CN110184275A - 一种nf1新突变致病基因及应用和试剂盒 - Google Patents

Abstract

本发明涉及一种和神经纤维瘤病1型疾病相关的NF1新突变致病基因，属于分子生物学领域，所述新突变致病基因的核酸样本与NF1基因如SEQ ID NO：1所示的核苷酸序列相比，核苷酸变化：c.738delA；还涉及到由新突变致病基因编码的突变体蛋白。本发明首次揭示了NF1基因c.738delA位点突变与神经纤维瘤病1型疾病相关，提供了新突变致病基因在制备检测或筛选NF1病变的试剂或试剂盒中的应用。进一步提供筛选神经纤维瘤病1型疾病的试剂盒，完善更多的神经纤维瘤病1型疾病突变基因，为患者及时发现及时治疗提供更可靠的指导。

Description

一种NF1新突变致病基因及应用和试剂盒

技术领域

本发明属于生物技术中突变基因领域，涉及一种NF1新突变致病基因及应用和试剂盒。

背景技术

神经纤维瘤病1型(NF1)是一种以遗传性疾病为特征的遗传性疾病，患有NF1的个体患各种肿瘤的风险增加。NF1基因是1型神经纤维瘤(neurofibromatosis type 1,NF1)患者生殖系中突变的抑癌基因，最常见的肿瘤包括神经纤维瘤、丛状神经纤维瘤、周围神经鞘恶性肿瘤和视神经胶质瘤；NF1的体细胞突变在癌症中很常见，包括黑色素瘤。

NF1的特征是皮肤和眼睛的色素病变，包括多个皮肤神经纤维瘤、咖啡-au-lait斑、腋窝腹股沟雀斑、脉络膜雀斑和虹膜Lisch结节。Lisch结节是眼睛的黑素细胞错构瘤，在虹膜上表现为界限清楚的圆顶状丘疹，透明至黄色或棕色，其组织学上表现为黑素细胞聚集，并与梭形细胞和肥大细胞混合。目前尚未明确Lisch结节导致的眼部并发症。不太常见但可能更严重的表现包括青光眼、视神经和其他中枢神经系统胶质瘤，恶性外周神经鞘瘤，脊柱侧凸，胫骨发育不良和血管病变。

儿童每年的眼科检查是对于该疾病的监控手段之一，由熟悉该疾病的医生每年进行体检，对儿童进行定期发展评估。NF1患者的视神经胶质瘤通常在6岁之前出现视力下降，突眼或斜视[Friedrich&Nuding 2016]。Lisch结节几乎可以在所有成人和不到一半小于5岁的NF1儿童中中看到。[Ragge等1993]。在标准眼科检查中无法看到脉络膜雀斑，但可以通过红外或近红外光扫描激光检眼镜，红外反射成像或光学相干断层扫描来观察脉络膜雀斑[Vagge等2016]，其病理改变是雪旺细胞增殖排列在轴突周围的同心环中，发生在大多数所有年龄的NF1患者中，并随着年龄的增长而增多。NF1的罕见眼部表现包括视网膜血管增生性肿瘤[Hood等人2009，Shields等人2014]和新生血管性青光眼[Elgi等人2010，Chiu等人2011，Al Freihi等人2013]。

NF1的诊断通常基于临床发现，但这种疾病的表现是非常多变的，即使在一个家庭中也可以出现多种临床表现。所以需要结合NF1的分子遗传学检测来明确诊断。NF1遗传方式为常染色体显性遗传。半数受影响的个体由于一种新的NF1致病变异而具有NF1。受影响个体的后代有50％的风险遗传变异的NF1等位基因。

NF1基因位于染色体17q11.2，全长为282 751bp，包含58个外显子，编码2839个氨基酸，编码神经纤维瘤蛋白，负责调控Ras蛋白转导信号。已经鉴定出数千种不同的NF1致病变异，包括错义/无义、剪接、小片段的插入/缺失、大片段的插入/缺失等各种突变类型，分布在整个编码区，大多数致病性变异是特定家族特有的。所以科研人员不遗余力的鉴定出每一个NF1致病变异的基因，以应用生物技术检测手段检查出NF1病变的可能性，及时的进行治疗干预，尽可能的减少患者视力衰退是当前科研人员迫不及待的心愿。

发明内容

有鉴于此，本发明的目的在于提供一种NF1新突变致病基因，以便更加完善NF1致病变异基因，在临床发现前期能通过生物基因筛查手段更及时的发现NF1病变的可能性，及时的进行治疗干预，让患者早日康复。

为达到上述目的，本发明提供如下技术方案：

1、一种NF1新突变致病基因，所述新突变致病基因的核酸样本与NF1基因如SEQ IDNO：1所示的核苷酸序列相比，核苷酸变化：c.738delA。

进一步，所述突变致病基因的核酸样本指人工分离或合成的单链DNA、双链DNA、RNA或DNA与RNA的聚合物。

2、本发明还提供一种视网膜变性疾病的NF1新突变体多肽，所述多肽的氨基酸序列与SEQ ID NO：2所示的氨基酸序列相比，氨基酸变化：p.E247KfsTer34。

进一步，所述多肽由权利要求1所述的基因编码的。

3、本发明还提供一种NF1新突变致病基因在制备检测或筛选NF1病变的试剂盒中的应用。所述新突变致病基因的核酸样本与NF1基因如SEQ ID NO：1所示的核苷酸序列相比，核苷酸变化：c.738delA。

4、本发明还提供一种筛选NF1病变的试剂盒，所述试剂盒包含检测NF1新突变致病基因的试剂，所述新突变致病基因与NF1基因如SEQ ID NO：1所示的核苷酸序列相比，具有核苷酸变化：c.738delA。

进一步，所述试剂盒检测的核酸样本指人工分离或合成的单链DNA、双链DNA、RNA或DNA与RNA的聚合物。

进一步，所述检测NF1新突变致病基因的试剂是核酸探针。

进一步，所述试剂盒还包括序列如SEQ ID NO：15和/或SEQ ID NO：16的引物。

进一步，所述试剂盒还包括序列如SEQ ID NO：3-SEQ ID NO：94的一个或多个引物。

进一步，所述检测NF1新突变致病基因的试剂是检测致病基因所编码的蛋白。

进一步，所述蛋白的氨基酸与SEQ ID NO：2所示的氨基酸序列相比，具有p.E247KfsTer34突变。

本发明的有益效果在于：本发明首次提供了NF1基因一个新的突变位点，与NF1野生型基因相比，核苷酸c.738delA位点突变，且与NF1疾病密切相关，并以此设计出直接检测该突变基因的试剂盒。本发明所提供了NF1基因一个新的突变位点更加完善NF1致病变异基因，为在临床发现前期能通过生物基因筛查手段更及时的发现NF1病变提供更多更有力的检测工具，能及时的进行治疗干预，让患者早日康复创造福音。本发明所提供了NF1基因一个新的突变位点，可以单独的开发直接检测该突变基因的试剂盒，也可以联合检测其他已知突变基因开发试剂盒。

本发明所述的试剂盒可以直接通过一代测序，减少繁琐程序，跳过高通量筛选，直接针对目的基因外显子全长测序，也可以直接针对28号外显子测序。本发明所提供的引物设计，PCR扩增加上一代测序对基因外显子突变进行检测，操作简单，技术成熟，成本极低，不受突变大小(20bp)影响，可对外显子纯合缺失及大片断缺失插入进行检测。

附图说明

为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：

图1为患者面部、胸部和腹部的牛奶咖啡斑，其中上图为患者胸部牛奶咖啡斑图，下图为患者腹部牛奶咖啡斑图。

图2为患者眼科检查结果，其中a和b分别为患者左眼和右眼眼底彩照，c和d分别为患者左眼和右眼NIR双眼近红外光眼底扫描。

图3为该患者右眼九方位眼底彩照和眼底血管荧光造影检查图。

图4为该患者父亲眼科检查结果，其中a和b分别为患者父亲左眼和右眼眼底彩照，眼底彩照显示双眼眼底未见明显异常；图4中c和d分别为患者父亲左眼和右眼NIR，NIR显示双眼后极部和中周部视网膜多灶团状和斑块状强反射。

图5为患者父亲背部照片。

图6为患者外显子8一代测序部分结果。

图7为该患者父亲在外显子8位点的验证。

图8为该患者母亲在外显子8位点的验证。

图9为该患者家系图，其中M为突变体，N为野生型。

具体实施方式

下面将结合附图，对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。

实施例1

获取生物样品

2018年，门诊收取一患者，临床表型为：新生血管性青光眼，视网膜血管增生性肿瘤，皮肤有牛奶咖啡斑，临床怀疑神经纤维瘤病1型。父亲及外婆皮肤有牛奶咖啡斑，视力正常，母亲和妹妹表型正常，样本为疾病患者及其父母的外周静脉血。所有参与本发明研究的家系成员均签署了知情同意书。(男孩，年龄7岁，(2011年)，父亲，年龄32岁，(1986年)；母亲：年龄29岁(1990年)；妹妹：年龄，4岁(2015)；外婆：57岁，(1962年)。

该患者面部、胸部和腹部均有大小不等的牛奶咖啡斑，如图1所示，其中上图为患者胸部牛奶咖啡斑图，下图为患者腹部牛奶咖啡斑图。

图2为该患者眼科检查结果，其中a和b分别为患者左眼和右眼眼底彩照，患者眼底彩照显示左眼眼底未见明显异常，患儿右眼因玻璃体浑浊眼底图像显示不清。图2中c和d分别为患者左眼和右眼NIR双眼近红外光眼底扫描(Near-infrared Reflectance,NIR)，显示左眼后极部和中周部视网膜多灶团状和斑块状强反射，右眼显示不清。图3为该患者右眼九方位眼底彩照和眼底血管荧光造影检查图，如图3左边图所示，右眼颞上方周边视网膜见灰白色斑块状结节样改变，融合成片，边界不清，其间隐约见形态异常血管。如图3右边图所示，FFA(眼底血管荧光造影)显示右眼颞上方周边部分视网膜血管轻微扩张，后期部分管壁荧光染色。

图4为该患者父亲眼科检查结果，其中a和b分别为患者父亲左眼和右眼眼底彩照，眼底彩照显示双眼眼底未见明显异常；图4中c和d分别为患者父亲左眼和右眼NIR，NIR显示双眼后极部和中周部视网膜多灶团状和斑块状强反射。

该患者父亲全身皮肤散在斑块状及椒盐状色素沉着改变(咖啡斑)，面部、颈部和背部均有，涉及患者隐私，只显示该患者父亲背部照片，如图5所示。

实施例2DNA提取

利用高盐沉淀法提取各血液样本中基因组DNA，利用分光光度计测量DNA的浓度及纯度，所得的每个标本基因组DNA的OD₂₆₀/OD₂₈₀均位于1.7-2.0之间，浓度不少于200ng/微升，总量不少于30微克。

实施例3引物设计及PCR反应

参考人类基因组序列数据库，设计得NF1基因1-58号外显子序列分别对应的PCR特异性引物对，具体内容如表1所示，1-58号外显子所对应的序列如表2所示。

表1 NF1基因1-58号外显子序列分别对应的PCR特异性引物对

表2 1-58号外显子所对应的基因

接着，分别按照以下配比配制各基因组DNA样本的PCR反应体系以及进行PCR反应，其中PCR使用的Promga品牌的Go Green Master Mix，lot号000242981。

按照表1针对NF1基因1-58号外显子序列，按表3配比分别配制各基因组DNA样本的各PCR反应体系，PCR反应体系为40ul。

按照表1针对NF1基因1-58号外显子序列，按表3配比分别配制各基因组DNA样本的各PCR反应体系，PCR反应体系为40ul。

表3：PCR反应体系(40ul)

再将以上配置好的各反应体系按照表4的反应条件进行PCR反应。

表4：PCR程序

实施例4Sanger法测序验证

将所获得的PCR扩增产物直接进行Sanger法测序验证。

PCR产物纯化：实施例3中得到的PCR产物经1％的琼脂糖凝胶电泳，160V,40-60Min。紫外透射仪下割下目的条带。切取的胶块质量应小于3g，于96孔板中按照胶块质量：Buffer GL＝1:3加入Buffer GL,盖上封口膜，65℃水浴12min，揭开封口膜，用连续加液器每孔加入100μl已经混匀的磁珠，磁珠吸附目的DNA。在磁力的作用下固定磁珠，Eluent或无菌水洗脱磁珠表面上目的DNA。

目的条带于96孔PCR板中进行测序PCR扩增：

测序反应PCR：采用正向或反向引物扩增

表4：测序PCR反应体系(5ul)

BDT(BDT原液:5x Buffer＝1:3)	1ul
		正向/反向引物(10P)	1ul
DNA模板(200ng/μl)	2ul
		ddH<sub>2</sub>O	1ul(add up to 5ul)

表5：PCR程序

测序扩增后的产物移至ABI3730测序仪，测序分析。使用CodonCode Aligner软件读取测序峰图，并将测序结果与参考序列比对。

结果如下：患者及患者父亲发现了核苷酸变异：738delA，导致后面的序列移码，对应氨基酸变化：p.E247KfsTer34。患者母亲核苷酸没有突变，为野生型。

图6为患者外显子8一代测序部分结果，其中在序列510位(核苷酸738位)碱基A丢失，由正常序列GCAGA变为GC_GA。

图7为该患者父亲在外显子8位点的验证；父亲验证在510位(核苷酸738位)碱基A丢失，由正常序列GCAGA变为GC_GA。

图8为该患者母亲在外显子8位点的验证；母亲验证在510位(核苷酸738位)为野生型。

图9为该患者家系图，其中M为突变体，N为野生型。

此方法可直接通过一代测序，直接针对目的基因外显子全长测序，能清楚知道外显子的情况，继而清楚NF1基因的突变情况可能导致的病变，简单直接有效。

最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。

序列表

<110> 中国人民解放军陆军军医大学第一附属医院

<120> 一种NF1新突变致病基因及应用和试剂盒

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<170> SIPOSequenceListing 1.0

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<212> DNA

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aatctctagc tcgctcgcgc tccctctccc cgggccgtgg aaaggatccc acttccggtg 60

gggtgtcatg gcggcgtctc ggactgtgat ggctgtgggg agacggcgct agtggggaga 120

gcgaccaaga ggccccctcc cctccccggg tccccttccc ctatccccct ccccccagcc 180

tccttgccaa cgcccccttt ccctctcccc ctcccgctcg gcgctgaccc cccatcccca 240

cccccgtggg aacactggga gcctgcactc cacagaccct ctccttgcct cttccctcac 300

ctcagcctcc gctccccgcc ctcttcccgg cccagggcgc cggcccaccc ttccctccgc 360

cgccccccgg ccgcggggag gacatggccg cgcacaggcc ggtggaatgg gtccaggccg 420

tggtcagccg cttcgacgag cagcttccaa taaaaacagg acagcagaac acacatacca 480

aagtcagtac tgagcacaac aaggaatgtc taatcaatat ttccaaatac aagttttctt 540

tggttataag cggcctcact actattttaa agaatgttaa caatatgaga atatttggag 600

aagctgctga aaaaaattta tatctctctc agttgattat attggataca ctggaaaaat 660

gtcttgctgg gcaaccaaag gacacaatga gattagatga aacgatgctg gtcaaacagt 720

tgctgccaga aatctgccat tttcttcaca cctgtcgtga aggaaaccag catgcagctg 780

aacttcggaa ttctgcctct ggggttttat tttctctcag ctgcaacaac ttcaatgcag 840

tctttagtcg catttctacc aggttacagg aattaactgt ttgttcagaa gacaatgttg 900

atgttcatga tatagaattg ttacagtata tcaatgtgga ttgtgcaaaa ttaaaacgac 960

tcctgaagga aacagcattt aaatttaaag ccctaaagaa ggttgcgcag ttagcagtta 1020

taaatagcct ggaaaaggca ttttggaact gggtagaaaa ttatccagat gaatttacaa 1080

aactgtacca gatcccacag actgatatgg ctgaatgtgc agaaaagcta tttgacttgg 1140

tggatggttt tgctgaaagc accaaacgta aagcagcagt ttggccacta caaatcattc 1200

tccttatctt gtgtccagaa ataatccagg atatatccaa agacgtggtt gatgaaaaca 1260

acatgaataa gaagttattt ctggacagtc tacgaaaagc tcttgctggc catggaggaa 1320

gtaggcagct gacagaaagt gctgcaattg cctgtgtcaa actgtgtaaa gcaagtactt 1380

acatcaattg ggaagataac tctgtcattt tcctacttgt tcagtccatg gtggttgatc 1440

ttaagaacct gctttttaat ccaagtaagc cattctcaag aggcagtcag cctgcagatg 1500

tggatctaat gattgactgc cttgtttctt gctttcgtat aagccctcac aacaaccaac 1560

actttaagat ctgcctggct cagaattcac cttctacatt tcactatgtg ctggtaaatt 1620

cactccatcg aatcatcacc aattccgcat tggattggtg gcctaagatt gatgctgtgt 1680

attgtcactc ggttgaactt cgaaatatgt ttggtgaaac acttcataaa gcagtgcaag 1740

gttgtggagc acacccagca atacgaatgg caccgagtct tacatttaaa gaaaaagtaa 1800

caagccttaa atttaaagaa aaacctacag acctggagac aagaagctat aagtatcttc 1860

tcttgtccat ggtgaaacta attcatgcag atccaaagct cttgctttgt aatccaagaa 1920

aacaggggcc cgaaacccaa ggcagtacag cagaattaat tacagggctc gtccaactgg 1980

tccctcagtc acacatgcca gagattgctc aggaagcaat ggaggctctg ctggttcttc 2040

atcagttaga tagcattgat ttgtggaatc ctgatgctcc tgtagaaaca ttttgggaga 2100

ttagctcaca aatgcttttt tacatctgca agaaattaac tagtcatcaa atgcttagta 2160

gcacagaaat tctcaagtgg ttgcgggaaa tattgatctg caggaataaa tttcttctta 2220

aaaataagca ggcagataga agttcctgtc actttctcct tttttacggg gtaggatgtg 2280

atattccttc tagtggaaat accagtcaaa tgtccatgga tcatgaagaa ttactacgta 2340

ctcctggagc ctctctccgg aagggaaaag ggaactcctc tatggatagt gcagcaggat 2400

gcagcggaac ccccccgatt tgccgacaag cccagaccaa actagaagtg gccctgtaca 2460

tgtttctgtg gaaccctgac actgaagctg ttctggttgc catgtcctgt ttccgccacc 2520

tctgtgagga agcagatatc cggtgtgggg tggatgaagt gtcagtgcat aacctcttgc 2580

ccaactataa cacattcatg gagtttgcct ctgtcagcaa tatgatgtca acaggaagag 2640

cagcacttca gaaaagagtg atggcactgc tgaggcgcat tgagcatccc actgcaggaa 2700

acactgaggc ttgggaagat acacatgcaa aatgggaaca agcaacaaag ctaatcctta 2760

actatccaaa agccaaaatg gaagatggcc aggctgctga aagccttcac aagaccattg 2820

ttaagaggcg aatgtcccat gtgagtggag gaggatccat agatttgtct gacacagact 2880

ccctacagga atggatcaac atgactggct tcctttgtgc ccttggggga gtgtgcctcc 2940

agcagagaag caattctggc ctggcaacct atagcccacc catgggtcca gtcagtgaac 3000

gtaagggttc tatgatttca gtgatgtctt cagagggaaa cgcagataca cctgtcagca 3060

aatttatgga tcggctgttg tccttaatgg tgtgtaacca tgagaaagtg ggacttcaaa 3120

tacggaccaa tgttaaggat ctggtgggtc tagaattgag tcctgctctg tatccaatgc 3180

tatttaacaa attgaagaat accatcagca agttttttga ctcccaagga caggttttat 3240

tgactgatac caatactcaa tttgtagaac aaaccatagc tataatgaag aacttgctag 3300

ataatcatac tgaaggcagc tctgaacatc tagggcaagc tagcattgaa acaatgatgt 3360

taaatctggt caggtatgtt cgtgtgcttg ggaatatggt ccatgcaatt caaataaaaa 3420

cgaaactgtg tcaattagtt gaagtaatga tggcaaggag agatgacctc tcattttgcc 3480

aagagatgaa atttaggaat aagatggtag aatacctgac agactgggtt atgggaacat 3540

caaaccaagc agcagatgat gatgtaaaat gtcttacaag agatttggac caggcaagca 3600

tggaagcagt agtttcactt ctagctggtc tccctctgca gcctgaagaa ggagatggtg 3660

tggaattgat ggaagccaaa tcacagttat ttcttaaata cttcacatta tttatgaacc 3720

ttttgaatga ctgcagtgaa gttgaagatg aaagtgcgca aacaggtggc aggaaacgtg 3780

gcatgtctcg gaggctggca tcactgaggc actgtacggt ccttgcaatg tcaaacttac 3840

tcaatgccaa cgtagacagt ggtctcatgc actccatagg cttaggttac cacaaggatc 3900

tccagacaag agctacattt atggaagttc tgacaaaaat ccttcaacaa ggcacagaat 3960

ttgacacact tgcagaaaca gtattggctg atcggtttga gagattggtg gaactggtca 4020

caatgatggg tgatcaagga gaactcccta tagcgatggc tctggccaat gtggttcctt 4080

gttctcagtg ggatgaacta gctcgagttc tggttactct gtttgattct cggcatttac 4140

tctaccaact gctctggaac atgttttcta aagaagtaga attggcagac tccatgcaga 4200

ctctcttccg aggcaacagc ttggccagta aaataatgac attctgtttc aaggtatatg 4260

gtgctaccta tctacaaaaa ctcctggatc ctttattacg aattgtgatc acatcctctg 4320

attggcaaca tgttagcttt gaagtggatc ctaccaggtt agaaccatca gagagccttg 4380

aggaaaacca gcggaacctc cttcagatga ctgaaaagtt cttccatgcc atcatcagtt 4440

cctcctcaga attcccccct caacttcgaa gtgtgtgcca ctgtttatac caggtggtta 4500

gccagcgttt ccctcagaac agcatcggtg cagtaggaag tgccatgttc ctcagattta 4560

tcaatcctgc cattgtctca ccgtatgaag cagggatttt agataaaaag ccaccaccta 4620

gaatcgaaag gggcttgaag ttaatgtcaa agatacttca gagtattgcc aatcatgttc 4680

tcttcacaaa agaagaacat atgcggcctt tcaatgattt tgtgaaaagc aactttgatg 4740

cagcacgcag gtttttcctt gatatagcat ctgattgtcc tacaagtgat gcagtaaatc 4800

atagtctttc cttcataagt gacggcaatg tgcttgcttt acatcgtcta ctctggaaca 4860

atcaggagaa aattgggcag tatctttcca gcaacaggga tcataaagct gttggaagac 4920

gaccttttga taagatggca acacttcttg catacctggg tcctccagag cacaaacctg 4980

tggcagatac acactggtcc agccttaacc ttaccagttc aaagtttgag gaatttatga 5040

ctaggcatca ggtacatgaa aaagaagaat tcaaggcttt gaaaacgtta agtattttct 5100

accaagctgg gacttccaaa gctgggaatc ctatttttta ttatgttgca cggaggttca 5160

aaactggtca aatcaatggt gatttgctga tataccatgt cttactgact ttaaagccat 5220

attatgcaaa gccatatgaa attgtagtgg accttaccca taccgggcct agcaatcgct 5280

ttaaaacaga ctttctctct aagtggtttg ttgtttttcc tggctttgct tacgacaacg 5340

tctccgcagt ctatatctat aactgtaact cctgggtcag ggagtacacc aagtatcatg 5400

agcggctgct gactggcctc aaaggtagca aaaggcttgt tttcatagac tgtcctggga 5460

aactggctga gcacatagag catgaacaac agaaactacc tgctgccacc ttggctttag 5520

aagaggacct gaaggtattc cacaatgctc tcaagctagc tcacaaagac accaaagttt 5580

ctattaaagt tggttctact gctgtccaag taacttcagc agagcgaaca aaagtcctag 5640

ggcaatcagt ctttctaaat gacatttatt atgcttcgga aattgaagaa atctgcctag 5700

tagatgagaa ccagttcacc ttaaccattg caaaccaggg cacgccgctc accttcatgc 5760

accaggagtg tgaagccatt gtccagtcta tcattcatat ccggacccgc tgggaactgt 5820

cacagcccga ctctatcccc caacacacca agattcggcc aaaagatgtc cctgggacac 5880

tgctcaatat cgcattactt aatttaggca gttctgaccc gagtttacgg tcagctgcct 5940

ataatcttct gtgtgcctta acttgtacct ttaatttaaa aatcgagggc cagttactag 6000

agacatcagg tttatgtatc cctgccaaca acaccctctt tattgtctct attagtaaga 6060

cactggcagc caatgagcca cacctcacgt tagaattttt ggaagagtgt atttctggat 6120

ttagcaaatc tagtattgaa ttgaaacacc tttgtttgga atacatgact ccatggctgt 6180

caaatctagt tcgtttttgc aagcataatg atgatgccaa acgacaaaga gttactgcta 6240

ttcttgacaa gctgataaca atgaccatca atgaaaaaca gatgtaccca tctattcaag 6300

caaaaatatg gggaagcctt gggcagatta cagatctgct tgatgttgta ctagacagtt 6360

tcatcaaaac cagtgcaaca ggtggcttgg gatcaataaa agctgaggtg atggcagata 6420

ctgctgtagc tttggcttct ggaaatgtga aattggtttc aagcaaggtt attggaagga 6480

tgtgcaaaat aattgacaag acatgcttat ctccaactcc tactttagaa caacatctta 6540

tgtgggatga tattgctatt ttagcacgct acatgctgat gctgtccttc aacaattccc 6600

ttgatgtggc agctcatctt ccctacctct tccacgttgt tactttctta gtagccacag 6660

gtccgctctc ccttagagct tccacacatg gactggtcat taatatcatt cactctctgt 6720

gtacttgttc acagcttcat tttagtgaag agaccaagca agttttgaga ctcagtctga 6780

cagagttctc attacccaaa ttttacttgc tgtttggcat tagcaaagtc aagtcagctg 6840

ctgtcattgc cttccgttcc agttaccggg acaggtcatt ctctcctggc tcctatgaga 6900

gagagacttt tgctttgaca tccttggaaa cagtcacaga agctttgttg gagatcatgg 6960

aggcatgcat gagagatatt ccaacgtgca agtggctgga ccagtggaca gaactagctc 7020

aaagatttgc attccaatat aatccatccc tgcaaccaag agctcttgtt gtctttgggt 7080

gtattagcaa acgagtgtct catgggcaga taaagcagat aatccgtatt cttagcaagg 7140

cacttgagag ttgcttaaaa ggacctgaca cttacaacag tcaagttctg atagaagcta 7200

cagtaatagc actaaccaaa ttacagccac ttcttaataa ggactcgcct ctgcacaaag 7260

ccctcttttg ggtagctgtg gctgtgctgc agcttgatga ggtcaacttg tattcagcag 7320

gtaccgcact tcttgaacaa aacctgcata ctttagatag tctccgtata ttcaatgaca 7380

agagtccaga ggaagtattt atggcaatcc ggaatcctct ggagtggcac tgcaagcaaa 7440

tggatcattt tgttggactc aatttcaact ctaactttaa ctttgcattg gttggacacc 7500

ttttaaaagg gtacaggcat ccttcacctg ctattgttgc aagaacagtc agaattttac 7560

atacactact aactctggtt aacaaacaca gaaattgtga caaatttgaa gtgaatacac 7620

agagcgtggc ctacttagca gctttactta cagtgtctga agaagttcga agtcgctgca 7680

gcctaaaaca tagaaagtca cttcttctta ctgatatttc aatggaaaat gttcctatgg 7740

atacatatcc cattcatcat ggtgaccctt cctataggac actaaaggag actcagccat 7800

ggtcctctcc caaaggttct gaaggatacc ttgcagccac ctatccaact gtcggccaga 7860

ccagtccccg agccaggaaa tccatgagcc tggacatggg gcaaccttct caggccaaca 7920

ctaagaagtt gcttggaaca aggaaaagtt ttgatcactt gatatcagac acaaaggctc 7980

ctaaaaggca agaaatggaa tcagggatca caacaccccc caaaatgagg agagtagcag 8040

aaactgatta tgaaatggaa actcagagga tttcctcatc acaacagcac ccacatttac 8100

gtaaagtttc agtgtctgaa tcaaatgttc tcttggatga agaagtactt actgatccga 8160

agatccaggc gctgcttctt actgttctag ctacactggt aaaatatacc acagatgagt 8220

ttgatcaacg aattctttat gaatacttag cagaggccag tgttgtgttt cccaaagtct 8280

ttcctgttgt gcataatttg ttggactcta agatcaacac cctgttatca ttgtgccaag 8340

atccaaattt gttaaatcca atccatggaa ttgtgcagag tgtggtgtac catgaagaat 8400

ccccaccaca ataccaaaca tcttacctgc aaagttttgg ttttaatggc ttgtggcggt 8460

ttgcaggacc gttttcaaag caaacacaaa ttccagacta tgctgagctt attgttaagt 8520

ttcttgatgc cttgattgac acgtacctgc ctggaattga tgaagaaacc agtgaagaat 8580

ccctcctgac tcccacatct ccttaccctc ctgcactgca gagccagctt agtatcactg 8640

ccaaccttaa cctttctaat tccatgacct cacttgcaac ttcccagcat tccccaggaa 8700

tcgacaagga gaacgttgaa ctctccccta ccactggcca ctgtaacagt ggacgaactc 8760

gccacggatc cgcaagccaa gtgcagaagc aaagaagcgc tggcagtttc aaacgtaata 8820

gcattaagaa gatcgtgtga agcttgcttg ctttcttttt taaaatcaac ttaacatggg 8880

ctcttcacta gtgacccctt ccctgtcctt gccctttccc cccatgttgt aatgctgcac 8940

ttcctgtttt ataatgaacc catccggttt gccatgttgc cagatgatca actcttcgaa 9000

gccttgccta aatttaatgc tgccttttct ttaacttttt ttcttctact tttggcgtgt 9060

atctggtata tgtaagtgtt cagaacaact gcaaagaaag tgggaggtca ggaaactttt 9120

aactgagaaa tctcaattgt aagagaggat gaattcttga atactgctac tactggccag 9180

tgatgaaagc catttgcaca gagctctgcc ttctgtggtt ttcccttctt catcctacag 9240

agtaaagtgt tagtcctatt tatacatttt tcaagataca agtttatgag agaaatagta 9300

ttataacccc agtatgttta atcttttagc tgtggacttt ttttttaacc gtacaaaact 9360

gaaagaacca tagaggtcaa gcctcagtga cttgacacca taaagccaca gacaaggtac 9420

ttggggggga gggcagggaa atttcatatt ttatagtgga ttcttaagaa atactaacac 9480

ttgagtatta gcaataatta caggaaaata agtgcgacca catatatctt aacattactg 9540

aattaaaact atggcttcta agtccttatc caaactcagt catccaaact agtttatttt 9600

tttctccagt tgattatctt ttaattttta attttgctaa aggtggtttt tttgtgtttt 9660

gttttttgta aaccaaaact atactaagta tagtaattat atatatatat atattttttc 9720

ccctccccct cttctttcct aactaattct gagcagggta atcagtgaac aaagtgttga 9780

aaattgttcc cagaaggtaa ttttcataga tgtttgcatt agctccatag caaaatggaa 9840

tggtacgtga catttagggt agctgatatt tttattttgt taaataattt ccaagaatag 9900

agtatggtgt atattataaa tttctttgat aagatgtatt ttgaatgtct tttaatcttc 9960

ctcctcctct ccaaaaaaat cagaaacctc tttaagaaaa catgtaggtt atatatgcta 10020

gaattgcatt taatcactgt gaaaagactg gtcagcctgc attagtatga cagtaggggg 10080

gctgttagaa ttgctgctat actggtggta tggattatca tggcattgga attttcatag 10140

taatgcagat ccaatttctt tgtggtacct gcagtttaca aaataatttg acttcagtga 10200

gcatattggt atctggatgt tccaatttag aactaaacca tatttattac aaaaagatat 10260

taatccctct actcccaggt tccctttata tgttaagata taatggcttt gaggggggaa 10320

aaaataaacc taggggagag gggagtttcc tgtagtgctg tttcattaga ggatttcagt 10380

aaattaaatt ccacagctaa ttcaataaat aatggtacat ttaagtgttc tgattttaat 10440

aatatatttc acatttatcc acacagtaac aatgtaatat gttaatgtaa ataaaattgg 10500

ttttgatact cagaaataac aagaatttaa ttttttaaat ttgtttacag tcctgggaaa 10560

agtaagaatt atttgccaaa ataagaggaa agaaaacctt agtattatta atgagtttac 10620

catagaattg ttggaaatac tgaagacagg tgcaatttac taaacttttg tttttaaact 10680

attgtagagg ctgcattaga agaaaatgtt tataatgaca gagcaactat gactatataa 10740

aaaagctgaa attagaactg tgtttagaaa tagatcagta acccagtgcc aaggatgcca 10800

agctgccacc atggtcttgg ctctcccaca acccagtgtt tctggggtaa gtttcacagt 10860

ttctaggccc tggaatagca ggcagtgtaa gcctttgata actttagttc gatgtttttc 10920

ttgtttttgt ttgttggttt ggtgcatatg atagtgggtg ttatgctatt ttgctcttcc 10980

catcaaaata aagaaacttc cagaggttta ctgttaaaaa tactgatatt tccataaacg 11040

ggtttaccaa gggtgtagta tttcataccg cctgaaatga tcagcattgg cacaaatcaa 11100

aattcagccg cctttgaaat gcaaaaatac ctttgactag taagtacatc ctaggagttt 11160

gaaaacttaa ctaaggttta aaatttacct tgtttaaaga acttctgact tttgaggaaa 11220

atctagcttt ccaagtaact aaaatgtaca tgagataaac ctctcaccac tatgtgtccc 11280

ttgagaaatg caacactttt ttagtcttca tacttgtaat ctataaaaga aattctgaag 11340

tttagaccaa gttgcccatt tctgcgtaat tgacataagt tctgttaaaa atattataag 11400

taattcgttt cggtttgtag atgtttcccc tgacttgtta aagaggaaac caggaactca 11460

gtcatgtttt tgtcctggat aatctacctg ttatgccagt actcccatcc gaggggcatg 11520

cccttagttg cccagatgga gatgcagttc agtagatttg gggcaaagtg gctacagctc 11580

tgtcttccat tcactcaaca cctgttcatg actgagccag gtgcccagga cacatcctaa 11640

acagtcagct tctatcctgt gtcctagttg gggagacaga gtgccagcca gcaaccctcc 11700

caggtttgta ggttttaggg gttttcagtt ttgtttgggt tttttgtttt ttgtttttgt 11760

ttctacatcc ttccccgact cccaggcata atgaggcatg tcttactcaa tgttatgcaa 11820

tggatttagg caaaaattca ttcttagtgt cagccacaca atttttttta atgcagtata 11880

ttcacctgta aatagtttgt gtaaaatttg acaaaaaaag tatatttact atactgtaaa 11940

tatatgtgat gatatattgt attattttgc ttttttgtaa agcagttagt tgctgcacat 12000

ggataacaac aaaaatttga ttattctcgt gttagtattg ttaacttctt tttgcgactg 12060

cgttacatca tttaaagaaa atgctgtgta ttgtaaactt aaattgtata tgataactta 12120

ctgtcctttc catccgggcc taaactttgg cagttccttt gtctacaacc ttgttaatac 12180

tgtaaacagt tgtacgccag caggaaaaat actgcccaac agacaaaatc gatcattgta 12240

ggggaaaatc atagaaatcc atttcagatc tttattgttc ctcaccccat tttcctcctt 12300

gtgtatgtac ttcccccacc cccctttttt taagtaaaat gtaaattcaa tctgctctaa 12360

gaaaaaaaaa aaaaaaaaaa a 12381

<210> 2

<211> 2839

<212> PRT

<213> Homo sapiens

<400> 2

Met Ala Ala His Arg Pro Val Glu Trp Val Gln Ala Val Val Ser Arg

1 5 10 15

Phe Asp Glu Gln Leu Pro Ile Lys Thr Gly Gln Gln Asn Thr His Thr

20 25 30

Lys Val Ser Thr Glu His Asn Lys Glu Cys Leu Ile Asn Ile Ser Lys

35 40 45

Tyr Lys Phe Ser Leu Val Ile Ser Gly Leu Thr Thr Ile Leu Lys Asn

50 55 60

Val Asn Asn Met Arg Ile Phe Gly Glu Ala Ala Glu Lys Asn Leu Tyr

65 70 75 80

Leu Ser Gln Leu Ile Ile Leu Asp Thr Leu Glu Lys Cys Leu Ala Gly

85 90 95

Gln Pro Lys Asp Thr Met Arg Leu Asp Glu Thr Met Leu Val Lys Gln

100 105 110

Leu Leu Pro Glu Ile Cys His Phe Leu His Thr Cys Arg Glu Gly Asn

115 120 125

Gln His Ala Ala Glu Leu Arg Asn Ser Ala Ser Gly Val Leu Phe Ser

130 135 140

Leu Ser Cys Asn Asn Phe Asn Ala Val Phe Ser Arg Ile Ser Thr Arg

145 150 155 160

Leu Gln Glu Leu Thr Val Cys Ser Glu Asp Asn Val Asp Val His Asp

165 170 175

Ile Glu Leu Leu Gln Tyr Ile Asn Val Asp Cys Ala Lys Leu Lys Arg

180 185 190

Leu Leu Lys Glu Thr Ala Phe Lys Phe Lys Ala Leu Lys Lys Val Ala

195 200 205

Gln Leu Ala Val Ile Asn Ser Leu Glu Lys Ala Phe Trp Asn Trp Val

210 215 220

Glu Asn Tyr Pro Asp Glu Phe Thr Lys Leu Tyr Gln Ile Pro Gln Thr

225 230 235 240

Asp Met Ala Glu Cys Ala Glu Lys Leu Phe Asp Leu Val Asp Gly Phe

245 250 255

Ala Glu Ser Thr Lys Arg Lys Ala Ala Val Trp Pro Leu Gln Ile Ile

260 265 270

Leu Leu Ile Leu Cys Pro Glu Ile Ile Gln Asp Ile Ser Lys Asp Val

275 280 285

Val Asp Glu Asn Asn Met Asn Lys Lys Leu Phe Leu Asp Ser Leu Arg

290 295 300

Lys Ala Leu Ala Gly His Gly Gly Ser Arg Gln Leu Thr Glu Ser Ala

305 310 315 320

Ala Ile Ala Cys Val Lys Leu Cys Lys Ala Ser Thr Tyr Ile Asn Trp

325 330 335

Glu Asp Asn Ser Val Ile Phe Leu Leu Val Gln Ser Met Val Val Asp

340 345 350

Leu Lys Asn Leu Leu Phe Asn Pro Ser Lys Pro Phe Ser Arg Gly Ser

355 360 365

Gln Pro Ala Asp Val Asp Leu Met Ile Asp Cys Leu Val Ser Cys Phe

370 375 380

Arg Ile Ser Pro His Asn Asn Gln His Phe Lys Ile Cys Leu Ala Gln

385 390 395 400

Asn Ser Pro Ser Thr Phe His Tyr Val Leu Val Asn Ser Leu His Arg

405 410 415

Ile Ile Thr Asn Ser Ala Leu Asp Trp Trp Pro Lys Ile Asp Ala Val

420 425 430

Tyr Cys His Ser Val Glu Leu Arg Asn Met Phe Gly Glu Thr Leu His

435 440 445

Lys Ala Val Gln Gly Cys Gly Ala His Pro Ala Ile Arg Met Ala Pro

450 455 460

Ser Leu Thr Phe Lys Glu Lys Val Thr Ser Leu Lys Phe Lys Glu Lys

465 470 475 480

Pro Thr Asp Leu Glu Thr Arg Ser Tyr Lys Tyr Leu Leu Leu Ser Met

485 490 495

Val Lys Leu Ile His Ala Asp Pro Lys Leu Leu Leu Cys Asn Pro Arg

500 505 510

Lys Gln Gly Pro Glu Thr Gln Gly Ser Thr Ala Glu Leu Ile Thr Gly

515 520 525

Leu Val Gln Leu Val Pro Gln Ser His Met Pro Glu Ile Ala Gln Glu

530 535 540

Ala Met Glu Ala Leu Leu Val Leu His Gln Leu Asp Ser Ile Asp Leu

545 550 555 560

Trp Asn Pro Asp Ala Pro Val Glu Thr Phe Trp Glu Ile Ser Ser Gln

565 570 575

Met Leu Phe Tyr Ile Cys Lys Lys Leu Thr Ser His Gln Met Leu Ser

580 585 590

Ser Thr Glu Ile Leu Lys Trp Leu Arg Glu Ile Leu Ile Cys Arg Asn

595 600 605

Lys Phe Leu Leu Lys Asn Lys Gln Ala Asp Arg Ser Ser Cys His Phe

610 615 620

Leu Leu Phe Tyr Gly Val Gly Cys Asp Ile Pro Ser Ser Gly Asn Thr

625 630 635 640

Ser Gln Met Ser Met Asp His Glu Glu Leu Leu Arg Thr Pro Gly Ala

645 650 655

Ser Leu Arg Lys Gly Lys Gly Asn Ser Ser Met Asp Ser Ala Ala Gly

660 665 670

Cys Ser Gly Thr Pro Pro Ile Cys Arg Gln Ala Gln Thr Lys Leu Glu

675 680 685

Val Ala Leu Tyr Met Phe Leu Trp Asn Pro Asp Thr Glu Ala Val Leu

690 695 700

Val Ala Met Ser Cys Phe Arg His Leu Cys Glu Glu Ala Asp Ile Arg

705 710 715 720

Cys Gly Val Asp Glu Val Ser Val His Asn Leu Leu Pro Asn Tyr Asn

725 730 735

Thr Phe Met Glu Phe Ala Ser Val Ser Asn Met Met Ser Thr Gly Arg

740 745 750

Ala Ala Leu Gln Lys Arg Val Met Ala Leu Leu Arg Arg Ile Glu His

755 760 765

Pro Thr Ala Gly Asn Thr Glu Ala Trp Glu Asp Thr His Ala Lys Trp

770 775 780

Glu Gln Ala Thr Lys Leu Ile Leu Asn Tyr Pro Lys Ala Lys Met Glu

785 790 795 800

Asp Gly Gln Ala Ala Glu Ser Leu His Lys Thr Ile Val Lys Arg Arg

805 810 815

Met Ser His Val Ser Gly Gly Gly Ser Ile Asp Leu Ser Asp Thr Asp

820 825 830

Ser Leu Gln Glu Trp Ile Asn Met Thr Gly Phe Leu Cys Ala Leu Gly

835 840 845

Gly Val Cys Leu Gln Gln Arg Ser Asn Ser Gly Leu Ala Thr Tyr Ser

850 855 860

Pro Pro Met Gly Pro Val Ser Glu Arg Lys Gly Ser Met Ile Ser Val

865 870 875 880

Met Ser Ser Glu Gly Asn Ala Asp Thr Pro Val Ser Lys Phe Met Asp

885 890 895

Arg Leu Leu Ser Leu Met Val Cys Asn His Glu Lys Val Gly Leu Gln

900 905 910

Ile Arg Thr Asn Val Lys Asp Leu Val Gly Leu Glu Leu Ser Pro Ala

915 920 925

Leu Tyr Pro Met Leu Phe Asn Lys Leu Lys Asn Thr Ile Ser Lys Phe

930 935 940

Phe Asp Ser Gln Gly Gln Val Leu Leu Thr Asp Thr Asn Thr Gln Phe

945 950 955 960

Val Glu Gln Thr Ile Ala Ile Met Lys Asn Leu Leu Asp Asn His Thr

965 970 975

Glu Gly Ser Ser Glu His Leu Gly Gln Ala Ser Ile Glu Thr Met Met

980 985 990

Leu Asn Leu Val Arg Tyr Val Arg Val Leu Gly Asn Met Val His Ala

995 1000 1005

Ile Gln Ile Lys Thr Lys Leu Cys Gln Leu Val Glu Val Met Met Ala

1010 1015 1020

Arg Arg Asp Asp Leu Ser Phe Cys Gln Glu Met Lys Phe Arg Asn Lys

1025 1030 1035 1040

Met Val Glu Tyr Leu Thr Asp Trp Val Met Gly Thr Ser Asn Gln Ala

1045 1050 1055

Ala Asp Asp Asp Val Lys Cys Leu Thr Arg Asp Leu Asp Gln Ala Ser

1060 1065 1070

Met Glu Ala Val Val Ser Leu Leu Ala Gly Leu Pro Leu Gln Pro Glu

1075 1080 1085

Glu Gly Asp Gly Val Glu Leu Met Glu Ala Lys Ser Gln Leu Phe Leu

1090 1095 1100

Lys Tyr Phe Thr Leu Phe Met Asn Leu Leu Asn Asp Cys Ser Glu Val

1105 1110 1115 1120

Glu Asp Glu Ser Ala Gln Thr Gly Gly Arg Lys Arg Gly Met Ser Arg

1125 1130 1135

Arg Leu Ala Ser Leu Arg His Cys Thr Val Leu Ala Met Ser Asn Leu

1140 1145 1150

Leu Asn Ala Asn Val Asp Ser Gly Leu Met His Ser Ile Gly Leu Gly

1155 1160 1165

Tyr His Lys Asp Leu Gln Thr Arg Ala Thr Phe Met Glu Val Leu Thr

1170 1175 1180

Lys Ile Leu Gln Gln Gly Thr Glu Phe Asp Thr Leu Ala Glu Thr Val

1185 1190 1195 1200

Leu Ala Asp Arg Phe Glu Arg Leu Val Glu Leu Val Thr Met Met Gly

1205 1210 1215

Asp Gln Gly Glu Leu Pro Ile Ala Met Ala Leu Ala Asn Val Val Pro

1220 1225 1230

Cys Ser Gln Trp Asp Glu Leu Ala Arg Val Leu Val Thr Leu Phe Asp

1235 1240 1245

Ser Arg His Leu Leu Tyr Gln Leu Leu Trp Asn Met Phe Ser Lys Glu

1250 1255 1260

Val Glu Leu Ala Asp Ser Met Gln Thr Leu Phe Arg Gly Asn Ser Leu

1265 1270 1275 1280

Ala Ser Lys Ile Met Thr Phe Cys Phe Lys Val Tyr Gly Ala Thr Tyr

1285 1290 1295

Leu Gln Lys Leu Leu Asp Pro Leu Leu Arg Ile Val Ile Thr Ser Ser

1300 1305 1310

Asp Trp Gln His Val Ser Phe Glu Val Asp Pro Thr Arg Leu Glu Pro

1315 1320 1325

Ser Glu Ser Leu Glu Glu Asn Gln Arg Asn Leu Leu Gln Met Thr Glu

1330 1335 1340

Lys Phe Phe His Ala Ile Ile Ser Ser Ser Ser Glu Phe Pro Pro Gln

1345 1350 1355 1360

Leu Arg Ser Val Cys His Cys Leu Tyr Gln Ala Thr Cys His Ser Leu

1365 1370 1375

Leu Asn Lys Ala Thr Val Lys Glu Lys Lys Glu Asn Lys Lys Ser Val

1380 1385 1390

Val Ser Gln Arg Phe Pro Gln Asn Ser Ile Gly Ala Val Gly Ser Ala

1395 1400 1405

Met Phe Leu Arg Phe Ile Asn Pro Ala Ile Val Ser Pro Tyr Glu Ala

1410 1415 1420

Gly Ile Leu Asp Lys Lys Pro Pro Pro Arg Ile Glu Arg Gly Leu Lys

1425 1430 1435 1440

Leu Met Ser Lys Ile Leu Gln Ser Ile Ala Asn His Val Leu Phe Thr

1445 1450 1455

Lys Glu Glu His Met Arg Pro Phe Asn Asp Phe Val Lys Ser Asn Phe

1460 1465 1470

Asp Ala Ala Arg Arg Phe Phe Leu Asp Ile Ala Ser Asp Cys Pro Thr

1475 1480 1485

Ser Asp Ala Val Asn His Ser Leu Ser Phe Ile Ser Asp Gly Asn Val

1490 1495 1500

Leu Ala Leu His Arg Leu Leu Trp Asn Asn Gln Glu Lys Ile Gly Gln

1505 1510 1515 1520

Tyr Leu Ser Ser Asn Arg Asp His Lys Ala Val Gly Arg Arg Pro Phe

1525 1530 1535

Asp Lys Met Ala Thr Leu Leu Ala Tyr Leu Gly Pro Pro Glu His Lys

1540 1545 1550

Pro Val Ala Asp Thr His Trp Ser Ser Leu Asn Leu Thr Ser Ser Lys

1555 1560 1565

Phe Glu Glu Phe Met Thr Arg His Gln Val His Glu Lys Glu Glu Phe

1570 1575 1580

Lys Ala Leu Lys Thr Leu Ser Ile Phe Tyr Gln Ala Gly Thr Ser Lys

1585 1590 1595 1600

Ala Gly Asn Pro Ile Phe Tyr Tyr Val Ala Arg Arg Phe Lys Thr Gly

1605 1610 1615

Gln Ile Asn Gly Asp Leu Leu Ile Tyr His Val Leu Leu Thr Leu Lys

1620 1625 1630

Pro Tyr Tyr Ala Lys Pro Tyr Glu Ile Val Val Asp Leu Thr His Thr

1635 1640 1645

Gly Pro Ser Asn Arg Phe Lys Thr Asp Phe Leu Ser Lys Trp Phe Val

1650 1655 1660

Val Phe Pro Gly Phe Ala Tyr Asp Asn Val Ser Ala Val Tyr Ile Tyr

1665 1670 1675 1680

Asn Cys Asn Ser Trp Val Arg Glu Tyr Thr Lys Tyr His Glu Arg Leu

1685 1690 1695

Leu Thr Gly Leu Lys Gly Ser Lys Arg Leu Val Phe Ile Asp Cys Pro

1700 1705 1710

Gly Lys Leu Ala Glu His Ile Glu His Glu Gln Gln Lys Leu Pro Ala

1715 1720 1725

Ala Thr Leu Ala Leu Glu Glu Asp Leu Lys Val Phe His Asn Ala Leu

1730 1735 1740

Lys Leu Ala His Lys Asp Thr Lys Val Ser Ile Lys Val Gly Ser Thr

1745 1750 1755 1760

Ala Val Gln Val Thr Ser Ala Glu Arg Thr Lys Val Leu Gly Gln Ser

1765 1770 1775

Val Phe Leu Asn Asp Ile Tyr Tyr Ala Ser Glu Ile Glu Glu Ile Cys

1780 1785 1790

Leu Val Asp Glu Asn Gln Phe Thr Leu Thr Ile Ala Asn Gln Gly Thr

1795 1800 1805

Pro Leu Thr Phe Met His Gln Glu Cys Glu Ala Ile Val Gln Ser Ile

1810 1815 1820

Ile His Ile Arg Thr Arg Trp Glu Leu Ser Gln Pro Asp Ser Ile Pro

1825 1830 1835 1840

Gln His Thr Lys Ile Arg Pro Lys Asp Val Pro Gly Thr Leu Leu Asn

1845 1850 1855

Ile Ala Leu Leu Asn Leu Gly Ser Ser Asp Pro Ser Leu Arg Ser Ala

1860 1865 1870

Ala Tyr Asn Leu Leu Cys Ala Leu Thr Cys Thr Phe Asn Leu Lys Ile

1875 1880 1885

Glu Gly Gln Leu Leu Glu Thr Ser Gly Leu Cys Ile Pro Ala Asn Asn

1890 1895 1900

Thr Leu Phe Ile Val Ser Ile Ser Lys Thr Leu Ala Ala Asn Glu Pro

1905 1910 1915 1920

His Leu Thr Leu Glu Phe Leu Glu Glu Cys Ile Ser Gly Phe Ser Lys

1925 1930 1935

Ser Ser Ile Glu Leu Lys His Leu Cys Leu Glu Tyr Met Thr Pro Trp

1940 1945 1950

Leu Ser Asn Leu Val Arg Phe Cys Lys His Asn Asp Asp Ala Lys Arg

1955 1960 1965

Gln Arg Val Thr Ala Ile Leu Asp Lys Leu Ile Thr Met Thr Ile Asn

1970 1975 1980

Glu Lys Gln Met Tyr Pro Ser Ile Gln Ala Lys Ile Trp Gly Ser Leu

1985 1990 1995 2000

Gly Gln Ile Thr Asp Leu Leu Asp Val Val Leu Asp Ser Phe Ile Lys

2005 2010 2015

Thr Ser Ala Thr Gly Gly Leu Gly Ser Ile Lys Ala Glu Val Met Ala

2020 2025 2030

Asp Thr Ala Val Ala Leu Ala Ser Gly Asn Val Lys Leu Val Ser Ser

2035 2040 2045

Lys Val Ile Gly Arg Met Cys Lys Ile Ile Asp Lys Thr Cys Leu Ser

2050 2055 2060

Pro Thr Pro Thr Leu Glu Gln His Leu Met Trp Asp Asp Ile Ala Ile

2065 2070 2075 2080

Leu Ala Arg Tyr Met Leu Met Leu Ser Phe Asn Asn Ser Leu Asp Val

2085 2090 2095

Ala Ala His Leu Pro Tyr Leu Phe His Val Val Thr Phe Leu Val Ala

2100 2105 2110

Thr Gly Pro Leu Ser Leu Arg Ala Ser Thr His Gly Leu Val Ile Asn

2115 2120 2125

Ile Ile His Ser Leu Cys Thr Cys Ser Gln Leu His Phe Ser Glu Glu

2130 2135 2140

Thr Lys Gln Val Leu Arg Leu Ser Leu Thr Glu Phe Ser Leu Pro Lys

2145 2150 2155 2160

Phe Tyr Leu Leu Phe Gly Ile Ser Lys Val Lys Ser Ala Ala Val Ile

2165 2170 2175

Ala Phe Arg Ser Ser Tyr Arg Asp Arg Ser Phe Ser Pro Gly Ser Tyr

2180 2185 2190

Glu Arg Glu Thr Phe Ala Leu Thr Ser Leu Glu Thr Val Thr Glu Ala

2195 2200 2205

Leu Leu Glu Ile Met Glu Ala Cys Met Arg Asp Ile Pro Thr Cys Lys

2210 2215 2220

Trp Leu Asp Gln Trp Thr Glu Leu Ala Gln Arg Phe Ala Phe Gln Tyr

2225 2230 2235 2240

Asn Pro Ser Leu Gln Pro Arg Ala Leu Val Val Phe Gly Cys Ile Ser

2245 2250 2255

Lys Arg Val Ser His Gly Gln Ile Lys Gln Ile Ile Arg Ile Leu Ser

2260 2265 2270

Lys Ala Leu Glu Ser Cys Leu Lys Gly Pro Asp Thr Tyr Asn Ser Gln

2275 2280 2285

Val Leu Ile Glu Ala Thr Val Ile Ala Leu Thr Lys Leu Gln Pro Leu

2290 2295 2300

Leu Asn Lys Asp Ser Pro Leu His Lys Ala Leu Phe Trp Val Ala Val

2305 2310 2315 2320

Ala Val Leu Gln Leu Asp Glu Val Asn Leu Tyr Ser Ala Gly Thr Ala

2325 2330 2335

Leu Leu Glu Gln Asn Leu His Thr Leu Asp Ser Leu Arg Ile Phe Asn

2340 2345 2350

Asp Lys Ser Pro Glu Glu Val Phe Met Ala Ile Arg Asn Pro Leu Glu

2355 2360 2365

Trp His Cys Lys Gln Met Asp His Phe Val Gly Leu Asn Phe Asn Ser

2370 2375 2380

Asn Phe Asn Phe Ala Leu Val Gly His Leu Leu Lys Gly Tyr Arg His

2385 2390 2395 2400

Pro Ser Pro Ala Ile Val Ala Arg Thr Val Arg Ile Leu His Thr Leu

2405 2410 2415

Leu Thr Leu Val Asn Lys His Arg Asn Cys Asp Lys Phe Glu Val Asn

2420 2425 2430

Thr Gln Ser Val Ala Tyr Leu Ala Ala Leu Leu Thr Val Ser Glu Glu

2435 2440 2445

Val Arg Ser Arg Cys Ser Leu Lys His Arg Lys Ser Leu Leu Leu Thr

2450 2455 2460

Asp Ile Ser Met Glu Asn Val Pro Met Asp Thr Tyr Pro Ile His His

2465 2470 2475 2480

Gly Asp Pro Ser Tyr Arg Thr Leu Lys Glu Thr Gln Pro Trp Ser Ser

2485 2490 2495

Pro Lys Gly Ser Glu Gly Tyr Leu Ala Ala Thr Tyr Pro Thr Val Gly

2500 2505 2510

Gln Thr Ser Pro Arg Ala Arg Lys Ser Met Ser Leu Asp Met Gly Gln

2515 2520 2525

Pro Ser Gln Ala Asn Thr Lys Lys Leu Leu Gly Thr Arg Lys Ser Phe

2530 2535 2540

Asp His Leu Ile Ser Asp Thr Lys Ala Pro Lys Arg Gln Glu Met Glu

2545 2550 2555 2560

Ser Gly Ile Thr Thr Pro Pro Lys Met Arg Arg Val Ala Glu Thr Asp

2565 2570 2575

Tyr Glu Met Glu Thr Gln Arg Ile Ser Ser Ser Gln Gln His Pro His

2580 2585 2590

Leu Arg Lys Val Ser Val Ser Glu Ser Asn Val Leu Leu Asp Glu Glu

2595 2600 2605

Val Leu Thr Asp Pro Lys Ile Gln Ala Leu Leu Leu Thr Val Leu Ala

2610 2615 2620

Thr Leu Val Lys Tyr Thr Thr Asp Glu Phe Asp Gln Arg Ile Leu Tyr

2625 2630 2635 2640

Glu Tyr Leu Ala Glu Ala Ser Val Val Phe Pro Lys Val Phe Pro Val

2645 2650 2655

Val His Asn Leu Leu Asp Ser Lys Ile Asn Thr Leu Leu Ser Leu Cys

2660 2665 2670

Gln Asp Pro Asn Leu Leu Asn Pro Ile His Gly Ile Val Gln Ser Val

2675 2680 2685

Val Tyr His Glu Glu Ser Pro Pro Gln Tyr Gln Thr Ser Tyr Leu Gln

2690 2695 2700

Ser Phe Gly Phe Asn Gly Leu Trp Arg Phe Ala Gly Pro Phe Ser Lys

2705 2710 2715 2720

Gln Thr Gln Ile Pro Asp Tyr Ala Glu Leu Ile Val Lys Phe Leu Asp

2725 2730 2735

Ala Leu Ile Asp Thr Tyr Leu Pro Gly Ile Asp Glu Glu Thr Ser Glu

2740 2745 2750

Glu Ser Leu Leu Thr Pro Thr Ser Pro Tyr Pro Pro Ala Leu Gln Ser

2755 2760 2765

Gln Leu Ser Ile Thr Ala Asn Leu Asn Leu Ser Asn Ser Met Thr Ser

2770 2775 2780

Leu Ala Thr Ser Gln His Ser Pro Gly Ile Asp Lys Glu Asn Val Glu

2785 2790 2795 2800

Leu Ser Pro Thr Thr Gly His Cys Asn Ser Gly Arg Thr Arg His Gly

2805 2810 2815

Ser Ala Ser Gln Val Gln Lys Gln Arg Ser Ala Gly Ser Phe Lys Arg

2820 2825 2830

Asn Ser Ile Lys Lys Ile Val

2835

Claims (10)

1.一种NF1新突变致病基因，其特征在于，所述新突变致病基因的核酸样本与NF1基因如SEQ ID NO：1所示的核苷酸序列相比，核苷酸变化：c.738delA。

2.权利要求1所述的NF1新突变致病基因，其特征在于，所述突变致病基因的核酸样本指人工分离或合成的单链DNA、双链DNA、RNA或DNA与RNA的聚合物。

3.一种视网膜变性疾病的NF1新突变体蛋白质，其特征在于，所述蛋白质的氨基酸序列与SEQ ID NO：2所示的氨基酸序列相比，氨基酸变化：p.E247KfsTer34。

4.根据权利3所述的蛋白质，其特征在于，所述蛋白质由权利要求1所述的基因编码的。

5.权利要求1或2所述的新突变致病基因在制备检测或筛选NF1病变的试剂盒中的应用。

6.筛选NF1病变的试剂盒的试剂盒，其特征在于，所述试剂盒包含检测权利要求1所述的NF1新突变致病基因的试剂，所述新突变致病基因与NF1基因如SEQ ID NO：1所示的核苷酸序列相比，具有核苷酸变化：c.738delA。

7.根据权利要求6所述的试剂盒，其特征在于，所述检测NF1新突变致病基因的试剂是核酸探针。

8.根据权利要求6所述的试剂盒，其特征在于，所述试剂盒包括序列如SEQ ID NO：15和/或SEQ ID NO：16的引物。

9.根据权利要求6所述的试剂盒，其特征在于，所述检测NF1新突变致病基因的试剂是检测致病基因所编码的蛋白。

10.根据权利要求9所述的试剂盒，其特征在于，所述蛋白的氨基酸与SEQ ID NO：2所示的氨基酸序列相比，具有p.E247KfsTer34突变。