JP2017006008A - Antibody and antiserum against mers - Google Patents
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- 208000025370 Middle East respiratory syndrome Diseases 0.000 claims abstract description 27
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 claims abstract description 16
- 241000272534 Struthio camelus Species 0.000 claims abstract description 16
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 13
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 13
- 101710198474 Spike protein Proteins 0.000 claims abstract description 11
- 230000003053 immunization Effects 0.000 claims abstract description 10
- 241000255789 Bombyx mori Species 0.000 claims abstract description 7
- 241000701447 unidentified baculovirus Species 0.000 claims abstract description 6
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 208000015181 infectious disease Diseases 0.000 abstract description 12
- 238000000034 method Methods 0.000 abstract description 5
- 102000018697 Membrane Proteins Human genes 0.000 abstract 1
- 108010052285 Membrane Proteins Proteins 0.000 abstract 1
- 239000000427 antigen Substances 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 210000002969 egg yolk Anatomy 0.000 description 7
- 241000287828 Gallus gallus Species 0.000 description 6
- 241000286209 Phasianidae Species 0.000 description 6
- 235000013330 chicken meat Nutrition 0.000 description 6
- 229940096437 Protein S Drugs 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 230000003449 preventive effect Effects 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 102000002322 Egg Proteins Human genes 0.000 description 4
- 108010000912 Egg Proteins Proteins 0.000 description 4
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- 239000000203 mixture Substances 0.000 description 4
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 3
- 101000908391 Homo sapiens Dipeptidyl peptidase 4 Proteins 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000271566 Aves Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
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- 235000009508 confectionery Nutrition 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
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- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007923 nasal drop Substances 0.000 description 1
- 229940100662 nasal drops Drugs 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
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- 208000011580 syndromic disease Diseases 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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Abstract
Description
本発明は、中東呼吸器症候群コロナウイルス（Ｍｉｄｄｌｅ Ｅａｓｔ ｒｅｓｐｉｒａｔｏｒｙ ｓｙｎｄｒｏｍｅ ｃｏｒｏｎａｖｉｒｕｓ：ＭＥＲＳ−ＣｏＶ）と反応する抗体および抗血清に関するものである。 The present invention relates to antibodies and antisera that react with Middle East respiratory syndrome coronavirus (MERS-CoV).
ＭＥＲＳは、２０１２年に初めて確認されたウイルス性の感染症である。主な症状は、発熱、咳、息切れなどである。２００２年から２００３年に発見されたＳＡＲＳ（Ｓｅｖｅｒｅ Ａｃｕｔｅ Ｒｅｓｐｉｒａｔｏｒｙ Ｓｙｎｄｒｏｍｅ：重症急性呼吸器症候群）と症状は似ている。しかし、ＳＡＲＳの感染後の死亡率が９％程度であるのに対し、ＭＥＲＳの死亡率は４０〜５０％と高く、危険な病気であるといえる。 MERS is a viral infection that was first identified in 2012. The main symptoms are fever, cough, shortness of breath. Symptoms are similar to SARS (Severe Accurate Respiratory Syndrome), which was discovered in 2002-2003. However, the mortality rate after infection with SARS is about 9%, whereas the mortality rate of MERS is as high as 40 to 50%, which can be said to be a dangerous disease.
ＭＥＲＳはＭＥＲＳコロナウイルス（Ｍｉｄｄｌｅ Ｅａｓｔ ｒｅｓｐｉｒａｔｏｒｙ ｓｙｎｄｒｏｍｅ ｃｏｒｏｎａｖｉｒｕｓ：ＭＥＲＳ−ＣｏＶ）によって引き起こされるということがわかっている。しかし、ＭＥＲＳの感染メカニズムについては、まだまだ不明な点が多く、直接作用する医薬組成物もまだ得られているとは言えない。 MERS has been found to be caused by MERS coronavirus (MERS-CoV). However, there are still many unclear points regarding the mechanism of MERS infection, and it cannot be said that a pharmaceutical composition that directly acts has been obtained.
特許文献１は、生体に広く分布しているＣＤ２６の抗体の特定部分のアミノ酸配列を認識する抗モノクロナール抗体がＭＥＲＳ−ＣｏＶとＣＤ２６発現細胞との結合を阻害することで、ＭＥＲＳへの感染予防又は治療剤を提供する。 Patent Document 1 discloses that anti-monoclonal antibody that recognizes an amino acid sequence of a specific portion of a CD26 antibody widely distributed in a living body inhibits binding between MERS-CoV and a CD26-expressing cell, thereby preventing infection with MERS. Alternatively, a therapeutic agent is provided.
特許文献１は、マウスから得たモノクロナール抗体を感染予防又は治療剤として用いる。しかし、特許文献１のモノクロナール抗体は、ＭＥＲＳ−ＣｏＶに直接結合するものではなく、また感染予防の直接実施例がないため、ＣＤ２６の発現細胞とＭＥＲＳ−ＣｏＶの結合が阻害されたということを示すに留まる。 Patent Document 1 uses a monoclonal antibody obtained from a mouse as a preventive or therapeutic agent for infection. However, the monoclonal antibody of Patent Document 1 does not directly bind to MERS-CoV, and since there is no direct example for preventing infection, the binding between CD26-expressing cells and MERS-CoV was inhibited. Stay on show.
本発明は、ＭＥＲＳ−ＣｏＶに直接作用し、宿主細胞との結合を阻害する抗体および抗血清を提供するものである。 The present invention provides antibodies and antisera that act directly on MERS-CoV and inhibit binding to host cells.
より具体的に本発明に係るＭＥＲＳ用抗体の製造方法は、
バキュロウイルスベクターにＭＥＲＳウイルスのスパイク蛋白質遺伝子を組み込み、カイコ細胞でリコンビナント蛋白質を得る工程と、
前記リコンビナント蛋白質を雌ダチョウに免疫する工程と、
前記免疫された雌ダチョウが産卵した卵からＩｇＹを精製する工程を含むことを特徴とする。
More specifically, the method for producing an antibody for MERS according to the present invention includes:
Incorporating a MERS virus spike protein gene into a baculovirus vector to obtain a recombinant protein in silkworm cells;
Immunizing female ostrich with the recombinant protein;
The method includes the step of purifying IgY from eggs laid by the immunized female ostrich.
また、本発明に係るＭＥＲＳ用抗血清の製造方法は、
バキュロウイルスベクターにＭＥＲＳウイルスのスパイク蛋白質遺伝子を組み込み、カイコ細胞でリコンビナント蛋白質を得る工程と、
前記リコンビナント蛋白質を雌ダチョウに免疫する工程と、
前記免疫された雌ダチョウから抗血清を取得する工程を含むことを特徴とする。
In addition, the method for producing an antiserum for MERS according to the present invention includes:
Incorporating a MERS virus spike protein gene into a baculovirus vector to obtain a recombinant protein in silkworm cells;
Immunizing female ostrich with the recombinant protein;
The method includes obtaining antiserum from the immunized female ostrich.
本発明に係るＭＥＲＳ用抗体およびＭＥＲＳ用工血清の製造方法では、ＭＥＲＳウイルスのスパイク蛋白質だけをカイコ細胞で作製し、抗原として利用するので、得られた抗体には毒性が混入する心配がない。 In the method for producing the MERS antibody and the MERS engineered serum according to the present invention, only the MERS virus spike protein is produced in silkworm cells and used as an antigen, so there is no concern that the obtained antibody is toxic.
また、本発明に係るＭＥＲＳ用抗体およびＭＥＲＳ用工血清の製造方法で作製される抗体および抗血清は、ダチョウを使用するので、大量に産生することができる。また、ダチョウは、抗原に対して反応性が高い抗体を産生するので、医薬組成物の生産性が高くなる。 Moreover, since the antibody and antiserum produced with the manufacturing method of the antibody for MERS and the engineering serum for MERS which concern on this invention use an ostrich, they can be produced in large quantities. In addition, ostriches produce antibodies that are highly reactive with antigens, which increases the productivity of pharmaceutical compositions.
以下に本発明に係るＭＥＲＳ用医薬組成物の製造方法について実施例を示し説明を行う。なお、以下の説明は、本発明の一実施形態および一実施例を例示するものであり、本発明が以下の説明に限定されるものではない。以下の説明は本発明の趣旨を逸脱しない範囲で改変することができる。 Examples of the method for producing a pharmaceutical composition for MERS according to the present invention will be described below. The following description exemplifies an embodiment and an example of the present invention, and the present invention is not limited to the following description. The following description can be modified without departing from the spirit of the present invention.
＜抗原＞
バキュロウイルスベクターにＭＥＲＳウイルス （ＨＣｏＶ−ＥＭＣ／２０１２）のスパイク蛋白遺伝子（Ｍｅｔ１−Ｔｒｐ１２９７））を組み込み、カイコ細胞（Ｓｆ９細胞）で作製したリコンビナント蛋白質を用いた。
<Antigen>
A spike protein gene (Met1-Trp1297) of MERS virus (HCoV-EMC / 2012) was incorporated into a baculovirus vector, and a recombinant protein produced in silkworm cells (Sf9 cells) was used.
＜抗体作製方法＞
成熟したメス鳥（ダチョウ、ニワトリ、ウズラ）を用いた。上記ＭＥＲＳスパイク蛋白液（蛋白量１００μｇ）をフロイントの完全アジュバント０．２ｍＬと混和し、ダチョウに初回免疫した。各抗原を個別に５羽のダチョウ、５羽のニワトリ、５羽のウズラに接種した。ダチョウもニワトリもウズラも同量の抗原を接種したことになる。
<Antibody production method>
Mature female birds (ostrich, chicken, quail) were used. The MERS spike protein solution (protein amount 100 μg) was mixed with 0.2 mL of Freund's complete adjuvant, and the ostrich was first immunized. Each antigen was individually inoculated into 5 ostriches, 5 chickens and 5 quails. Ostriches, chickens and quails have been vaccinated with the same amount of antigen.
初回免疫後、２週目と４週目に５０μｇの抗原とフロイントの不完全アジュバントの混和液を、各鳥に追加免疫した。初回免疫後８週目に得られた各鳥からの血清分離、および卵黄から卵黄抗体（ＩｇＹ）を精製した。 After the first immunization, each bird was boosted with a mixture of 50 μg of antigen and Freund's incomplete adjuvant at 2 and 4 weeks. Serum separation from each bird obtained 8 weeks after the first immunization, and yolk antibody (IgY) was purified from the yolk.
＜ＥＬＩＳＡ法＞
得られた血清およびＩｇＹのＭＥＲＳスパイク蛋白対する反応性をＥＬＩＳＡにより検証した。 ９６穴ＥＬＩＳＡプレートの各穴にＭＥＲＳスパイク蛋白（リコンビナント蛋白）をそれぞれ２μｇを別々に固層化した（室温で４時間）。
<ELISA method>
The reactivity of the obtained serum and IgY to the MERS spike protein was verified by ELISA. 2 μg of MERS spike protein (recombinant protein) was separately solidified in each hole of the 96-well ELISA plate (4 hours at room temperature).
その後、ダチョウ抗体（各５羽のダチョウから得た卵黄からの抗体の混合物）、ニワトリ抗体（各５羽のニワトリから得た卵黄からの抗体の混合物）、ウズラ抗体（各５羽のウズラから得た卵黄からの抗体の混合物）の段階希釈液（血清は原液）（ＩｇＹ原液は２ｍｇ／ｍＬ）を各穴に滴下し、室温で１時間反応させた。 Then ostrich antibody (mixture of antibodies from egg yolk obtained from each of five ostriches), chicken antibody (mixture of antibodies from egg yolk obtained from each of five chickens), quail antibody (obtained from five quail each) A mixture of antibodies from egg yolk) was added dropwise to each well (serum was a stock solution) (IgY stock solution was 2 mg / mL) and allowed to react at room temperature for 1 hour.
洗浄後、各抗体に対するＨＲＰ標識２次抗体を室温で１時間反応させた。十分な洗浄後、ペルオキシダーゼ用発色キット（Ｓ−Ｂｉｏ ＳＵＭＩＬＯＮ）を用いてプレートリーダーにて吸光度（４５０ｎｍ）を測定した。免疫前の各鳥種の卵黄抗体の２倍以上の吸光度値を示す最大希釈倍率をＥＬＩＳＡ値として示した。表１に結果を示す。 After washing, an HRP-labeled secondary antibody for each antibody was reacted at room temperature for 1 hour. After sufficient washing, absorbance (450 nm) was measured with a plate reader using a peroxidase coloring kit (S-Bio SUMILON). The maximum dilution factor showing the absorbance value of 2 times or more of the yolk antibody of each bird species before immunization was shown as the ELISA value. Table 1 shows the results.
表１を参照して、ＭＥＲＳのスパイクタンパク質（ リコンビナント蛋白）を免疫することにより、ダチョウ、ニワトリ、ウズラに高感度の抗血清および卵黄ＩｇＹ抗体が作製されることが判明した。特に、各鳥種類には同量の抗原を免疫したのにもかかわらず、巨大なダチョウが最も反応性が高い抗体が産生されている。これは、少量の抗原でも高感度の抗体が産生できることを意味する。 Referring to Table 1, it was found that immunization with MERS spike protein (recombinant protein) produced highly sensitive antiserum and egg yolk IgY antibody in ostrich, chicken and quail. In particular, despite the immunization of the same amount of antigen in each bird species, the most reactive antibody is produced by the giant ostrich. This means that a highly sensitive antibody can be produced even with a small amount of antigen.
本発明に係る抗体および抗血清は、ＭＥＲＳの感染予防・治療薬（点鼻薬、ネブライザー液、注射薬）、ＭＥＲＳの感染予防用スプレー剤、ＭＥＲＳの感染予防用マスク、ＭＥＲＳの感染予防用エアコンフィルターおよび噴霧剤、ＭＥＲＳの感染予防防疫服、ＭＥＲＳの感染予防用の食品（キャンディーやガムなど）、ＭＥＲＳの診断薬に利用することができる。 The antibody and antiserum according to the present invention include MERS infection preventive / therapeutic drugs (nasal drops, nebulizer solution, injection), MERS infection preventive spray, MERS infection preventive mask, MERS infection preventive air conditioner filter Further, it can be used for sprays, MERS infection prevention and prevention clothing, MERS infection prevention foods (candy, gum, etc.), and MERS diagnostic agents.
Claims (2)
前記リコンビナント蛋白質を雌ダチョウに免疫する工程と、
前記免疫された雌ダチョウが産卵した卵からＩｇＹを精製する工程を含むことを特徴とするＭＥＲＳ用抗体の製造方法。 Incorporating a MERS virus spike protein gene into a baculovirus vector to obtain a recombinant protein in silkworm cells;
Immunizing female ostrich with the recombinant protein;
A method for producing an antibody for MERS, comprising the step of purifying IgY from an egg laid by the immunized female ostrich.
前記リコンビナント蛋白質を雌ダチョウに免疫する工程と、
前記免疫された雌ダチョウから抗血清を取得する工程を含むことを特徴とするＭＥＲＳ用抗血清の製造方法。 Incorporating a MERS virus spike protein gene into a baculovirus vector to obtain a recombinant protein in silkworm cells;
Immunizing female ostrich with the recombinant protein;
A method for producing antiserum for MERS, comprising the step of obtaining antiserum from the immunized female ostrich.
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| US11131672B1 (en) | 2021-01-29 | 2021-09-28 | King Abdulaziz University | Method for detecting MERS-CoV in Camilidae |
| US11319382B1 (en) * | 2021-06-28 | 2022-05-03 | King Abdulaziz University | Methods for producing and using IgY antibodies targeting the middle east respiratory syndrome coronavirus spike protein to treat or prevent MERS-CoV infection |
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