JP2016060743A - 日本ウナギの一本鎖の卵胞刺激ホルモンポリペプチド、これに対する抗体及びこれらの用途 - Google Patents
日本ウナギの一本鎖の卵胞刺激ホルモンポリペプチド、これに対する抗体及びこれらの用途 Download PDFInfo
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Abstract
Description
日本ウナギの脳下垂体を回収し、サムブルックら(Sambrook,et al.Molecular cloning:A Laboratory manual.2ed.Vol.1.pp.101−104,Cold Spring Harbor Laboratory Press,1989)の方法によってトータルRNAを分離した後、1st strand cDNAを合成した。前記合成されたcDNAからFSH遺伝子をクローニングするため、後述するようにPCRプライマーを合成し、これを利用したPCR方法を通じてFSH遺伝子の増幅を試した。βサブユニット(単体)を増幅するため、次の配列を有するPCRプライマーFSHb F(5´‐T GAA TTC ATG CAT CTG GCT GTC ACA GCG‐3´)及びFSHb R(5´‐GTT GTT GGG ATA CTG GGT CAG ACA GCC TGA‐3´)を合成した。また、αサブユニット(単体)を増幅するため、FSHa F(5´‐TGT CTG ACC CAC TAT CCC AAC AAC GAA ATG‐3´)及びFSHa R(5´‐AC GTC GAC TTA AAA TTT GTG GTA GTA GCA‐3´)を使用した。PCR方法により、前記プライマーを用いて日本ウナギ由来のcDNAからFSH遺伝子のβ381bp、α279bpの断片を取得した(図1)。
前記実施例1において確認されたFSH遺伝子を利用し、再組合タンパク質を生産するためにFSH遺伝子の発現ベクターを作製した。FSH遺伝子の5´部分にNdeI認識部位の塩基配列を添加した日本ウナギのFSH NdeIプライマー(5´‐AGT CAT ATG TGT GGT CTC GCC AAC‐3´)及び3´部分にXhoI認識部位の塩基配列を添加した日本ウナギのFSH XhoIプライマー(5´‐CTC GAG TTA AAA TTT GTG GTA GTA GCA‐3´)を合成し、前記プライマーと日本ウナギのFSH一本鎖のDNAとを利用し、PCRを通じて増幅されたDNA断片を取得した。そして、取得した断片を大腸菌用発現ベクターであるpREST(Invitrogen社、米国)のNdeI/XhoI制限酵素の認識部位に連結し、FSH再組合発現ベクターを作製した。このように作製されたFSH再組合発現ベクターの地図を図6に示す。
前記実施例2において作製された発現ベクターを供給源方法に沿って大腸菌(Escherichia coli)に導入し、形質を転換させ、そのうちから安定したクローンを選別した。前記形質転換された大腸菌のうち、安定したクローンを選別するため、アンピシリン(ampicillin、50μg/ml)が添付されたLB培地に接種し、37°Cの条件で培養器で1日培養し、生存した細胞を選別した。前記で作製された形質転換体のうち、3つのクローンを対象にタンパク質の生産を確認するため、その中から最も多くのタンパク質を生産するクローンを用いて大量に培養し、Ni‐NTA sepharose精製方法を通じてタンパク質を精製した(図7及び図8)。
前記精製された再組合された一本鎖の日本ウナギのFSHの50μgを3匹のBalb/cマウス(6〜8週齢)に2週毎に3回、免疫を行なった。最後の免疫から1週間後に血液を採取し、ELISAで抗体の力価を分析した。そして、骨髄細胞との融合3日前に20μgのFSHを尾血管(intravenous)に投与し、最も高い抗体力価を表すマウスの脾臓を粉砕し、マウスの骨髄細胞と50%のポリエチレングリコール(PEG)1500(Sigma‐Aldrich、Germany)を利用して融合した。HAT培養液(Sigma‐Aldrich,Germany)を利用してハイブリドーマ細胞(hybridoma cells)を選別し、選別された培養上層を希釈し、ELISA方法を利用してクローンを分離した(図9)。分離された6つの抗体上層と抗原との結合力を分析するため、ウェスタンブロッティングを行なった(図10)。前記6つの抗体のうち、#5、#11、#14に対しHi‐Trap protein G column(GE Healthcare、Uppsala、Sweden)を利用し、培養上層から精製を行なった。培養上層を0.45μmフィルタリングした後、pHを7.5に固定し、溶出はGlycine HCI(0.1M、pH=2.7)を用いて行なった。溶出された抗体は、PBS(pH=7.5)に投石した。前記精製された抗体の同種型(Isotype)を分析した結果、重鎖(Heavy Chain)はIgG2bタイプであり、軽鎖(light chain)はkappaタイプであった(図11及び下記表1)。
図12に示した構築手順を参照し、前記で作製されたFSH抗体を用いて、動物細胞(CHO)から生産された再組合体日本ウナギの一本鎖FSHを対象にウェスタンブロッティングを行った(図13)。11番抗体に対しては、N‐linked糖鎖を除去した後、ウェスタンブロッティングを行なった(図14)。図13に示すように#5、#11は一本鎖FSHのα単体と結合し、#14は一本鎖FSHのβ単体と結合することが確認された。動物細胞で生産された一本鎖FSHとLHは、約34kDAのタンパク質を検出することができた。
前記で作製された3つの抗体を利用し、HRPラベリングをした後、最適の結合状態を確認した結果、まず、#5抗体をプレートにコーティングした後、HRPラベリングした#11抗体を利用した場合に、最も最適のスタンダードカーブが確認できた(表2、表3及び図15、図15は表3の結果をグラフに示したものである)。
前記で作製された抗体を利用し、HRPラベリングをした抗体の希釈倍率によってスタンダードカーブを分析した結果、図16に示すように100倍、200倍、400倍、800倍、1600倍及び3200倍の分析においても、十分な濃度増加によりdose‐responseカーブが確認できた。したがって、再組合体日本ウナギの定量分析においてHRPラベリングした11番抗体は、3200倍まで希釈し、検出することができた。
前記で確立されたELISAシステムを活用し、スタンダード濃度0から200ng/mlまで用いてCHO細胞で生産した日本ウナギの卵胞刺激ホルモンと黄体形成ホルモン、そして昆虫細胞であるバキュロウイルシ細胞で生産した卵胞刺激ホルモンと黄体形成ホルモンを分析した結果を図17に示す。分析した4つの再組合体は全てスタンダードカーブ中で検出されたため、開発されたELISAシステムは、日本ウナギの一本鎖の卵胞刺激ホルモンと黄体形成ホルモンの定量分析に正常に利用できるようになった。
Claims (12)
- 日本ウナギ(Anguilla japonica)の卵胞刺激ホルモンのαサブユニットとβサブユニットとを一本鎖に融合した、配列番号:1のアミノ酸配列からなるポリペプチドを含む再組合された日本ウナギの一本鎖の卵胞刺激ホルモンのポリペプチド。
- 請求項1に記載されている再組合された日本ウナギの一本鎖の卵胞刺激ホルモンのポリペプチドをコーディングする、配列番号:2の塩基配列からなる核酸。
- 請求項1に記載されている核酸が挿入された再組合発現ベクター。
- 請求項3に記載されている再組合発現ベクターによって形質転換されている宿主細胞。
- 前記宿主細胞は、バクテリア細胞、昆虫細胞及び脊椎動物の細胞を含む、請求項4に記載の宿主細胞。
- 請求項2に記載されている核酸分子を宿主細胞内に導入し、コーディングされた、再組合された日本ウナギの一本鎖の卵胞刺激ホルモンのポリペプチドが発現できる条件下で培養し、再組合された日本ウナギの一本鎖の卵胞刺激ホルモンタンパク質を製造する方法。
- 請求項1に記載されている再組合された日本ウナギの一本鎖の卵胞刺激ホルモンのポリペプチドを有効成分として含有する日本ウナギの性成熟誘導用ホルモン組成物。
- 請求項1に記載されている再組合された日本ウナギの一本鎖の卵胞刺激ホルモンのポリペプチドを特異的に認識する抗体。
- 前記抗体は、単クローン抗体である、請求項8に記載の抗体。
- 請求項8または請求項9に記載されている抗体を含む日本ウナギの卵胞刺激ホルモンを検出するための組成物。
- 請求項8または請求項9に記載されている抗体を生物学的試料と接触させて抗原‐抗体複合体の形成を検出し、前記生物学的試料に日本ウナギの卵胞刺激ホルモンを検出する方法。
- 前記抗原‐抗体複合体の形成は、酵素免疫吸着法(ELISA)、放射免疫測定法(RIA)、免疫蛍光法(IFA)、ウェスタンブロッティング及び流動細胞分析法のうち、少なくとも1つの方法によって検出される、請求項11に記載の方法。
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WO2010050554A1 (ja) * | 2008-10-31 | 2010-05-06 | 東レ株式会社 | ヒトcxcl1タンパク質の免疫学的測定方法 |
JP2011168562A (ja) * | 2010-02-22 | 2011-09-01 | Fisheries Research Agency | 魚類gthタンパク質および該タンパク質を用いる魚類の成熟誘導方法 |
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WO2010050554A1 (ja) * | 2008-10-31 | 2010-05-06 | 東レ株式会社 | ヒトcxcl1タンパク質の免疫学的測定方法 |
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