具体实施方式

下面对本发明做进一步说明：

核酸，所述核酸具有下列目标片段，

所述目标片段与野生型FLNC基因相比(一种如SEQ ID NO.1)，所述目 标片段具有c.6451G>A突变；优选的，所述核酸为DNA，在一些情况下含有 该目标片段的RNA也在本发明范围内。

多肽，与野生型FLNC(一种如SEQ ID NO.2)相比，所述多肽具有 p.G2151S突变。

基因突变，与野生型FLNC基因相比，所述突变基因具有c.6451G>A突 变。

上述野生型FLNC，当FLNC基因其他位点单核苷酸或氨基酸序列位点发 生突变时，不影响本发明所涉及检测位点的保护，在目标位点突变与本发明 相同前提下，其他位点相对于SEQ ID NO.1或SEQ ID NO.2发生变化时也应 当在本发明范围内。

生物模型在制备心肌病和/或防治试剂中的应用，所述生物模型至少包括 下述之一

a.前述的具有c.6451G>A突变的核酸，

b.前述的具有p.G2151S突变的多肽，

c.前述的具有c.6451G>A突变的基因；

优选的，所述心肌病为限制性心肌病。

当在制备筛查试剂使用时，参考后续具体使用；当在制备防治试剂中使 用时，一些方式为作为药物的作用靶点，或其他生物药物制备过程中作为测 试、提取等步骤中应用，主要作为制药过程中的一些辅助试剂，比如在制药 过程的测试抑制剂作用效果中。

筛查心肌病生物样品的药剂，包含有

能检测FLNC基因突变体的试剂，与野生型FLNC基因相比，所述FLNC 基因突变体具有c.6451G>A突变；

优选的，所述FLNC基因突变体至少为下述之一

a.前述的具有c.6451G>A突变的核酸，

b.前述的具有p.G2151S突变的多肽，

c.前述的具有c.6451G>A突变的基因；

优选的，所述心肌病为限制性心肌病。

一些方式中，所述试剂含有核酸探针或引物；

优选的，所述引物包括

序列为GGCACCTACATCATCAACATCAA的正向引物，和/或

序列为CCATCAGTTAAACCTCCTCCTCTG的反向引物；

核酸探针一种方式可采用相近基因检测探针，可行即可，不做特别限定。

构建体，包括前述的核酸或前述的基因突变。

重组细胞，所述重组细胞通过前述构建体转化受体细胞或表达前述所述 的多肽而得。

用于制备防治限制性心肌病药物的重组蛋白，由前述的重组细胞表达。

抑制剂在制备防治限制性心肌病药物中的应用，所述抑制剂对至少下述 之一具有抑制作用：

a.FLNC基因的c.6451G>A突变，

b.FLNC基因多肽的p.G2151S突变。

防治心肌病药物，至少含有下述之一

a.FLNC基因的c.6451G>A突变抑制剂，

b.FLNC基因多肽的p.G2151S突变抑制剂；

优选的，所述心肌病为限制性心肌病，更优选为限制性舒张功能障碍。

其中，前述抑制剂包含现有客观可行药物试剂的同样也应当包含后续新 研制的一些药物试剂。

基因片段在制备心肌病药物中的应用，所述基因片段为FLNC基因的 c.6451位点单核苷酸为G的基因片段，优选的，所述心肌病为限制性心肌病。

防治心肌病药物，包括基因载体，所述基因载体含有

能将FLNC基因的c.6451位点单核苷酸A替换为单核苷酸G并表达的基 因片段，优选的，所述心肌病为限制性心肌病；基因载体一些表现形式为质 粒，腺病毒载体。通过替换突变基因，使其恢复成正常基因序列实现治疗， 起到防、治目的。目前，基因载体优选考虑已知且成熟的，当然未来新出现 能获得认可的也应当在本发明范围内。

下面结合一具体的案例进行说明：包含1名临床确诊的RCM患者在内的 RCM家系。首先通过病史采集、心电图以及超声心动图等检查方法对RCM 进行诊断，先证者即RCM患者进行全外显子测序，对测序获得的突变进行筛 选：(1)根据测序数据质量和突变位置和功能进行初步筛选；(2)根据突变 频率通过千人基因组计划数据库(http://www.1000genomes.org/)、ESP6500数 据库、ExAC(Exome Aggregation Consortium)数据库进行进一步筛选；(3) 通过软件SIFT、Mutation Taster以及PolyPhen-2等对突变进行功能预测和保 守性注释，筛选出高致病性的突变；(4)通过Sanger测序法对家系中的其他 成员进行突变筛查；(5)通过查询OMIM、PubMed等数据库对家系致病性突 变进行查新。

一、采集家系临床和外周血样本

在获得所有家系成员的知情同意后，采用一次性采血针和柠檬酸钠采血 管收集所有家系成员的外周血各5-10mL，在管壁做好标记后放4度冰箱以备 后续提取基因组DNA。家系图谱如图1，图中正方形代表男性，圆形代表女 性，黑色代表患者，箭头代表先证者，斜线代表离世。

二、全基因组DNA提取与质量检测

使用天根公司的TIANamp血液基因组DNA提取试剂盒，按照说明书提 取外周血基因组DNA。

利用NanoDrop2000分光光度计对上述提取的DNA样本进行浓度及纯度 测定。

三、全外显子测序

选取RCM患者的外周血基因组DNA，交送上海伯豪生物技术有限公司 进行全外显子测序，测序主要流程包括DNA质量检测、测序文库构建和测序 原始数据处理。具体如下：纯化后的DNA经2.0Fluorometer和1％琼 脂糖凝胶电泳进行质检，分析DNA降解程度以及是否有污染，DNA样品检 测合格后，利用超声波破碎方法将DNA破碎至180bp-300bp左右,并连上接头, 通过PCR构建捕获文库，利用Illumina HiSeq X测序仪进行双端150bp测序， 测序过程由Illumina提供的数据收集软件进行控制，采用主流的基因组比对软 件BWA将过滤后得到的高质量reads与人类参考基因组序列(UCSC hg19) 进行比对，并利用GAKT软件进行变异检测。

四、数据分析

根据突变频率通过千人基因组计划数据库(http://www.1000genomes.org/)、ESP6500数据库、ExAC(Exome Aggregation Consortium)Kaviar数据库等多 种数据库进行进一步筛选。

五、Sanger测序验证

利用Sanger测序将通过全外显子测序获得的候选突变位点在家系成员中 进行筛查，一方面可以进一步明确突变在该家系的致病性，另一方面可以对 家系中携带致病基因突变的表型正常的个体做到早期诊断。Sanger测序主要 实验流程如下：

1.突变引物设计

根据基因突变在基因组中的位置，在Ensembl数据库 (http://www.ensembl.org/)查询突变所在的人基因组序列，用获得的突变序 列，在引物设计软件PrimerPremier 5进行引物设计。用设计好的引物，在UCSC 网站上进行电子PCR，PCR结果显示可扩增出目的片段且条带单一，说明引 物特异性较好。

引物序列

正向：GGCACCTACATCATCAACATCAA，

反向：CCATCAGTTAAACCTCCTCCTCTG。

产物长度：773。

2.PCR扩增

扩增体系如下：

扩增步骤如图4中所示。

3.测序

扩增的家系DNA目的片段委托擎科生物公司进行Sanger测序。使用 Chromas2对测序结果进行分析，通过与正常序列对比找到基因突变。结果如 图2。

六、突变分析

经过系统查询，未在HGMD数据库，千人基因组计划数据库 (http://www.1000genomes.org/)、ESP6500数据库、ExAC(Exome Aggregation Consortium)Kaviar数据库中找到基因突变FLNC(c.6451G>A，p.G2151S)的相 关记录，Pubmed数据库中也没有该突变与RCM的相关文献报道，且患者父 母均不携带该突变。说明该突变是一个新发突变。如图3中，FLNC蛋白第 2151位的甘氨酸变成丝氨酸，该突变在物种间具有保守性。如图3中，通过 软件SIFT、Mutation Taster以及PolyPhen-2等对突变进行功能预测发现该突 变具有致病性。

美国医学遗传学与基因组学学会(ACMG)联合分子病理学学会(AMP) 发布了基因变异的解读标准及指导原则。指南提出的建议可应用于临床实验 室的各种基因检测方法，包括基因分型、单基因、基因panel、外显子组和基 因组。本指南建议使用特定标准术语来描述孟德尔疾病相关的基因变异—— “致病”、“可能致病”、“意义不明确”、“可能良性”和“良性”。此外，本指 南描述了基于典型的数据类型(如人群数据，计算数据，功能数据，共分离 数据)对变异进行五级分类的标准过程。目前该指南是国内外遗传检测机构 出具基因检测报告的重要标准。通过该ACMG/AMP 2015指南对本发明的基 因突变的临床评估为致病性。评估依据为：本发明为PS2 PM6新发变异，新 发变异是指患者自身携带的变异，而患者父母不带该变异。

本领域的技术人员可以明确，在不脱离本发明的总体精神以及构思的情 形下，可以做出对于以上实施例的各种变型。其均落入本发明的保护范围之 内。本发明的保护方案以本发明所附的权利要求书为准。

序列表

<110> 华中科技大学同济医学院附属协和医院

<120> FLNC基因突变体及其应用

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2520

<212> DNA

<213> 人(Huma)

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aactccccca ccaggtgatg actccatgag gacctcacag ctgaatgtgg gcacctccac 60

ggacgtgtca ctgaagatca ccgagagtga tctgagccag ctgaccgcca gcatccgtgc 120

cccctcgggc aacgaggagc cctgcctgct gaagcgcctg cccaaccggc acattggtga 180

gcgtggggcc tcacggggac ctcaggggtg ggggcccaca ggatgctctg cctaacaccc 240

actttccaca gggatctcct tcacccccaa ggaggtcggg gagcacgtgg tgagcgtgcg 300

caagagtggc aagcatgtca ccaacagccc cttcaagatc ctggtggggc catctgagat 360

cggggacgcc agcaaggtgc gggtctgggg caaggggctt tccgagggac acacattcca 420

ggtggcagag ttcatcgtgg acactcgcaa tgcaggtacc tcctgcccca gagagccccc 480

attccagcgg gtgcctccca caggcacttg tcctcgtcct gcccagcacc cccttggccg 540

cactctctcc tccctgaaac ttcctgacca gtctgcgtca ggattcgcat tctggggccc 600

cttgcgggaa agtgaatggc cccgcatcag ttctctccct tttaagagaa agctcagctg 660

tcctgagttt ctgtccctcc cttgctcact ggaatccaag aggcttacct tagggaattt 720

tccagaccgc ctgtcccgtg gtgcccccgc tcctcccact gagccatttt tgttagtggt 780

cactacacac atcggtgccc attctgggtg gagcctgcag tctggggaga ggaaagcatt 840

gtggcttggc cagcctagga ctgagggaga tgtgttcctt gctttccccc aggttatggg 900

ggcttggggc tgagtattga aggcccaagc aaggtggaca tcaactgtga ggacatggag 960

gacgggacat gcaaagtcac ctactgcccc accgagcccg gcacctacat catcaacatc 1020

aagtttgctg acaagcacgt gcctggtaag gctctgggca gaggtcggtg gcgagagaca 1080

gggaggccag gaggctgggg ctctgaggtt cctgacccac cctttgtccc cacttcagga 1140

agccccttca ctgtgaaggt gaccggcgag ggccgcatga aggagagcat cacccggcgg 1200

agacaggcac cttccatcgc caccatcggc agcacctgtg acctcaacct caagatccca 1260

ggtagaagcc tggaggaccc tgggtggggc gggtggtggg agagggctgg cccgggccag 1320

agcccacctg tcgggcctcc accctgcttc ctcacccctc gcttccctcc ctcaccctgg 1380

ctcccttgac cacacaggaa actggttcca gatggtgtct gcccaggagc gcctgacacg 1440

caccttcaca cgcagcagcc acacctacac ccgcacggag cgcacggaga tcagcaagac 1500

gcggggcggg gagacaaagc gcgaggtgcg ggtggaggag tccacccagg tcggcgggga 1560

ccccttccct gctgtgtttg gggacttcct gggccgggag cgcctgggat ccttcggcag 1620

catcacccgg cagcaggagg gtgagcaccg cacactgggc cggccgggtc ctcacggcgg 1680

gatgggaggg tgctgcggac caggcttgat gctggcagac tggccccgaa ggccagggca 1740

ggtctgagca gaggaggagg tttaactgat gggggaggga agggccaggg ctaggaggaa 1800

tcccagtgtt gccctgacat cccccaaacc ctgcaggtga ggccagctct caggacatga 1860

ctgcacaggt gaccagccca tcgggcaagg tggaagccgc agagatcgtc gagggcgagg 1920

acagcgccta cagcgtgcgc tttgtgcccc aggaaatggg gccccatacg gtcgctgtca 1980

agtaccgtgg ccagcacgtg cccggcagcc cctttcagtt cactgtgggg ccgctgggtg 2040

aaggtggtgc ccacaaggtg cgggccggag gcacagggct ggagcgaggt gtggccggcg 2100

tgccaggtaa ggggcaggtg gccaggagtg gggatgaagt cagggcagcc agtgtgaggg 2160

gcgatgatgc tgaagtccac taccttgcct gtccccagcc gagttcagca tctggacccg 2220

ggaggctggc gctgggggcc tgtccattgc tgtggagggt cctagcaaag cggagattgc 2280

atttgaggat cgcaaagatg gctcctgcgg cgtctcctat gtcgtccagg aaccaggtgg 2340

gcgtccacac tggcagtggg gctgggcctg cctgaccttc cagactgggt ttctgcccac 2400

tggccaggca ggagatgctt ggggccacag aactcccctc cccggagccc cctgctcttc 2460

ctctgccccg tctccctcta ccccacaccc tcagaaacat gtgtctgcct ccagatctga 2520

<210> 2

<211> 2692

<212> PRT

<213> 人(Huma)

<400> 2

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

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Ala Val Arg Tyr Met Pro Pro Glu Glu Gly Pro Tyr Lys Val Asp Ile

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Thr Tyr Asp Gly His Pro Val Pro Gly Ser Pro Phe Ala Val Glu Gly

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Val Leu Pro Pro Asp Pro Ser Lys Val Cys Ala Tyr Gly Pro Gly Leu

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Lys Gly Gly Leu Val Gly Thr Pro Ala Pro Phe Ser Ile Asp Thr Lys

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Gly Ala Gly Thr Gly Gly Leu Gly Leu Thr Val Glu Gly Pro Cys Glu

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Ala Lys Ile Glu Cys Gln Asp Asn Gly Asp Gly Ser Cys Ala Val Ser

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Tyr Leu Pro Thr Glu Pro Gly Glu Tyr Thr Ile Asn Ile Leu Phe Ala

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Glu Ala His Ile Pro Gly Ser Pro Phe Lys Ala Thr Ile Arg Pro Val

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Phe Asp Pro Ser Lys Val Arg Ala Ser Gly Pro Gly Leu Glu Arg Gly

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

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Glu Val Leu Ile His Asn Asn Ala Asp Gly Thr Tyr His Ile Thr Tyr

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His Pro Val Pro Lys Phe Pro Thr Arg Val His Val Gln Pro Ala Val

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Asp Thr Ser Gly Val Lys Val Ser Gly Pro Gly Val Glu Pro His Gly

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

1285 1290 1295

Gly Ala Lys Thr Asp Thr Tyr Val Thr Asp Asn Gly Asp Gly Thr Tyr

1300 1305 1310

Arg Val Gln Tyr Thr Ala Tyr Glu Glu Gly Val His Leu Val Glu Val

1315 1320 1325

Leu Tyr Asp Glu Val Ala Val Pro Lys Ser Pro Phe Arg Val Gly Val

1330 1335 1340

Thr Glu Gly Cys Asp Pro Thr Arg Val Arg Ala Phe Gly Pro Gly Leu

1345 1350 1355 1360

Glu Gly Gly Leu Val Asn Lys Ala Asn Arg Phe Thr Val Glu Thr Arg

1365 1370 1375

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

1380 1385 1390

Ala Lys Met Ser Cys Lys Asp Asn Lys Asp Gly Ser Cys Thr Val Glu

1395 1400 1405

Tyr Ile Pro Phe Thr Pro Gly Asp Tyr Asp Val Asn Ile Thr Phe Gly

1410 1415 1420

Gly Arg Pro Ile Pro Gly Ser Pro Phe Arg Val Pro Val Lys Asp Val

1425 1430 1435 1440

Val Asp Pro Gly Lys Val Lys Cys Ser Gly Pro Gly Leu Gly Ala Gly

1445 1450 1455

Val Arg Ala Arg Val Pro Gln Thr Phe Thr Val Asp Cys Ser Gln Ala

1460 1465 1470

Gly Arg Ala Pro Leu Gln Val Ala Val Leu Gly Pro Thr Gly Val Ala

1475 1480 1485

Glu Pro Val Glu Val Arg Asp Asn Gly Asp Gly Thr His Thr Val His

1490 1495 1500

Tyr Thr Pro Ala Thr Asp Gly Pro Tyr Thr Val Ala Val Lys Tyr Ala

1505 1510 1515 1520

Asp Gln Glu Val Pro Arg Ser Pro Phe Lys Ile Lys Val Leu Pro Ala

1525 1530 1535

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

1540 1545 1550

Gly Ile Pro Ala Ser Leu Pro Val Glu Phe Thr Ile Asp Ala Arg Asp

1555 1560 1565

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

1570 1575 1580

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

1585 1590 1595 1600

Ser Tyr Leu Pro Asp Met Ser Gly Arg Tyr Thr Ile Thr Ile Lys Tyr

1605 1610 1615

Gly Gly Asp Glu Ile Pro Tyr Ser Pro Phe Arg Ile His Ala Leu Pro

1620 1625 1630

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

1635 1640 1645

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

1650 1655 1660

Val Ile Thr Val Asp Ala Lys Ala Ala Gly Glu Gly Lys Val Thr Cys

1665 1670 1675 1680

Thr Val Ser Thr Pro Asp Gly Ala Glu Leu Asp Val Asp Val Val Glu

1685 1690 1695

Asn His Asp Gly Thr Phe Asp Ile Tyr Tyr Thr Ala Pro Glu Pro Gly

1700 1705 1710

Lys Tyr Val Ile Thr Ile Arg Phe Gly Gly Glu His Ile Pro Asn Ser

1715 1720 1725

Pro Phe His Val Leu Ala Thr Glu Glu Pro Val Val Pro Val Glu Pro

1730 1735 1740

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

1745 1750 1755 1760

Gln Lys Gly Glu Leu Thr Gly Glu Val Arg Met Pro Ser Gly Lys Thr

1765 1770 1775

Ala Arg Pro Asn Ile Thr Asp Asn Lys Asp Gly Thr Ile Thr Val Arg

1780 1785 1790

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

1795 1800 1805

Gly Asn His Ile Pro Gly Ser Pro Leu Gln Phe Tyr Val Asp Ala Ile

1810 1815 1820

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

1825 1830 1835 1840

Val Asn Lys Pro Ala Thr Phe Thr Ile Val Thr Lys Asp Ala Gly Glu

1845 1850 1855

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

1860 1865 1870

Cys Lys Asp Asn Lys Asp Gly Thr Cys Thr Val Ser Tyr Leu Pro Thr

1875 1880 1885

Ala Pro Gly Asp Tyr Ser Ile Ile Val Arg Phe Asp Asp Lys His Ile

1890 1895 1900

Pro Gly Ser Pro Phe Thr Ala Lys Ile Thr Gly Asp Asp Ser Met Arg

1905 1910 1915 1920

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

1925 1930 1935

Thr Glu Ser Asp Leu Ser Gln Leu Thr Ala Ser Ile Arg Ala Pro Ser

1940 1945 1950

Gly Asn Glu Glu Pro Cys Leu Leu Lys Arg Leu Pro Asn Arg His Ile

1955 1960 1965

Gly Ile Ser Phe Thr Pro Lys Glu Val Gly Glu His Val Val Ser Val

1970 1975 1980

Arg Lys Ser Gly Lys His Val Thr Asn Ser Pro Phe Lys Ile Leu Val

1985 1990 1995 2000

Gly Pro Ser Glu Ile Gly Asp Ala Ser Lys Val Arg Val Trp Gly Lys

2005 2010 2015

Gly Leu Ser Glu Gly His Thr Phe Gln Val Ala Glu Phe Ile Val Asp

2020 2025 2030

Thr Arg Asn Ala Gly Tyr Gly Gly Leu Gly Leu Ser Ile Glu Gly Pro

2035 2040 2045

Ser Lys Val Asp Ile Asn Cys Glu Asp Met Glu Asp Gly Thr Cys Lys

2050 2055 2060

Val Thr Tyr Cys Pro Thr Glu Pro Gly Thr Tyr Ile Ile Asn Ile Lys

2065 2070 2075 2080

Phe Ala Asp Lys His Val Pro Gly Ser Pro Phe Thr Val Lys Val Thr

2085 2090 2095

Gly Glu Gly Arg Met Lys Glu Ser Ile Thr Arg Arg Arg Gln Ala Pro

2100 2105 2110

Ser Ile Ala Thr Ile Gly Ser Thr Cys Asp Leu Asn Leu Lys Ile Pro

2115 2120 2125

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

2130 2135 2140

Phe Thr Arg Ser Ser His Thr Tyr Thr Arg Thr Glu Arg Thr Glu Ile

2145 2150 2155 2160

Ser Lys Thr Arg Gly Gly Glu Thr Lys Arg Glu Val Arg Val Glu Glu

2165 2170 2175

Ser Thr Gln Val Gly Gly Asp Pro Phe Pro Ala Val Phe Gly Asp Phe

2180 2185 2190

Leu Gly Arg Glu Arg Leu Gly Ser Phe Gly Ser Ile Thr Arg Gln Gln

2195 2200 2205

Glu Gly Glu Ala Ser Ser Gln Asp Met Thr Ala Gln Val Thr Ser Pro

2210 2215 2220

Ser Gly Lys Val Glu Ala Ala Glu Ile Val Glu Gly Glu Asp Ser Ala

2225 2230 2235 2240

Tyr Ser Val Arg Phe Val Pro Gln Glu Met Gly Pro His Thr Val Ala

2245 2250 2255

Val Lys Tyr Arg Gly Gln His Val Pro Gly Ser Pro Phe Gln Phe Thr

2260 2265 2270

Val Gly Pro Leu Gly Glu Gly Gly Ala His Lys Val Arg Ala Gly Gly

2275 2280 2285

Thr Gly Leu Glu Arg Gly Val Ala Gly Val Pro Ala Glu Phe Ser Ile

2290 2295 2300

Trp Thr Arg Glu Ala Gly Ala Gly Gly Leu Ser Ile Ala Val Glu Gly

2305 2310 2315 2320

Pro Ser Lys Ala Glu Ile Ala Phe Glu Asp Arg Lys Asp Gly Ser Cys

2325 2330 2335

Gly Val Ser Tyr Val Val Gln Glu Pro Gly Asp Tyr Glu Val Ser Ile

2340 2345 2350

Lys Phe Asn Asp Glu His Ile Pro Asp Ser Pro Phe Val Val Pro Val

2355 2360 2365

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

2370 2375 2380

Glu Thr Gly Leu Lys Val Asn Gln Pro Ala Ser Phe Ala Val Gln Leu

2385 2390 2395 2400

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

2405 2410 2415

Ala Val Glu Glu Cys Tyr Val Ser Glu Leu Asp Ser Asp Lys His Thr

2420 2425 2430

Ile Arg Phe Ile Pro His Glu Asn Gly Val His Ser Ile Asp Val Lys

2435 2440 2445

Phe Asn Gly Ala His Ile Pro Gly Ser Pro Phe Lys Ile Arg Val Gly

2450 2455 2460

Glu Gln Ser Gln Ala Gly Asp Pro Gly Leu Val Ser Ala Tyr Gly Pro

2465 2470 2475 2480

Gly Leu Glu Gly Gly Thr Thr Gly Val Ser Ser Glu Phe Ile Val Asn

2485 2490 2495

Thr Leu Asn Ala Gly Ser Gly Ala Leu Ser Val Thr Ile Asp Gly Pro

2500 2505 2510

Ser Lys Val Gln Leu Asp Cys Arg Glu Cys Pro Glu Gly His Val Val

2515 2520 2525

Thr Tyr Thr Pro Met Ala Pro Gly Asn Tyr Leu Ile Ala Ile Lys Tyr

2530 2535 2540

Gly Gly Pro Gln His Ile Val Gly Ser Pro Phe Lys Ala Lys Val Thr

2545 2550 2555 2560

Gly Pro Arg Leu Ser Gly Gly His Ser Leu His Glu Thr Ser Thr Val

2565 2570 2575

Leu Val Glu Thr Val Thr Lys Ser Ser Ser Ser Arg Gly Ser Ser Tyr

2580 2585 2590

Ser Ser Ile Pro Lys Phe Ser Ser Asp Ala Ser Lys Val Val Thr Arg

2595 2600 2605

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

2610 2615 2620

Val Asp Cys Ser Lys Ala Gly Thr Asn Met Met Met Val Gly Val His

2625 2630 2635 2640

Gly Pro Lys Thr Pro Cys Glu Glu Val Tyr Val Lys His Met Gly Asn

2645 2650 2655

Arg Val Tyr Asn Val Thr Tyr Thr Val Lys Glu Lys Gly Asp Tyr Ile

2660 2665 2670

Leu Ile Val Lys Trp Gly Asp Glu Ser Val Pro Gly Ser Pro Phe Lys

2675 2680 2685

Val Lys Val Pro

2690