CN113980113A - Kcnq4功能增强型突变体及其在治疗听力损失中的应用 - Google Patents
Kcnq4功能增强型突变体及其在治疗听力损失中的应用 Download PDFInfo
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- CN113980113A CN113980113A CN202111204216.6A CN202111204216A CN113980113A CN 113980113 A CN113980113 A CN 113980113A CN 202111204216 A CN202111204216 A CN 202111204216A CN 113980113 A CN113980113 A CN 113980113A
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Abstract
本发明公开了一种KCNQ4功能增强型突变体及其在治疗听力损失中的应用。本申请的第一方面,提供一种KCNQ4突变蛋白,该KCNQ4突变蛋白在野生型KCNQ4的氨基酸序列中具有至少一种突变位点。根据本申请实施例的KCNQ4突变蛋白,至少具有如下有益效果:申请人在通过电生理技术进行KCNQ4基因的功能研究中发现,KCNQ4的43个突变位点具有功能增强的特征,这些KCNQ4功能增强型突变体表现为能够挽救功能缺失型突变体的显性抑制作用,因而可以基于这些错义突变位点的突变蛋白进行听力损失的治疗。
Description
技术领域
本申请涉及基因技术领域,尤其是涉及KCNQ4功能增强型突变体及其在治疗听力损失中的应用。
背景技术
感觉神经性听力损失(Sensorineural hearing loss,SNHL)是最常见的感觉缺陷,严重影响正常的生活和社交。SNHL的病因多种多样,包括生物因素,如先天性耳蜗发育不良、随年龄增长而引起的老年性耳聋,由于感染或病毒性疾患及耳毒性药物引起的耳蜗感受器细胞和听神经纤维退行性变化;环境因素,常见且波及面较大的,是长期在噪声环境下工作,因噪声引起的耳聋,由极强烈的冲击声引起的爆震性耳聋。耳聋助听器和人工耳蜗植入是治疗感觉神经性听觉丧失的主要方法,但治疗结果存在高度的异质性且无法恢复由听力影响带来的其它功能区域的损伤。因此开展针对遗传性耳聋的基因治疗研究十分必要。
典型的电压门控钾通道KCNQ4(potassium voltage gated channel subfamily Qmember 4,Gene ID:9132)包括4个亚基,每个亚基包含六个跨膜螺旋(S1-S6)和胞内的N端、C端。四个亚基形成一个功能性离子通道,在电压刺激下产生外向电流。KCNQ4是引起常染色体显性遗传非综合征型耳聋的基因。KCNQ4主要表达在耳蜗的感觉外毛细胞上,KCNQ4基因敲除小鼠表现为外毛细胞的退化和渐进性耳聋。根据gnomAD数据库,大约每100人中就有1人携带KCNQ4错义突变(不包含等位基因频率频率大于0.1%的错义突变)。KCNQ4蛋白有695个氨基酸,经计算,所有可能的错义单核苷酸突变总共4085种。目前,KCNQ4中已有研究报道的错义突变有40多个。这些报道的错义突变中,38个显示可能与常染色体显性非综合征型渐进性听力损失相关。由于KCNQ4错义突变引起的听力损失多为显性遗传,传统的基因治疗导入野生型的基因不能抑制显性致病基因,从而没有治疗效果。因此,有必要从KCNQ4出发,寻找能够治疗感觉神经性听力损失的新的靶点。
发明内容
本申请旨在至少解决现有技术中存在的技术问题之一。为此,本申请提出一种KCNQ4功能增强型突变体,基于该功能增强型突变体可以制备能够治疗感觉神经性听力损失的产品。
本申请的第一方面,提供一种KCNQ4突变蛋白,该KCNQ4突变蛋白在野生型KCNQ4的氨基酸序列中具有以下至少一种突变位点:R72C、K78E、N85D、N85T、N85Y、N89D、V90A、V90G、R93L、R93Q、V114A、L132I、E136D、C156G、C156S、C157S、C158F、C158W、R159G、R159H、R159L、R159P、R159S、R161G、F174V、D218E、R219P、T223A、L227V、V231L、W242C、W242L、V326I、S371R、Y556F、K559R、V561A、S566P、G568R、G568S、L573R、G574R、D584Y;
其中,野生型KCNQ4的氨基酸序列如SEQ ID No.1所示:
maeapprrlglgpppgdapraelvaltavqseqgeaggggsprrlgllgsplppgaplpgpgsgsgsacgqrssaahkryrrlqnwvynvlerprgwafvyhvfifllvfsclvlsvlstiqehqelanecllilefvmivvfgleyivrvwsagcccryrgwqgrfrfarkpfcvidfivfvasvaviaagtqgnifatsalrsmrflqilrmvrmdrrggtwkllgsvvyahskelitawyigflvlifasflvylaekdansdfssyadslwwgtitlttigygdktphtwlgrvlaagfallgisffalpagilgsgfalkvqeqhrqkhfekrrmpaanliqaawrlystdmsrayltatwyyydsilpsfrelallfehvqrarngglrplevrrapvpdgapsryppvatchrpgstsfcpgessrmgikdrirmgssqrrtgpskqhlapptmptspsseqvgeatsptkvqkswsfndrtrfraslrlkprtsaedapseevaeeksyqceltvddimpavktvirsirilkflvakrkfketlrpydvkdvieqysaghldmlgrikslqtrvdqivgrgpgdrkarekgdkgpsdaevvdeismmgrvvkvekqvqsiehkldlllgfysrclrsgtsaslgavqvplfdpditsdyhspvdhedisvsaqtlsisrsvstnmd(SEQ ID No.1)。
根据本申请实施例的KCNQ4突变蛋白,至少具有如下有益效果:
申请人在通过电生理技术进行KCNQ4基因的功能研究中发现,KCNQ4的43个突变位点具有功能增强的特征,这些KCNQ4功能增强性突变体表现为能够挽救功能缺失型突变体的显性抑制作用,因而可以基于这些错义突变位点的突变蛋白进行听力损失的治疗。
在本申请的一些实施方式中,突变位点为R159H。
本申请的第二方面,提供编码前述的KCNQ4突变蛋白的核酸分子。传统的基因治疗导入野生型的KCNQ4基因不能抑制显性致病基因,从而没有治疗效果。而基于本方案可以通过CRISPR/Cas技术、TALENs技术、ZFNs技术等导入具有上述错义突变位点的突变型的KCNQ4基因,从而起到改善听力损失的效果。
本申请的第三方面,提供重组载体,该重组载体包括前述的核酸分子。重组载体的具体类型包括但不限于腺病毒表达载体、腺病毒相关病毒表达载体、慢病毒表达载体、质粒载体等。
本申请的第四方面,提供重组细胞,该重组细胞包括前述的核酸分子。
本申请的第五方面,提供前述的KCNQ4突变蛋白,或核酸分子,或重组载体,或重组细胞在制备治疗听力损失的药物中的应用。申请人在实验过程中发现,KCNQ4的这些功能增强性突变体表现为能够挽救功能缺失型突变体的显性抑制作用,在噪声暴露实验中,可以明显改善野生型KCNQ4耳聋模型的听力恢复能力,因而可以用于制备能够治疗听力损失的药物。例如,利用rAAV作为治疗载体装载上述的突变型的核酸分子作为治疗性的基因序列,进一步还可以添加基因表达的调控元件(如启动子、多聚腺苷酸等),以此来对野生型KCNQ4所引起的耳聋进行治疗以改善其听力状况。
在本申请的一些实施方式中,听力损失为感觉神经性听力损失。
在本申请的一些实施方式中,听力损失为噪声引起的听力损失。
在本申请的一些实施方式中,噪声包括高频噪声。
在本申请的一些实施方式中,高频噪声的频率为16kHz以上,优选的,高频噪声的频率在24kHz以上,进一步在36kHz以上。
本申请的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本申请的实践了解到。
附图说明
图1是本申请的实施例2中的R159H突变蛋白和野生型KCNQ4蛋白的离子通道活性检测结果。其中,a表示细胞中只转入野生型的KCNQ4质粒,b表示细胞中只转入错义突变R159H的KCNQ4质粒,c表示细胞中转入1:1的野生型KCNQ4质粒和错义突变R159H的KCNQ4质粒。
图2是本申请的实施例2中错义突变蛋白和野生型KCNQ4蛋白的离子通道活性检测的统计结果。其中,测试细胞中转入了野生型KCNQ4质粒或错义突变的KCNQ4质粒。
图3是本申请的实施例2中错义突变蛋白和野生型KCNQ4蛋白的离子通道活性检测的统计结果。其中,测试细胞中转入了野生型KCNQ4质粒或1:1的野生型KCNQ4质粒和错义突变的KCNQ4质粒。
图4是本申请的实施例3中噪声暴露试验的检测结果。其中,a为暴露后24小时的检测结果,b为暴露后7天的检测结果,c为暴露后14天的检测结果。横轴为暴露时所用白噪声的频率,纵轴为检测时间点对应的听力阈值相对于暴露前的听力阈值的变化量。
具体实施方式
以下将结合实施例对本申请的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本申请的目的、特征和效果。显然,所描述的实施例只是本申请的一部分实施例,而不是全部实施例,基于本申请的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本申请保护的范围。
下面详细描述本申请的实施例,描述的实施例是示例性的,仅用于解释本申请,而不能理解为对本申请的限制。
在本申请的描述中,若干的含义是一个以上,多个的含义是两个以上,大于、小于、超过等理解为不包括本数,以上、以下、以内等理解为包括本数。如果有描述到第一、第二只是用于区分技术特征为目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量或者隐含指明所指示的技术特征的先后关系。
本申请的描述中,参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本申请的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
实施例1
本实施例提供一种KCNQ4错义突变位点的点突变质粒,以野生型KCNQ4为模板DNA,利用以下各组点突变引物通过PCR以及同源重组获得KCNQ4错义突变质粒,质粒通过Sanger测序进行鉴定,经过比对,插入片段序列和目的片段一致,质粒构建成功。
实验所用的KCNQ4的CDS(NP_004691.2,a亚型)购买自GeneCopoeia(载体为EX-U0210-M98-5)。
具体序列如下:
ATGGCTGAGGCTCCTCCGCGACGACTCGGACTCGGTCCCCCTCCCGGAGATGCTCCAAGGGCGGAACTTGTGGCGCTGACGGCTGTACAATCAGAGCAGGGTGAAGCTGGGGGTGGAGGGTCTCCTCGACGGTTGGGATTGCTGGGCTCCCCACTCCCACCTGGCGCACCTCTCCCTGGGCCAGGAAGCGGTTCCGGGTCTGCGTGCGGCCAGCGCAGCAGCGCGGCTCACAAAAGATATAGGCGACTCCAGAACTGGGTCTACAACGTGCTTGAGAGGCCTAGGGGCTGGGCCTTTGTGTACCACGTGTTCATTTTCCTCTTGGTCTTCTCATGCCTGGTTCTGAGTGTACTGAGCACTATACAGGAGCACCAGGAGCTCGCAAACGAGTGCCTGCTCATCTTGGAGTTCGTAATGATCGTCGTCTTCGGCCTTGAGTATATAGTACGGGTGTGGAGCGCCGGATGCTGTTGCAGGTATCGGGGTTGGCAGGGCCGGTTTCGCTTTGCTCGAAAACCCTTCTGCGTCATTGACTTCATCGTTTTCGTGGCAAGCGTCGCCGTTATTGCTGCCGGAACTCAGGGCAACATATTTGCCACCAGCGCTCTGAGAAGCATGAGGTTCCTTCAGATTCTGAGGATGGTAAGGATGGATAGACGGGGAGGGACGTGGAAGCTCCTCGGGAGTGTGGTGTACGCGCACTCAAAAGAGCTCATCACGGCTTGGTACATTGGATTTCTTGTCCTGATCTTCGCGAGCTTTCTGGTTTATCTGGCGGAAAAGGACGCGAATAGCGACTTTAGCAGCTATGCCGACAGCCTTTGGTGGGGAACAATCACGCTTACGACCATCGGCTACGGCGATAAAACCCCGCACACATGGTTGGGTAGAGTGCTGGCAGCCGGATTCGCACTGTTGGGCATCAGCTTCTTCGCATTGCCTGCCGGTATCTTGGGCAGCGGCTTTGCACTGAAGGTTCAAGAGCAGCACAGGCAGAAGCACTTCGAGAAGCGCCGCATGCCAGCTGCGAACTTGATTCAGGCAGCGTGGAGGCTGTATTCTACCGATATGTCACGGGCGTACCTGACAGCAACATGGTACTACTACGATAGCATTCTGCCGAGTTTCAGAGAGCTGGCCCTCCTGTTTGAACATGTGCAGAGGGCACGCAACGGAGGGCTGCGACCATTGGAGGTCCGGCGAGCCCCAGTACCCGACGGGGCACCCTCTCGATACCCCCCTGTAGCAACGTGTCACAGGCCCGGAAGTACAAGCTTCTGTCCTGGGGAGTCATCCCGGATGGGTATCAAGGACCGGATACGCATGGGTTCTTCCCAGAGACGAACGGGCCCTTCAAAACAGCAGCTCGCTCCGCCCACAATGCCTACGAGTCCCAGCTCCGAGCAGGTCGGCGAGGCGACCTCACCGACGAAGGTACAAAAGTCATGGTCATTCAACGACAGGACGAGGTTCAGGGCTTCCCTGCGACTGAAGCCAAGAACATCCGCCGAGGATGCGCCCAGCGAGGAAGTTGCGGAAGAGAAGAGCTACCAGTGTGAGCTTACTGTGGACGACATCATGCCCGCTGTAAAAACCGTGATCCGCAGCATACGGATACTGAAGTTTCTCGTTGCGAAGCGCAAATTCAAGGAAACGCTCCGGCCGTACGACGTAAAGGATGTTATAGAACAGTACTCTGCGGGCCACTTGGACATGTTGGGACGGATAAAGTCCCTGCAAACCAGGGTGGACCAGATCGTTGGACGCGGTCCGGGGGATCGCAAAGCACGCGAAAAAGGAGATAAGGGGCCTTCCGATGCCGAGGTTGTGGATGAGATTTCTATGATGGGGCGAGTAGTGAAAGTGGAAAAGCAGGTGCAATCCATTGAACACAAGCTCGACCTTCTCCTGGGATTCTACTCTAGGTGCCTCCGCAGTGGAACTAGCGCGTCTCTCGGTGCCGTGCAGGTTCCCCTTTTCGATCCGGACATCACCTCTGACTATCACTCTCCCGTCGACCACGAAGACATCAGCGTAAGTGCGCAGACTCTGAGCATTAGCCGCTCTGTGAGCACGAACATGGACTAG(SEQ ID No.88)。
根据所购KCNQ4质粒序列设计并合成点突变基因的引物如下:
R72C:
Forward primer:CCGGGTCTGCGTGCGGCCAG TGCAGCAGCGCGGCTCACAAAAG(SEQ IDNo.2),
Reverse primer:CTGGCCGCACGCAGACCCGGAAC(SEQ ID No.3);
K78E:
Forward primer:AGCGCAGCAGCGCGGCTCAC GAGAGATATAGGCGACTCCAG(SEQ IDNo.4),
Reverse primer:GTGAGCCGCGCTGCTGCGCTGGCCG(SEQ ID No.5);
N85D:
Forward primer:AAAGATATAGGCGACTCCAG GACTGGGTCTACAACGTGCTTGAG(SEQ IDNo.6),
Reverse primer:CTGGAGTCGCCTATATCTTTTGTG(SEQ ID No.7);
N85T:
Forward primer:AAAGATATAGGCGACTCCAG ACCTGGGTCTACAACGTGCTTGAG(SEQ IDNo.8),
Reverse primer:CTGGAGTCGCCTATATCTTTTGTG(SEQ ID No.9);
N85Y:
Forward primer:AAAGATATAGGCGACTCCAG TACTGGGTCTACAACGTGCTTGAG(SEQ IDNo.10),
Reverse primer:CTGGAGTCGCCTATATCTTTTGTG(SEQ ID No.11);
N89D:
Forward primer:GACTCCAGAACTGGGTCTAC GACGTGCTTGAGAGGCCTAGG(SEQ IDNo.12),
Reverse primer:GTAGACCCAGTTCTGGAGTCGCC(SEQ ID No.13);
V90A:
Forward primer:TCCAGAACTGGGTCTACAAC GCGCTTGAGAGGCCTAGGGGCTG(SEQ IDNo.14),
Reverse primer:GTTGTAGACCCAGTTCTGGAGTC(SEQ ID No.15);
V90G:
Forward primer:TCCAGAACTGGGTCTACAAC GGGCTTGAGAGGCCTAGGGGCTG(SEQ IDNo.16),
Reverse primer:GTTGTAGACCCAGTTCTGGAGTC(SEQ ID No.17);
R93L:
Forward primer:GGGTCTACAACGTGCTTGAG CTGCCTAGGGGCTGGGCCT(SEQ IDNo.18),
Reverse primer:CTCAAGCACGTTGTAGACCCAG(SEQ ID No.19);
R93Q:
Forward primer:GGGTCTACAACGTGCTTGAG CAGCCTAGGGGCTGGGCCT(SEQ IDNo.20),
Reverse primer:CTCAAGCACGTTGTAGACCCAG(SEQ ID No.21);
V114A:
Forward primer:TCTTGGTCTTCTCATGCCTG GCGCTGAGTGTACTGAGCACTATAC(SEQ IDNo.22),
Reverse primer:CAGGCATGAGAAGACCAAGAGG(SEQ ID No.23);
L132I:
Forward primer:AATGTCCTGAATGGAATTGC ATCCTCATCTTGGAGTTCGTA(SEQ IDNo.24),
Reverse primer:GCAATTCCATTCAGGACATT(SEQ ID No.25);
E136D:
Forward primer:GGAATTGCCTGCTCATCTTG GACTTCGTAATGATCGTCGTC(SEQ IDNo.26),
Reverse primer:CAAGATGAGCAGGCAATTCCA(SEQ ID No.27);
C156G:
Forward primer:GTACGGGTGTGGAGCGCCGGAGGCTGTTGCAGGTATCGGGG(SEQ IDNo.28),
Reverse primer:TCCGGCGCTCCACACCCGTAC(SEQ ID No.29);
C156S:
Forward primer:GTACGGGTGTGGAGCGCCGGAAGCTGTTGCAGGTATCGGGG(SEQ IDNo.30),
Reverse primer:TCCGGCGCTCCACACCCGTAC(SEQ ID No.31);
C157S:
Forward primer:CGGGTGTGGAGCGCCGGATGC AGCTGCAGGTATCGGGGTTGG(SEQ IDNo.32),
Reverse primer:GCATCCGGCGCTCCACACCCG(SEQ ID No.33);
C158F:
Forward primer:GTGTGGAGCGCCGGATGCTGT TTCAGGTATCGGGGTTGGCAG(SEQ IDNo.34),
Reverse primer:ACAGCATCCGGCGCTCCACAC(SEQ ID No.35);
C158W:
Forward primer:GTGTGGAGCGCCGGATGCTGT TGGAGGTATCGGGGTTGGCAG(SEQ IDNo.36),
Reverse primer:ACAGCATCCGGCGCTCCACAC(SEQ ID No.37);
R159G:
Forward primer:TGGAGCGCCGGATGCTGTTGC GGGTATCGGGGTTGGCAGG(SEQ IDNo.38),
Reverse primer:GCAACAGCATCCGGCGCTCCACAC(SEQ ID No.39);
R159H:
Forward primer:TGGAGCGCCGGATGCTGTTGC CACTATCGGGGTTGGCAGG(SEQ IDNo.40),
Reverse primer:GCAACAGCATCCGGCGCTCCACAC(SEQ ID No.41);
R159L:
Forward primer:TGGAGCGCCGGATGCTGTTGC CTCTATCGGGGTTGGCAGG(SEQ IDNo.42),
Reverse primer:GCAACAGCATCCGGCGCTCCACAC(SEQ ID No.43);
R159P:
Forward primer:TGGAGCGCCGGATGCTGTTGC CCCTATCGGGGTTGGCAGG(SEQ IDNo.44),
Reverse primer:GCAACAGCATCCGGCGCTCCACAC(SEQ ID No.45);
R159S:
Forward primer:TGGAGCGCCGGATGCTGTTGC AGCTATCGGGGTTGGCAGG(SEQ IDNo.46),
Reverse primer:GCAACAGCATCCGGCGCTCCACAC(SEQ ID No.47);
R161G:
Forward primer:GCCGGATGCTGTTGCAGGTAT GGAGGTTGGCAGGGCCGGTTTC(SEQ IDNo.48),
Reverse primer:ATACCTGCAACAGCATCCGGCGC(SEQ ID No.49);
F174V:
Forward primer:TTCGCTTTGCTCGAAAACCC GTCTGCGTCATTGACTTCATCG(SEQ IDNo.50),
Reverse primer:GGGTTTTCGAGCAAAGCGAAAC(SEQ ID No.51);
D218E:
Forward primer:TTCTGAGGATGGTAAGGATG GAAAGACGGGGAGGGACGTG(SEQ IDNo.52),
Reverse primer:CATCCTTACCATCCTCAGAATCTG(SEQ ID No.53);
R219P:
Forward primer:TGAGGATGGTAAGGATGGAT CCCCGGGGAGGGACGTG(SEQ ID No.54),
Reverse primer:ATCCATCCTTACCATCCTCAG(SEQ ID No.55);
T223A:
Forward primer:GGATGGATAGACGGGGAGGG GCGTGGAAGCTCCTCGGGAGT(SEQ IDNo.56),
Reverse primer:CCCTCCCCGTCTATCCATCCT(SEQ ID No.57);
L227V:
Forward primer:CGGGGAGGGACGTGGAAGCTC GTCGGGAGTGTGGTGTACGC(SEQ IDNo.58),
Reverse primer:GAGCTTCCACGTCCCTCCCCG(SEQ ID No.59);
V231L:
Forward primer:TGGAAGCTCCTCGGGAGTGTG CTGTACGCGCACTCAAAAGAG(SEQ IDNo.60),
Reverse primer:CACACTCCCGAGGAGCTTCCA(SEQ ID No.61);
W242C:
Forward primer:CAAAAGAGCTCATCACGGCT TGTTACATTGGATTTCTTGTC(SEQ IDNo.62),
Reverse primer:AGCCGTGATGAGCTCTTTTGAG(SEQ ID No.63);
W242L:
Forward primer:CAAAAGAGCTCATCACGGCT TTGTACATTGGATTTCTTGTC(SEQ IDNo.64),
Reverse primer:AGCCGTGATGAGCTCTTTTGAG(SEQ ID No.65);
V326I:
Forward primer:GGCAGCGGCTTTGCACTGAAG ATTCAAGAGCAGCACAGGCAGAAG(SEQ IDNo.66),
Reverse primer:CTTCAGTGCAAAGCCGCTGCC(SEQ ID No.67);
S371R:
Forward primer:CAGCAACATGGTACTACTACGAT CGTATTCTGCCGAGTTTCAGAGAG(SEQID No.68),
Reverse primer:ATCGTAGTAGTACCATGTTGCTG(SEQ ID No.69);
Y556F:
Forward primer:TTCAAGGAAACGCTCCGGCCG TTCGACGTAAAGGATGTTATAG(SEQ IDNo.70),
Reverse primer:CGGCCGGAGCGTTTCCTTGAA(SEQ ID No.71);
K559R:
Forward primer:ACGCTCCGGCCGTACGACGTAAGGGATGTTATAGAACAGTAC(SEQ IDNo.72),
Reverse primer:TACGTCGTACGGCCGGAGCGT(SEQ ID No.73);
V561A:
Forward primer:CGGCCGTACGACGTAAAGGAT GCCATAGAACAGTACTCTGCG(SEQ IDNo.74),
Reverse primer:ATCCTTTACGTCGTACGGCCG(SEQ ID No.75);
S566P:
Forward primer:AAGGATGTTATAGAACAGTAC CCAGCGGGCCACTTGGACAT(SEQ IDNo.76),
Reverse primer:GTACTGTTCTATAACATCCTT(SEQ ID No.77);
G568R:
Forward primer:GTTATAGAACAGTACTCTGCG CGCCACTTGGACATGTTGG(SEQ IDNo.78),
Reverse primer:CGCAGAGTACTGTTCTATAAC(SEQ ID No.79);
G568S:
Forward primer:GTTATAGAACAGTACTCTGCG AGCCACTTGGACATGTTGG(SEQ IDNo.80),
Reverse primer:CGCAGAGTACTGTTCTATAAC(SEQ ID No.81);
L573R:
Forward primer:TCTGCGGGCCACTTGGACATG CGGGGACGGATAAAGTCCCT(SEQ IDNo.82),
Reverse primer:CATGTCCAAGTGGCCCGCAGA(SEQ ID No.83);
G574R:
Forward primer:GCGGGCCACTTGGACATGTTG CGCCGGATAAAGTCCCTGCA(SEQ IDNo.84),
Reverse primer:CAACATGTCCAAGTGGCCCGC(SEQ ID No.85);
D584Y:
Forward primer:AAGTCCCTGCAAACCAGGGTG TACCAGATCGTTGGACGC(SEQ IDNo.86),
Reverse primer:CACCCTGGTTTGCAGGGACTT(SEQ ID No.87)。
实施例2:突变基因表达及功能测试
使用Lipofectamine3000试剂将野生型KCNQ4和上述KCNQ4错义突变质粒分别瞬时转染至中国仓鼠卵巢细胞系(CHO)中表达正常或突变的KCNQ4蛋白。
通过全细胞电压钳实验检测野生型和各个KCNQ4突变蛋白的离子通道活性。使用protocol如下:将细胞钳制在-80mV,给予细胞从-100mV至+40mV的阶梯电压,增量为20mV,持续时间1s,检测不同电压下细胞被激发出的最大电流。R159H突变质粒及其对照组的试验结果如图1所示,其中,a表示细胞中只转入野生型的KCNQ4质粒,b表示细胞中只转入错义突变R159H的KCNQ4质粒,模拟人KCNQ4基因的两个等位基因同时发生该错义突变;c表示细胞中转入野生型KCNQ4质粒和错义突变R159H的KCNQ4质粒,且转入比例为1:1,模拟人的KCNQ4基因中一个等位基因发生该错义突变,一个等位基因正常。从图中可以看出,只转入野生型的细胞的电流值最低,而两个等位基因同时发生该错义突变R159H后,细胞的电流值最高,两等位基因中仅有一个发生R159H错义突变的细胞的电流值在两者之间。因此,通过上述错义突变位点的引入,细胞电流变大,且具有等位基因数量的依赖性。上述结果表明,这种错义突变可以使KCNQ4具有更高的离子通道活性。
对在记录的最高电压下的电流值进行了统计,即在+40mV时,野生型和功能增强型错义突变的外向整流全细胞电流的统计结果如图2和图3所示。其中,图2表示测试细胞中转入了野生型KCNQ4质粒(对照组WT)或错义突变的KCNQ4质粒(相应突变组)。图3表示细胞中,测试细胞中转入了野生型KCNQ4质粒(对照组WT)或1:1的野生型KCNQ4质粒和错义突变的KCNQ4质粒(相应突变+WT组)。可以看出,相对于对照组(WT),两等位基因中的一个引入上述突变位点后,全细胞电流有明显增加;而两等位基因均引入上述突变位点后,全细胞电流出现了更显著的增加。综合上述实验结果可以看出,本申请实施例所公开的43种错义突变可以使KCNQ4具有更高的离子通道活性,具有明显的功能增强的特征,可以显著改善野生型KCNQ4的显性抑制作用。由于该电压门控通道与耳聋有关,推测这些错义突变位点也可以改善野生型KCNQ4引起的听力损失状况。因此,进一步研究上述这些错义突变与耳聋的关系。
实施例3:突变噪声暴露实验
选择6-7周龄的雄性小鼠,实验前检查小鼠外耳,排除有外耳道疾病以及中耳炎症小鼠。并对余下小鼠作为野生型小鼠(WT组)进行听力检测。同时取相同小鼠采用CRISPR/Cas9技术构建R160H/+小鼠作为R160H组(小鼠的R160H相对应于人类R159H)。在暴露前经检测WT组和R160H组小鼠的听力水平相近,随后将WT组和R160H组的小鼠通过噪声暴露仪(110dB白噪声)进行持续2小时的暴露,模拟噪声引起的SNHL耳聋,并在24小时、7天后和14天后分别对小鼠进行听力检测。具体测试方法如下:
测听前使用经典的1.25%阿佛丁(2,2,2-三溴乙醇)进行麻醉。麻醉后,在25℃黑暗环境下,将记录电极置于小鼠两侧耳廓前缘皮下,参考电极置于测试耳耳后皮下,接地电极置于另外一侧耳耳后皮下,发声喇叭距外耳道口约0.5cm。采用短声(Click)和短纯音,刺激声强度自90dB SPL开始,以10dB逐渐递减,直至检测不出重复的ABR波形,再向上递增5dB,直至能检测出重复的ABR波形,此刺激声强度即为小鼠的听力阈值。
结果如图4所示,其中,a为暴露后24小时的检测结果,b为暴露后7天的检测结果,c为暴露后14天的检测结果。横轴为暴露时所用白噪声的频率,纵轴为检测时间点对应的听力阈值相对于暴露前的听力阈值的变化量。参考图4,野生型小鼠和R160H/+小鼠原本听力水平相近,而在噪声暴露后都表现出一定的听力阈值上升。同时可以看到,相较于低频噪声,高频噪声(≥16kHz)所造成的小鼠的听力损失更为严重,暴露后听力阈值上升得更为明显。其中,32kHz噪声暴露下,小鼠的听力阈值的变化高达50dB左右。在噪声暴露7天后,R160H/+小鼠高频率下的听力阈值的下降相对于野生型小鼠而言下降得更为明显,说明两组小鼠之间的听力恢复速度存在显著差异,R160H/+小鼠要明显快于野生型小鼠。同理,在噪声暴露后第14天,R160H/+小鼠的恢复速度就更加快于野生型小鼠,听力阈值的变化更大。
实施例4
采用CRISPR/Cas9技术分别构建实施例1中公开的除R159H以外的其它42种错义突变(或对应这些人类错义突变位点的小鼠突变位点)的小鼠,采用实施例3中的方法检验噪声暴露下听力恢复情况,结果与实施例3类似,包含这些错义突变的小鼠相较于野生型小鼠具有更快的恢复速度,在噪声暴露后第7天和第14天,听力阈值的变化显著高于对照组。
上面结合实施例对本申请作了详细说明,但是本申请不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本申请宗旨的前提下作出各种变化。此外,在不冲突的情况下,本申请的实施例及实施例中的特征可以相互组合。
SEQUENCE LISTING
<110> 深圳湾实验室
<120> KCNQ4功能增强型突变体及其在治疗听力损失中的应用
<130> 1
<160> 88
<170> PatentIn version 3.5
<210> 1
<211> 695
<212> PRT
<213> homo sapiens
<400> 1
Met Ala Glu Ala Pro Pro Arg Arg Leu Gly Leu Gly Pro Pro Pro Gly
1 5 10 15
Asp Ala Pro Arg Ala Glu Leu Val Ala Leu Thr Ala Val Gln Ser Glu
20 25 30
Gln Gly Glu Ala Gly Gly Gly Gly Ser Pro Arg Arg Leu Gly Leu Leu
35 40 45
Gly Ser Pro Leu Pro Pro Gly Ala Pro Leu Pro Gly Pro Gly Ser Gly
50 55 60
Ser Gly Ser Ala Cys Gly Gln Arg Ser Ser Ala Ala His Lys Arg Tyr
65 70 75 80
Arg Arg Leu Gln Asn Trp Val Tyr Asn Val Leu Glu Arg Pro Arg Gly
85 90 95
Trp Ala Phe Val Tyr His Val Phe Ile Phe Leu Leu Val Phe Ser Cys
100 105 110
Leu Val Leu Ser Val Leu Ser Thr Ile Gln Glu His Gln Glu Leu Ala
115 120 125
Asn Glu Cys Leu Leu Ile Leu Glu Phe Val Met Ile Val Val Phe Gly
130 135 140
Leu Glu Tyr Ile Val Arg Val Trp Ser Ala Gly Cys Cys Cys Arg Tyr
145 150 155 160
Arg Gly Trp Gln Gly Arg Phe Arg Phe Ala Arg Lys Pro Phe Cys Val
165 170 175
Ile Asp Phe Ile Val Phe Val Ala Ser Val Ala Val Ile Ala Ala Gly
180 185 190
Thr Gln Gly Asn Ile Phe Ala Thr Ser Ala Leu Arg Ser Met Arg Phe
195 200 205
Leu Gln Ile Leu Arg Met Val Arg Met Asp Arg Arg Gly Gly Thr Trp
210 215 220
Lys Leu Leu Gly Ser Val Val Tyr Ala His Ser Lys Glu Leu Ile Thr
225 230 235 240
Ala Trp Tyr Ile Gly Phe Leu Val Leu Ile Phe Ala Ser Phe Leu Val
245 250 255
Tyr Leu Ala Glu Lys Asp Ala Asn Ser Asp Phe Ser Ser Tyr Ala Asp
260 265 270
Ser Leu Trp Trp Gly Thr Ile Thr Leu Thr Thr Ile Gly Tyr Gly Asp
275 280 285
Lys Thr Pro His Thr Trp Leu Gly Arg Val Leu Ala Ala Gly Phe Ala
290 295 300
Leu Leu Gly Ile Ser Phe Phe Ala Leu Pro Ala Gly Ile Leu Gly Ser
305 310 315 320
Gly Phe Ala Leu Lys Val Gln Glu Gln His Arg Gln Lys His Phe Glu
325 330 335
Lys Arg Arg Met Pro Ala Ala Asn Leu Ile Gln Ala Ala Trp Arg Leu
340 345 350
Tyr Ser Thr Asp Met Ser Arg Ala Tyr Leu Thr Ala Thr Trp Tyr Tyr
355 360 365
Tyr Asp Ser Ile Leu Pro Ser Phe Arg Glu Leu Ala Leu Leu Phe Glu
370 375 380
His Val Gln Arg Ala Arg Asn Gly Gly Leu Arg Pro Leu Glu Val Arg
385 390 395 400
Arg Ala Pro Val Pro Asp Gly Ala Pro Ser Arg Tyr Pro Pro Val Ala
405 410 415
Thr Cys His Arg Pro Gly Ser Thr Ser Phe Cys Pro Gly Glu Ser Ser
420 425 430
Arg Met Gly Ile Lys Asp Arg Ile Arg Met Gly Ser Ser Gln Arg Arg
435 440 445
Thr Gly Pro Ser Lys Gln His Leu Ala Pro Pro Thr Met Pro Thr Ser
450 455 460
Pro Ser Ser Glu Gln Val Gly Glu Ala Thr Ser Pro Thr Lys Val Gln
465 470 475 480
Lys Ser Trp Ser Phe Asn Asp Arg Thr Arg Phe Arg Ala Ser Leu Arg
485 490 495
Leu Lys Pro Arg Thr Ser Ala Glu Asp Ala Pro Ser Glu Glu Val Ala
500 505 510
Glu Glu Lys Ser Tyr Gln Cys Glu Leu Thr Val Asp Asp Ile Met Pro
515 520 525
Ala Val Lys Thr Val Ile Arg Ser Ile Arg Ile Leu Lys Phe Leu Val
530 535 540
Ala Lys Arg Lys Phe Lys Glu Thr Leu Arg Pro Tyr Asp Val Lys Asp
545 550 555 560
Val Ile Glu Gln Tyr Ser Ala Gly His Leu Asp Met Leu Gly Arg Ile
565 570 575
Lys Ser Leu Gln Thr Arg Val Asp Gln Ile Val Gly Arg Gly Pro Gly
580 585 590
Asp Arg Lys Ala Arg Glu Lys Gly Asp Lys Gly Pro Ser Asp Ala Glu
595 600 605
Val Val Asp Glu Ile Ser Met Met Gly Arg Val Val Lys Val Glu Lys
610 615 620
Gln Val Gln Ser Ile Glu His Lys Leu Asp Leu Leu Leu Gly Phe Tyr
625 630 635 640
Ser Arg Cys Leu Arg Ser Gly Thr Ser Ala Ser Leu Gly Ala Val Gln
645 650 655
Val Pro Leu Phe Asp Pro Asp Ile Thr Ser Asp Tyr His Ser Pro Val
660 665 670
Asp His Glu Asp Ile Ser Val Ser Ala Gln Thr Leu Ser Ile Ser Arg
675 680 685
Ser Val Ser Thr Asn Met Asp
690 695
<210> 2
<211> 43
<212> DNA
<213> 人工序列
<400> 2
ccgggtctgc gtgcggccag tgcagcagcg cggctcacaa aag 43
<210> 3
<211> 23
<212> DNA
<213> 人工序列
<400> 3
ctggccgcac gcagacccgg aac 23
<210> 4
<211> 41
<212> DNA
<213> 人工序列
<400> 4
agcgcagcag cgcggctcac gagagatata ggcgactcca g 41
<210> 5
<211> 25
<212> DNA
<213> 人工序列
<400> 5
gtgagccgcg ctgctgcgct ggccg 25
<210> 6
<211> 44
<212> DNA
<213> 人工序列
<400> 6
aaagatatag gcgactccag gactgggtct acaacgtgct tgag 44
<210> 7
<211> 24
<212> DNA
<213> 人工序列
<400> 7
ctggagtcgc ctatatcttt tgtg 24
<210> 8
<211> 44
<212> DNA
<213> 人工序列
<400> 8
aaagatatag gcgactccag acctgggtct acaacgtgct tgag 44
<210> 9
<211> 24
<212> DNA
<213> 人工序列
<400> 9
ctggagtcgc ctatatcttt tgtg 24
<210> 10
<211> 44
<212> DNA
<213> 人工序列
<400> 10
aaagatatag gcgactccag tactgggtct acaacgtgct tgag 44
<210> 11
<211> 24
<212> DNA
<213> 人工序列
<400> 11
ctggagtcgc ctatatcttt tgtg 24
<210> 12
<211> 41
<212> DNA
<213> 人工序列
<400> 12
gactccagaa ctgggtctac gacgtgcttg agaggcctag g 41
<210> 13
<211> 23
<212> DNA
<213> 人工序列
<400> 13
gtagacccag ttctggagtc gcc 23
<210> 14
<211> 43
<212> DNA
<213> 人工序列
<400> 14
tccagaactg ggtctacaac gcgcttgaga ggcctagggg ctg 43
<210> 15
<211> 23
<212> DNA
<213> 人工序列
<400> 15
gttgtagacc cagttctgga gtc 23
<210> 16
<211> 43
<212> DNA
<213> 人工序列
<400> 16
tccagaactg ggtctacaac gggcttgaga ggcctagggg ctg 43
<210> 17
<211> 23
<212> DNA
<213> 人工序列
<400> 17
gttgtagacc cagttctgga gtc 23
<210> 18
<211> 39
<212> DNA
<213> 人工序列
<400> 18
gggtctacaa cgtgcttgag ctgcctaggg gctgggcct 39
<210> 19
<211> 22
<212> DNA
<213> 人工序列
<400> 19
ctcaagcacg ttgtagaccc ag 22
<210> 20
<211> 39
<212> DNA
<213> 人工序列
<400> 20
gggtctacaa cgtgcttgag cagcctaggg gctgggcct 39
<210> 21
<211> 22
<212> DNA
<213> 人工序列
<400> 21
ctcaagcacg ttgtagaccc ag 22
<210> 22
<211> 45
<212> DNA
<213> 人工序列
<400> 22
tcttggtctt ctcatgcctg gcgctgagtg tactgagcac tatac 45
<210> 23
<211> 22
<212> DNA
<213> 人工序列
<400> 23
caggcatgag aagaccaaga gg 22
<210> 24
<211> 41
<212> DNA
<213> 人工序列
<400> 24
aatgtcctga atggaattgc atcctcatct tggagttcgt a 41
<210> 25
<211> 20
<212> DNA
<213> 人工序列
<400> 25
gcaattccat tcaggacatt 20
<210> 26
<211> 41
<212> DNA
<213> 人工序列
<400> 26
ggaattgcct gctcatcttg gacttcgtaa tgatcgtcgt c 41
<210> 27
<211> 21
<212> DNA
<213> 人工序列
<400> 27
caagatgagc aggcaattcc a 21
<210> 28
<211> 41
<212> DNA
<213> 人工序列
<400> 28
gtacgggtgt ggagcgccgg aggctgttgc aggtatcggg g 41
<210> 29
<211> 21
<212> DNA
<213> 人工序列
<400> 29
tccggcgctc cacacccgta c 21
<210> 30
<211> 41
<212> DNA
<213> 人工序列
<400> 30
gtacgggtgt ggagcgccgg aagctgttgc aggtatcggg g 41
<210> 31
<211> 21
<212> DNA
<213> 人工序列
<400> 31
tccggcgctc cacacccgta c 21
<210> 32
<211> 42
<212> DNA
<213> 人工序列
<400> 32
cgggtgtgga gcgccggatg cagctgcagg tatcggggtt gg 42
<210> 33
<211> 21
<212> DNA
<213> 人工序列
<400> 33
gcatccggcg ctccacaccc g 21
<210> 34
<211> 42
<212> DNA
<213> 人工序列
<400> 34
gtgtggagcg ccggatgctg tttcaggtat cggggttggc ag 42
<210> 35
<211> 21
<212> DNA
<213> 人工序列
<400> 35
acagcatccg gcgctccaca c 21
<210> 36
<211> 42
<212> DNA
<213> 人工序列
<400> 36
gtgtggagcg ccggatgctg ttggaggtat cggggttggc ag 42
<210> 37
<211> 21
<212> DNA
<213> 人工序列
<400> 37
acagcatccg gcgctccaca c 21
<210> 38
<211> 40
<212> DNA
<213> 人工序列
<400> 38
tggagcgccg gatgctgttg cgggtatcgg ggttggcagg 40
<210> 39
<211> 24
<212> DNA
<213> 人工序列
<400> 39
gcaacagcat ccggcgctcc acac 24
<210> 40
<211> 40
<212> DNA
<213> 人工序列
<400> 40
tggagcgccg gatgctgttg ccactatcgg ggttggcagg 40
<210> 41
<211> 24
<212> DNA
<213> 人工序列
<400> 41
gcaacagcat ccggcgctcc acac 24
<210> 42
<211> 40
<212> DNA
<213> 人工序列
<400> 42
tggagcgccg gatgctgttg cctctatcgg ggttggcagg 40
<210> 43
<211> 24
<212> DNA
<213> 人工序列
<400> 43
gcaacagcat ccggcgctcc acac 24
<210> 44
<211> 40
<212> DNA
<213> 人工序列
<400> 44
tggagcgccg gatgctgttg cccctatcgg ggttggcagg 40
<210> 45
<211> 24
<212> DNA
<213> 人工序列
<400> 45
gcaacagcat ccggcgctcc acac 24
<210> 46
<211> 40
<212> DNA
<213> 人工序列
<400> 46
tggagcgccg gatgctgttg cagctatcgg ggttggcagg 40
<210> 47
<211> 24
<212> DNA
<213> 人工序列
<400> 47
gcaacagcat ccggcgctcc acac 24
<210> 48
<211> 43
<212> DNA
<213> 人工序列
<400> 48
gccggatgct gttgcaggta tggaggttgg cagggccggt ttc 43
<210> 49
<211> 23
<212> DNA
<213> 人工序列
<400> 49
atacctgcaa cagcatccgg cgc 23
<210> 50
<211> 42
<212> DNA
<213> 人工序列
<400> 50
ttcgctttgc tcgaaaaccc gtctgcgtca ttgacttcat cg 42
<210> 51
<211> 22
<212> DNA
<213> 人工序列
<400> 51
gggttttcga gcaaagcgaa ac 22
<210> 52
<211> 40
<212> DNA
<213> 人工序列
<400> 52
ttctgaggat ggtaaggatg gaaagacggg gagggacgtg 40
<210> 53
<211> 24
<212> DNA
<213> 人工序列
<400> 53
catccttacc atcctcagaa tctg 24
<210> 54
<211> 37
<212> DNA
<213> 人工序列
<400> 54
tgaggatggt aaggatggat ccccggggag ggacgtg 37
<210> 55
<211> 21
<212> DNA
<213> 人工序列
<400> 55
atccatcctt accatcctca g 21
<210> 56
<211> 41
<212> DNA
<213> 人工序列
<400> 56
ggatggatag acggggaggg gcgtggaagc tcctcgggag t 41
<210> 57
<211> 21
<212> DNA
<213> 人工序列
<400> 57
ccctccccgt ctatccatcc t 21
<210> 58
<211> 41
<212> DNA
<213> 人工序列
<400> 58
cggggaggga cgtggaagct cgtcgggagt gtggtgtacg c 41
<210> 59
<211> 21
<212> DNA
<213> 人工序列
<400> 59
gagcttccac gtccctcccc g 21
<210> 60
<211> 42
<212> DNA
<213> 人工序列
<400> 60
tggaagctcc tcgggagtgt gctgtacgcg cactcaaaag ag 42
<210> 61
<211> 21
<212> DNA
<213> 人工序列
<400> 61
cacactcccg aggagcttcc a 21
<210> 62
<211> 41
<212> DNA
<213> 人工序列
<400> 62
caaaagagct catcacggct tgttacattg gatttcttgt c 41
<210> 63
<211> 22
<212> DNA
<213> 人工序列
<400> 63
agccgtgatg agctcttttg ag 22
<210> 64
<211> 41
<212> DNA
<213> 人工序列
<400> 64
caaaagagct catcacggct ttgtacattg gatttcttgt c 41
<210> 65
<211> 22
<212> DNA
<213> 人工序列
<400> 65
agccgtgatg agctcttttg ag 22
<210> 66
<211> 45
<212> DNA
<213> 人工序列
<400> 66
ggcagcggct ttgcactgaa gattcaagag cagcacaggc agaag 45
<210> 67
<211> 21
<212> DNA
<213> 人工序列
<400> 67
cttcagtgca aagccgctgc c 21
<210> 68
<211> 47
<212> DNA
<213> 人工序列
<400> 68
cagcaacatg gtactactac gatcgtattc tgccgagttt cagagag 47
<210> 69
<211> 23
<212> DNA
<213> 人工序列
<400> 69
atcgtagtag taccatgttg ctg 23
<210> 70
<211> 43
<212> DNA
<213> 人工序列
<400> 70
ttcaaggaaa cgctccggcc gttcgacgta aaggatgtta tag 43
<210> 71
<211> 21
<212> DNA
<213> 人工序列
<400> 71
cggccggagc gtttccttga a 21
<210> 72
<211> 42
<212> DNA
<213> 人工序列
<400> 72
acgctccggc cgtacgacgt aagggatgtt atagaacagt ac 42
<210> 73
<211> 21
<212> DNA
<213> 人工序列
<400> 73
tacgtcgtac ggccggagcg t 21
<210> 74
<211> 42
<212> DNA
<213> 人工序列
<400> 74
cggccgtacg acgtaaagga tgccatagaa cagtactctg cg 42
<210> 75
<211> 21
<212> DNA
<213> 人工序列
<400> 75
atcctttacg tcgtacggcc g 21
<210> 76
<211> 41
<212> DNA
<213> 人工序列
<400> 76
aaggatgtta tagaacagta cccagcgggc cacttggaca t 41
<210> 77
<211> 21
<212> DNA
<213> 人工序列
<400> 77
gtactgttct ataacatcct t 21
<210> 78
<211> 40
<212> DNA
<213> 人工序列
<400> 78
gttatagaac agtactctgc gcgccacttg gacatgttgg 40
<210> 79
<211> 21
<212> DNA
<213> 人工序列
<400> 79
cgcagagtac tgttctataa c 21
<210> 80
<211> 40
<212> DNA
<213> 人工序列
<400> 80
gttatagaac agtactctgc gagccacttg gacatgttgg 40
<210> 81
<211> 21
<212> DNA
<213> 人工序列
<400> 81
cgcagagtac tgttctataa c 21
<210> 82
<211> 41
<212> DNA
<213> 人工序列
<400> 82
tctgcgggcc acttggacat gcggggacgg ataaagtccc t 41
<210> 83
<211> 21
<212> DNA
<213> 人工序列
<400> 83
catgtccaag tggcccgcag a 21
<210> 84
<211> 41
<212> DNA
<213> 人工序列
<400> 84
gcgggccact tggacatgtt gcgccggata aagtccctgc a 41
<210> 85
<211> 21
<212> DNA
<213> 人工序列
<400> 85
caacatgtcc aagtggcccg c 21
<210> 86
<211> 39
<212> DNA
<213> 人工序列
<400> 86
aagtccctgc aaaccagggt gtaccagatc gttggacgc 39
<210> 87
<211> 21
<212> DNA
<213> 人工序列
<400> 87
caccctggtt tgcagggact t 21
<210> 88
<211> 2088
<212> DNA
<213> 人工序列
<400> 88
atggctgagg ctcctccgcg acgactcgga ctcggtcccc ctcccggaga tgctccaagg 60
gcggaacttg tggcgctgac ggctgtacaa tcagagcagg gtgaagctgg gggtggaggg 120
tctcctcgac ggttgggatt gctgggctcc ccactcccac ctggcgcacc tctccctggg 180
ccaggaagcg gttccgggtc tgcgtgcggc cagcgcagca gcgcggctca caaaagatat 240
aggcgactcc agaactgggt ctacaacgtg cttgagaggc ctaggggctg ggcctttgtg 300
taccacgtgt tcattttcct cttggtcttc tcatgcctgg ttctgagtgt actgagcact 360
atacaggagc accaggagct cgcaaacgag tgcctgctca tcttggagtt cgtaatgatc 420
gtcgtcttcg gccttgagta tatagtacgg gtgtggagcg ccggatgctg ttgcaggtat 480
cggggttggc agggccggtt tcgctttgct cgaaaaccct tctgcgtcat tgacttcatc 540
gttttcgtgg caagcgtcgc cgttattgct gccggaactc agggcaacat atttgccacc 600
agcgctctga gaagcatgag gttccttcag attctgagga tggtaaggat ggatagacgg 660
ggagggacgt ggaagctcct cgggagtgtg gtgtacgcgc actcaaaaga gctcatcacg 720
gcttggtaca ttggatttct tgtcctgatc ttcgcgagct ttctggttta tctggcggaa 780
aaggacgcga atagcgactt tagcagctat gccgacagcc tttggtgggg aacaatcacg 840
cttacgacca tcggctacgg cgataaaacc ccgcacacat ggttgggtag agtgctggca 900
gccggattcg cactgttggg catcagcttc ttcgcattgc ctgccggtat cttgggcagc 960
ggctttgcac tgaaggttca agagcagcac aggcagaagc acttcgagaa gcgccgcatg 1020
ccagctgcga acttgattca ggcagcgtgg aggctgtatt ctaccgatat gtcacgggcg 1080
tacctgacag caacatggta ctactacgat agcattctgc cgagtttcag agagctggcc 1140
ctcctgtttg aacatgtgca gagggcacgc aacggagggc tgcgaccatt ggaggtccgg 1200
cgagccccag tacccgacgg ggcaccctct cgataccccc ctgtagcaac gtgtcacagg 1260
cccggaagta caagcttctg tcctggggag tcatcccgga tgggtatcaa ggaccggata 1320
cgcatgggtt cttcccagag acgaacgggc ccttcaaaac agcagctcgc tccgcccaca 1380
atgcctacga gtcccagctc cgagcaggtc ggcgaggcga cctcaccgac gaaggtacaa 1440
aagtcatggt cattcaacga caggacgagg ttcagggctt ccctgcgact gaagccaaga 1500
acatccgccg aggatgcgcc cagcgaggaa gttgcggaag agaagagcta ccagtgtgag 1560
cttactgtgg acgacatcat gcccgctgta aaaaccgtga tccgcagcat acggatactg 1620
aagtttctcg ttgcgaagcg caaattcaag gaaacgctcc ggccgtacga cgtaaaggat 1680
gttatagaac agtactctgc gggccacttg gacatgttgg gacggataaa gtccctgcaa 1740
accagggtgg accagatcgt tggacgcggt ccgggggatc gcaaagcacg cgaaaaagga 1800
gataaggggc cttccgatgc cgaggttgtg gatgagattt ctatgatggg gcgagtagtg 1860
aaagtggaaa agcaggtgca atccattgaa cacaagctcg accttctcct gggattctac 1920
tctaggtgcc tccgcagtgg aactagcgcg tctctcggtg ccgtgcaggt tccccttttc 1980
gatccggaca tcacctctga ctatcactct cccgtcgacc acgaagacat cagcgtaagt 2040
gcgcagactc tgagcattag ccgctctgtg agcacgaaca tggactag 2088
Claims (10)
1.KCNQ4突变蛋白,其特征在于,在野生型KCNQ4的氨基酸序列中具有以下至少一种突变位点:R72C、K78E、N85D、N85T、N85Y、N89D、V90A、V90G、R93L、R93Q、V114A、L132I、E136D、C156G、C156S、C157S、C158F、C158W、R159G、R159H、R159L、R159P、R159S、R161G、F174V、D218E、R219P、T223A、L227V、V231L、W242C、W242L、V326I、S371R、Y556F、K559R、V561A、S566P、G568R、G568S、L573R、G574R、D584Y;
其中,所述野生型KCNQ4的氨基酸序列如SEQ ID No.1所示。
2.根据权利要求1所述的KCNQ4突变蛋白,其特征在于,所述突变位点为R159H。
3.编码权利要求1或2任一项所述的KCNQ4突变蛋白的核酸分子。
4.重组载体,其特征在于,包括权利要求3所述的核酸分子。
5.重组细胞,其特征在于,包括权利要求3所述的核酸分子。
6.权利要求1或2所述的KCNQ4突变蛋白,或权利要求3所述的核酸分子,或权利要求4所述的重组载体,或权利要求5所述的重组细胞在制备治疗听力损失的药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述听力损失为感觉神经性听力损失。
8.根据权利要求6所述的应用,其特征在于,所述听力损失为噪声引起的听力损失。
9.根据权利要求8所述的应用,其特征在于,所述噪声包括高频噪声。
10.根据权利要求9所述的应用,其特征在于,所述高频噪声的频率为16kHz以上。
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CN109554464A (zh) * | 2018-12-29 | 2019-04-02 | 中国人民解放军第四军医大学 | 非综合征性常染色体显性遗传性耳聋致病基因kcnq4突变检测用试剂盒 |
Non-Patent Citations (2)
Title |
---|
LI T等: "NP_004691.2", GENBANK, 25 June 2021 (2021-06-25), pages 1 - 3 * |
李丽娜等: "KCNQ4基因的多态性与高频听力下降的研究", 中国听力语言康复科学杂志, no. 06, 15 November 2007 (2007-11-15), pages 12 - 14 * |
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