JP2021006811A - 魚介類アレルギーの診断の方法及び製品 - Google Patents
魚介類アレルギーの診断の方法及び製品 Download PDFInfo
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Abstract
Description
a. 担体、これは試料が該担体に接触するときに、試料からの少なくとも1つの抗体を捕捉する手段を含む、
b. 検出可能な手段が担体に接触するときに、担体によって捕捉された抗体に結合することができる検出可能な手段、特に、該検出可能な手段は、担体上に捕捉された抗体に結合することができる標識二次抗体である、
c. 任意選択的に、担体及び検出可能な手段から任意の試料を、好ましくは洗浄により除去する手段、
d. 検出可能な手段の存在を検出し、結果を電気シグナルに変換するための検出デバイス、及び
e. 任意選択的に、検出デバイスから電子シグナルを受信し、バックグラウンドで検出されたシグナルのレベルとの比較、又は健康な対象由来の試料で得られた入力参照値との比較によって、シグナルのレベルがイカアレルギーの可能性の増加、特にエビアレルギーよりイカアレルギーの可能性が高いことを示しているかを測定する手段、
を含む、アレルギーの可能性の増加を示す1つ又は複数の抗体を検出するための患者からの試料を分析する装置を提供する。
本発明は、一連の新規の核酸配列及びポリペプチド配列を含み、より具体的には以下を含む。
ATGAGCCATCATCATCATCATCATCATCATTTGGAAGTGCTGTTTCAGGGTCCATCCATGTCTCAACTGACCAAAGACGAAATTGAGGAAGTCCGTGAAGTGTTTGACCTCTTCGACTTTTGGGATGGTCGTGATGGTGATGTTGACGCTGCGAAAGTTGGCGATCTGTTACGCTGTTTAGGGATGAATCCAACCGAAGCTCAGGTACACCAACATGGAGGCACGAAGAAAATGGGCGAGAAAGCGTATAAACTGGAAGAGATTCTGCCGATTTATGAGGAAATGAGCTCCAAAGATACTGGCACAGCAGCGGACGAATTCATGGAAGCCTTCAAAACGTTTGATCGTGAAGGTCAGGGTTTGATCAGTTCAGCCGAAATTCGGAATGTGCTGAAAATGCTTGGCGAACGCATTACCGAGGATCAGTGCAACGATATCTTCACCTTTTGCGACATTCGCGAAGATATCGATGGGAACATCAAATACGAGGATCTGATGAAGAAGGTGATGGCAGGACCTTTTCCGGACAAATCGGATTAA
ATGAGCCATCATCATCATCATCATCATCATTTGGAAGTGCTGTTTCAGGGTCCATCCATGGCGGAAGAAGCTCCTCGTCGCGTGAAACTGTCACAACGCCAGATGCAGGAGCTGAAAGAAGCGTTTACCATGATTGATCAGGACCGTGATGGGTTCATTGGCATGGAAGATCTGAAAGACATGTTCAGCTCTTTAGGACGGGTACCACCCGATGACGAACTGAATGCCATGCTGAAAGAATGCCCGGGTCAACTCAATTTCACGGCTTTTCTGACCTTGTTTGGGGAGAAAGTGAGTGGTACTGACCCGGAAGATGCACTTCGCAATGCCTTTTCGATGTTCGACGAAGATGGTCAGGGCTTTATCCCGGAAGATTATCTGAAAGACTTGCTGGAAAATATGGGCGATAACTTTTCCAAAGAGGAGATTAAGAACGTCTGGAAAGATGCACCGTTAAAGAACAAACAGTTCAACTACAACAAGATGGTTGACATCAAAGGCAAAGCGGAGGATGAAGATTAA
MSHHHHHHHHLEVLFQGPSMDAIKKKMQAMKLEKDNAMDRADTLEQQNKEANNRAEKSEEEVHNLQKRMQQLENDLDQVQESLLKANIQLVEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERMRKVLENRSLSDEERMDALENQLKEARFLAEEADRKYDEVARKLAMVEADLERAEERAETGESKIVELEEELRVVGNNLKSLEVSEEKANQREEAYKEQIKTLTNKLKAAEARAEFAERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGY
MDAIKKKMQAMKLEKDNAMDRADTLEQQNKEANNRAEKSEEEVHNLQKRMQQLENDLDQVQESLLKANIQLVEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERMRKVLENRSLSDEERMDALENQLKEARFLAEEADRKYDEVARKLAMVEADLERAEERAETGESKIVELEEELRVVGNNLKSLEVSEEKANQREEAYKEQIKTLTNKLKAAEARAEFAERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGY
MSHHHHHHHHLEVLFQGPSMDAIKKKMLAMKMEKEVATDKAEQTEQSLRDLEDAKNKIEEDLSTLQKKYANLENDFDNANEQLTAANTNLEASEKRVAECESEIQGLNRRIQLLEEDLERSEERFSSAQSKLEDASKAADESERGRKVLENRSQGDEERIDLLEKQLEEAKWIAEDADRKFDEAARKLAITEVDLERAEARLEAAEAKIVELEEELKVVGNNMKSLEISEQEASQREDSYEETIRDLTHRLKEAENRAAEAERTVSKLQKEVDRLEDELLAEKERYKTISDELDQTFAELAGY
MDAIKKKMLAMKMEKEVATDKAEQTEQSLRDLEDAKNKIEEDLSTLQKKYANLENDFDNANEQLTAANTNLEASEKRVAECESEIQGLNRRIQLLEEDLERSEERFSSAQSKLEDASKAADESERGRKVLENRSQGDEERIDLLEKQLEEAKWIAEDADRKFDEAARKLAITEVDLERAEARLEAAEAKIVELEEELKVVGNNMKSLEISEQEASQREDSYEETIRDLTHRLKEAENRAAEAERTVSKLQKEVDRLEDELLAEKERYKTISDELDQTFAELAGY
MSRKSGSRSSSKRSKKSGGGSNVFDMFTQRQVAEFKEGFQLMDRDKDGVIGKTDLRGTFDEIGRIATDQELDEMLADAPAPINFTMLLNMFAERQTGESDDDDVVAKAFLAFADEEGNIDCDTFRHALMTWGDKFSSQEADDALDQMDIDDGGKIDVQGVIQMLTAGGGDDAAAEEA
SQLTKDEIEEVREVFDLFDFWDGRDGDVDAAKVGDLLRCLGMNPTEAQVHQHGGTKKMGEKAYKLEEILPIYEEMSSKDTGTAADEFMEAFKTFDREGQGLISSAEIRNVLKMLGERITEDQCNDIFTFCDIREDIDGNIKYEDLMKKVMAGPFPDKSD
MSHHHHHHHHLEVLFQGPSMSQLTKDEIEEVREVFDLFDFWDGRDGDVDAAKVGDLLRCLGMNPTEAQVHQHGGTKKMGEKAYKLEEILPIYEEMSSKDTGTAADEFMEAFKTFDREGQGLISSAEIRNVLKMLGERITEDQCNDIFTFCDIREDIDGNIKYEDLMKKVMAGPFPDKSD
AEEAPRRVKLSQRQMQELKEAFTMIDQDRDGFIGMEDLKDMFSSLGRVPPDDELNAMLKECPGQLNFTAFLTLFGEKVSGTDPEDALRNAFSMFDEDGQGFIPEDYLKDLLENMGDNFSKEEIKNVWKDAPLKNKQFNYNKMVDIKGKAEDED
MSHHHHHHHHLEVLFQGPSMAEEAPRRVKLSQRQMQELKEAFTMIDQDRDGFIGMEDLKDMFSSLGRVPPDDELNAMLKECPGQLNFTAFLTLFGEKVSGTDPEDALRNAFSMFDEDGQGFIPEDYLKDLLENMGDNFSKEEIKNVWKDAPLKNKQFNYNKMVDIKGKAEDED
GGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGTCTCGCAAAAGTGGCTCACGTTCCAGCTCCAAACGCAGCAAGAAATCGGGTGGTGGCTCGAATGTCTTTGACATGTTCACGCAACGCCAAGTTGCGGAGTTCAAAGAAGGGTTTCAGCTGATGGATCGCGACAAAGATGGGGTGATTGGCAAAACCGATCTGCGTGGTACCTTTGACGAGATTGGCCGCATTGCGACCGATCAGGAACTGGATGAGATGCTTGCAGATGCCCCAGCACCGATCAATTTCACGATGCTCCTGAACATGTTTGCGGAACGTCAGACTGGCGAATCTGACGACGATGACGTGGTTGCCAAAGCGTTCTTAGCCTTTGCCGATGAAGAGGGAAACATCGATTGCGATACCTTTCGGCATGCTCTGATGACTTGGGGCGACAAGTTCAGCAGTCAAGAAGCGGATGATGCCTTGGATCAGATGGACATTGATGACGGCGGGAAAATCGACGTACAGGGTGTGATTCAGATGCTGACAGCTGGTGGAGGTGATGATGCAGCAGCGGAAGAAGCTCTCGAGGTCCTGTTCCAAGGACCCCACCACCATCACCATCACTGATCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATA
GGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAGCCATCATCATCATCATCATCATCATTTGGAAGTGCTGTTTCAGGGTCCATCCATGGATGCCATCAAGAAGAAGATGCAGGCCATGAAACTGGAGAAAGACAACGCCATGGATCGTGCGGATACACTGGAACAGCAGAACAAAGAAGCGAATAATCGTGCGGAAAAGTCTGAAGAAGAAGTGCATAATCTGCAGAAACGCATGCAACAGTTGGAAAACGATCTGGATCAAGTGCAGGAAAGTCTCCTGAAAGCGAACATTCAGCTGGTAGAGAAAGACAAAGCGCTTTCGAATGCAGAAGGCGAAGTTGCCGCCTTGAATCGTCGCATTCAACTGTTGGAAGAAGATCTGGAACGCTCAGAAGAGCGGTTAAATACCGCCACCACCAAATTAGCTGAAGCAAGCCAAGCAGCCGACGAATCTGAACGTATGCGCAAAGTGCTGGAGAATCGCTCACTCTCGGATGAAGAACGCATGGACGCGTTAGAGAACCAGCTGAAAGAGGCTCGTTTTCTCGCGGAGGAAGCTGACCGGAAATACGATGAAGTGGCGCGCAAACTGGCCATGGTTGAGGCCGACCTTGAGCGCGCTGAAGAACGCGCAGAGACTGGTGAGAGCAAAATCGTAGAACTGGAAGAGGAGTTACGTGTCGTTGGCAATAACCTGAAATCCCTGGAAGTTTCCGAGGAGAAAGCTAACCAGCGCGAAGAAGCGTATAAAGAACAGATCAAGACTCTGACGAACAAACTGAAAGCGGCAGAGGCACGTGCGGAATTTGCAGAACGTAGCGTGCAAAAGCTGCAGAAAGAAGTCGATCGCTTGGAAGATGAACTGGTCAACGAGAAAGAAAAGTACAAATCGATTACCGATGAACTCGACCAAACGTTCAGCGAACTTAGTGGGTATTAATCGAGTAAGTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATA
GGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAGCCATCATCATCATCATCATCATCATTTGGAAGTGCTGTTTCAGGGTCCATCCATGGATGCCATCAAGAAGAAAATGCTGGCTATGAAAATGGAGAAAGAGGTTGCAACCGACAAAGCGGAGCAAACCGAACAGTCACTGCGTGATCTGGAAGATGCGAAGAACAAAATCGAAGAAGATCTGAGCACATTGCAGAAGAAATATGCCAACCTCGAAAACGATTTCGACAATGCGAATGAACAGCTGACTGCAGCGAACACGAACTTAGAAGCGTCCGAGAAACGTGTCGCAGAATGCGAATCGGAGATTCAAGGCCTTAATCGGCGCATTCAGCTGCTTGAAGAGGACTTAGAGCGTAGTGAAGAACGCTTTTCTAGCGCTCAGAGCAAACTGGAAGATGCCTCGAAAGCCGCTGATGAATCCGAACGTGGTCGCAAAGTGCTGGAGAATCGCAGTCAAGGGGACGAAGAACGCATTGATCTGCTCGAGAAACAGTTGGAAGAAGCGAAATGGATTGCCGAAGATGCGGATCGCAAATTCGACGAAGCTGCGCGTAAACTGGCCATTACCGAAGTCGATCTCGAACGTGCGGAAGCACGTTTGGAAGCAGCTGAGGCGAAAATCGTGGAACTGGAGGAGGAACTGAAAGTAGTTGGCAACAACATGAAATCGCTGGAAATCTCAGAGCAGGAAGCTTCTCAACGTGAGGACAGCTATGAGGAAACCATTCGCGATCTTACTCATCGCCTGAAAGAGGCCGAAAATCGGGCAGCAGAAGCCGAACGCACAGTGTCCAAACTGCAGAAAGAGGTTGATCGCTTAGAGGACGAACTGCTGGCCGAAAAGGAACGCTACAAGACCATCAGTGACGAATTGGATCAGACGTTTGCGGAATTAGCGGGTTATTAATGATCGAGTAAGTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATA
GGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAGCCATCATCATCATCATCATCATCATTTGGAAGTGCTGTTTCAGGGTCCATCCATGTCTCAACTGACCAAAGACGAAATTGAGGAAGTCCGTGAAGTGTTTGACCTCTTCGACTTTTGGGATGGTCGTGATGGTGATGTTGACGCTGCGAAAGTTGGCGATCTGTTACGCTGTTTAGGGATGAATCCAACCGAAGCTCAGGTACACCAACATGGAGGCACGAAGAAAATGGGCGAGAAAGCGTATAAACTGGAAGAGATTCTGCCGATTTATGAGGAAATGAGCTCCAAAGATACTGGCACAGCAGCGGACGAATTCATGGAAGCCTTCAAAACGTTTGATCGTGAAGGTCAGGGTTTGATCAGTTCAGCCGAAATTCGGAATGTGCTGAAAATGCTTGGCGAACGCATTACCGAGGATCAGTGCAACGATATCTTCACCTTTTGCGACATTCGCGAAGATATCGATGGGAACATCAAATACGAGGATCTGATGAAGAAGGTGATGGCAGGACCTTTTCCGGACAAATCGGATTAATGATCGAGTAAGTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATA
GGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAGCCATCATCATCATCATCATCATCATTTGGAAGTGCTGTTTCAGGGTCCATCCATGGCGGAAGAAGCTCCTCGTCGCGTGAAACTGTCACAACGCCAGATGCAGGAGCTGAAAGAAGCGTTTACCATGATTGATCAGGACCGTGATGGGTTCATTGGCATGGAAGATCTGAAAGACATGTTCAGCTCTTTAGGACGGGTACCACCCGATGACGAACTGAATGCCATGCTGAAAGAATGCCCGGGTCAACTCAATTTCACGGCTTTTCTGACCTTGTTTGGGGAGAAAGTGAGTGGTACTGACCCGGAAGATGCACTTCGCAATGCCTTTTCGATGTTCGACGAAGATGGTCAGGGCTTTATCCCGGAAGATTATCTGAAAGACTTGCTGGAAAATATGGGCGATAACTTTTCCAAAGAGGAGATTAAGAACGTCTGGAAAGATGCACCGTTAAAGAACAAACAGTTCAACTACAACAAGATGGTTGACATCAAAGGCAAAGCGGAGGATGAAGATTAATGATCGAGTAAGTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATA
MSRKSGSRSSSKRSKKSGGGSNVFDMFTQRQVAEFKEGFQLMDRDKDGVIGKTDLRGTFDEIGRIATDQELDEMLADAPAPINFTMLLNMFAERQTGESDDDDVVAKAFLAFADEEGNIDCDTFRHALMTWGDKFSSQEADDALDQMDIDDGGKIDVQGVIQMLTAGGGDDAAAEEALEVLFQGPHHHHHH
GGTKKMGE
SSKDTGTA
DREGQ
GGTKKMGEKAYKLEEILPIYEEMSSKDTGTAADEFMEAFKTFDREGQ
QGPSMA
KVSGTDPE
DREGQ
QELKEAFTMIDQDRDGFIGMEDLKDMFSSLGRVPPDDELNAMLKECPGQLNFT
DESERGRKVLENRSQGDEER
IDLLEKQLEEAKWIAEDADR
KFDEAARKLAITEVDLERAEARLE
KEVDRLEDELLAEKERYKTISDELDQTFAELAGY
ALRNAFSMFD
NAFSMF
GTDPEDALRNAFSMF
TDPEDALRNAFSMFD
DPEDALRNAFSMFDE
PEDALRNAFSMFDED
EDALRNAFSMFDEDG
DALRNAFSMFDEDGQ
ALRNAFSMFDEDGQG
LRNAFSMFDEDGQGF
RNAFSMFDEDGQGFI
NAFSMFDEDGQGFIP
FIPEDYLKDL
IPEDYLKDL
EDGQGFIPEDYLKDL
DGQGFIPEDYLKDLL
GQGFIPEDYLKDLLE
QGFIPEDYLKDLLEN
GFIPEDYLKDLLENM
FIPEDYLKDLLENMG
IPEDYLKDLLENMGD
FSKEEIKNVWKD
GDNFSKEEIKNVWKD
DNFSKEEIKNVWKDA
NFSKEEIKNVWKDAP
FSKEEIKNVWKDAPL
GSGSGSGAEEAPRRV
SGSGSGAEEAPRRVK
GSGSGAEEAPRRVKL
SGSGAEEAPRRVKLS
GSGAEEAPRRVKLSQ
SGAEEAPRRVKLSQR
GAEEAPRRVKLSQRQ
AEEAPRRVKLSQRQM
EEAPRRVKLSQRQMQ
EAPRRVKLSQRQMQE
APRRVKLSQRQMQEL
PRRVKLSQRQMQELK
RRVKLSQRQMQELKE
RVKLSQRQMQELKEA
VKLSQRQMQELKEAF
KLSQRQMQELKEAFT
LSQRQMQELKEAFTM
SQRQMQELKEAFTMI
QRQMQELKEAFTMID
RQMQELKEAFTMIDQ
QMQELKEAFTMIDQD
MQELKEAFTMIDQDR
QELKEAFTMIDQDRD
ELKEAFTMIDQDRDG
LKEAFTMIDQDRDGF
KEAFTMIDQDRDGFI
EAFTMIDQDRDGFIG
AFTMIDQDRDGFIGM
FTMIDQDRDGFIGME
TMIDQDRDGFIGMED
MIDQDRDGFIGMEDL
IDQDRDGFIGMEDLK
DQDRDGFIGMEDLKD
QDRDGFIGMEDLKDM
DRDGFIGMEDLKDMF
RDGFIGMEDLKDMFS
DGFIGMEDLKDMFSS
GFIGMEDLKDMFSSL
FIGMEDLKDMFSSLG
IGMEDLKDMFSSLGR
GMEDLKDMFSSLGRV
MEDLKDMFSSLGRVP
EDLKDMFSSLGRVPP
DLKDMFSSLGRVPPD
LKDMFSSLGRVPPDD
KDMFSSLGRVPPDDE
DMFSSLGRVPPDDEL
MFSSLGRVPPDDELN
FSSLGRVPPDDELNA
SSLGRVPPDDELNAM
SLGRVPPDDELNAML
LGRVPPDDELNAMLK
GRVPPDDELNAMLKE
RVPPDDELNAMLKEC
VPPDDELNAMLKECP
PPDDELNAMLKECPG
PDDELNAMLKECPGQ
DDELNAMLKECPGQL
DELNAMLKECPGQLN
ELNAMLKECPGQLNF
LNAMLKECPGQLNFT
NAMLKECPGQLNFTA
AMLKECPGQLNFTAF
MLKECPGQLNFTAFL
LKECPGQLNFTAFLT
KECPGQLNFTAFLTL
ECPGQLNFTAFLTLF
CPGQLNFTAFLTLFG
PGQLNFTAFLTLFGE
GQLNFTAFLTLFGEK
QLNFTAFLTLFGEKV
LNFTAFLTLFGEKVS
NFTAFLTLFGEKVSG
FTAFLTLFGEKVSGT
TAFLTLFGEKVSGTD
AFLTLFGEKVSGTDP
FLTLFGEKVSGTDPE
LTLFGEKVSGTDPED
TLFGEKVSGTDPEDA
LFGEKVSGTDPEDAL
FGEKVSGTDPEDALR
GEKVSGTDPEDALRN
EKVSGTDPEDALRNA
KVSGTDPEDALRNAF
VSGTDPEDALRNAFS
SGTDPEDALRNAFSM
GTDPEDALRNAFSMF
TDPEDALRNAFSMFD
DPEDALRNAFSMFDE
PEDALRNAFSMFDED
EDALRNAFSMFDEDG
DALRNAFSMFDEDGQ
ALRNAFSMFDEDGQG
LRNAFSMFDEDGQGF
RNAFSMFDEDGQGFI
NAFSMFDEDGQGFIP
AFSMFDEDGQGFIPE
FSMFDEDGQGFIPED
SMFDEDGQGFIPEDY
MFDEDGQGFIPEDYL
FDEDGQGFIPEDYLK
DEDGQGFIPEDYLKD
EDGQGFIPEDYLKDL
DGQGFIPEDYLKDLL
GQGFIPEDYLKDLLE
QGFIPEDYLKDLLEN
GFIPEDYLKDLLENM
FIPEDYLKDLLENMG
IPEDYLKDLLENMGD
PEDYLKDLLENMGDN
EDYLKDLLENMGDNF
DYLKDLLENMGDNFS
YLKDLLENMGDNFSK
LKDLLENMGDNFSKE
KDLLENMGDNFSKEE
DLLENMGDNFSKEEI
LLENMGDNFSKEEIK
LENMGDNFSKEEIKN
ENMGDNFSKEEIKNV
NMGDNFSKEEIKNVW
MGDNFSKEEIKNVWK
GDNFSKEEIKNVWKD
DNFSKEEIKNVWKDA
NFSKEEIKNVWKDAP
FSKEEIKNVWKDAPL
SKEEIKNVWKDAPLK
KEEIKNVWKDAPLKN
EEIKNVWKDAPLKNK
EIKNVWKDAPLKNKQ
IKNVWKDAPLKNKQF
KNVWKDAPLKNKQFN
NVWKDAPLKNKQFNY
VWKDAPLKNKQFNYN
WKDAPLKNKQFNYNK
KDAPLKNKQFNYNKM
DAPLKNKQFNYNKMV
APLKNKQFNYNKMVD
PLKNKQFNYNKMVDI
LKNKQFNYNKMVDIK
KNKQFNYNKMVDIKG
NKQFNYNKMVDIKGK
KQFNYNKMVDIKGKA
QFNYNKMVDIKGKAE
FNYNKMVDIKGKAED
NYNKMVDIKGKAEDE
YNKMVDIKGKAEDED
NKMVDIKGKAEDEDG
KMVDIKGKAEDEDGS
MVDIKGKAEDEDGSG
VDIKGKAEDEDGSGS
DIKGKAEDEDGSGSG
IKGKAEDEDGSGSGS
KGKAEDEDGSGSGSG
<Tod p MLC1及びTod p MLC2のクローニング、組換え発現及び精製>
リンカー及びHisタグの配列に連結される上記のタンパク質のコーディングDNA配列を、遺伝子合成によって生成し(Eurofins Genomics GmbH)、ポリメラーゼ連鎖反応によって増幅し、pBAD/TOPO(登録商標)ThioFusion(商標)発現キット(Invitrogen)を使用して組換えベクターpEt24dにサブクローニングした。得られた配列番号12(エビMLC、Lit v 3.0101をコードするインサートを含む)、配列番号13(エビTM、Pen a 1.0101をコードするインサートを含む)、配列番号14(イカTM、Sep l1.0101をコードするインサートを含む)、配列番号15(イカMLC1をコードするインサートを含む)及び配列番号16(イカMLC2をコードするインサートを含む)を使用した。この融合タンパク質を、製造業者によって記載のとおり、大腸菌(E.coli)で発現させ、ニッケルマトリックスにおけるアフィニティークロマトグラフィー(IMAC)によって単離した。
EUROLINEラインブロットは、ニトロセルロースメンブレン(Whatmanから市販されている)上に、精密ディスペンサー(Zeta Corporationから市販されている)を使用し、単離された単一融合タンパク質の配列番号3(Hisタグを有するエビTM Pen a 1.0101)、配列番号5(Sep I 1.0101、Hisタグを有するイカTM)、配列番号17(Lit v 3.0101、Hisタグを有するエビMLC2)、配列番号9(Hisタグを有するイカMLC1)及び配列番号11(Hisタグを有するイカMLC2)をコーティングすることによって生成した。製造業者の指示の推奨のとおり、ブロッキング及び洗浄の後に、単一のタンパク質を含有するメンブレンを3mmのストリップに整列及び切断し、マルチパラメータ試験を行った。最終的な配置を図1に示す。
EUROLINEストリップを、製造業者(EUROIMMUN Medizinische Labordiagnostika AG、リューベック、ドイツ)の指示(製品番号DP3110−1601E)の推奨のとおり、インキュベートした。詳細には、ストリップを、洗浄緩衝液において1mlの1/11(v/v)の希釈患者試料(25人のイカアレルギー患者及び42人のイカ耐性患者)と、室温にて16時間インキュベートした。患者の試料を除去した後、ストリップを1ストリップあたり1mlの洗浄緩衝液で5分間、3回洗浄し、続いてAPとコンジュゲートした1mlの抗ヒトIgE抗体と1時間インキュベートした。その後、ストリップを再度洗浄緩衝液で5回洗浄し、次いでBCIP/NBT基質で10分間インキュベートした。
アッセイ性能統計は、患者の食品チャレンジの結果に基づいてTP:真陽性試験結果のパーセンテージ、TN:真陰性試験結果のパーセンテージ、FP:偽陽性試験結果のパーセンテージ、FN:偽陰性試験結果のパーセンテージの定義で計算した。
単一アレルゲン成分に対するイカアレルギーに基づく感作の予測が可能かどうかを特定するために、様々なエビ及びイカのタンパク質を含有するEUROLINE多重パラメータストリップを、25人のイカアレルギーの小児及び42人のイカ耐性の小児からの試料とインキュベートした。
Ehlersらの記載のとおり(Ehlers,A.M.,Klinge,M.,Suer,W.,Weimann,Y.,Knulst,A.C.,Besa,F.,Le,T.M.,Otten,H.G.(2019)Ara h 7 isoforms share many linear epitopes:Are 3D epitopes crucial to elucidate divergent abilities?,Clin Exp Allergy 49(11),1512−1519)、配列番号10を示す15個のアミノ酸配列の重複ペプチドを含むペプチドマイクロアレイチップを製造し、インキュベートし、1個のオフセットを選択し、3重に測定を実施した。使用されるペプチドは、配列番号56〜配列番号208を含む。各ペプチドは15個のアミノ酸を含み、オフセットは1個であった。測定を3重に実施した。
アレルギー患者からの血清の反応性と耐性患者からの血清の反応性との間に、明確な区別が示された。
Claims (16)
- 対象由来の試料中のイカMLC1若しくはイカMLC2又はそのバリアントを含む群からのポリペプチドに対する抗体を特異的に捕捉する手段を含む、診断的に有用な担体。
- 前記担体が、ビーズ、試験ストリップ、マイクロタイタープレート、マイクロアレイ、ブロットを含む群から、好ましくはウエスタンブロット、ラインブロット及びドットブロット、ガラス表面、スライド、バイオチップ及びメンブレンを含む群から選択され、最も好ましくはマイクロタイタープレート又はラインブロットである、請求項1に記載の担体。
- 前記担体が、イカTMに対する抗体を捕捉する手段をさらに含む、請求項1又は2に記載の担体。
- 請求項1〜3のいずれか1項に記載の診断的に有用な担体を含むキットであって、好ましくは、キャリブレーター、好ましくはキャリブレーターのセット、洗浄緩衝液、及びIgE抗体、好ましくは標識二次抗体を検出する手段を含む群から1つ又は複数を、好ましくは全てをさらに含む、キット。
- イカMLC1又はイカMLC2に対する抗体の存在又は非存在を、対象由来の試料において検出するステップを含む方法。
- イカTMに対する抗体の存在又は非存在を、前記試料において検出するステップをさらに含む、請求項5に記載の方法。
- イカMLC1若しくはイカMLC2又はそのバリアントを含み、好ましくはアジュバントをさらに含む、医薬組成物。
- イカMLC1若しくはイカMLC2又はそのバリアントを含むポリペプチドであって、単離又は固定化されたポリペプチド。
- 前記ポリペプチドが、前記ポリペプチドに特異的に結合する抗体、好ましくはIgEクラス抗体との複合体である、請求項8に記載のポリペプチド。
- 前記ポリペプチドが、真核細胞で発現されたポリペプチドである、請求項1〜9のいずれか1項に記載の担体、キット、方法、組成物又はポリペプチド。
- アレルギーの診断のための、請求項8〜10のいずれか1項に記載のポリペプチド、請求項1〜3のいずれか1項に記載の担体、又は請求項4に記載のキットの使用。
- 好ましくはアレルギーの診断のための、診断キットの製造のための、請求項8〜11のいずれか1項に記載のポリペプチド、又は請求項1〜3のいずれか1項に記載の担体、又はイカMLC1若しくはイカMLC2に対する患者の試料からのIgE抗体に特異的に結合するポリペプチドの使用。
- 前記アレルギーが、貝類アレルギーである、好ましくはイカアレルギーである、請求項11に記載の使用。
- 前記診断が、貝類アレルギーの鑑別診断であり、好ましくはエビアレルギーとイカアレルギーとの間で区別される、請求項12又は13に記載の使用。
- ポリペプチドを含む医療デバイス又は診断デバイスに、好ましくは前記ポリペプチドに対し既知の濃度で、抗体、好ましくは前記ポリペプチドに対するIgE抗体、を含む溶液を接触させるステップを含む方法であって、前記ポリペプチドが、イカMLC1及びイカMLC2又はそれらのバリアントを含む群から選択される、方法。
- イカMLC1若しくはイカMLC2又はそのバリアントを含むポリペプチドに特異的に結合する抗体、好ましくはIgEクラス抗体であって、好ましくは単離された抗体。
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Non-Patent Citations (4)
Title |
---|
MAITA, T. ET AL.: "Amino Acid Sequence of The Regulatory Light Chain of Squid Mantle Muscle Myosin", JOURNAL OF BIOCHEMISTRY, JPN6023018389, 1 November 1987 (1987-11-01), JP, ISSN: 0005056033 * |
MOTOYAMA, K. ET AL.: "Cephalopod Tropomyosins: Identification as Major Allergens and Molecular Cloning", FOOD AND CHEMICAL TOXICOLOGY, vol. 44, no. 12, JPN6023018390, December 2006 (2006-12-01), pages 1997 - 2002, XP025065461, ISSN: 0005056034, DOI: 10.1016/j.fct.2006.06.018 * |
WATANABE, B. ET AL.: "Amino-Acid Sequence of LC-1 Light Chain of Squid Mantle Muscle Myosin", BIOLOGICAL CHEMISTRY, vol. 367, no. 2, JPN6023018388, October 1986 (1986-10-01), pages 1025 - 1032, ISSN: 0005056032 * |
吉岡武也,木下康宣: "生鮮スルメイカ保管中に起こる性状変化に関する研究II −物性測定法の検討と保管中の変化−", 北海道道立工業技術センター研究報告, vol. 9, JPN6023018387, 2006, pages 15 - 20, ISSN: 0005056031 * |
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