抗假肥大型肌营养不良症特异性抗体Anti5～7

摘要

抗Dystrophin特异性抗体Anti5—7,涉及利用基因工程技术制备特异性抗体,其目的为利用基因工程技术克隆表达了DMD基团节段cDNA5.8—7.0kb,并制备出具有抗Dystrophin特异性的抗体Anti5—7,所选用的表达基因对应于DMD基因缺失突变热点区;并正好包含着Dystrophin蛋白分子上一段功能不明的蛋白酶敏感区对应的编码部位的特点,因此能精确检出Dystrophin蛋白质或量的改变,可同时应用于基础研究及临床诊断。

主权项

1.抗Dystrophin特异性抗体Anti5～7，其制备方法为：首先对DMD基因cDNA5～7kb的片段进行了测序，然后对片段两端克隆位点进行修饰，以原开放读框克隆入原核表达载体，同时在其5′端融合了10个氨基酸的碱基，然后在大肠杆菌中进行诱导表达，电洗脱纯化表达蛋白，与福氏佐剂混合后免疫大白兔而制得.DMD cDNA5-7蛋白的基因序列及氨基酸序列为：------------------------------------------------------------GlnTyrLysArgGlnAlaAspAspLeuLeuLysCysLeuAspAspIleGluLys          66          78          90         102CAGTACAAGAGGCAGGCTGATGATCTCCTGAAATGCTTGGATGACATTGAAAAAGTCATGTTCTCCGTCCGACTACTAGAGGACTTTACGAACCTACTGTAACTTTTT ------------------------------------------------------------LysLeuAlaSerLeuProGluProArgAspGLuArgLysIleLysGluIleAsp         120         132         144         156AAATTAGCCAGCCTACCTGAGCCCAGAGATGAAAGGAAAATAAAGGAAATTGATTTTAATCGGTCGGATGGACTCGGGTCTCTACTTTCCTTTTATTTCCTTTAACTA ------------------------------------------------------------ArgGluLeuGlnLysLysLysGluGluLeuAsnAlaValArgArgGlnAlaGlu         174         186         198         210CGGGAATTGCAGAAGAAGAAAGAGGAGCTGAATGCAGTGCGTAGGCAAGCTGAGGCCCTTAACGTCTTCTTCTTTCTCCTCGACTTACGTCACGCATCCGTTCGACTC ------------------------------------------------------------GlyLeuSerGluAspGlyAlaAlaMetAlaValGluProThrGlnIleGlnLeu         228         240         252         264GGCTTGTCTGAGGATGGGGCCGCAATGGCAGTGGAGCCAACTCAGATCCAGCTCCCGAACAGACTCCTACCCCGGCGTTACCGTCACCTCGGTTGAGTCTAGGTCGAG ------------------------------------------------------------SerLysArgTrpArgGluIleGluSerLysPheAlaGlnPheArgArgLeuAsn         282         294         306         318AGCAAGCGCTGGCGGGAAATTGAGAGCAAATTTGCTCAGTTTCGAAGACTCAACTCGTTCGCGACCGCCCTTTAACTCTCGTTTAAACGAGTCAAAGCTTCTGAGTTG ------------------------------------------------------------PheAlaGlnIleHisThrValArgGluGluThrMetMetValMetThrGluAsp         336         348         360         372TTTGCACAAATTCACACTGTCCGTGAAGAAACGATGATGGTGATGACTGAAGACAAACGTGTTTAAGTGTGACAGGCACTTCTTTGCTACTACCACTACTGACTTCTG ------------------------------------------------------------MetProLeuGluIleSerTyrValProSerThrTyrLeuThrGluIleThrHis         390         402         414         426ATGCCTTTGGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATTACGGAAACCTTTAAAGAATACACGGAAGATGAATAAACTGACTTTAGTGAGTA ------------------------------------------------------------ValSerGlnAlaLeuLeuGluValGluGlnLeuLeuAsnAlaProAspLeuCys         444         456         468         480GTCTCACAAGCCCTATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTCAGAGTGTTCGGGATAATCTTCACCTTGTTGAAGAGTTACGAGGACTGGAGACA ------------------------------------------------------------AlaLysAspPheGluAspLeuPheLysGlnGluGluSerLeuLysAsnIleLys         498         510         522         534GCTAAGGACTTTGAAGATCTCTTTAAGCAAGAGGAGTCTCTGAAGAATATAAAACGATTCCTGAAACTTCTAGAGAAATTCGTTCTCCTCAGAGACTTCTTATATTTT ------------------------------------------------------------AspSerLeuGlnGlnSerSerGlyArgIleAspIleIleHisSerLysLysThr         552         564         576         588GATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGACACTATCAGATGTTGTTTCGAGTCCAGCCTAACTGTAATAAGTATCGTTCTTCTGT ------------------------------------------------------------AlaAlaLeuGlnSerAlaThrProValGluArgValLysLeuGlnGluAlaLeu         606         618         630         642GCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCCGTCGTAACGTTTCACGTTGCGGACACCTTTCCCACTTCGATGTCCTTCGAGAG ------------------------------------------------------------SerGlnLeuAspPheGlnTrpGluLysValAsnLysMetTyrLysAspArgGln         660         672         684         696TCCCAGCTTGATTTCCAATGGGAAAAAGTTAACAAAATGTACAAGGACCGACAAAGGGTCGAACTAAAGGTTACCCTTTTTCAATTGTTTTACATGTTCCTGGCTGTT ------------------------------------------------------------GlyArgPheAspArgSerValGluLysTrpArgArgPheHisTyrAspIleLys         714         726         733         750GGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAAAGCCCGCTAAACTGTCTAGACAACTCTTTACCGCCGCAAAAGTAATACTATATTTC ------------------------------------------------------------IlePheAsnGlnTrpLeuThrGluAlaGluGlnPheLeuArgLysThrGlnIle         768         780         792         804ATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTTATAAATTAGTCACCGATTGTCTTCGACTTGTCAAAGAGTCTTTCTGTGTTTAA ------------------------------------------------------------ProGluAsnTrpGluHisAlaLysTyrLysTrpTyrLeuLysGluLeuGlnAsp         822         834         846         858CCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCCAGGATGGACTCTTAACCCTTGTACGATTTATGTTTACCATAGAATTCCTTGAGGTCCTA ------------------------------------------------------------GlyIleGlyGlnArgGlnThrValValArgThrLeuAsnAlaThrGlyGluGlu         876         888         900         912GGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGAATGCAACTGGGGAAGAACCGTAACCCGTCGCCGTTTGACAACAGTCTTGTAACTTACGTTGACCCCTTCTT ------------------------------------------------------------IleIleGlnGlnSerSerLysThrAspAlaSerIleLeuGlnGluLysLeuGly         930         942         954         966ATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGATATTAAGTCGTTAGGAGTTTTTGTCTACGGTCATAAGATGTCCTTTTTAACCCT ------------------------------------------------------------SerLeuAsnLeuArgTrpGlnGluValCysLysGlnLeuSerAspArgLysLys         984         996        1008        1020AGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGTCGGACTTAGACGCCACCGTCCTCCAGACGTTTGTCGACAGTCTGTCTTTTTTC ------------------------------------------------------------ArgLeuGluGluGlnLysAsnIleLeuSerGluPheGlnArgAspLeuAsnGlu        1038        1050        1062        1074AGGCTAGAAGAACAAAAGAATATCTTGTCAGAATTTCAAAGAGATTTAAATGAATCCGATCTTCTTGTTTTCTTATAGAACAGTCTTAAAGTTTCTCTAAATTTACTT ------------------------------------------------------------PheValLeuTrpLeuGluGluAlaAspAsnIleAlaSerIleProLeuGluPro        1092        1104        1116        1128TTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAACCTAAACAAAATACCAACCTCCTTCGTCTATTGTAACGATCATAGGGTGAACTTGGA ------------------------------------------------------------GlyLysGluGlnGlnLeuLysGluLysLeuGluGlnValLysLeuLeuValGlu        1146        1158        1170        1182GGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAGTTACTGGTGGAACCTTTTCTCGTCGTTGATTTTCTTTTCGAACTCGTTCAGTTCAATGACCACCTT ------------------------------------------------------------