CN109438566A - 阿尔茨海默病突变蛋白、突变基因及其医药用途 - Google Patents
阿尔茨海默病突变蛋白、突变基因及其医药用途 Download PDFInfo
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Abstract
本发明属于生物医药领域,涉及一种阿尔茨海默病突变蛋白、突变基因及其医药用途。具体地,本发明涉及家族性阿尔茨海默病致病基因PSEN1编码的早老素蛋白1(presenilin 1)3个新突变位点。更具体地,本发明涉及一种蛋白,其氨基酸序列如SEQ ID NOs:1-3中任一序列所示。本发明人在中国EOFAD中发现PSEN1 3个新突变,它们与各家系AD的发病密切相关,具有应用于制备治疗和/或预防或者诊断阿尔茨海默病的药物或者试剂的潜力。
Description
技术领域
本发明属于生物医药领域,涉及一种阿尔茨海默病突变蛋白、突变基因及其医药用途。具体地,本发明涉及家族性阿尔茨海默病致病基因PSEN1编码的早老素蛋白1(presenilin 1)3个新突变位点。
背景技术
阿尔茨海默病(Alzheimer's disease,AD)是痴呆最常见的类型,主要表现为以记忆障碍为特征的进行性全面认知功能减退。从遗传学角度,AD可分为家族性AD(familialAD,FAD)和散发性AD(sporadic AD,SAD)。FAD呈家族聚集性,发病年龄(age at onset,AAO)较早,疾病进程较快,临床症状较严重。
FAD与SAD存在共同的病理特点:细胞外淀粉样老年斑(senile plaque,SP)和细胞内神经原纤维缠结(neurofibrillary tangles,NFTs)。淀粉样蛋白级联假说仍是AD发病的主流学说1,目前认为较易聚集的可溶性的淀粉样蛋白具有更大的神经毒性2,3。
研究报道,FAD相关的致病基因突变可导致较长肽段Aβ42/Aβ43的绝对或相对升高4。早老素1基因(presenilin 1,简称为PSEN1)、早老素2基因(presenilin2,简称为PSEN2)和淀粉样前体蛋白基因(amyloidprecursor protein,简称为APP)是已知的3个重要的阿尔茨海默病致病基因,其中PSEN1是最常见的一种。
PSEN1是常染色体显性遗传AD最常见的致病基因,目前世界范围内报道的PSEN1突变种类200余种(https://www.alzforum.org),国内仅有数例报道5-12。已报道的200余种突变类型大多为错义突变,也有少数缺失和(或)插入等突变类型。大多数PSEN1突变为致病性突变,也有部分为基因多态性或致病性不明。
由于不同的基因背景和(或)环境因素,不同的PSEN1突变位点或同一位点突变为不同的氨基酸类型,携带PSEN1突变的AD患者的临床表现具有较大的异质性。因此,发现携带PSEN1新突变的FAD有助于丰富FAD的临床表型和扩大FAD的突变谱,为开发防治阿尔茨海默病的药物和方法提供新的途径。
发明内容
本发明人对满足纳入和排除标准的FAD家系成员收集临床资料、影像学检查和血液标本。对3个早发性家族性阿尔茨海默病(Early onset Family Alzheimer’s disease,EOFAD)家系成员进行已知致病基因(APP,PSEN1,PSEN2)测序以筛查突变。对发现的PSEN1新突变位点进行突变信息分析。构建表达野生型和突变型PSEN1的体外细胞模型(HEK293/APPPsw),检测PSEN1新突变对Aβ产生的影响。
本发明人在3个EOFAD中发现了PSEN1的3个新突变位点PSEN1 G111V、PSEN1 M139L和PSEN1 L172W。本发明人发现,该3个PSEN1突变在体外细胞水平均可导致Aβ产生的改变。3个新突变PSEN1 G111V、PSEN1 M139L和PSEN1 L172W为很可能的致病突变,可能与早发AD有关。
由此提供了下述发明:
本发明的一个方面涉及一种突变的蛋白,其为野生型人PSEN1蛋白发生选自如下的任意1个、任意2个或者3个突变:
G111V、M139L和L172W;
优选地,所述野生型人PSEN1蛋白的氨基酸序列如SEQ ID NO:7所示;
优选地,所述蛋白的氨基酸序列如SEQ ID NOs:1-3中任一序列所示。
表述形式G111V、M139L或L172W具有本领域技术人员知悉的含义,例如,分别是指野生型人PSEN1蛋白的第111个氨基酸由甘氨酸(G)突变为缬氨酸(V),第139位的甲硫氨酸(M)突变为亮氨酸(L),第172位的亮氨酸(L)突变为色氨酸(W)。
PSEN1 G111V的氨基酸序列如下:467aa
MTELPAPLSYFQNAQMSEDNHLSNTVRSQNDNRERQEHNDRRSLGHPEPLSNGRPQGNSRQVVEQDEEEDEELTLKYGAKHVIMLFVPVTLCMVVVVATIKSVSFYTRKDQLIYTPFTEDTETVGQRALHSILNAAIMISVIVVMTILLVVLYKYRCYKVIHAWLIISSLLLLFFFSFIYLGEVFKTYNVAVDYITVALLIWNFGVVGMISIHWKGPLRLQQAYLIMISALMALVFIKYLPEWTAWLILAVISVYDLVAVLCPKGPLRMLVETAQERNETLFPALIYSSTMVWLVNMAEGDPEAQRRVSKNSKYNAESTERESQDTVAENDDGGFSEEWEAQRDSHLGPHRSTPESRAAVQELSSSILAGEDPEERGVKLGLGDFIFYSVLVGKASATASGDWNTTIACFVAILIGLCLTLLLLAIFKKALPALPISITFGLVFYFATDYLVQPFMDQLAFHQFYI(SEQ ID NO:1)
PSEN1 M139L的氨基酸序列如下:467aa
MTELPAPLSYFQNAQMSEDNHLSNTVRSQNDNRERQEHNDRRSLGHPEPLSNGRPQGNSRQVVEQDEEEDEELTLKYGAKHVIMLFVPVTLCMVVVVATIKSVSFYTRKDGQLIYTPFTEDTETVGQRALHSILNAAIISVIVVMTILLVVLYKYRCYKVIHAWLIISSLLLLFFFSFIYLGEVFKTYNVAVDYITVALLIWNFGVVGMISIHWKGPLRLQQAYLIMISALMALVFIKYLPEWTAWLILAVISVYDLVAVLCPKGPLRMLVETAQERNETLFPALIYSSTMVWLVNMAEGDPEAQRRVSKNSKYNAESTERESQDTVAENDDGGFSEEWEAQRDSHLGPHRSTPESRAAVQELSSSILAGEDPEERGVKLGLGDFIFYSVLVGKASATASGDWNTTIACFVAILIGLCLTLLLLAIFKKALPALPISITFGLVFYFATDYLVQPFMDQLAFHQFYI(SEQ ID NO:2)
PSEN1 L172W的氨基酸序列如下:467aa
MTELPAPLSYFQNAQMSEDNHLSNTVRSQNDNRERQEHNDRRSLGHPEPLSNGRPQGNSRQVVEQDEEEDEELTLKYGAKHVIMLFVPVTLCMVVVVATIKSVSFYTRKDGQLIYTPFTEDTETVGQRALHSILNAAIMISVIVVMTILLVVLYKYRCYKVIHAWLIISSLLLFFFSFIYLGEVFKTYNVAVDYITVALLIWNFGVVGMISIHWKGPLRLQQAYLIMISALMALVFIKYLPEWTAWLILAVISVYDLVAVLCPKGPLRMLVETAQERNETLFPALIYSSTMVWLVNMAEGDPEAQRRVSKNSKYNAESTERESQDTVAENDDGGFSEEWEAQRDSHLGPHRSTPESRAAVQELSSSILAGEDPEERGVKLGLGDFIFYSVLVGKASATASGDWNTTIACFVAILIGLCLTLLLLAIFKKALPALPISITFGLVFYFATDYLVQPFMDQLAFHQFYI(SEQ ID NO:3)
在本发明的一些实施方式中,所述蛋白为分离的蛋白。
在本发明的一些实施方式中,两个突变是G111V和M139L。
在本发明的一些实施方式中,两个突变是M139L和L172W。
在本发明的一些实施方式中,两个突变是G111V和L172W。
本发明的另一方面涉及一种核酸分子,其编码本发明所述的蛋白。
编码PSEN1 G111V的核苷酸序列如下:1404bp
ATGACAGAGTTACCTGCACCGTTGTCCTACTTCCAGAATGCACAGATGTCTGAGGACAACCACCTGAGCAATACTGTACGTAGCCAGAATGACAATAGAGAACGGCAGGAGCACAACGACAGACGGAGCCTTGGCCACCCTGAGCCATTATCTAATGGACGACCCCAGGGTAACTCCCGGCAGGTGGTGGAGCAAGATGAGGAAGAAGATGAGGAGCTGACATTGAAATATGGCGCCAAGCATGTGATCATGCTCTTTGTCCCTGTGACTCTCTGCATGGTGGTGGTCGTGGCTACCATTAAGTCAGTCAGCTTTTATACCCGGAAGGATGGCAGCTAATCTATACCCCATTCACAGAAGATACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATTCTGAATGCTGCCATCATGATCAGTGTCATTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGCTTATTATATCATCTCTATTGTTGCTGTTCTTTTTTTCATTCATTTACTTGGGGGAAGTGTTTAAAACCTATAACGTTGCTGTGGACTACATTACTGTTGCACTCCTGATCTGGAATTTTGGTGTGGTGGGAATGATTTCCATTCACTGGAAAGGTCCACTTCGACTCCAGCAGGCATATCTCATTATGATTAGTGCCCTCATGGCCCTGGTGTTTATCAAGTACCTCCCTGAATGGACTGCGTGGCTCATCTTGGCTGTGATTTCAGTATATGATTTAGTGGCTGTTTTGTGTCCGAAAGGTCCACTTCGTATGCTGGTTGAAACAGCTCAGGAGAGAAATGAAACGCTTTTTCCAGCTCTCATTTACTCCTCAACAATGGTGTGGTTGGTGAATATGGCAGAAGGAGACCCGGAAGCTCAAAGGAGAGTATCCAAAAATTCCAAGTATAATGCAGAAAGCACAGAAAGGGAGTCACAAGACACTGTTGCAGAGAATGATGATGGCGGGTTCAGTGAGGAATGGGAAGCCCAGAGGGACAGTCATCTAGGGCCTCATCGCTCTACACCTGAGTCACGAGCTGCTGTCCAGGAACTTTCCAGCAGTATCCTCGCTGGTGAAGACCCAGAGGAAAGGGGAGTAAAACTTGGATTGGGAGATTTCATTTTCTACAGTGTTCTGGTTGGTAAAGCCTCAGCAACAGCCAGTGGAGACTGGAACACAACCATAGCCTGTTTCGTAGCCATATTAATTGGTTTGTGCCTTACATTATTACTCCTTGCCATTTTCAAGAAAGCATTGCCAGCTCTTCCAATCTCCATCACCTTTGGGCTTGTTTTCTACTTTGCCACAGATTATCTTGTACAGCCTTTTATGGACCAATTAGCATTCCATCAATTTTATATCTAG(SEQ ID NO:4)
编码PSEN1 M139L的核苷酸序列如下:1404bp
ATGACAGAGTTACCTGCACCGTTGTCCTACTTCCAGAATGCACAGATGTCTGAGGACAACCACCTGAGCAATACTGTACGTAGCCAGAATGACAATAGAGAACGGCAGGAGCACAACGACAGACGGAGCCTTGGCCACCCTGAGCCATTATCTAATGGACGACCCCAGGGTAACTCCCGGCAGGTGGTGGAGCAAGATGAGGAAGAAGATGAGGAGCTGACATTGAAATATGGCGCCAAGCATGTGATCATGCTCTTTGTCCCTGTGACTCTCTGCATGGTGGTGGTCGTGGCTACCATTAAGTCAGTCAGCTTTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCACAGAAGATACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATTCTGAATGCTGCCATCTGATCAGTGTCATTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGCTTATTATATCATCTCTATTGTTGCTGTTCTTTTTTTCATTCATTTACTTGGGGGAAGTGTTTAAAACCTATAACGTTGCTGTGGACTACATTACTGTTGCACTCCTGATCTGGAATTTTGGTGTGGTGGGAATGATTTCCATTCACTGGAAAGGTCCACTTCGACTCCAGCAGGCATATCTCATTATGATTAGTGCCCTCATGGCCCTGGTGTTTATCAAGTACCTCCCTGAATGGACTGCGTGGCTCATCTTGGCTGTGATTTCAGTATATGATTTAGTGGCTGTTTTGTGTCCGAAAGGTCCACTTCGTATGCTGGTTGAAACAGCTCAGGAGAGAAATGAAACGCTTTTTCCAGCTCTCATTTACTCCTCAACAATGGTGTGGTTGGTGAATATGGCAGAAGGAGACCCGGAAGCTCAAAGGAGAGTATCCAAAAATTCCAAGTATAATGCAGAAAGCACAGAAAGGGAGTCACAAGACACTGTTGCAGAGAATGATGATGGCGGGTTCAGTGAGGAATGGGAAGCCCAGAGGGACAGTCATCTAGGGCCTCATCGCTCTACACCTGAGTCACGAGCTGCTGTCCAGGAACTTTCCAGCAGTATCCTCGCTGGTGAAGACCCAGAGGAAAGGGGAGTAAAACTTGGATTGGGAGATTTCATTTTCTACAGTGTTCTGGTTGGTAAAGCCTCAGCAACAGCCAGTGGAGACTGGAACACAACCATAGCCTGTTTCGTAGCCATATTAATTGGTTTGTGCCTTACATTATTACTCCTTGCCATTTTCAAGAAAGCATTGCCAGCTCTTCCAATCTCCATCACCTTTGGGCTTGTTTTCTACTTTGCCACAGATTATCTTGTACAGCCTTTTATGGACCAATTAGCATTCCATCAATTTTATATCTAG(SEQ ID NO:5)
编码PSEN1 L172W的核苷酸序列如下:1404bp
ATGACAGAGTTACCTGCACCGTTGTCCTACTTCCAGAATGCACAGATGTCTGAGGACAACCACCTGAGCAATACTGTACGTAGCCAGAATGACAATAGAGAACGGCAGGAGCACAACGACAGACGGAGCCTTGGCCACCCTGAGCCATTATCTAATGGACGACCCCAGGGTAACTCCCGGCAGGTGGTGGAGCAAGATGAGGAAGAAGATGAGGAGCTGACATTGAAATATGGCGCCAAGCATGTGATCATGCTCTTTGTCCCTGTGACTCTCTGCATGGTGGTGGTCGTGGCTACCATTAAGTCAGTCAGCTTTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCACAGAAGATACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATTCTGAATGCTGCCATCATGATCAGTGTCATTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGCTTATTATATCATCTCTATGTTGCTGTTCTTTTTTTCATTCATTTACTTGGGGGAAGTGTTTAAAACCTATAACGTTGCTGTGGACTACATTACTGTTGCACTCCTGATCTGGAATTTTGGTGTGGTGGGAATGATTTCCATTCACTGGAAAGGTCCACTTCGACTCCAGCAGGCATATCTCATTATGATTAGTGCCCTCATGGCCCTGGTGTTTATCAAGTACCTCCCTGAATGGACTGCGTGGCTCATCTTGGCTGTGATTTCAGTATATGATTTAGTGGCTGTTTTGTGTCCGAAAGGTCCACTTCGTATGCTGGTTGAAACAGCTCAGGAGAGAAATGAAACGCTTTTTCCAGCTCTCATTTACTCCTCAACAATGGTGTGGTTGGTGAATATGGCAGAAGGAGACCCGGAAGCTCAAAGGAGAGTATCCAAAAATTCCAAGTATAATGCAGAAAGCACAGAAAGGGAGTCACAAGACACTGTTGCAGAGAATGATGATGGCGGGTTCAGTGAGGAATGGGAAGCCCAGAGGGACAGTCATCTAGGGCCTCATCGCTCTACACCTGAGTCACGAGCTGCTGTCCAGGAACTTTCCAGCAGTATCCTCGCTGGTGAAGACCCAGAGGAAAGGGGAGTAAAACTTGGATTGGGAGATTTCATTTTCTACAGTGTTCTGGTTGGTAAAGCCTCAGCAACAGCCAGTGGAGACTGGAACACAACCATAGCCTGTTTCGTAGCCATATTAATTGGTTTGTGCCTTACATTATTACTCCTTGCCATTTTCAAGAAAGCATTGCCAGCTCTTCCAATCTCCATCACCTTTGGGCTTGTTTTCTACTTTGCCACAGATTATCTTGTACAGCCTTTTATGGACCAATTAGCATTCCATCAATTTTATATCTAG(SEQ ID NO:6)
在本发明的一些实施方式中,所述核酸分子,其如SEQ ID NOs:4-6中任一序列所示。
在本发明的一些实施方式中,所述核酸分子为分离的核酸分子。
本发明还涉及一种核酸构建体,其含本发明的核酸分子;优选地,所述核酸构建体为重组载体;优选地,所述重组载体为重组表达载体。
本发明还涉及一种重组宿主细胞,其表达本发明的蛋白,或者含有本发明的核酸分子或者本发明的核酸构建体。
本发明的再一方面涉及一种组合物,其包含本发明的蛋白、本发明的核酸分子、本发明的核酸构建体或者本发明的重组宿主细胞。可选地,所述组合物还包含一种或多种药学上可接受的辅料。
本发明的再一方面涉及一种单克隆抗体或其抗原结合片段,其能够特异性地结合本发明的蛋白。
本发明的再一方面涉及一种偶联物,其包含本发明的单克隆抗体或其抗原结合片段以及偶联部分,其中,所述偶联部分为可检测的标记;优选地,所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及一种试剂盒,其包含本发明的单克隆抗体或其抗原结合片段,或者包括本发明的偶联物;
优选地,所述试剂盒还包括第二抗体,其特异性识别所述单克隆抗体或其抗原结合片段;任选地,所述第二抗体还包括可检测的标记,例如放射性同位素、荧光物质、发光物质、有色物质或酶;
可选地,所述试剂盒还包含说明书。
本发明的再一方面涉及一种引物或探针,其能够特异性地结合本发明的核酸分子;
优选地,所述探针的5’端标记有荧光报告基团,3’端标记有荧光淬灭基团;
优选地,所述荧光报告基团选自FAM、Hex、VIC、ROX和Cy5;
优选地,所述荧光淬灭基团选自BHQ1、TAMRA、JOE、BHQ2和BHQ3。
本发明的再一方面涉及一种试剂盒,其包含本发明的引物或探针;
可选地,所述试剂盒还包含说明书。
本发明的再一方面涉及选自如下的(1)-(6)项中的任意一项在制备治疗和/或预防或者诊断阿尔茨海默病的药物中的用途;或者在制备降低Aβ水平的药物中的用途;或者在制备筛选药物的模型例如细胞模型或者动物模型中的用途,所述药物用于治疗和/或预防和/或诊断阿尔茨海默病:
(1)本发明的蛋白;
(2)本发明的核酸分子;
(3)抑制或阻断本发明的蛋白的药物,例如本发明的单克隆抗体或其抗原结合片段;
(4)抑制或降低本发明的核酸分子的表达水平的药物;例如siRNA如shRNA,或者为用于CRISPR/Cas9系统的guide RNA;
(5)将本发明的蛋白修复为野生型人PSEN1蛋白(例如SEQ ID NO:7所示)的药物;
(6)将本发明的核酸分子修复为野生型人PSEN1基因(例如SEQ ID NO:8所示)的药物;
(7)检测本发明的蛋白的药物,例如本发明的单克隆抗体或其抗原结合片段或者本发明的偶联物;
(8)检测本发明的核酸分子的表达水平的药物,例如本发明的引物或探针;
优选地,所述阿尔茨海默病为家族性阿尔茨海默病;优选地,所述阿尔茨海默病为早发性家族性阿尔茨海默病;
优选地,所述,所述Aβ为Aβ40和/或Aβ42。
上述第(5)项或(6)项中,所述药物可以包括设计的引物,将突变的基因修复为野生型人PSEN1基因,通过本领域技术人员知悉的基因转染或者转导手段,转入到细胞中表达野生型蛋白。
野生型人PSEN1蛋白的氨基酸序列如下:467aa
MTELPAPLSYFQNAQMSEDNHLSNTVRSQNDNRERQEHNDRRSLGHPEPLSNGRPQGNSRQVVEQDEEEDEELTLKYGAKHVIMLFVPVTLCMVVVVATIKSVSFYTRKDGQLIYTPFTEDTETVGQRALHSILNAAIMISVIVVMTILLVVLYKYRCYKVIHAWLIISSLLLLFFFSFIYLGEVFKTYNVAVDYITVALLIWNFGVVGMISIHWKGPLRLQQAYLIMISALMALVFIKYLPEWTAWLILAVISVYDLVAVLCPKGPLRMLVETAQERNETLFPALIYSSTMVWLVNMAEGDPEAQRRVSKNSKYNAESTERESQDTVAENDDGGFSEEWEAQRDSHLGPHRSTPESRAAVQELSSSILAGEDPEERGVKLGLGDFIFYSVLVGKASATASGDWNTTIACFVAILIGLCLTLLLLAIFKKALPALPISITFGLVFYFATDYLVQPFMDQLAFHQFYI(SEQ ID NO:7)
野生型人PSEN1基因的读码框序列如下:1404bp
ATGACAGAGTTACCTGCACCGTTGTCCTACTTCCAGAATGCACAGATGTCTGAGGACAACCACCTGAGCAATACTGTACGTAGCCAGAATGACAATAGAGAACGGCAGGAGCACAACGACAGACGGAGCCTTGGCCACCCTGAGCCATTATCTAATGGACGACCCCAGGGTAACTCCCGGCAGGTGGTGGAGCAAGATGAGGAAGAAGATGAGGAGCTGACATTGAAATATGGCGCCAAGCATGTGATCATGCTCTTTGTCCCTGTGACTCTCTGCATGGTGGTGGTCGTGGCTACCATTAAGTCAGTCAGCTTTTATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCACAGAAGATACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATTCTGAATGCTGCCATCATGATCAGTGTCATTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGCTTATTATATCATCTCTATTGTTGCTGTTCTTTTTTTCATTCATTTACTTGGGGGAAGTGTTTAAAACCTATAACGTTGCTGTGGACTACATTACTGTTGCACTCCTGATCTGGAATTTTGGTGTGGTGGGAATGATTTCCATTCACTGGAAAGGTCCACTTCGACTCCAGCAGGCATATCTCATTATGATTAGTGCCCTCATGGCCCTGGTGTTTATCAAGTACCTCCCTGAATGGACTGCGTGGCTCATCTTGGCTGTGATTTCAGTATATGATTTAGTGGCTGTTTTGTGTCCGAAAGGTCCACTTCGTATGCTGGTTGAAACAGCTCAGGAGAGAAATGAAACGCTTTTTCCAGCTCTCATTTACTCCTCAACAATGGTGTGGTTGGTGAATATGGCAGAAGGAGACCCGGAAGCTCAAAGGAGAGTATCCAAAAATTCCAAGTATAATGCAGAAAGCACAGAAAGGGAGTCACAAGACACTGTTGCAGAGAATGATGATGGCGGGTTCAGTGAGGAATGGGAAGCCCAGAGGGACAGTCATCTAGGGCCTCATCGCTCTACACCTGAGTCACGAGCTGCTGTCCAGGAACTTTCCAGCAGTATCCTCGCTGGTGAAGACCCAGAGGAAAGGGGAGTAAAACTTGGATTGGGAGATTTCATTTTCTACAGTGTTCTGGTTGGTAAAGCCTCAGCAACAGCCAGTGGAGACTGGAACACAACCATAGCCTGTTTCGTAGCCATATTAATTGGTTTGTGCCTTACATTATTACTCCTTGCCATTTTCAAGAAAGCATTGCCAGCTCTTCCAATCTCCATCACCTTTGGGCTTGTTTTCTACTTTGCCACAGATTATCTTGTACAGCCTTTTATGGACCAATTAGCATTCCATCAATTTTATATCTAG(SEQ ID NO:8)
本发明的再一方面涉及一种治疗和/或预防阿尔茨海默病的方法或者一种降低Aβ(例如Aβ40和/或Aβ42)水平的方法,包括减少或者降低受试者中的SEQ ID NOs:1-3中任一序列所示蛋白的水平的步骤,或者包括减少或者降低受试者中的SEQ ID NOs:4-6中任一序列所示核酸分子的水平的步骤。在本发明的一个实施方案中,所述方法包括将SEQ IDNOs:1-3中任一序列所示的蛋白修正为相应的野生型蛋白的步骤。在本发明的一个实施方案中,所述方法包括将SEQ ID NOs:4-6中任一序列所示的核酸分子修正为相应的野生型核酸分子的步骤。
本发明的再一方面涉及一种诊断阿尔茨海默病的方法,包括检测受试者是否存在SEQ ID NOs:1-3中任一序列所示的蛋白的步骤,或者包括检测受试者是否存在SEQ IDNOs:4-6中任一序列所示的核酸分子的步骤;如果存在所述蛋白或核酸分子,则诊断为阳性。
本发明的再一方面涉及一种筛选治疗和/或预防阿尔茨海默病的药物的方法,包括检测候选药物是否减少或者降低受试者中或者细胞中的SEQ ID NOs:1-3中任一序列所示的蛋白的水平的步骤,或者包括检测候选药物是否减少或者降低受试者中或者细胞中的SEQ ID NOs:4-6中任一序列所示的核酸分子的水平的步骤。如果受试者中或者细胞中的所述蛋白或核酸的水平降低或者减少,则作为阳性药物。可选地,以不加入候选药物的细胞作为对照。
受试者中或者细胞中的所述蛋白或核酸的水平降低或者减少,可以是相对于向受试者给药之前的该蛋白或核酸的水平,或者相对于向细胞中加入候选药物之前的该蛋白或核酸的水平。
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时,为了更好地理解本发明,下面提供相关术语的定义和解释。
本发明中,术语“分离的”或“被分离的”指的是,从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分,那么可能是其所处的天然环境发生了改变,或从天然环境下分离出该物质,或二者情况均有发生。例如,某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽,而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质,也不排除存在不影响物质活性的其它不纯物质。
本发明中,术语“核酸构建体”,在文中定义为单链或双链核酸分子,优选是指人工构建的核酸分子。可选地,所述核酸构建体还包含有可操作地连接的1个或多个调控序列。
本发明中,术语“可操作地连接”是指两个或多个核苷酸区域或核酸序列的功能性的空间排列。所述“可操作地连接”可以通过基因重组的手段实现。
本发明中,术语“载体”指的是,可将抑制某蛋白的多核苷酸插入其中的一种核酸运载工具。举例来说,载体包括:质粒;噬菌粒;柯斯质粒;人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC);噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件。
本发明中,术语“宿主细胞”指的是导入载体的细胞,包括如下许多细胞类型,如大肠杆菌或枯草菌等原核细胞,如酵母细胞或曲霉菌等真菌细胞,如S2果蝇细胞或Sf9等昆虫细胞,或者如纤维原细胞,CHO细胞,COS细胞,NSO细胞,HeLa细胞,BHK细胞,HEK 293细胞或人细胞的动物细胞。
本发明中,术语“单克隆抗体”或“单抗”是指,来自一群高度同源的抗体分子中的一个抗体或抗体的一个片断,也即除可能自发出现的自然突变外,一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的,其通常包含至少2种或更多种的不同抗体,这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature,256:495,1975),但也可采用重组DNA技术获得(如参见U.S.P 4,816,567)。
本发明中,术语“抗原结合片段”是指包含全长抗体的片段的多肽,其保持特异性结合全长抗体所结合的相同抗原的能力,和/或与全长抗体竞争对抗原的特异性结合,其也被称为“抗原结合部分”。通常参见,Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版,Raven Press,N.Y.(1989),其以其全文通过引用合并入本文,用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下,抗原结合片段包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如,scFv)、嵌合抗体、双抗体(diabody)和这样的多肽,其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
在一些情况下,抗原结合片段是单链抗体(例如,scFv),其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见,例如,Bird等人,Science242:423 426(1988)和Huston等人,Proc.Natl.Acad.Sci.USA 85:5879 5883(1988))。此类scFv分子可具有一般结构:NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如,可使用具有氨基酸序列(GGGGS)4的接头,但也可使用其变体(Holliger等人(1993),Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995),Protein Eng.8:725-731,Choi等人(2001),Eur.J.Immunol.31:94-106,Hu等人(1996),Cancer Res.56:3055-3061,Kipriyanov等人(1999),J.Mol.Biol.293:41-56和Roovers等人(2001),Cancer Immunol.描述。
本发明中,术语“APP(beta淀粉样蛋白前体,amyloid precursor protein)”编码Aβ前体蛋白,是Aβ的直接来源,APP经β-分泌酶剪切后产生C99,C99继而被γ-分泌酶剪切产生Aβ。PSEN1和PSEN2基因分别编码早老素1和早老素2,是γ-分泌酶的催化亚单位。由此可见,APP、PSEN1、PSEN2基因编码的蛋白都位于APP酶切通路上,突变可能是通过影响APP的酶切位点和早老素蛋白的结构,从而影响APP的酶切过程,导致Aβ生成异常。
本发明中,术语“Aβ”是APP依次经β-分泌酶和γ-分泌酶酶切的代谢产物,是包含约40个氨基酸的多肽。Aβ40(含40个氨基酸)和Aβ42(含42个氨基酸)是Aβ的主要类型,Aβ40约占80%-90%,Aβ42约占5%-10%。Aβ42比Aβ40疏水性更强,更易聚集形成斑块,是形成脑内淀粉样斑块(amyloid plaque)的主要类型。Aβ异常聚集形成淀粉样斑块的核心,是AD的主要病理特征之一。
Aβ40的氨基酸序列如下:(40aa)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV(SEQ ID NO:9)
Aβ42的氨基酸序列如下:(42aa)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(SEQ ID NO:10)
本发明中,术语“有效量”是指可在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的剂量。
术语“疾病和/或病症”是指所述受试者的一种身体状态,该身体状态与本发明所述疾病和/或病症有关。
术语“受试者”可以指患者或者其它接受本发明药物组合物以治疗、预防、减轻和/或缓解本发明所述疾病或病症的动物,特别是哺乳动物,例如人、狗、猴、牛、马等。
发明的有益效果
本发明人在中国EOFAD中发现PSEN1 3个新突变,它们与各家系AD的发病密切相关,具有应用于制备治疗和/或预防或者诊断阿尔茨海默病的药物或者试剂的潜力。
附图说明
图1A-图1B:PSEN1 G111V家系图与测序图。其中:
图1A,家系1携带p.G111V(c.332G>T)突变。先证者用箭头指示,黑色实心符号表示患病成员,白色空心符号表示未受累成员,方型表示男性,圆圈表示女性,斜杠表示已故成员;
图1B,测序色谱图显示先证者携带杂合PSEN1 c.332G>T突变(参考序列:NM_000021)。
图2A-图2B:PSEN1 M139L家系图与测序图。其中:
图2A,家系2携带p.M139L(c.415A>T)突变。先证者用箭头指示,黑色实心符号表示患病成员,白色空心符号表示未受累成员,方型表示男性,圆圈表示女性,斜杠表示已故成员;
图2B,测序色谱图显示先证者携带杂合PSEN1 c.415 A>T突变(参考序列:NM_000021)。
图3A-图3B:PSEN1 L172W家系图与测序图。其中:
图3A,家系3携带p.L172W(c.515 T>G)突变。先证者用箭头指示,黑色实心符号表示患病成员,白色空心符号表示未受累成员,方型表示男性,圆圈表示女性,斜杠表示已故成员;
图3B,测序色谱图显示先证者携带杂合PSEN1 c.515 T>G突变(参考序列:NM_000021)。
图4A-图4C:家系患病成员MRI。其中:
图4A,家系1(PSEN1 G111V)先证者头部失状位,海马相和水平位MRI平扫:双侧海马萎缩,右侧为著;
图4B,家系2(PSEN1 M139L)III-8成员头部失状位,海马相和水平位MRI平扫:双侧海马萎缩;
图4C,家系3(PSEN1 L172W)先证者头部失状位,海马相和水平位MRI平扫:双侧海马轻度萎缩,额顶颞部皮层萎缩,左侧为著。
图5A-图5C:细胞上清中各组Aβ40和Aβ42水平及Aβ42/Aβ40比例。病毒感染HEK23-APP695sw细胞48h细胞上清中Aβ40、Aβ42浓度定量分析。柱代表3次独立实验的平均值±标准误。P值代表与WT组比较的Tukey’s检验结果,*指P<0.05。其中:
图5A,细胞上清Aβ40浓度;
图5B,细胞上清Aβ42浓度;
图5C,细胞上清Aβ42/Aβ40比值。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:阿尔茨海默病致病基因的筛查和测序
1.实验方法
本研究经首都医科大学宣武医院伦理委员会批准。每位受试者或其法定监护人签署书面知情同意书。
家系通过宣武医院认知专科门诊或中国阿尔茨海默病登记网(Chinese FamilyAlzheimer’s disease Network,CFAN)收集。家系纳入标准为家系中至少2位患病成员,且满足连续2代一级亲属关系。AD的诊断标准为符合2011 NIA-AA很可能AD诊断标准13。EOAD为发病年龄<65岁。至少2位有经验的神经内科主治医师及以上职称进行诊断。家系成员收集个人信息、现病史、家族史、既往史、用药情况、体格检查、成套的神经心理评估量表及脑部磁共振扫描。确定出3个家系,均为EOFAD。
对家系的先证者采用Sanger测序法进行AD已知致病基因(APP、PSEN1和PSEN2)突变筛查,即对APP 16-17外显子、PSEN1的3-12外显子和PSEN2的3-12外显子进行测序。对于在先证者检测出的基因突变,在家系成员中采用直接测序法对该突变位点进行检测。同时,在100名散发性晚发AD患者和100名认知正常人中对AD已知致病基因(APP、PSEN1和PSEN2)进行筛查。
2.实验结果
2.1家系临床特点
3个FAD家系满足EOFAD诊断标准,家系的临床特点:
家系1(图1A)患病成员累及2代2人,发病年龄53-63岁,平均AAO 58岁。先证者发病年龄53岁,记忆障碍逐渐加重伴执行功能减退,工作任务完成质量下降,后办理提前离退。55岁诊断为“轻度认知功能障碍(mild cognitive impairement,MCI)”。先证者母亲已故。约62岁出现记忆力减退,近事遗忘,逐渐加重伴执行功能、空间技能减退,约70岁左右进展至痴呆,后期自理能力逐渐丧失,卧床、二便障碍。79岁因“肺病”去世。该家系患病成员符合典型AD的临床特征,以记忆障碍起病,逐渐累及其他认知域。先证者脑部磁共振影像示双侧海马萎缩(图4A)。
家系2(图2A)患病成员累及3代11人,发病年龄45-56岁,平均AAO 53岁。先证者45岁出现记忆力下降,近记忆为主,并逐渐出现执行功能减退,48岁不能正常上班,生活尚可自理。49岁时出现视空间障碍,外出迷路,穿衣分不清前后、正反面,逐渐加重并出现行为异常,随地大小便,偶有胡言乱语、情绪激动、打骂人,并存在不知饥饿。50岁时因摔倒脊柱骨折后卧床至今,目前四肢不能活动、不能翻身,言语不能,二便失禁。先证者大哥约55岁时出现记忆力下降,存在视空间障碍和行为异常,饮食无节制,存在激越行为,临终前淡漠状态。62岁去世。先证者二哥现62岁,55岁左右开始出现记忆力下降,目前仅知道部分家人的名字,远记忆轻度受损,伴执行功能、视空间功能减退,伴行为异常和性格改变,存在捡拾癖,饮食单一且无节制。近2年来出现站立时无缓冲,双脚交叉摔倒,平素动作显笨拙。走路时间长时可出现身体向一侧偏斜,偶有夜间肌阵挛发作。自理能力明显下降。头部MRI平扫示双侧海马萎缩(图4B)。先证者三哥现59岁,约51岁出现记忆力下降,有激越行为:哭喊大叫、打人,不听从建议(洗澡不让脱裤子)。目前淡漠状态,不言语,在家里来回走,但找不到自己想去的地方,困了就随时随地躺下睡,吃饭为给什么吃什么,二便失禁。该家系患病成员符合典型AD的临床特征,以记忆障碍起病,逐渐累及其他认知域,病情进展较为迅速。同时,该家系患病成员视空间功能障碍和性格行为改变较为突出。
家系3(图3A)患病成员累及2代2人,发病年龄49-70岁,平均AAO 59.5岁。先证者49岁起病,主要表现为近记忆力下降合并性格改变,病情逐渐进展出现手部震颤、行走不稳等运动症状,日常生活能力下降。头部MRI示脑萎缩、海马萎缩、额顶叶多发小腔隙灶(图4C)。头部FDG-PETCT:双侧额叶葡萄糖代谢一致性减低,左侧顶叶、枕叶、颞叶皮层局部葡萄糖代谢较对侧减低。先证者父亲72岁起病,82岁进展为“痴呆”,83岁去世。
2.2DNA测序
在家系1的先证者的血液样本中检测出杂合错义突变PSEN1 G111V(c.332G>T,NM_000021)(图1B)。
在家系2的先证者中发现杂合错义突变PSEN1 M139L(c.415A>T,NM_000021)(图2B)。同时,在家系2的成员III-7,III-8,III-9,III-10(该4位家系成员均为患病个体)中检测出该同一个错义突变,而在家系2成员III-12(认知正常)未检测出该突变。
在家系3先证者的DNA样本中检出杂合错义突变PSEN1 L172W(c.515T>G,NM_000021)(图3B)。
在100名散发晚发AD患者和100名认知正常人未发现上述3种突变(PSEN1 G111V、PSEN1 M139L和PSEN1 172W)。
实施例2:阿尔茨海默病相关突变蛋白和突变基因的功能分析
1.实验方法
对在FAD中筛查出的突变,进行突变信息分析,包括在家系内进行疾病突变的共分离分析14,在人群数据库[Exome Aggregation Consortium(ExAC,http://exac.broadinstitute.org/),Exome Variant Server(http://evs.gs.washington.edu/EVS)and 1000Genomes Project(http://browser.1000genomes.org)]和疾病数据库(http://www.ncbi.nlm.nih.gov/clinvar,ClinVar)中进行该突变位点频率的查找,运用计算机软件预测系统[PANTHER(http://www.pantherdb.org/tools/csnpScoreForm.jsp),Mutation Taster(http://mutationassessor.org),and PolyPhen-2(http://genetics.bwh.harvard.edu/pph2)]对突变位点进行致病性预测。
2.实验结果
PSEN1 G111V:在人群数据库ExAC,Exome Variant Server和千人基因组数据库中均未发现该突变,在疾病数据库ClinVar中未发现该突变。PSEN1 G111V突变位于PSEN1第一疏水环,该家系发病年龄为53-63岁,临床特征和影像学表现符合典型AD的特点。既往报道,携带第一疏水环上的突变的家系多为超早发家系,且病情进展迅速,常伴有肌阵挛、癫痫、痉挛性截瘫等运动症状和行为异常、性格改变等疑似额颞叶痴呆的临床表型15。如位于111位附近的L113Q16和L113P17突变。3个计算机预测软件对该突变的预测结果分别为:Mutation Taster:disease causing,PANTHER:probably damaging,Polyphen-2:probablydamaging.PSEN1 G111V错义突变在人群数据库和疾病数据库中均未发现,3个计算机软件预测该突变均可能是致病性突变。
PSEN1 M139L:在人群数据库ExAC,Exome Variant Server和千人基因组数据库中均未发现该突变,在疾病数据库ClinVar中未发现该突变。PSEN1 M139L突变位于PSEN1第二跨膜区,该区域是PSEN1突变热点区域之一15,18。该突变在人群数据库和疾病数据库中均未报道,3个计算机软件预测该突变军可能导致AD发病。3个计算机预测软件对该突变的预测结果分别为:Mutation Taster:disease causing,PANTHER:probably damaging,Polyphen-2:probably damaging.运用已报道的疾病突变共分离计算公式,其概率N=(1/2)6=1/64,提示该突变为强致病性突变。该家系发病成员众多,不同个体间临床表型相似,且与既往该位点其他突变类型的临床特点相似19-23家系内疾病突变共分离分析提示该突变为强致病性突变。综上,PSEN1 M139L为很可能导致AD致病的新突变类型。
PSEN1 L172W:在人群数据库中ExAC,Exome Variant Server和千人基因组数据库中均未发现该突变,在疾病数据库ClinVar中未发现该突变。PSEN1 L172W突变位于PSEN1第三跨膜区,该家系先证者发病年龄49岁,以记忆障碍伴性格改变为特征起病,临床表型兼具AD与FTD的临床特点,且随着病情进展出现震颤、共济失调等运动症状,与附近既往报道的突变PSEN1 L173F(G>C)相似24。先证者头部MRI和FDG-PETCT显示左侧大脑皮层萎缩严重和代谢减低,但双侧海马萎缩程度相对较轻,说明该患者脑部病变除海马外主要累及左侧皮层。既往PSEN1突变也有类似报道17,25。3个计算机预测软件对该突变的预测结果分别为:Mutation Taster:polymorphism,PANTHER:probably damaging,Polyphen-2:probablydamaging。PSEN1 L172W在人群数据库和疾病数据库中未见报道,至少2个计算机预测软件提示该突变为致病性突变,该突变附近位点均为致病性突变,且患者发病年龄较早、病情较重24,26,27,提示该区域可能为PSEN1重要的功能区。
实施例3:细胞学实验
1.实验方法
1.1慢病毒表达质粒的构建
构建表达野生型人源PSEN1的慢病毒表达质粒pLVX-PSEN1-IRES-ZsGreen1,以测序正确的质粒pLVX-PSEN1为模板分别进行定点突变构建表达PSEN1 G111V、PSEN1 M139L和PSEN1 L172W的突变质粒。
pLVX-IRES-ZsGreen1空质粒(Clontech,Catalog No.632187)、包装质粒psPAX2(Addgene#12260),包装质粒pMD2.G(Addgene plasmid#12259)。插入的野生型PSEN1序列来源于本发明人所在实验室构建的pCDNA 3.1-EGFP-PSEN128。
具体步骤如下:
1.1.1.引物设计:
载体构建引物如下面的表1所示,其中下划线部分为酶切位点。
表1
点突变的引物如下面的表2所示。其中下划线部分表示碱基突变位点。
表2
1.1.2.PCR
利用TransStart FastPfu Fly DNA Polymerase扩增目的片段PSEN1,以构建成功的质粒pLVX-PSEN1-IRES-ZsGreen1为模板进行点突变。具体操作如下:
PCR反应体系如下面的表3所示。
表3
PCR反应条件如下:
载体构建
点突变及缺失:
1.1.3.酶切回收质粒pLVX-IRES-ZsGreen1,双酶切体系如下面的表4所示。
表4
成分 | 体积 |
pLVX-IRES-ZsGreen1 | 1μg |
FlyCut BamHI | 1μl |
FlyCut EcoRI | 1μl |
10×FlyCut Buffer | 5μl |
ddH<sub>2</sub>O to final volume | 50μl |
1.1.4.胶回收
(1)取5μl PCR产物进行1.5%TAE琼脂糖凝胶电泳检测扩增结果;向剩余扩增PSEN1的PCR产物中加入1μl DMT Enzyme,37℃消化1h;
(2)使用EasyPureQuick Gel Extraction Kit(Code#EG101)纯化回收目的片段PSEN1和双酶切后的质粒pLVX-IRES-ZsGreen1,详细步骤参照说明书。
1.1.5.使用pEASY-Uni Seamless Cloning and Assembly Kit做连接,反应体系如下面的表5所示。
表5
成分 | 体积 |
2×Assembly Mix | 5μl |
pLVX-IRES-ZsGreen1 | 4μl |
PSEN1 | 1μl |
连接产物用于下面的转化。
1.1.6.转化
(1)取5μl PCR产物进行1.5%TAE琼脂糖凝胶电泳检测点突扩增结果;
(2)向扩增的PCR产物中加入1μl DMT Enzyme,37℃消化1h;
(3)取10μl消化后的PCR产物或者连接产物转化Trans1-T1(ps1-WT质粒扩增产物)和TransStbl3(载体构建连接产物和pLVX-PSEN1质粒扩增产物)感受态细胞(北京全式金公司),转化条件:冰浴30min后,42℃热激30s(Trans1-T1)/45s(TransStbl3),迅速置于冰上2min,加入500μl液体LB培养基中于37℃摇床振荡培养1h后,4000rpm离心1min,留150μl重悬菌体,将菌液全涂于含Amp+(终浓度:100μg/ml)的LB抗性平板上,37℃恒温过夜培养约16h;
1.1.7.次日早晨从培养箱中取出平板,随机挑取3个单克隆摇菌,然后送测序公司测序,将鉴定正确的突变质粒对应的重组菌加入甘油(终浓度:30%)保存。
1.1.8将甘油菌解冻,溶于LB抗性培养液(1%胰蛋白胨,0.5%酵母粉,1%NaCl,NaOH 0.04ml/L)中,过夜摇菌。次日进行质粒大提,采用无内毒素质粒大提试剂盒(天根生化科技北京有限公司,DP117)。质粒提取完毕后酶标仪检测浓度,分装后-80℃储存。
1.2慢病毒质粒的包装与扩增
通过脂质体转染法共转染慢病毒表达质粒pLVX-IRES-ZsGreen1(步骤1.1.8制得)以及慢病毒包装质粒psPAX2和pMD2G至HEK293T细胞(获自北京全式金生物技术有限公司),转染6小时后更换培养基。分别在转染30h和54h时收集细胞培养上清,3000rpm离心5分钟以去除细胞碎片,采用0.45μm滤膜过滤上清,收集后分装冻存于80℃备用。
1.3细胞培养与转染
将稳定表达APP KM670/671NL(APP-swe)的HEK293细胞(制备方法参考王琦.Tyro3受体的表达对Aβ产生的影响及其与阿尔茨海默病的相关性研究;2011)在含10%胎牛血清(Gibco,Grand Island,NY,USA)的Dulbecco改良的Eagle培养基(DMEM)中,37℃,5%CO 2的潮湿培养箱中培养。对于慢病毒转导,细胞生长至40%-50%汇合并在第二天用每2cm2的30μl慢病毒溶液(前面步骤1.2制得)感染。24小时后,弃去病毒溶液并用新鲜培养基替换,再过48小时更换新鲜培养基,继续培养细胞48小时后收取细胞上清,3000rpm离心5分钟,离心后上清分装冻于-80℃。
病毒感染细胞72H后,目的蛋白的表达基本稳定。在显微镜下观察荧光蛋白的表达,对于每组细胞,随机选择3个视野,使用ImageJ软件计算荧光强度并计数细胞个数,每个细胞的平均荧光强度用于指示病毒的转染效率。同时,收取细胞总蛋白对目的蛋白PSEN1进行western检测,比较各组目的蛋白的表达量。具有相同转染效率的各组细胞用于后续实验和结果的分析。
1.4ELISA方法检测Aβ
根据酶联免疫吸附试剂盒(enzyme-linked immunosorbent assay,ELISA)(IBL,Hamburg,Germany)说明书测定步骤1.3中的细胞上清液中的Aβ40、Aβ42浓度,计算Aβ42/Aβ40的比值。细胞上清蛋白总浓度通过BCA法测定,Aβ40、Aβ42浓度依据蛋白总浓度进行校正。
1.5统计分析
采用SPSS 23.0统计分析软件进行数据处理。所有计量资料采用平均值±标准误(mean±SEM)表示。多组数据比较采用单因素方差分析one-way ANOVA,组间比较采用posthoc Tukey’s test。P<0.05时,即认为有统计学差异。采用Graphpad prism 7软件绘制统计图。
2.实验结果
在表达野生型和突变型PSEN1的体外细胞模型HEK293/APPswe的培养基中检测Aβ40,Aβ42的产生(图5A-5C)。其中,PSEN1 M139V为阳性对照组。与PSEN1 WT相比:
PSEN1 G111V和PSEN1 M139L的Aβ40的产量降低,Aβ42的生成不变,而Aβ42/Aβ40的比例升高;提示PSEN1 G111V突变和PSEN1 M139L突变很可能与该家系AD的发病相关;
PSEN1 L172W的Aβ40的产量降低,Aβ42的生成不变,Aβ42/Aβ40的比例有升高的趋势。
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28.方伯言.中国人家族性阿尔茨海默病早老素1基因突变病理功能研究[博士]:首都医科大学;2006.
尽管本发明的具体实施方式已经得到详细的描述,本领域技术人员将会理解。根据已经公开的所有教导,可以对那些细节进行各种修改和替换,这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
SEQUENCE LISTING
<110> 首都医科大学宣武医院
<120> 阿尔茨海默病突变蛋白、突变基因及其医药用途
<130> IDC180312
<160> 20
<170> PatentIn version 3.2
<210> 1
<211> 467
<212> PRT
<213> Artificial
<220>
<223> PSEN1 G111V的氨基酸序列
<400> 1
Met Thr Glu Leu Pro Ala Pro Leu Ser Tyr Phe Gln Asn Ala Gln Met
1 5 10 15
Ser Glu Asp Asn His Leu Ser Asn Thr Val Arg Ser Gln Asn Asp Asn
20 25 30
Arg Glu Arg Gln Glu His Asn Asp Arg Arg Ser Leu Gly His Pro Glu
35 40 45
Pro Leu Ser Asn Gly Arg Pro Gln Gly Asn Ser Arg Gln Val Val Glu
50 55 60
Gln Asp Glu Glu Glu Asp Glu Glu Leu Thr Leu Lys Tyr Gly Ala Lys
65 70 75 80
His Val Ile Met Leu Phe Val Pro Val Thr Leu Cys Met Val Val Val
85 90 95
Val Ala Thr Ile Lys Ser Val Ser Phe Tyr Thr Arg Lys Asp Val Gln
100 105 110
Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr Glu Thr Val Gly Gln Arg
115 120 125
Ala Leu His Ser Ile Leu Asn Ala Ala Ile Met Ile Ser Val Ile Val
130 135 140
Val Met Thr Ile Leu Leu Val Val Leu Tyr Lys Tyr Arg Cys Tyr Lys
145 150 155 160
Val Ile His Ala Trp Leu Ile Ile Ser Ser Leu Leu Leu Leu Phe Phe
165 170 175
Phe Ser Phe Ile Tyr Leu Gly Glu Val Phe Lys Thr Tyr Asn Val Ala
180 185 190
Val Asp Tyr Ile Thr Val Ala Leu Leu Ile Trp Asn Phe Gly Val Val
195 200 205
Gly Met Ile Ser Ile His Trp Lys Gly Pro Leu Arg Leu Gln Gln Ala
210 215 220
Tyr Leu Ile Met Ile Ser Ala Leu Met Ala Leu Val Phe Ile Lys Tyr
225 230 235 240
Leu Pro Glu Trp Thr Ala Trp Leu Ile Leu Ala Val Ile Ser Val Tyr
245 250 255
Asp Leu Val Ala Val Leu Cys Pro Lys Gly Pro Leu Arg Met Leu Val
260 265 270
Glu Thr Ala Gln Glu Arg Asn Glu Thr Leu Phe Pro Ala Leu Ile Tyr
275 280 285
Ser Ser Thr Met Val Trp Leu Val Asn Met Ala Glu Gly Asp Pro Glu
290 295 300
Ala Gln Arg Arg Val Ser Lys Asn Ser Lys Tyr Asn Ala Glu Ser Thr
305 310 315 320
Glu Arg Glu Ser Gln Asp Thr Val Ala Glu Asn Asp Asp Gly Gly Phe
325 330 335
Ser Glu Glu Trp Glu Ala Gln Arg Asp Ser His Leu Gly Pro His Arg
340 345 350
Ser Thr Pro Glu Ser Arg Ala Ala Val Gln Glu Leu Ser Ser Ser Ile
355 360 365
Leu Ala Gly Glu Asp Pro Glu Glu Arg Gly Val Lys Leu Gly Leu Gly
370 375 380
Asp Phe Ile Phe Tyr Ser Val Leu Val Gly Lys Ala Ser Ala Thr Ala
385 390 395 400
Ser Gly Asp Trp Asn Thr Thr Ile Ala Cys Phe Val Ala Ile Leu Ile
405 410 415
Gly Leu Cys Leu Thr Leu Leu Leu Leu Ala Ile Phe Lys Lys Ala Leu
420 425 430
Pro Ala Leu Pro Ile Ser Ile Thr Phe Gly Leu Val Phe Tyr Phe Ala
435 440 445
Thr Asp Tyr Leu Val Gln Pro Phe Met Asp Gln Leu Ala Phe His Gln
450 455 460
Phe Tyr Ile
465
<210> 2
<211> 467
<212> PRT
<213> Artificial
<220>
<223> PSEN1 M139L的氨基酸序列
<400> 2
Met Thr Glu Leu Pro Ala Pro Leu Ser Tyr Phe Gln Asn Ala Gln Met
1 5 10 15
Ser Glu Asp Asn His Leu Ser Asn Thr Val Arg Ser Gln Asn Asp Asn
20 25 30
Arg Glu Arg Gln Glu His Asn Asp Arg Arg Ser Leu Gly His Pro Glu
35 40 45
Pro Leu Ser Asn Gly Arg Pro Gln Gly Asn Ser Arg Gln Val Val Glu
50 55 60
Gln Asp Glu Glu Glu Asp Glu Glu Leu Thr Leu Lys Tyr Gly Ala Lys
65 70 75 80
His Val Ile Met Leu Phe Val Pro Val Thr Leu Cys Met Val Val Val
85 90 95
Val Ala Thr Ile Lys Ser Val Ser Phe Tyr Thr Arg Lys Asp Gly Gln
100 105 110
Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr Glu Thr Val Gly Gln Arg
115 120 125
Ala Leu His Ser Ile Leu Asn Ala Ala Ile Leu Ile Ser Val Ile Val
130 135 140
Val Met Thr Ile Leu Leu Val Val Leu Tyr Lys Tyr Arg Cys Tyr Lys
145 150 155 160
Val Ile His Ala Trp Leu Ile Ile Ser Ser Leu Leu Leu Leu Phe Phe
165 170 175
Phe Ser Phe Ile Tyr Leu Gly Glu Val Phe Lys Thr Tyr Asn Val Ala
180 185 190
Val Asp Tyr Ile Thr Val Ala Leu Leu Ile Trp Asn Phe Gly Val Val
195 200 205
Gly Met Ile Ser Ile His Trp Lys Gly Pro Leu Arg Leu Gln Gln Ala
210 215 220
Tyr Leu Ile Met Ile Ser Ala Leu Met Ala Leu Val Phe Ile Lys Tyr
225 230 235 240
Leu Pro Glu Trp Thr Ala Trp Leu Ile Leu Ala Val Ile Ser Val Tyr
245 250 255
Asp Leu Val Ala Val Leu Cys Pro Lys Gly Pro Leu Arg Met Leu Val
260 265 270
Glu Thr Ala Gln Glu Arg Asn Glu Thr Leu Phe Pro Ala Leu Ile Tyr
275 280 285
Ser Ser Thr Met Val Trp Leu Val Asn Met Ala Glu Gly Asp Pro Glu
290 295 300
Ala Gln Arg Arg Val Ser Lys Asn Ser Lys Tyr Asn Ala Glu Ser Thr
305 310 315 320
Glu Arg Glu Ser Gln Asp Thr Val Ala Glu Asn Asp Asp Gly Gly Phe
325 330 335
Ser Glu Glu Trp Glu Ala Gln Arg Asp Ser His Leu Gly Pro His Arg
340 345 350
Ser Thr Pro Glu Ser Arg Ala Ala Val Gln Glu Leu Ser Ser Ser Ile
355 360 365
Leu Ala Gly Glu Asp Pro Glu Glu Arg Gly Val Lys Leu Gly Leu Gly
370 375 380
Asp Phe Ile Phe Tyr Ser Val Leu Val Gly Lys Ala Ser Ala Thr Ala
385 390 395 400
Ser Gly Asp Trp Asn Thr Thr Ile Ala Cys Phe Val Ala Ile Leu Ile
405 410 415
Gly Leu Cys Leu Thr Leu Leu Leu Leu Ala Ile Phe Lys Lys Ala Leu
420 425 430
Pro Ala Leu Pro Ile Ser Ile Thr Phe Gly Leu Val Phe Tyr Phe Ala
435 440 445
Thr Asp Tyr Leu Val Gln Pro Phe Met Asp Gln Leu Ala Phe His Gln
450 455 460
Phe Tyr Ile
465
<210> 3
<211> 467
<212> PRT
<213> Artificial
<220>
<223> PSEN1 L172W的氨基酸序列
<400> 3
Met Thr Glu Leu Pro Ala Pro Leu Ser Tyr Phe Gln Asn Ala Gln Met
1 5 10 15
Ser Glu Asp Asn His Leu Ser Asn Thr Val Arg Ser Gln Asn Asp Asn
20 25 30
Arg Glu Arg Gln Glu His Asn Asp Arg Arg Ser Leu Gly His Pro Glu
35 40 45
Pro Leu Ser Asn Gly Arg Pro Gln Gly Asn Ser Arg Gln Val Val Glu
50 55 60
Gln Asp Glu Glu Glu Asp Glu Glu Leu Thr Leu Lys Tyr Gly Ala Lys
65 70 75 80
His Val Ile Met Leu Phe Val Pro Val Thr Leu Cys Met Val Val Val
85 90 95
Val Ala Thr Ile Lys Ser Val Ser Phe Tyr Thr Arg Lys Asp Gly Gln
100 105 110
Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr Glu Thr Val Gly Gln Arg
115 120 125
Ala Leu His Ser Ile Leu Asn Ala Ala Ile Met Ile Ser Val Ile Val
130 135 140
Val Met Thr Ile Leu Leu Val Val Leu Tyr Lys Tyr Arg Cys Tyr Lys
145 150 155 160
Val Ile His Ala Trp Leu Ile Ile Ser Ser Leu Trp Leu Leu Phe Phe
165 170 175
Phe Ser Phe Ile Tyr Leu Gly Glu Val Phe Lys Thr Tyr Asn Val Ala
180 185 190
Val Asp Tyr Ile Thr Val Ala Leu Leu Ile Trp Asn Phe Gly Val Val
195 200 205
Gly Met Ile Ser Ile His Trp Lys Gly Pro Leu Arg Leu Gln Gln Ala
210 215 220
Tyr Leu Ile Met Ile Ser Ala Leu Met Ala Leu Val Phe Ile Lys Tyr
225 230 235 240
Leu Pro Glu Trp Thr Ala Trp Leu Ile Leu Ala Val Ile Ser Val Tyr
245 250 255
Asp Leu Val Ala Val Leu Cys Pro Lys Gly Pro Leu Arg Met Leu Val
260 265 270
Glu Thr Ala Gln Glu Arg Asn Glu Thr Leu Phe Pro Ala Leu Ile Tyr
275 280 285
Ser Ser Thr Met Val Trp Leu Val Asn Met Ala Glu Gly Asp Pro Glu
290 295 300
Ala Gln Arg Arg Val Ser Lys Asn Ser Lys Tyr Asn Ala Glu Ser Thr
305 310 315 320
Glu Arg Glu Ser Gln Asp Thr Val Ala Glu Asn Asp Asp Gly Gly Phe
325 330 335
Ser Glu Glu Trp Glu Ala Gln Arg Asp Ser His Leu Gly Pro His Arg
340 345 350
Ser Thr Pro Glu Ser Arg Ala Ala Val Gln Glu Leu Ser Ser Ser Ile
355 360 365
Leu Ala Gly Glu Asp Pro Glu Glu Arg Gly Val Lys Leu Gly Leu Gly
370 375 380
Asp Phe Ile Phe Tyr Ser Val Leu Val Gly Lys Ala Ser Ala Thr Ala
385 390 395 400
Ser Gly Asp Trp Asn Thr Thr Ile Ala Cys Phe Val Ala Ile Leu Ile
405 410 415
Gly Leu Cys Leu Thr Leu Leu Leu Leu Ala Ile Phe Lys Lys Ala Leu
420 425 430
Pro Ala Leu Pro Ile Ser Ile Thr Phe Gly Leu Val Phe Tyr Phe Ala
435 440 445
Thr Asp Tyr Leu Val Gln Pro Phe Met Asp Gln Leu Ala Phe His Gln
450 455 460
Phe Tyr Ile
465
<210> 4
<211> 1404
<212> DNA
<213> Artificial
<220>
<223> 编码PSEN1 G111V的核苷酸序列
<400> 4
atgacagagt tacctgcacc gttgtcctac ttccagaatg cacagatgtc tgaggacaac 60
cacctgagca atactgtacg tagccagaat gacaatagag aacggcagga gcacaacgac 120
agacggagcc ttggccaccc tgagccatta tctaatggac gaccccaggg taactcccgg 180
caggtggtgg agcaagatga ggaagaagat gaggagctga cattgaaata tggcgccaag 240
catgtgatca tgctctttgt ccctgtgact ctctgcatgg tggtggtcgt ggctaccatt 300
aagtcagtca gcttttatac ccggaaggat gtgcagctaa tctatacccc attcacagaa 360
gataccgaga ctgtgggcca gagagccctg cactcaattc tgaatgctgc catcatgatc 420
agtgtcattg ttgtcatgac tatcctcctg gtggttctgt ataaatacag gtgctataag 480
gtcatccatg cctggcttat tatatcatct ctattgttgc tgttcttttt ttcattcatt 540
tacttggggg aagtgtttaa aacctataac gttgctgtgg actacattac tgttgcactc 600
ctgatctgga attttggtgt ggtgggaatg atttccattc actggaaagg tccacttcga 660
ctccagcagg catatctcat tatgattagt gccctcatgg ccctggtgtt tatcaagtac 720
ctccctgaat ggactgcgtg gctcatcttg gctgtgattt cagtatatga tttagtggct 780
gttttgtgtc cgaaaggtcc acttcgtatg ctggttgaaa cagctcagga gagaaatgaa 840
acgctttttc cagctctcat ttactcctca acaatggtgt ggttggtgaa tatggcagaa 900
ggagacccgg aagctcaaag gagagtatcc aaaaattcca agtataatgc agaaagcaca 960
gaaagggagt cacaagacac tgttgcagag aatgatgatg gcgggttcag tgaggaatgg 1020
gaagcccaga gggacagtca tctagggcct catcgctcta cacctgagtc acgagctgct 1080
gtccaggaac tttccagcag tatcctcgct ggtgaagacc cagaggaaag gggagtaaaa 1140
cttggattgg gagatttcat tttctacagt gttctggttg gtaaagcctc agcaacagcc 1200
agtggagact ggaacacaac catagcctgt ttcgtagcca tattaattgg tttgtgcctt 1260
acattattac tccttgccat tttcaagaaa gcattgccag ctcttccaat ctccatcacc 1320
tttgggcttg ttttctactt tgccacagat tatcttgtac agccttttat ggaccaatta 1380
gcattccatc aattttatat ctag 1404
<210> 5
<211> 1404
<212> DNA
<213> Artificial
<220>
<223> 编码PSEN1 M139L的核苷酸序列
<400> 5
atgacagagt tacctgcacc gttgtcctac ttccagaatg cacagatgtc tgaggacaac 60
cacctgagca atactgtacg tagccagaat gacaatagag aacggcagga gcacaacgac 120
agacggagcc ttggccaccc tgagccatta tctaatggac gaccccaggg taactcccgg 180
caggtggtgg agcaagatga ggaagaagat gaggagctga cattgaaata tggcgccaag 240
catgtgatca tgctctttgt ccctgtgact ctctgcatgg tggtggtcgt ggctaccatt 300
aagtcagtca gcttttatac ccggaaggat gggcagctaa tctatacccc attcacagaa 360
gataccgaga ctgtgggcca gagagccctg cactcaattc tgaatgctgc catcttgatc 420
agtgtcattg ttgtcatgac tatcctcctg gtggttctgt ataaatacag gtgctataag 480
gtcatccatg cctggcttat tatatcatct ctattgttgc tgttcttttt ttcattcatt 540
tacttggggg aagtgtttaa aacctataac gttgctgtgg actacattac tgttgcactc 600
ctgatctgga attttggtgt ggtgggaatg atttccattc actggaaagg tccacttcga 660
ctccagcagg catatctcat tatgattagt gccctcatgg ccctggtgtt tatcaagtac 720
ctccctgaat ggactgcgtg gctcatcttg gctgtgattt cagtatatga tttagtggct 780
gttttgtgtc cgaaaggtcc acttcgtatg ctggttgaaa cagctcagga gagaaatgaa 840
acgctttttc cagctctcat ttactcctca acaatggtgt ggttggtgaa tatggcagaa 900
ggagacccgg aagctcaaag gagagtatcc aaaaattcca agtataatgc agaaagcaca 960
gaaagggagt cacaagacac tgttgcagag aatgatgatg gcgggttcag tgaggaatgg 1020
gaagcccaga gggacagtca tctagggcct catcgctcta cacctgagtc acgagctgct 1080
gtccaggaac tttccagcag tatcctcgct ggtgaagacc cagaggaaag gggagtaaaa 1140
cttggattgg gagatttcat tttctacagt gttctggttg gtaaagcctc agcaacagcc 1200
agtggagact ggaacacaac catagcctgt ttcgtagcca tattaattgg tttgtgcctt 1260
acattattac tccttgccat tttcaagaaa gcattgccag ctcttccaat ctccatcacc 1320
tttgggcttg ttttctactt tgccacagat tatcttgtac agccttttat ggaccaatta 1380
gcattccatc aattttatat ctag 1404
<210> 6
<211> 1404
<212> DNA
<213> Artificial
<220>
<223> 编码PSEN1 L172W的核苷酸序列
<400> 6
atgacagagt tacctgcacc gttgtcctac ttccagaatg cacagatgtc tgaggacaac 60
cacctgagca atactgtacg tagccagaat gacaatagag aacggcagga gcacaacgac 120
agacggagcc ttggccaccc tgagccatta tctaatggac gaccccaggg taactcccgg 180
caggtggtgg agcaagatga ggaagaagat gaggagctga cattgaaata tggcgccaag 240
catgtgatca tgctctttgt ccctgtgact ctctgcatgg tggtggtcgt ggctaccatt 300
aagtcagtca gcttttatac ccggaaggat gggcagctaa tctatacccc attcacagaa 360
gataccgaga ctgtgggcca gagagccctg cactcaattc tgaatgctgc catcatgatc 420
agtgtcattg ttgtcatgac tatcctcctg gtggttctgt ataaatacag gtgctataag 480
gtcatccatg cctggcttat tatatcatct ctatggttgc tgttcttttt ttcattcatt 540
tacttggggg aagtgtttaa aacctataac gttgctgtgg actacattac tgttgcactc 600
ctgatctgga attttggtgt ggtgggaatg atttccattc actggaaagg tccacttcga 660
ctccagcagg catatctcat tatgattagt gccctcatgg ccctggtgtt tatcaagtac 720
ctccctgaat ggactgcgtg gctcatcttg gctgtgattt cagtatatga tttagtggct 780
gttttgtgtc cgaaaggtcc acttcgtatg ctggttgaaa cagctcagga gagaaatgaa 840
acgctttttc cagctctcat ttactcctca acaatggtgt ggttggtgaa tatggcagaa 900
ggagacccgg aagctcaaag gagagtatcc aaaaattcca agtataatgc agaaagcaca 960
gaaagggagt cacaagacac tgttgcagag aatgatgatg gcgggttcag tgaggaatgg 1020
gaagcccaga gggacagtca tctagggcct catcgctcta cacctgagtc acgagctgct 1080
gtccaggaac tttccagcag tatcctcgct ggtgaagacc cagaggaaag gggagtaaaa 1140
cttggattgg gagatttcat tttctacagt gttctggttg gtaaagcctc agcaacagcc 1200
agtggagact ggaacacaac catagcctgt ttcgtagcca tattaattgg tttgtgcctt 1260
acattattac tccttgccat tttcaagaaa gcattgccag ctcttccaat ctccatcacc 1320
tttgggcttg ttttctactt tgccacagat tatcttgtac agccttttat ggaccaatta 1380
gcattccatc aattttatat ctag 1404
<210> 7
<211> 467
<212> PRT
<213> Homo sapiens
<400> 7
Met Thr Glu Leu Pro Ala Pro Leu Ser Tyr Phe Gln Asn Ala Gln Met
1 5 10 15
Ser Glu Asp Asn His Leu Ser Asn Thr Val Arg Ser Gln Asn Asp Asn
20 25 30
Arg Glu Arg Gln Glu His Asn Asp Arg Arg Ser Leu Gly His Pro Glu
35 40 45
Pro Leu Ser Asn Gly Arg Pro Gln Gly Asn Ser Arg Gln Val Val Glu
50 55 60
Gln Asp Glu Glu Glu Asp Glu Glu Leu Thr Leu Lys Tyr Gly Ala Lys
65 70 75 80
His Val Ile Met Leu Phe Val Pro Val Thr Leu Cys Met Val Val Val
85 90 95
Val Ala Thr Ile Lys Ser Val Ser Phe Tyr Thr Arg Lys Asp Gly Gln
100 105 110
Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr Glu Thr Val Gly Gln Arg
115 120 125
Ala Leu His Ser Ile Leu Asn Ala Ala Ile Met Ile Ser Val Ile Val
130 135 140
Val Met Thr Ile Leu Leu Val Val Leu Tyr Lys Tyr Arg Cys Tyr Lys
145 150 155 160
Val Ile His Ala Trp Leu Ile Ile Ser Ser Leu Leu Leu Leu Phe Phe
165 170 175
Phe Ser Phe Ile Tyr Leu Gly Glu Val Phe Lys Thr Tyr Asn Val Ala
180 185 190
Val Asp Tyr Ile Thr Val Ala Leu Leu Ile Trp Asn Phe Gly Val Val
195 200 205
Gly Met Ile Ser Ile His Trp Lys Gly Pro Leu Arg Leu Gln Gln Ala
210 215 220
Tyr Leu Ile Met Ile Ser Ala Leu Met Ala Leu Val Phe Ile Lys Tyr
225 230 235 240
Leu Pro Glu Trp Thr Ala Trp Leu Ile Leu Ala Val Ile Ser Val Tyr
245 250 255
Asp Leu Val Ala Val Leu Cys Pro Lys Gly Pro Leu Arg Met Leu Val
260 265 270
Glu Thr Ala Gln Glu Arg Asn Glu Thr Leu Phe Pro Ala Leu Ile Tyr
275 280 285
Ser Ser Thr Met Val Trp Leu Val Asn Met Ala Glu Gly Asp Pro Glu
290 295 300
Ala Gln Arg Arg Val Ser Lys Asn Ser Lys Tyr Asn Ala Glu Ser Thr
305 310 315 320
Glu Arg Glu Ser Gln Asp Thr Val Ala Glu Asn Asp Asp Gly Gly Phe
325 330 335
Ser Glu Glu Trp Glu Ala Gln Arg Asp Ser His Leu Gly Pro His Arg
340 345 350
Ser Thr Pro Glu Ser Arg Ala Ala Val Gln Glu Leu Ser Ser Ser Ile
355 360 365
Leu Ala Gly Glu Asp Pro Glu Glu Arg Gly Val Lys Leu Gly Leu Gly
370 375 380
Asp Phe Ile Phe Tyr Ser Val Leu Val Gly Lys Ala Ser Ala Thr Ala
385 390 395 400
Ser Gly Asp Trp Asn Thr Thr Ile Ala Cys Phe Val Ala Ile Leu Ile
405 410 415
Gly Leu Cys Leu Thr Leu Leu Leu Leu Ala Ile Phe Lys Lys Ala Leu
420 425 430
Pro Ala Leu Pro Ile Ser Ile Thr Phe Gly Leu Val Phe Tyr Phe Ala
435 440 445
Thr Asp Tyr Leu Val Gln Pro Phe Met Asp Gln Leu Ala Phe His Gln
450 455 460
Phe Tyr Ile
465
<210> 8
<211> 1404
<212> DNA
<213> Homo sapiens
<400> 8
atgacagagt tacctgcacc gttgtcctac ttccagaatg cacagatgtc tgaggacaac 60
cacctgagca atactgtacg tagccagaat gacaatagag aacggcagga gcacaacgac 120
agacggagcc ttggccaccc tgagccatta tctaatggac gaccccaggg taactcccgg 180
caggtggtgg agcaagatga ggaagaagat gaggagctga cattgaaata tggcgccaag 240
catgtgatca tgctctttgt ccctgtgact ctctgcatgg tggtggtcgt ggctaccatt 300
aagtcagtca gcttttatac ccggaaggat gggcagctaa tctatacccc attcacagaa 360
gataccgaga ctgtgggcca gagagccctg cactcaattc tgaatgctgc catcatgatc 420
agtgtcattg ttgtcatgac tatcctcctg gtggttctgt ataaatacag gtgctataag 480
gtcatccatg cctggcttat tatatcatct ctattgttgc tgttcttttt ttcattcatt 540
tacttggggg aagtgtttaa aacctataac gttgctgtgg actacattac tgttgcactc 600
ctgatctgga attttggtgt ggtgggaatg atttccattc actggaaagg tccacttcga 660
ctccagcagg catatctcat tatgattagt gccctcatgg ccctggtgtt tatcaagtac 720
ctccctgaat ggactgcgtg gctcatcttg gctgtgattt cagtatatga tttagtggct 780
gttttgtgtc cgaaaggtcc acttcgtatg ctggttgaaa cagctcagga gagaaatgaa 840
acgctttttc cagctctcat ttactcctca acaatggtgt ggttggtgaa tatggcagaa 900
ggagacccgg aagctcaaag gagagtatcc aaaaattcca agtataatgc agaaagcaca 960
gaaagggagt cacaagacac tgttgcagag aatgatgatg gcgggttcag tgaggaatgg 1020
gaagcccaga gggacagtca tctagggcct catcgctcta cacctgagtc acgagctgct 1080
gtccaggaac tttccagcag tatcctcgct ggtgaagacc cagaggaaag gggagtaaaa 1140
cttggattgg gagatttcat tttctacagt gttctggttg gtaaagcctc agcaacagcc 1200
agtggagact ggaacacaac catagcctgt ttcgtagcca tattaattgg tttgtgcctt 1260
acattattac tccttgccat tttcaagaaa gcattgccag ctcttccaat ctccatcacc 1320
tttgggcttg ttttctactt tgccacagat tatcttgtac agccttttat ggaccaatta 1380
gcattccatc aattttatat ctag 1404
<210> 9
<211> 40
<212> PRT
<213> Artificial
<220>
<223> Aβ40的氨基酸序列
<400> 9
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys
1 5 10 15
Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile
20 25 30
Gly Leu Met Val Gly Gly Val Val
35 40
<210> 10
<211> 42
<212> PRT
<213> Artificial
<220>
<223> Aβ42的氨基酸序列
<400> 10
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys
1 5 10 15
Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile
20 25 30
Gly Leu Met Val Gly Gly Val Val Ile Ala
35 40
<210> 11
<211> 42
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 11
gatctatttc cggtgaattc atgacagagt tacctgcacc gt 42
<210> 12
<211> 44
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 12
gagggagagg ggcgggatcc ctagatataa aattgatgga atgc 44
<210> 13
<211> 31
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 13
atacccggaa ggatgtgcag ctaatctata c 31
<210> 14
<211> 31
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 14
acatccttcc gggtataaaa gctgactgac t 31
<210> 15
<211> 31
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 15
ctgaatgctg ccatcttgat cagtgtcatt g 31
<210> 16
<211> 31
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 16
agatggcagc attcagaatt gagtgcaggg c 31
<210> 17
<211> 31
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 17
ctgaatgctg ccatcgtgat cagtgtcatt g 31
<210> 18
<211> 31
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 18
cgatggcagc attcagaatt gagtgcaggg c 31
<210> 19
<211> 34
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 19
ttatatcatc tctatggttg ctgttctttt tttc 34
<210> 20
<211> 31
<212> DNA
<213> Artificial
<220>
<223> 引物
<400> 20
catagagatg atataataag ccaggcatgg a 31
Claims (11)
1.一种突变的蛋白,其为野生型人PSEN1蛋白发生选自如下的任意1个、任意2个或者3个突变:
G111V、M139L和L172W;
优选地,所述野生型人PSEN1蛋白的氨基酸序列如SEQ ID NO:7所示;
优选地,所述蛋白的氨基酸序列如SEQ ID NOs:1-3中任一序列所示。
2.一种核酸分子,其编码权利要求1所述的蛋白;优选地,所述核酸分子如SEQ ID NOs:4-6中任一序列所示。
3.一种核酸构建体,其含有权利要求2所述的核酸分子;优选地,所述核酸构建体为重组载体;优选地,所述重组载体为重组表达载体。
4.一种重组宿主细胞,其表达权利要求1所述的蛋白,或者含有权利要求2所述的核酸分子或者权利要求3所述的核酸构建体。
5.一种组合物,其包含权利要求1所述的蛋白、权利要求2所述的核酸分子、权利要求3所述的核酸构建体或者权利要求4所述的重组宿主细胞;可选地,所述组合物还包含一种或多种药学上可接受的辅料。
6.一种单克隆抗体或其抗原结合片段,其能够特异性地结合权利要求1所述的蛋白。
7.一种偶联物,其包含权利要求6所述的单克隆抗体或其抗原结合片段以及偶联部分,其中,所述偶联部分为可检测的标记;优选地,所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
8.一种引物或探针,其能够特异性地结合权利要求2所述的核酸分子;
优选地,所述探针的5’端标记有荧光报告基团,3’端标记有荧光淬灭基团;
优选地,所述荧光报告基团选自FAM、Hex、VIC、ROX和Cy5;
优选地,所述荧光淬灭基团选自BHQ1、TAMRA、JOE、BHQ2和BHQ3。
9.一种试剂盒,其包含权利要求6所述的单克隆抗体或其抗原结合片段、权利要求7所述的偶联物或者包含权利要求8所述的引物或探针;
优选地,当包含权利要求6所述的单克隆抗体或其抗原结合片段或者权利要求7所述的偶联物时,所述试剂盒还包括第二抗体,其特异性识别所述单克隆抗体或其抗原结合片段;任选地,所述第二抗体还包括可检测的标记,例如放射性同位素、荧光物质、发光物质、有色物质或酶;
可选地,所述试剂盒还包含说明书。
10.选自如下的(1)-(6)项中的任意一项在制备治疗和/或预防或者诊断阿尔茨海默病的药物中的用途;或者在制备降低Aβ水平的药物中的用途;或者在制备筛选药物的模型例如细胞模型或者动物模型中的用途,所述药物用于治疗和/或预防和/或诊断阿尔茨海默病:
(1)权利要求1所述的蛋白;
(2)权利要求2所述的核酸分子;
(3)抑制或阻断权利要求1所述的蛋白的药物,例如权利要求6所述的单克隆抗体或其抗原结合片段;
(4)抑制或降低权利要求2所述的核酸分子的表达水平的药物;
(5)将本发明的蛋白修复为野生型人PSEN1蛋白(例如SEQ ID NO:7所示)的药物;
(6)将本发明的核酸分子修复为野生型人PSEN1基因(例如SEQ ID NO:8所示)的药物;
(7)检测权利要求1所述的蛋白的药物,例如权利要求6所述的单克隆抗体或其抗原结合片段或者权利要求7所述的偶联物;
(8)检测权利要求2所述的核酸分子的表达水平的药物,例如权利要求8所述的引物或探针;
优选地,所述阿尔茨海默病为家族性阿尔茨海默病;优选地,所述阿尔茨海默病为早发性家族性阿尔茨海默病;
优选地,所述,所述Aβ为Aβ40和/或Aβ42。
11.一种筛选治疗和/或预防阿尔茨海默病的药物的方法,包括检测候选药物是否减少或者降低受试者中或者细胞中的SEQ ID NOs:1-3中任一序列所示的蛋白的水平的步骤,或者包括检测候选药物是否减少或者降低受试者中或者细胞中的SEQ ID NOs:4-6中任一序列所示的核酸分子的水平的步骤;
如果受试者中或者细胞中的所述蛋白或核酸的水平降低或者减少,则作为阳性药物;
可选地,以不加入候选药物的细胞作为对照。
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