JP6795211B2 - 分泌型免疫グロブリンA(sIgA)結合核酸分子、sIgA分析用センサ、およびsIgAの分析方法 - Google Patents
分泌型免疫グロブリンA(sIgA)結合核酸分子、sIgA分析用センサ、およびsIgAの分析方法 Download PDFInfo
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- JP6795211B2 JP6795211B2 JP2018539512A JP2018539512A JP6795211B2 JP 6795211 B2 JP6795211 B2 JP 6795211B2 JP 2018539512 A JP2018539512 A JP 2018539512A JP 2018539512 A JP2018539512 A JP 2018539512A JP 6795211 B2 JP6795211 B2 JP 6795211B2
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- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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Description
前記核酸分子が、前記本発明のsIgA結合核酸分子であり、
前記検出工程において、前記試料中のsIgAと前記核酸分子とを結合させて、前記結合により、前記試料中のsIgAを検出することを特徴とする。
(a)配列番号1〜12のいずれかの塩基配列からなるポリヌクレオチド
(a1)配列番号13、14、または15の塩基配列からなるポリヌクレオチド
(a2)配列番号16または17の塩基配列からなるポリヌクレオチド
(a3)配列番号18または19の塩基配列からなるポリヌクレオチド
(a4)配列番号20または21の塩基配列からなるポリヌクレオチド
本発明のsIgA結合核酸分子(以下、「核酸分子」ともいう)は、前述のように、sIgAに対する解離定数が、37.7nM以下の核酸分子であることを特徴とする。
(a)配列番号1〜12のいずれかの塩基配列からなるポリヌクレオチド
(a1)配列番号13、14、または15の塩基配列からなるポリヌクレオチド
(a2)配列番号16または17の塩基配列からなるポリヌクレオチド
(a3)配列番号18または19の塩基配列からなるポリヌクレオチド
(a4)配列番号20または21の塩基配列からなるポリヌクレオチド
(b)前記(a)のいずれかの塩基配列において、1もしくは数個の塩基が欠失、置換、挿入および/または付加された塩基配列からなり、前記sIgAに結合するポリヌクレオチド
(c)前記(a)のいずれかの塩基配列に対して、80%以上の同一性を有する塩基配列からなり、前記sIgAに結合するポリヌクレオチド
(d)前記(a)のいずれかの塩基配列からなるポリヌクレオチドに対してストリンジェントな条件下でハイブリダイズするポリヌクレオチドに、相補的な塩基配列からなり、sIgAに結合するポリヌクレオチド
[ヌクレオチド残基]−[リンカー]−[アデニン残基]
[ヌクレオチド残基]−[リンカー]−[グアニン残基]
[ヌクレオチド残基] =C-C(=O)-NH-(CH2)n- [アデニン残基]
[ヌクレオチド残基] =C-C(=O)-NH-(CH2)n- [グアニン残基]
[ヌクレオチド残基] -C=C-C(=O)-NH-(CH2)n- [アデニン残基]
[ヌクレオチド残基] =C-C(=O)-NH-CH2-CH2- [アデニン残基]
[ヌクレオチド残基] =C-C(=O)-NH-CH2-CH2- [グアニン残基]
[ヌクレオチド残基] -C=C-C(=O)-NH-CH2-CH2- [アデニン残基]
本発明の分析用センサは、前述のように、sIgAの分析用センサであって、前記本発明の核酸分子を含むことを特徴とする。本発明の分析用センサは、前記本発明の核酸分子を含んでいればよく、その他の構成は、特に制限されない。本発明の分析用センサを使用すれば、例えば、前記核酸分子と前記sIgAとを結合させることで、前述のように、前記sIgAを検出できる。
本発明の分析方法は、前述のように、試料と核酸分子とを接触させ、前記試料中のsIgAを検出する工程を含み、前記核酸分子が、前記本発明のsIgA結合核酸分子であり、前記検出工程において、前記試料中のsIgAと前記核酸分子とを結合させて、前記結合により、前記試料中のsIgAを検出することを特徴とする。本発明の分析方法は、前記本発明の核酸分子を使用することが特徴であって、その他の工程および条件等は、特に制限されない。また、本発明の分析方法は、前記本発明の核酸分子として、前記本発明のsIgA分析用センサを使用してもよい。
本発明の検出キットは、前記本発明のsIgA結合核酸分子を含むことを特徴とする。本発明の検出キットは、前記本発明の核酸分子を含んでいればよく、その他の構成は何ら制限されない。本発明の検出キットを使用すれば、前述のように、例えば、前記sIgAの検出等を行うことができる。
以下に示す合成例により、MK4を調製した。
収量:261mg 収率:60%
ESI-MS (positive ion mode) m/z, found = 481.2, calculated for [(M+H)+] = 481.2
found = 503.1, calculated for [(M+Na)+] = 503.2
1HNMR (400 MHz, DMSO-d6) δ8.22 (1H, m), 8.11 (1H, s), 8.10 (1H, s), 7.87 (1H, s), 7.63 (1H, d), 6.52 (1H, q), 6.35 (1H, d), 5.27 (1H, s), 3.82 (1H, m), 2.18 (1H, m)
収量:30.0μmol 収率:13.4%
ESI-MS (negative ion mode) m/z, found = 559.1, calculated for [(M-H)-] = 559.2
理論収量:30.03μmol
収量:3.33μmol 収率:11.1%
ESI-MS (negative ion mode) m/z, found = 719.0, calculated for [(M-H)-] = 719.1
以下に示す合成例により、NG7を調製した。
収量:3.573g 収率:97.4%
ESI-MS (positive ion mode) m/z, found= 161.4, calculated for [(M+H)+] = 161.1
1HNMR(400 MHz, CDCl3) δ3.13 (2H, q) 2.76 (2H, t) 1.41 (9H, s)
収量:720mg 収率:79%
ESI-MS (positive ion mode) m/z, found = 171.0, calculated for [(M+H)+] = 171.0
found = 193.1, calculated for [(M+Na)+] = 193.0
found = 209.1, calculated for [(M+K)+] = 209.0
ESI-MS (negative ion mode) m/z, found = 169.0, calculated for [(M-H)-] = 518.0
1HNMR (400 MHz, DMSO-d6) δ8.29 (1H, s)
収量:1.117mg 収率:74%
ESI-MS (positive ion mode) m/z, found = 171.0, calculated for [(M+H)+] = 171.0,
found = 193.1, calculated for [(M+Na)+] = 193.0,
found = 209.1, calculated for [(M+K)+] = 209.0,
ESI-MS (negative ion mode) m/z, found = 169.0, calculated for [(M-H)-] = 518.0
1HNMR (400 MHz, CD3OD) δ7.73 (1H, s), 3.45 (2H, m), 3.30 (2H,s), 1.39 (9H, s)
収量:467mg 収率:89.1%
ESI-MS (positive ion mode) m/z, found = 195.1, calculated for [(M+H)+] = 195.1,
found = 217.2, calculated for [(M+Na)+] = 217.1,
found = 233.0, calculated for [(M+K)+] = 233.1
1HNMR (400 MHz, D2O) δ7.83 (1H, s), 3.55 (2H, t), 3.11 (2H, t)
収量:147mg 収率:91%
ESI-MS (positive ion mode) m/z, found = 475.1, calculated for [(M+H)+] = 475.2,
found = 497.2, calculated for [(M+Na)+] = 497.2,
ESI-MS (negative ion mode) m/z, found = 473.1, calculated for [(M-H)-] = 473.2
1HNMR (400 MHz, DMSO-d6) δ8.27 (1H, s), 8.20 (1H, s), 7.10 (1H,s), 7.05 (1H, s), 6.13 (1H, t), 5.25 (1H, d), 5.16 (1H, m), 4.09 (1H, m), 3.79 (1H, m), 3.60 (2H, m), 3.16 (2H, d), 2.14 (2H, m)
収量:41.49μmol 収率:19.5%
ESI-MS (negative ion mode) m/z, found = 553.1, calculated for [(M-H)-] = 553.1
理論収量:78.65μmol
収量:19.55μmol 収率:24.9%
ESI-MS (negative ion mode) m/z, found = 712.9, calculated for [(M-H)-] = 713.1
本例では、配列番号1〜21のアプタマーについて、sIgAに対する結合能および動態パラメータを、SPRにより確認した。
下記ポリヌクレオチドを合成し、実施例のアプタマーとした。配列番号1〜4のアプタマー(以下、「MK4アプタマー」ともいう)は、下記表3において下線で示すアデニンを含むヌクレオチド残基が、前記化学式(1)で示すヌクレオチド残基である。また、配列番号8〜12および16〜21のアプタマー(以下、「KS9アプタマー」ともいう)は、下記表3において下線で示すチミンを含むヌクレオチド残基が、前記化学式(2)で示すヌクレオチド残基であり、配列番号5〜7および13〜15のアプタマー(以下、「NG7アプタマー」ともいう)は、下記表3において下線で示すチミンを含むヌクレオチド残基が、前記化学式(3)で示すヌクレオチド残基である。
市販のヒトsIgA(IgA (Secretory) ,Human、MP Biomedicals, LLC-Cappel Products社製、カタログ番号: #55905)を、試料として、以下の試験に使用した。
結合能の解析には、ProteON XPR36(BioRad社)を、その使用説明書にしたがって使用した。
5’-GGTAACGCCCAGTCTAGGTCATTTG-(N)30-GTTACGGGAGCCTGCACTTAATG-3’(配列番号22)
前記試料中のsIgAの濃度を、12.5、25、50、100、または200nmol/Lとした以外は、前記(3)と同様にして、結合量の相対値(RU)を測定した。そして、前記結合量の相対値に基づき、前記sIgA結合核酸分子と前記sIgAとの解離定数を算出した。この結果を下記表4に示す。下記表4に示すように、いずれのアプタマーも解離定数が37.7nM以下であり、特に、配列番号2、配列番号3、配列番号9、配列番号11、配列番号15、配列番号19、および配列番号21の核酸分子は、解離定数が、8nM以下であり、極め優れたsIgAとの結合能を有することがわかった。
配列番号3、5および9のアプタマーを用い、試料として、400nmol/LとなるようにsIgA、ヒトIgG−Fc(比較例3−3、BETHYL社製、カタログ番号:P-80-104)、未標識ヒトIgG(比較例3−4、BECKMAN COULTER社製、カタログ番号:731696)、未標識ヒトIgG−Fc(比較例3−5、BECKMAN COULTER社製、カタログ番号:731703)またはヒトIgG1κ(比較例3−6、Southern Biotech社製、カタログ番号:0151K-01)を含む試料を用いた以外は、前記(3)と同様にして、結合量の相対値を求めた。
本例では、配列番号5、9および11のアプタマーについて、sIgAに対する結合能を、磁気ビーズによるプルダウンにより確認した。
磁気ビーズの表面にストレプトアビジン(SA)が結合したSAビーズ(Invitrogen社製、商品名:MyOne−SA C1)に、前記配列番号5、9または11のアプタマーを結合させ、アプタマー結合ビーズを作製した。具体的には、まず、前記アプタマーに100%相補的な相補鎖を調製した。他方、前記アプタマーの5’領域の配列(配列番号23、5’-GGATACCTTAACGCCGCCTATTG-3’)を準備し、その5’末端をビオチン化して、ビオチン化プライマーを調製した。そして、前記相補鎖を鋳型として、前記ビオチン化プライマーを用い、PCRによる増幅を行い、5’末端がビオチン化された前記アプタマーを合成した。前記合成された前記アプタマーと前記相補鎖とからなる二本鎖に対して、前記SAビーズを反応させ、前記二本鎖におけるビオチンと前記SAビーズのアビジンとを結合させた。つぎに、前記二本鎖とSAビーズとが結合した複合体を、NaOHでアルカリ処理し、前記二本鎖の解離を行うことによって、前記相補鎖を除去した。これによって、前記SAビーズに、ビオチン−アビジン結合により前記ビオチン化アプタマーが結合した、前記アプタマー結合ビーズを作製した。
5μgのヒトsIgAまたはヒトの唾液を、試料として、以下の実験に使用した。
前記アプタマー結合ビーズ(終濃度10mg/ml)と前記試料(sIgA:終濃度50μg/mL、唾液:終濃度90%)とを、SB1T緩衝液(40mmol/L HEPES、125mmol/L NaCl、5mmol/L KCl、1mmol/L MgCl2および0.01% Tween(登録商標)20、pH7.4)中で混合し、この反応液を、室温で30分間反応させた。前記反応液を遠心して前記ビーズを回収し、前記SB1T緩衝液で3回遠心洗浄した。前記アプタマーがsIgAに結合する場合、前記ビーズには、前記アプタマーを介してsIgAが結合していることになる。そこで、前記ビーズをSDSバッファー緩衝液に混合し、95℃で10分間、加熱処理することによって、前記ビーズからsIgAを遊離させた。そして、加熱処理後の前記SDS緩衝液から、前記ビーズを除去して、PAGEL(C520L、アトー社)を用いてSDS−PAGEに供した。泳動用のバッファーは、前記SDSバッファーを使用した。前記SDSバッファーの組成は、25mmol/L Tris、192mmol/L グリシン、0.1% SDSとした。
Claims (3)
- 下記(a)のポリヌクレオチドを含む、sIgA結合核酸分子:
(a)配列番号9、11、15、および21のいずれかの塩基配列からなるポリヌクレオチド;
配列番号9、11、および21の塩基配列において、下記表1において、下線で示すチミンを含むヌクレオチド残基が、下記化学式(2)で示すヌクレオチド残基であり、
配列番号15の塩基配列において、下記表1において、下線で示すチミンを含むヌクレオチド残基が、下記化学式(3)で示すヌクレオチド残基である。
- 請求項1に記載の分泌型免疫グロブリンA(sIgA)結合核酸分子を含むことを特徴とする、sIgA分析用センサ。
- 試料と核酸分子とを接触させ、前記試料中の分泌型免疫グロブリンA(sIgA)を検出する工程を含み、
前記核酸分子が、請求項1に記載のsIgA結合核酸分子であり、
前記検出工程において、前記試料中のsIgAと前記核酸分子とを結合させて、前記結合により、前記試料中のsIgAを検出することを特徴とする、sIgAの分析方法。
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WO2018051581A1 (ja) * | 2016-09-15 | 2018-03-22 | Necソリューションイノベータ株式会社 | ヌクレオシド誘導体またはその塩、ポリヌクレオチドの合成試薬、ポリヌクレオチドの製造方法、ポリヌクレオチド、および結合核酸分子の製造方法 |
WO2018096831A1 (ja) | 2016-11-28 | 2018-05-31 | Necソリューションイノベータ株式会社 | ヌクレオシド誘導体またはその塩、ポリヌクレオチドの合成試薬、ポリヌクレオチドの製造方法、ポリヌクレオチド、および結合核酸分子の製造方法 |
CN115260261A (zh) * | 2021-04-30 | 2022-11-01 | 苏州诺维康生物科技有限公司 | 一种荧光染料修饰的脱氧核苷固相载体的制备方法 |
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JPH10239312A (ja) * | 1997-02-28 | 1998-09-11 | Toyota Central Res & Dev Lab Inc | 感性の計測方法及び簡易感性計測キット |
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EP1151000A2 (en) * | 1999-02-12 | 2001-11-07 | Oklahoma Medical Research Foundation | Polymeric immunoglobulin receptor (pigr)-binding domains and methods of use therefor |
CA2592965A1 (en) * | 1999-06-25 | 2001-01-04 | Basf Aktiengesellschaft | Corynebacterium glutamicum genes encoding metabolic pathway proteins |
JP4623825B2 (ja) * | 1999-12-16 | 2011-02-02 | 協和発酵バイオ株式会社 | 新規ポリヌクレオチド |
AU2013273727A1 (en) * | 2006-10-27 | 2014-01-16 | Lpath, Inc. | Compositions and methods for treating ocular diseases and conditions |
JP5812478B2 (ja) * | 2011-08-12 | 2015-11-11 | 国立大学法人群馬大学 | 抗癌剤結合性核酸アプタマー及びその利用 |
BR112015009138A2 (pt) * | 2012-10-23 | 2020-10-20 | Caris Life Sciences Switzerland Holdings, S.A.R.L. | métodos para caracterizar um câncer |
US10385090B2 (en) | 2013-10-31 | 2019-08-20 | National University Corporation Gunma University | Nucleotide derivative or salt thereof, nucleotide-derived 5′-phosphate ester or salt thereof, nucleotide-derived 3′-phosphoramidite compound or salt thereof, and polynucleotide |
JP6442941B2 (ja) * | 2014-09-10 | 2018-12-26 | 国立大学法人群馬大学 | 血管内皮細胞増殖因子結合性核酸アプタマー及びその利用 |
JP2016180892A (ja) | 2015-03-24 | 2016-10-13 | 三菱化学株式会社 | 静電荷像現像用トナーの製造方法 |
CN106636114A (zh) * | 2016-05-08 | 2017-05-10 | 杨岭燕 | 一种用于检测妇科慢性盆腔炎的试剂盒及其检测方法 |
CN109715807B (zh) * | 2016-09-15 | 2022-05-17 | 日本电气方案创新株式会社 | 分泌型免疫球蛋白A(sIgA)结合核酸分子、sIgA分析用传感器和sIgA分析方法 |
WO2018052063A1 (ja) * | 2016-09-15 | 2018-03-22 | Necソリューションイノベータ株式会社 | ヌクレオシド誘導体またはその塩、ポリヌクレオチドの合成試薬、ポリヌクレオチドの製造方法、ポリヌクレオチド、および結合核酸分子の製造方法 |
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