 (en) | 1993-12-15 | 1994-12-15 | Method for culturing pathogenic non-acid-fast bacteria |
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| JP3733152B2 true JP3733152B2 (en) | 2006-01-11 |
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