JP2008169142A - Method for producing antibody to food poisoning bacterium - Google Patents
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- JP2008169142A JP2008169142A JP2007003306A JP2007003306A JP2008169142A JP 2008169142 A JP2008169142 A JP 2008169142A JP 2007003306 A JP2007003306 A JP 2007003306A JP 2007003306 A JP2007003306 A JP 2007003306A JP 2008169142 A JP2008169142 A JP 2008169142A
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- 206010016952 Food poisoning Diseases 0.000 title claims abstract description 16
- 208000019331 Foodborne disease Diseases 0.000 title claims abstract description 16
- 241000894006 Bacteria Species 0.000 title claims abstract description 13
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 9
- 235000013601 eggs Nutrition 0.000 claims abstract description 25
- 241000272534 Struthio camelus Species 0.000 claims abstract description 18
- 241000271566 Aves Species 0.000 claims abstract description 7
- 238000009007 Diagnostic Kit Methods 0.000 claims abstract description 3
- 210000002969 egg yolk Anatomy 0.000 claims description 18
- 241000271567 Struthioniformes Species 0.000 claims description 6
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 claims 2
- 238000000034 method Methods 0.000 abstract description 4
- 241000124008 Mammalia Species 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract description 3
- 208000004739 Egg Hypersensitivity Diseases 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 229940039227 diagnostic agent Drugs 0.000 abstract 1
- 239000000032 diagnostic agent Substances 0.000 abstract 1
- 241000287828 Gallus gallus Species 0.000 description 15
- 235000013330 chicken meat Nutrition 0.000 description 15
- 102000002322 Egg Proteins Human genes 0.000 description 13
- 108010000912 Egg Proteins Proteins 0.000 description 13
- 235000013345 egg yolk Nutrition 0.000 description 12
- 239000000427 antigen Substances 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 206010020751 Hypersensitivity Diseases 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000004062 sedimentation Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 206010039438 Salmonella Infections Diseases 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000000172 allergic effect Effects 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 210000000991 chicken egg Anatomy 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 206010039447 salmonellosis Diseases 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010016946 Food allergy Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000001775 anti-pathogenic effect Effects 0.000 description 1
- 230000003935 attention Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 201000010860 egg allergy Diseases 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 230000010429 evolutionary process Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229940037467 helicobacter pylori Drugs 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 230000017448 oviposition Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Landscapes
- Feed For Specific Animals (AREA)
- Fodder In General (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
本発明は食中毒菌に対する抗体の産生方法に関する。より詳しくは鳥類、特にダチョウを用いその卵から食中毒菌の抗体を産生する方法に関するものである。 The present invention relates to a method for producing an antibody against food poisoning bacteria. More specifically, the present invention relates to a method for producing antibodies against food poisoning bacteria from eggs using birds, particularly ostriches.
哺乳類と比べ鳥類は大変高い免疫能力を持つことが知られている。これは鳥類が哺乳類とは全く異なった進化の過程を経てきているからといわれている。また鳥類の雌が有する抗体は卵となって体外に排出されるので、抗体の作製にも利用されている。 Birds are known to have much higher immunity than mammals. This is said to be because birds have undergone a completely different evolutionary process from mammals. In addition, the antibodies possessed by avian females are excreted out of the body as eggs, so they are also used for the production of antibodies.
鶏は従来からこのような抗体産生のために検討され、実際に利用されている動物である。例えば、鶏に抗原を接種し、抗原に特異的な抗体を鶏体内に形成させる。抗体が形成された後、この鶏が産卵した卵の全卵、卵黄または卵白より特異的抗体を含有する材料を大量に得る方法が開示されている（特許文献１参照）。 Chickens are animals that have been studied and used in practice for the production of such antibodies. For example, an antigen is inoculated into a chicken and an antibody specific for the antigen is formed in the chicken. A method for obtaining a large amount of a material containing a specific antibody from the whole egg, egg yolk or egg white of the egg laid by the chicken after the antibody is formed is disclosed (see Patent Document 1).
しかし、鶏の卵では量産性に課題があると考え、より量産性を向上させた抗体の産生方法が提案された。特許文献２は、大腸菌、ヘリコバクター・ピロリおよびサルモネラ菌を抗原化させて、幼いひよこに３種の抗原を同時に接種することで、抗−病原性バクテリア抗体を１つの卵に共存させる技術を開示している。すなわち、複数の抗原によって免疫させた鶏に複数の抗体を作らせ、１つの鶏卵から複数の抗体を得ようとするものである。
鶏は１つの卵が小さく、大量に抗体を産生する場合は多くの鶏を用いる必要がある。鶏は個体が違えば免疫力も異なり、産生される抗体もそれぞれ異なる。従って同じ抗原を用いても免疫した個体が異なれば、全くの同一物ではない。それゆえ、得られた抗体は個体の違いによるロット差が発生してしまうという課題があった。抗体を利用する場合はできるだけ、均一な品質でロット間による違いのないものが望ましい。 A chicken has a small egg, and it is necessary to use a large number of chickens if it produces antibodies in large quantities. Different chickens have different immunity and different antibodies are produced. Therefore, even if the same antigen is used, if the immunized individuals are different, they are not exactly the same. Therefore, the obtained antibody has a problem that lot differences due to individual differences occur. When using antibodies, it is desirable that they are as uniform as possible with no difference between lots.
また、鶏卵に対するアレルギーを有する人は比較的多いので、鶏卵から得ることのできる特異的抗体含有物を経口摂取し、予防若しくは治療効果を図る場合は、アレルギー反応に注意しなければならないという課題もある。 In addition, since there are relatively many people who are allergic to chicken eggs, there is a problem that when taking a specific antibody-containing substance obtained from chicken eggs orally and aiming at preventive or therapeutic effects, attention must be paid to allergic reactions. is there.
本発明はかかる課題の解決のために想到されたものである。本発明では鳥類としてダチョウ（Struthio camelus）を用いて、その卵から抗体を得る。また、その卵から特異的抗体含有物を得る。 The present invention has been conceived for solving such problems. In the present invention, an ostrich (Struthio camelus) is used as a bird, and an antibody is obtained from the egg. Further, a specific antibody-containing product is obtained from the egg.
ダチョウの卵は鶏卵のおよそ２０〜３０倍であり、その卵から得ることのできる抗体の量も同等倍である。従って、１羽のダチョウから大量の抗体を得ることができ、これはすなわちロット間の差を少なくすることができるという効果を得る。 Ostrich eggs are approximately 20-30 times that of chicken eggs, and the amount of antibody that can be obtained from the eggs is equivalent. Therefore, a large amount of antibody can be obtained from one ostrich, which means that the difference between lots can be reduced.
また、ダチョウの卵は鶏卵と比較しアレルギーが少ないという特徴がある。従って、卵から得られた抗体含有物を経口摂取し、予防若しくは治療効果を図る場合には、アレルギー反応というリスクを回避しやすいという効果を得る。 In addition, ostrich eggs are less allergic than chicken eggs. Therefore, when an antibody-containing product obtained from an egg is orally ingested to achieve a preventive or therapeutic effect, an effect of easily avoiding the risk of an allergic reaction is obtained.
ダチョウに抗原を免疫すれば血液に特異抗体が産生され、そのうちＩｇＹが卵黄に移行し、産卵と同時に排出される。この卵黄よりＩｇＹを抽出すれば特異的で高感度の抗体を大量に得ることができ、それを診断・検査薬や治療薬として利用できる。一個の卵黄から２〜４ｇのＩｇＹの精製が可能であり、１羽のダチョウから年間４００ｇの抗体を得ることが可能である。つまり１羽で約４０００万人分以上のロット差の少ない診断キットの作製が可能となる。多数のダチョウを使用すれば莫大な量の抗体の生産も可能であり、工業用としての利用価値がある。 When an ostrich is immunized with an antigen, specific antibodies are produced in the blood, of which IgY moves to the yolk and is excreted simultaneously with egg laying. If IgY is extracted from this egg yolk, a large amount of a specific and highly sensitive antibody can be obtained, which can be used as a diagnostic / testing agent or therapeutic agent. It is possible to purify 2 to 4 g of IgY from one egg yolk and obtain 400 g of antibody per year from one ostrich. In other words, it is possible to produce a diagnostic kit with a small lot difference of about 40 million or more in one bird. If a large number of ostriches are used, an enormous amount of antibody can be produced, which is useful for industrial use.
ここでは、食中毒菌として有名サルモネラ・エンテリティヂス（Salmonella Enteritidis 以下ＳＥと呼ぶ）に対する抗体をダチョウの卵黄にて作製した。 Here, an antibody against Salmonella Enteritidis (hereinafter referred to as SE), which is famous as a food poisoning fungus, was produced in ostrich egg yolk.
市販のＳＥ抗原ワクチン（商品名 レイヤーミューンＳＥ）（第一製薬社製）１ｍｌを産卵ダチョウに隔週免疫（筋肉内注射）し、初回免疫より4週目の卵黄を採取した。卵黄の精製は以下のように行う。 A commercially available SE antigen vaccine (trade name: Layer Mun SE) (Daiichi Pharmaceutical Co., Ltd.) (1 ml) was immunized biweekly (intramuscular injection) to laying ostriches, and yolks were collected at 4 weeks from the first immunization. Purification of egg yolk is performed as follows.
まず、卵黄を5倍量のＴＢＳ（２０ｍＭＴｒｉｓ−ＨＣｌ、０．１５Ｍ ＮａＣｌ，０．５％ＮａＮ３）と同量の１０％デキストラン硫酸／ＴＢＳを加え２０分攪拌する。 First, 5 times the amount of TBS (20 mM Tris-HCl, 0.15 M NaCl, 0.5% NaN3) and the same amount of 10% dextran sulfate / TBS are added to the yolk and stirred for 20 minutes.
次に、１ＭＣａＣｌ２／ＴＢＳを卵黄と同量加え攪拌し、１２時間静置する。その後、１５０００ｒｐｍで２０分遠心し上清を回収する。 Next, 1MCaCl2 / TBS is added in the same amount as egg yolk, stirred and allowed to stand for 12 hours. Thereafter, the supernatant is recovered by centrifugation at 15000 rpm for 20 minutes.
さらに、最終濃度４０％になるように硫酸アンモニウムを加え４℃で１２時間静置する。静置後、１５０００ｒｐｍで２０分遠心し、沈殿物を回収する。最後に、卵黄と同量のＴＢＳに再懸濁し、ＴＢＳにて透析する。この課程により９０％以上の純度のＩｇＹの回収が可能となった。１個の卵黄より２〜４ｇのＩｇＹを精製することができた。 Further, ammonium sulfate is added so that the final concentration is 40%, and the mixture is allowed to stand at 4 ° C. for 12 hours. After standing, the mixture is centrifuged at 15000 rpm for 20 minutes to collect the precipitate. Finally, it is resuspended in the same amount of TBS as egg yolk and dialyzed with TBS. This process enabled the recovery of 90% or higher purity IgY. 2 to 4 g of IgY could be purified from one egg yolk.
図１は、ＳＥ非接種（１羽）と接種ダチョウ（免疫４週目）（４羽）の卵黄抽出液に対して寒天ゲル内沈降反応を用いて抗体の存在を調べた結果の写真である。写真中央（Ａ）が抗原として用いたインフルエンザウイルスＨＡ抗原の結果である。抗原が存在すると白色の沈降線が出現する。 FIG. 1 is a photograph of the results of examining the presence of antibodies using an agar gel precipitation reaction on the yolk extract of SE non-inoculated (1) and inoculated ostrich (4 weeks after immunization) (4). . The center of the photograph (A) is the result of the influenza virus HA antigen used as the antigen. When the antigen is present, a white sedimentation line appears.
図１で１はリン酸緩衝液（ＰＢＳ），２はＳＥ非接種ダチョウの卵黄、３〜６はＳＥ接種ダチョウ（免疫４週目）４羽の卵黄抽出液を添加したものである。ＰＢＳおよび非接種ダチョウでは卵黄は白色の沈降線は出現せず、抗体が存在しないと結論できる。一方、接種ダチョウ４羽の場合は全て白色の沈降性が出現した。従ってＳＥを接種した全てのダチョウの卵で、抗体の存在が確認できた。 In FIG. 1, 1 is a phosphate buffer (PBS), 2 is an egg yolk of an SE non-inoculated ostrich, 3 to 6 are SE-inoculated ostriches (4 weeks after immunization) and 4 yolk extracts are added. In PBS and non-inoculated ostrich, egg yolk does not show a white sedimentation line and it can be concluded that no antibody is present. On the other hand, in the case of 4 inoculated ostriches, white sedimentation appeared. Therefore, the presence of the antibody could be confirmed in all ostrich eggs inoculated with SE.
1羽のダチョウメスから4月から９月までの産卵期に約１００個の卵が得られることより、最大約４００ｇの抗体（ＩｇＹ）が得られる。同じ個体から得られる抗体は、卵が変わっても同じ抗体であるので、ロット間の違いのない抗体を大量に産生することができる。また、ここで得られた抗体は、精製しサルモネラ感染患者（食中毒）の治療薬として応用可能である。 Since about 100 eggs are obtained from one ostrich female during the spawning season from April to September, a maximum of about 400 g of antibody (IgY) can be obtained. Since antibodies obtained from the same individual are the same antibody even if the egg changes, it is possible to produce a large amount of antibodies with no difference between lots. Further, the antibody obtained here can be purified and applied as a therapeutic agent for Salmonella-infected patients (food poisoning).
また、抗体を精製せずに卵黄を特異的抗体含有物として食用に用いてもサルモネラの感染予防、治療として応用可能である。この場合そのままでもよいが、粉末乾燥させて用いてもよい。特にダチョウ卵は卵アレルギー性が極めて少ないので、鶏卵アレルギー患者においても服用可能と考えられる。 In addition, even if egg yolk is used as a food containing a specific antibody without purifying the antibody, it can be applied for prevention and treatment of Salmonella infection. In this case, it may be used as it is, but it may be used after powder drying. In particular, ostrich eggs are extremely low in egg allergy, so it can be taken even in chicken allergy patients.
さらに、卵黄粉末を産卵鶏の飼料に含有させれば、鶏のサルモネラ感染も予防でき、サルモネラ非含有の鶏卵の産生にもできる。 Furthermore, if egg yolk powder is contained in the laying hen's feed, it is possible to prevent the Salmonella infection of the chicken and to produce a salmonella-free chicken egg.
Claims (4)
前記食中毒菌を接種した個体が産卵した卵の卵黄からＩｇＹを抽出する工程とを含む食中毒菌に対する抗体の産生方法。 Inoculating avian females with food poisoning bacteria;
A method for producing an antibody against food poisoning bacteria, comprising the step of extracting IgY from the yolk of eggs laid by an individual inoculated with said food poisoning bacteria.
A specific antibody-containing product comprising at least a part of eggs laid by an individual who inoculates an ostrich female with a food-poisoning bacterium and ingested the food-poisoning bacterium.
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| WO2013027356A1 (en) * | 2011-08-19 | 2013-02-28 | オーストリッチファーマ株式会社 | Antibody and antibody-containing composition |
| JP2013163662A (en) * | 2012-02-10 | 2013-08-22 | Ostrich Pharma Kk | Antibody-including pollen allergy protective material |
| WO2019088230A1 (en) * | 2017-11-02 | 2019-05-09 | オーストリッチファーマ株式会社 | Ostrich antibody for bacterial infectious diseases |
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| JP2011020927A (en) * | 2009-07-13 | 2011-02-03 | Ostrich Pharma Kk | Antibody produced from ostrich and filter using the same |
| EP3406716A1 (en) * | 2011-08-19 | 2018-11-28 | Ostrich Pharma KK | Antibody and antibody-containing composition |
| US11041016B2 (en) | 2011-08-19 | 2021-06-22 | Ostrich Pharma Kk | Compositions containing anti-HIV ostrich antibodies |
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| US9828419B2 (en) | 2011-08-19 | 2017-11-28 | Ostrich Pharma Kk | Antibody and antibody-containing composition |
| US10106599B2 (en) | 2011-08-19 | 2018-10-23 | Ostrich Pharma Kk | Antibody and antibody-containing composition |
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| JP2013163662A (en) * | 2012-02-10 | 2013-08-22 | Ostrich Pharma Kk | Antibody-including pollen allergy protective material |
| WO2019087372A1 (en) * | 2017-11-02 | 2019-05-09 | オーストリッチファーマ株式会社 | Ostrich antibody for bacterial infectious diseases |
| WO2019088230A1 (en) * | 2017-11-02 | 2019-05-09 | オーストリッチファーマ株式会社 | Ostrich antibody for bacterial infectious diseases |
| JPWO2019088230A1 (en) * | 2017-11-02 | 2020-11-26 | オーストリッチファーマ株式会社 | Ostrich antibody for bacterial infections |
| JP7075671B2 (en) | 2017-11-02 | 2022-05-26 | オーストリッチファーマ株式会社 | Ostrich antibody for bacterial infectious diseases |
| JP2022093735A (en) * | 2017-11-02 | 2022-06-23 | オーストリッチファーマ株式会社 | Ostrich antibody for bacterial infectious diseases |
| JP7329279B2 (en) | 2017-11-02 | 2023-08-18 | オーストリッチファーマ株式会社 | ostrichantibody for bacterial infections |
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