CN108315430B - Pdl1 snp基因型作为预测乳腺癌新辅助化疗疗效标记物的用途 - Google Patents
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Abstract
本发明提供了PDL1 SNP基因型在制备诊断预测乳腺癌新辅助化疗疗效标记物的用途。还提供了PDL1 SNP基因型作为标记物在制备诊断预测乳腺癌新辅助化疗疗效的试剂中的用途。还提供了一种用于检测PDL1 SNP基因型的试剂盒，含有SEQ ID NO.1~18所示的引物。本发明发现在所有乳腺癌人群以及HR阳性的人群中，PDL1基因非编码区SNP rs4143815 C至G改变后，GC基因型患者较GG和CC基因型患者更容易达到pCR。通过病人血液中的PDL1基因非编码区SNP的检测，尽早的发现GC基因型患者，进行新辅助化疗，以便达到病理的完全缓解，提高生存的时间。
Description
技术领域
本发明属于医学检测领域，涉及一种预测乳腺癌新辅助化疗疗效的标记物，具体来说是PDL1 SNP基因型作为预测乳腺癌新辅助化疗疗效标记物的用途。
背景技术
乳腺癌是全球女性最常见的恶性肿瘤之一。根据最新全球肿瘤流行病统计数据(GLOBOCAN)报道，仅2012年一年，全球就有167万例新发乳腺癌病例，并有52.2万人死于乳腺癌。同年中国乳腺癌的年龄标准化发病率达到了十万分之21.6人。中国的乳腺癌发病率虽然仍低于发达国家(北美和北欧等国家高达十万分之100人)，但从上世纪八十年代起一直呈上升趋势，增长速度是全球平均水平的2倍多。在我国东南沿海等经济发达地区，乳腺癌已经成为女性发病率第一的恶性肿瘤。
居高不下的发病率吸引了全球医务工作者对乳腺癌的关注。多年来，乳腺癌的治疗模式已从十九世纪单一的手术治疗，逐渐转变成为现在手术、化疗、放疗、靶向治疗相结合的综合治疗模式。其中，乳腺癌新辅助化疗(Neoadjuvant chemotherapy，NAT)也已经成为乳腺癌治疗的常规项目。新辅助化疗不仅仅可以为局部晚期患者降低肿瘤负荷，提供手术机会和保乳机会，另一方面还可以早期评估患者对治疗的反应性，从而调整后续治疗，预测患者的远期预后。新辅助化疗的病理完全缓解(Pathological complete response，pCR)指治疗后手术标本的原发灶，或原发灶加腋下***，无肿瘤细胞残留或仅有导管内癌成分残留。多项大型临床试验证实，在NAT中取得pCR的患者的预后远好于没有取得pCR的患者。然而，并不是所有患者都可以在相同的NAT治疗中取得pCR结果，因此，如何早期预测患者对NAT的治疗反应，发现pCR的预测指标便成为了现阶段研究的重点和热点。
细胞程式死亡-配体1(Programmed death-ligand-1，PDL1)是一种细胞膜跨膜糖蛋白。该蛋白主要表达在T细胞、NK细胞、树突样细胞以及肿瘤细胞细胞膜表面，其功能主要与免疫抑制相关。细胞程式死亡1(Programmed death-1，PD1)则主要表达在T细胞膜表面，与其配体PDL1相结合后，可以引起T细胞凋亡以及诱导T细胞向抑制T细胞分化。先天性PDL1表达缺失常与自身免疫性疾病相关，比如***性红斑狼疮。而后天性的PDL1表达改变，则和肿瘤的免疫逃逸相关。
基础实验提示，PDL1受多种细胞通路调节及影响，比如MAPK以及PI3K/AKT通路。表观遗传学方面，PDL1也受到许多miRNA(Micro-Ribonucleic acid，miRNA)调控，从而参与上皮细胞-间充质转化(Epithelial-Mesenchymal Transition，EMT)等肿瘤发展进程。另外，在胃癌以及肺癌的研究中还有报道显示，PDL1非编码区的单核苷酸多态性(SingleNucleotide Polymorphism，SNP)同时与肿瘤发病风险和预后相关。
SNP是指基因组中特定碱基的多态性改变，与基因突变不同，一个位点上的某一突变碱基需要在人群中达到一定出现频率(大于1/1000)才能被称之为SNP。人群中SNP变化往往和疾病发病、药物代谢与疗效相关。根据SNP位点在基因组上的分布不同可以将其分为编码区SNP和非编码区SNP。编码区SNP可分为同义替换和非同义替换。两者可以分别通过直接改变蛋白产物或是影响翻译进程，最终影响蛋白功能。而非编码区的SNP虽然不参与蛋白合成，但是可以通过影响基因剪切、转录因子结合、mRNA降解等多种生物学过程从而影响细胞功能。非编码区SNP突变常被报道与肿瘤发病相关。前文所述PDL1SNP在胃癌与肺癌中的临床价值正是非编码区SNP体现的。
申请人开展的紫杉醇联合顺铂周疗的新辅助临床试验(SHPD001及SHPD002)已获得了初步结果，展现出了较高的pCR率。其中，在总人群中pCR率为34.4％，在人表皮生长因子受体2(Human epidermal growth factor 2，HER2)阳性型乳腺癌和三阴型乳腺癌中更是高达64.7％和52.4％，这种疗效一般认为和节拍化疗以及免疫调节相关。然而，在激素受体(Hormone receptor，HR)阳性患者中，pCR率则不太理想，因此我们更希望在这部分患者中找到pCR的预测指标，从而提高治疗效果。有文献提示，顺铂可以提高肿瘤细胞PDL1的表达水平，这一药物也正是我科NAT临床试验患者所采用的化疗药物之一。
发明内容
本发明的目的在于提供PDL1 SNP基因型作为预测乳腺癌新辅助化疗疗效标记物的用途，所述的这种用途要解决现有技术中没有合适的生物标记来预测乳腺癌患者新辅助化疗疗效的技术问题。
本发明提供了PDL1 SNP基因型在制备诊断预测乳腺癌新辅助化疗疗效标记物的用途。
进一步的，在所述的PDL1 SNP基因型中，rs4143815C>G。
进一步的，所述的乳腺癌为激素受体阳性。
本发明还提供了PDL1 SNP基因型作为标记物在制备诊断预测乳腺癌新辅助化疗疗效的试剂中的用途。
本发明还提供了PDL1 SNP基因作为标记物在制备诊断预测乳腺癌新辅助化疗疗效的试剂中的用途，所述的PDL1 SNP基因的序列如SEQ ID NO.20所示。
本发明还提供了一种用于检测PDL1 SNP基因型的试剂盒，含有SEQ ID NO.2～19所示的引物。
本发明通过检测患者新辅助化疗前的全血标本中的SNP基因型，如果发现PDL1基因非编码区SNP rs4143815C至G的改变，则表示该患者更容易达到pCR；从而预测到该患者对治疗具有敏感性和耐受性。GC基因型患者较GG和CC基因型患者更容易达到pCR；而GG基因型患者较GC和CC基因型患者更容易出现DFS事件，生存较差。
本发明和已有技术相比，其技术进步是显著的。本发明发现在所有人群以及HR阳性的人群中，PDL1基因非编码区SNP rs4143815C至G改变后，GC基因型患者较GG和CC基因型患者更容易达到pCR；而GG基因型患者较GC和CC基因型患者更容易出现DFS事件，生存较差。通过病人血液中的PDL1基因非编码区SNP的检测，尽早的发现GC基因型患者，这样可以进行新辅助化疗，以便可以达到病理的完全缓解，提高生存的时间。如果发现新辅助化疗效果不好的，采用手术等其它的手段，避免了过度治疗，不会延误手术时机。
附图说明
图1显示了不同PDL1 SNP基因型患者的无病生存时间；Log-rank P value＝0.01。
图2显示了HR阳性亚组中不同SNP基因型患者的无病生存时间，Log-rank p＝0.003。
具体实施方式
实施例1
为了明确PDL1基因(如SEQ ID NO.1所示)非编码区SNP基因型对乳腺癌新辅助化疗敏感性的预测作用，尤其是为了明确其在HR阳性乳腺癌中的预测作用；同时，观察不同基因型患者对治疗的耐受性和安全性。我们假设在所有人群以及HR阳性乳腺癌中，PDL1 SNP基因型可以预测患者对治疗的敏感性和耐受性，并通过检测患者新辅助化疗前的全血标本中的SNP基因型进行敏感性分析。
1材料与方法
1.1主要仪器
1.2主要试剂盒耗材
1.3入组患者与标本采集
所有入选的病例来自于上海交通大学医学院附属仁济医院乳腺外科的两个临床试验入组的患者。两个临床试验分别在ClinicalTrials.gov网站上注册为SHPD001(NCT02199418)，SHPD002(NCT02221999)。两组患者皆接受紫杉醇联合顺铂的单周新辅助化疗方案，其中SHPD002患者同时随机接受内分泌治疗。
留取患者治疗前全血标本用于后续SNP检测。整理统计患者基本信息和肿瘤数据，包括患者年龄、月经状态、家族史、术前原发肿瘤大小、穿刺标本雌孕激素受体、穿刺标本HER-2受体、化疗期间不良反应及化疗后是否PCR。两个临床试验皆已通过上海交通大学医学院附属仁济医院伦理委员会审批，所有患者皆签署知情同意书，自愿入组并为相关医学研究提供标本及临床资料。
1.4SNP位点选择
通过公共数据库(NCBI/TargentScan)，按以下标准选取所检测的SNP位点:A.罕见等位基因频率(Minor Alleles Frequency，MAF)>0.05且B.位于PDL1基因的3’UTR区，或者位于PDL1基因其他非编码区域但是在别的肿瘤中报道过存在预测或预后价值。共计9个SNP位点入选。标本DNA提取、SNP基因型检测皆由上海邃志生物科技公司完成。
1.5DNA提取：采用TIANamp Genomic DNA Kit试剂盒进行。
A.在样本血液中(200μl)加入20μl Proteinase K溶液，混匀。
B.混合液中加入200μl缓冲液GB，70℃x 10min。
C.上步所得混合液中加入200μl 100％乙醇，振荡15s。
D.将以上溶液加入一个吸附柱CB3中，12000rpm离心30s，弃废液，将吸附柱CB3置于收集管。
E.向吸附柱CB3中加入500μl缓冲液，12000rpm离心30s，弃废液，将吸附柱CB3置于收集管。
F.向吸附柱CB3中加入600μl漂洗液PW，12000rpm离30s，弃废液，将吸附柱CB3置于收集管。
G.重复操作步骤F。
H.将吸附柱CB3放回收集管中，12000rpm离心2min，倒掉废液。将吸附柱CB3置于室温放置数分钟晾干。
I.将吸附柱CB3放在另一干净离心管中，向吸附膜中间悬空滴加100μl洗脱缓冲液TE，室温放置2-5min，12000rpm离心2min，将溶液收集于离心管中。
1.6PCR反应
下述的引物通过Assay Design Suite V2.0 Online在线设计。
引物序列：
A.以384孔板为例，在每孔中加入1μl DNA样品，离心后备用。
B.以Qiagen公司试剂盒进行PCR，反应体系如下：
C.在每孔DNA样品中加入4μl反应体系，将384孔板置入PCR仪，反应程序如下：
D.PCR结束后，384孔板备用。
1.7SAP处理
A.以SEQUENOM试剂盒进行SAP反应液配置，体系如下：
B.在384孔板PCR反应后体系中每孔加入2μl SAP反应体系。
C.进行SAP程序如下：
D.SAP结束后，384孔板备用。
1.8延伸反应
A.以SEQUENOM公司试剂盒配制iPlex反应体系，反应体系如下：
B.在384孔板SAP反应后体系中每孔加入2μl延伸反应体系。
C.进行延伸程序如下：
D.延伸结束后，384孔板备用。
1.9产物纯化
A.将少许树脂放在384孔树脂刮板上，刮除多余树脂使其均匀覆盖，室温放置20min。
B.延伸后的384孔板1000rpm离心1min，每孔加25μl去离子水，倒置于树脂板。反扣树脂板，使树脂落入384孔板中，封膜。C.以384孔板的长轴为轴心，翻转384孔板20min，3500rpm离心5min备用。
1.10SNP基因型检测
A.Nanodispenser SpectroCHIP芯片点样；
将检测样品从384孔反应板转移到表面覆盖基质的MassARRAY SpectroCHIP芯片。
B.MassARRAY Analyzer Compac质谱检测；
将样品转移到SpectroCHIP芯片后，即可放入质谱仪进行检测，每个检测点只需3-5秒，全自动分析。
C.TYPER软件分析实验结果，获得分型数据。
1.11统计学分析
SNP基因型的哈迪-温伯格平衡通过卡方检验完成。不同SNP间的连锁不平衡分析通过HaploView软件(http://broad.mit.edu/mpg/haploview)完成。对SNP基因型进行分析时，共建立了四种模型：假设某一SNP位点上，“T”为常见等位基因，而“A”为罕见/突变等位基因，那么显性模型比较TT vs TA+AA的差别；隐性模型比较TT+TA vs AA的差别；共显性模型比较TT+AA vs TA的差别；累加模型比较TT vs TA vs AA的差别。
PDL1SNP位和副反应间的相关关系通过Spearman相关分析进行计算；PDL1 SNP不同基因型和pCR间的相关性通过logistic回归模型进行单因素及多因素分析。本发明使用无病生存(DFS)进行生存状态评估，定义为从手术切除病灶到以下任一事件发生：1、远处转移；2、同侧区域局部复发；3、对侧乳腺癌；4、死亡。不同组间生存曲线对比通过Kaplan-Meier方法绘制，并通过log-rank检验比较生存率差别，COX风险模型进行多因素调整、计算HR及95％CI。
本发明通过STATA 12.0以及SPSS 23.0对数据进行分析，双侧p<0.05为存在统计学差异。
2实验结果
2.1 PDL1 SNP表达的预测价值
本发明一共检测了PDL1基因非编码区的9个SNP位点，每个位点的位置和基因型频率见表1。其中rs7864231和rs10217310两个位点由于未能测到突变型，故在文中未进行后续分析。其余的7个位点经过检验，都符合哈迪温伯格平衡，故纳入后续分析。通过多因素logistic回归分析，我们发现PDL1的一个非编码区SNP位点，rs4143815C到G改变(rs4143815C>G)的累加模型(OR1＝9.284，p1＝0.049；OR2＝13.501，p2＝0.023)和共显性模型(OR＝2.800，p＝0.042)与患者新辅助治疗pCR相关(表1)。通过进一步计算，该改变的显性模型还与PR表达相关(p＝0.043)。
表1不同SNP基因型和pCR的关系
a：p<0.05，有统计学差异。
b：多因素分析调整了年龄、肿瘤大小、ER、PR、HER2和ki67。
2.2 PDL1 SNP表达的预后价值
中位随访18个月时，存在rs4143815C>G(SEQ ID NO.20所示)的患者展现了不同的DFS。其中，Kaplan-Meier图显示，GG基因型的患者生存劣于GC+CC型的患者(Log-rank p＝0.01；图1)。COX多因素风险模型也提示rs4143815GG基因型患者复发风险较高(HR＝20.098，95％CI 2.774-145.596；p＝0.003；详见表2)，为DFS的独立预测因素。另外，患者的年龄(HR＝0.125，95％CI 0.024-0.656；p＝0.014)、ki67状态(HR＝1.050，95％CI 1.015-1.088；p＝0.005)、术后***状态(HR＝9.750，95％1.894-50.194；p＝0.006)也是DFS的独立预测因素。
表2 PDL1 SNP基因型与生存关系(COX多因素风险模型)
a：p<0.05，有统计学差异。
2.3副反应分析
对不同rs4143815基因型的患者进行副反应分析，发现在CC+CG与GG患者相比更容易出现中性粒细胞减少(rho＝-0.183，p＝0.032)，而不容易出现水肿(rho＝0.179，p＝0.036)的不良反应。而CC+GG与CG患者相比，更容易出现谷草转氨酶升高(rho＝-0.186，p＝0.030)。具体不良反应谱见表3。
表3不同rs4143815基因型患者的副反应
Table 3Adverse events according to different genotype of rs4143815
a：p<0.05，有统计学差异。
2.4亚组分析
亚组分析中，我们在HR阳性乳腺癌中观察到了与总人群类似的结论。
在预测pCR方面，HR阳性乳腺癌中多因素logistic回归分析显示，rs4143815C>G的患者GC基因型比GG+CC型易于pCR(OR＝2.362,p＝0.022；表4)。
在生存方面，HR阳性乳腺癌中Kaplan-Meier图显示GG基因型生存劣于GC+CC型组患者(Log-rank p＝0.003；图2)。COX多因素风险模型则提示rs4143815C>G的患者GG基因型比GC+CC型复发风险较高(HR＝10.781，p＝0.008；表4)。在HER2阳性BC中未观察到PDL1的预测价值，由于TNBC乳腺癌患者例数较少，未能进行有效的亚组分析。
表4HR阳性亚组中PDL1SNP基因型与pCR及DFS关系
a：p<0.05，有统计学差异。
3结论
我们在本发明中讨论了患者PDL1 SNP不同基因型对新辅助化疗pCR的预测价值以及远期的预后价值。这是首次在乳腺癌患者中报道PDL1 SNP基因型的新辅助化疗的预测和预后价值，也是首次在HR阳性乳腺癌中报道了PDL1 SNP基因型的预测和预后价值。同时还是首次观察到了PDL1 SNP基因型和新辅助化疗副反应间的关系。
我们发现在所有人群以及HR阳性的人群中，PDL1基因非编码区SNP rs4143815C至G改变后，GC基因型患者都较GG和CC基因型患者更容易达到pCR；而GG基因型患者较GC和CC基因型患者更容易出现DFS事件，生存较差。
Rs4143815位于PDL1基因非编码3’UTR区。非编码区的SNP虽然不参与蛋白合成，但是可以通过影响基因剪切、转录因子结合、mRNA降解等多种生物学过程从而影响细胞功能。本发明发现了rs4143815这一位点的临床预测价值。一方面，该位点的基因多态性变化引起PDL1蛋白表达的变化，从而影响细胞功能。在IFNγ的诱导下，rs4143815CC基因型携带者体内巨噬细胞/单核细胞表面的PDL1蛋白表达远高于GG基因型个体。
序列表
<110> 上海交通大学医学院附属仁济医院
<120> PDL1 SNP基因型作为预测乳腺癌新辅助化疗疗效标记物的用途
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20065
<212> DNA
<213> PDL1基因(Homo sapiens)
<400> 1
ggcgcaacgc tgagcagctg gcgcgtcccg cgcggcccca gttctgcgca gcttcccgag 60
gctccgcacc agccgcgctt ctgtccgcct gcaggtaggg agcgttgttc ctccgcgggt 120
gcccacggcc cagtatctct ggctagctcg ctgggcactt taggacggag ggtctctaca 180
ccctttcttt gggatggaga gaggagaagg gaaagggaac gcgatggtct agggggcagt 240
agagccaatt acctgttggg gttaataaga acaggcaatg catctggcct tcctccaggc 300
gcgattcagt tttgctctaa aaataattta tacctctaaa aataaataag ataggtagta 360
taggataggt agtcattctt atgcgactgt gtgttcagaa tatagctctg atgctaggct 420
ggaggtctgg acacgggtcc aagtccaccg ccagctgctt gctagtaaca tgacttgtgt 480
aagttatccc agctgcagca tctaagtaag tctcttcctg cgctaagcag gtccaggatc 540
cctgaacgga atttatttgc tctgtccatt ctgagaaccc aaaggagtcc taaaagagga 600
atggaggagc ctaagaataa aaatagtata ataaaacatt tcttagacac attgaccttg 660
gcctatgtca aagttcagtc tgggtttgtc ttataacaca aggagtaaaa gtaccattgt 720
tctacctctt tttttaatac ttgaaaaaaa tttactgtgg atgcttttct atgaattaaa 780
taaccttcta aaaaatgttt tcattgctgc attcgattag attgggtaac taaatgaaat 840
taattcctca ctgttgggta taaaggttat ttacagtggt tctgtcttag ccattcactg 900
aactcattgc atatatatct ctggaatatt gctgattgtt tccttcaagt aaacttagaa 960
gtgtaactac ttagtcaaag agcctgaata ttttaaaggc cttttgaaga aaactgaaaa 1020
tgctttccag aaaggatgta tcagttgaca atgacagtcg tcaacagtat ttaaggagaa 1080
ctatgatact ctgaagaaaa acttagcctt tctcagtaaa agtaggtagg cagaggccac 1140
atgacagcag ttagagtgtg gtcttcaagg aagtcacaga aatactgtgg ggaattgaaa 1200
ccccatgtgg aaaatgtaca agagtgtctc agtgtgactg agaaggaggt tgggcatggg 1260
gtttcatgga gtttaataaa gtttggtcac ttagtagagg tttaataaat caactgtctt 1320
aatctttgat cctacttaag aatttttttt ttgtttttgt agagatgggg ctcttgttat 1380
gttgcccagg ctgttctcga actcctagcc tcaggcgatc ctccctcctc aggctccaga 1440
agtcctggga ttactggcgg gagccaccat gcaggcctct tgctcctact tttgagaaag 1500
gaagtttaac cggttttttt tgtctttttt tttttttttt tgagacagag tctcactctg 1560
ttgcccatgc tggagtgcag tggtgcaatc tcagctcact gcctcccggg ttcaagtgat 1620
tctcctgcct cagcctcccg agtagctggg actacaggca cctgccacca cgcccagcta 1680
atttttgtat ttttagtaga aatggggttt caccatattg gccaggctga tctcgaactc 1740
ctgacctcag gtgatccgcc tgcctcggcc tcccaaagtg ctgggattac aggcatgagc 1800
cactgctcct ggctgcttaa ctttttctct atctcatcct cctacccatc ctacccttgg 1860
aagatagaga agtagtatta gttccatagt gttatactgg gcttccccca gggacaaacc 1920
cacttcccca acctgaatga gccatcactt cttccccagt ttacatttca ttgctcttta 1980
aatgtctcca ttcggatatg ggaattcaca tatggtcata attcttacct gaagaagatg 2040
tcagtcttct tctcttagac caactgccct gatatgaggt ttagaggtta aagaacatgt 2100
gtgtatttac atgatctttg tattctgcct tttcgtccct cactaatgac agctgcaccc 2160
caaggaaatg gagctgtgga agagagggtt tgataagaaa ttaagtaaat attggatcta 2220
atccatcacc ctccaggaag cctttattac tcctaaaaat ttcaaccaaa ttcattaaag 2280
gacaagaact ccaccagagt aggccataaa cattggcaaa attagttgta atccatgact 2340
agatttaatg tccctttgtt ttattcccat atggttataa tgctttgctt ggcattaggg 2400
gtattttaag ttttcttctg cctagtaagt gaatttgtgt ttataataca ataatcataa 2460
aatatcacat taatatttta taactgtaca gttataaaat attttataag taatatttat 2520
attttataag taatatttta taactgtaca gttaactctg gcccaaggaa aagatagtct 2580
gatagatgct gcagccccat tttagcaaat gtgacctcac aggcctgaat gccatcgcta 2640
ttccacatct acaggataga cggaaaggaa agaaataaaa aaataggtac ctaacactgg 2700
caagaggatg atgactcatg ttatttcact taaccttttt atcttttaac atgaaggact 2760
catacaggtt gataagaaac cagtgacata aacagaccaa aaaatgatca gatctttcaa 2820
attagcaaaa aaataatatt ttttaaacaa tgggtgaaaa tacagtgtaa cagtaccaat 2880
tatcaacatg tgttgagaac cagaaaaatg ttctttttct ttgatcagca acactatttg 2940
ggaaaatcta tcctcagggc ctagcctggg gccctggcac acagtaggca ctcaacgaat 3000
atttgctgaa cacacaaata cttatgatat tttaaaaaat tggcaacaat ctgataccta 3060
acaatagagg gattaaatat tatggaactg ttaaataaga tgcttatgaa taccatgcag 3120
taagatgggc aatatttatg ccataagctt taatgaaaca aatgggtatt aaatgtatga 3180
taaggttata aattactttt taaaagatta cagggaaaaa aattgaaaga tatacactga 3240
aatgtttttt gctcacagtg gtgacaaggt ttctcagcac tggcactgtt gacgttttag 3300
gctgtatgtc tttgctgtgg gaggctggcc tgtgcactgc agggtgtttg gcagcactct 3360
tggcctctgc ccctagatag caatagcagt cctccctcaa ccagcccaat tttgacaacc 3420
aaaaatgttt ccaggcatca ccagatgctc cctgggtgag agtgatgaaa tagtagggga 3480
ttttcccctt cttttcttat tttctgtaat tccattatat tactttaata ataaagaaaa 3540
aaacataaaa aataaacgaa tgttattatt ctacgtcagt ttggatgttt ggactccatt 3600
ttggggttct ttccattata tcacttggtc tgctaaacat tctacggttt ggtaaggtga 3660
agtgattcat gaaattttgg ttttattttt ttcctgatac taaaaataaa acattctttc 3720
acttggaaat ttggacacag aacaccaaaa aaaatccata atctcatctc tctttttctg 3780
tcttttcctt ccttttttcc ctttaaaaac aataaagagt gaaacctacc tgttctccct 3840
ctaatttaat tcctaaatat aatcactgtc aatatcttgg acatttcctg tgtctaaaca 3900
cacacacaca cttttttttt tcagcaaaag tggatttctg ctacatgtag tgttctgcaa 3960
cttactttct atgtgtttac aaaatcagta catgtacata tgctgaattc agtccttaat 4020
ggtattatat tttgtgaata taccaaaatt tgtttaacca cttagacaat ctaggatatt 4080
ctcagtttgc tgttatgagc aatgctcttc ctttacatat acagacatat atatatatat 4140
gtgtgtgtgt gtgtttttgt tttagtagga tagatttcta ggagagggtg aaaggtctta 4200
tgacatccgc atttacgatt gtaataggaa gtatcaaagt gccccctaaa gaaaaaaatc 4260
ctcccattag tgggtaagaa agcctatttg ttcatatctt cacaaacact aaatattaga 4320
aatatttaca attgtggtca agctcataag tgaaaatggt atttcatatc ttatattttt 4380
tattgtgaga ttgaacatct ttcatatgtt tacatgtcac ctgtatttct tattctctga 4440
actatatgtt atgacctttc actttttttc ctcatgggtt atgtgtagtt tgtatagttg 4500
tcttattgat tgttaggagc tatttatata ttaggaacat taatctcctg tcttatatat 4560
acgtggcatc gattagttga tcatttgtga gttcatgtct gtatacaaag attggagagg 4620
cactaagagg gaaaacttac ctctttctta tcaaagtttg taaatatatg tataacagaa 4680
gagggagaaa atattaataa atgcacagat tggctgaaat agagtataaa tcttttactc 4740
ccctacttca acataaactg caaaaggaga gtgacttttc tttcactctg acttccgtat 4800
tcctcatgct taaaatagtg cctagcacag aagaggtgct caatcagtgt ttgctaaacg 4860
aaataattag tcacatttca agcaggatga ctaaatgaag aatagaatct aggcagatac 4920
tctggaagag tggctgtgag tcattcatat cttagtatga attagtcaaa tccaactctc 4980
tccccttccc actccccact gttagtagaa gaatctgttt attgagagaa tagatttata 5040
atttagaata agtgagaggg gcagaagagg agattttgaa ggatggcacc tgaaggagga 5100
ctagcatggc tgagacagtg aagtggaagc cttgaatagc taaagggtaa gatgaaagta 5160
tttagctgta gggggaaaaa gcattgacag gttggaaaag taaaagtcag attctccttg 5220
ctctgaaatt ttgtacaggg caggttctac taggtatgtt acaatgcaga aaaaacatga 5280
aataattgag aggaatttgg tgcaatatta tcttcttggc ttcttttgag tgggcagatt 5340
tttttcacgg cctgtaacta taataaattt gaaacttctc atcttttagt aacttttttc 5400
acttaagttt atgtggctgt gggcaatgga atgaagatat tgaacttcca attccctgtt 5460
gggtttccac aattacaagt caatcatgac tggttattag aagactattt cagttagaac 5520
caccaagtcc catattgtca tattgtatgt ttaattatta agtgaagcag tcttcttttc 5580
gtgttttcca taattagggc attccagaaa gatgaggata tttgctgtct ttatattcat 5640
gacctactgg catttgctga acggtaagac accaaatcct tccattaggt tctatatttt 5700
aaatatttta accatgagtt taaaactaaa atgatcattt aaaatgcatg caattttctt 5760
atagagagaa cattctattc tttcttctac tttacacaat ggcaaagtct tctttctact 5820
ttacgcaatg ataaagttac ctgtgtcatt ttgtaaaaat atagagaata tagacaaatt 5880
gaaagacaca aaataatcta ttacccattt cccagggtta actactgaaa atatctgggg 5940
aaatggcctg tatgtataca tttatttgtt tgctttcaac aaggccaaga tcctttgatc 6000
tttcagtctt ggttgctctg tgacatgcct ttcctgatga ggatacttta aggaagaatt 6060
gtaagataca tggaaaatgt caggctaaca cagtactggc atcaccctgt gctctttcct 6120
gaactccata ccaatgtact tcttgccaga aaactgatca aaagtttagg gaagtaaaaa 6180
gagatgactg ttagaatcta ccattccctc tatgtaggaa gcaaataggt gtcctgtcaa 6240
aggacattct ggggatgtct acatgaaacc aagtctccct ggttgtaagg actccatctc 6300
catataatat ttatacagta atatatgttt ataaattgtg ggggcaactt gtttagctaa 6360
ttttattatt ctgctattgg gacactgtgt ctcagcatga gatatagtgt cccaaaacat 6420
atttcaagcc cattggataa aatatgtgtt tagcaagttc ttaaatataa tgataacata 6480
accgaccaga taaagtgatt tataaacgct gtgccaattt tgtaaatgtt tcgaggaatt 6540
ttcccttttc tgaagattgt ccttctttct ttttagcatt tactgtcacg gttcccaagg 6600
acctatatgt ggtagagtat ggtagcaata tgacaattga atgcaaattc ccagtagaaa 6660
aacaattaga cctggctgca ctaattgtct attgggaaat ggaggataag aacattattc 6720
aatttgtgca tggagaggaa gacctgaagg ttcagcatag tagctacaga cagagggccc 6780
ggctgttgaa ggaccagctc tccctgggaa atgctgcact tcagatcaca gatgtgaaat 6840
tgcaggatgc aggggtgtac cgctgcatga tcagctatgg tggtgccgac tacaagcgaa 6900
ttactgtgaa agtcaatggt aagaattatt atagatgaga ggcctgatct ttattgaaaa 6960
catattccaa gtgttgaaga cttttcattc ttgtaagtcc atacttattt tcaaacagaa 7020
cagcatagtc tgttcattca ttcattcaat tcatgaattc attcacataa ttatccaatt 7080
tcttgagcac ctatttgata gtcactggaa atccagagac aaacaacaca gagccatgtt 7140
ctacagtatg tacagttttc caaaaagaat ttctagtctt tactttttta ttacaaatgg 7200
aatacgtata cttgcaaata attcagatac tgtggaagag atcaaatgaa ttgcaaaagt 7260
gtccctcctc ccttcaccac tatctcccat ggcatgcaga gagagtaacc attatttgtg 7320
tgtccctcca gaaatttttt tattcaacta ctattttttt attttattag gtccgtcagt 7380
tttccttttt tgagcctctc tatatcaaat gcaaataaat atattcagaa caaaccccac 7440
tgtaaggttc acattaaaaa agacttgaag tcaccctatg aagacaaaaa ataatcacat 7500
taagtgtgaa agaacctatt cttccagtac aggataagcc atacttactg ggcatatatt 7560
catcttgaaa atctatactg atgttgtctt ggggaattga aaaggaacta ggagtgttag 7620
ttcctcggta ttgacccaca gttatgttat caggtcactt gagttcaaag ttttgtgttg 7680
gcactagcta agtaaaggaa aacacctctg ctttcattgt tgagtttcac agaattgaga 7740
gctgaaagga tcccaggcag gagcagctaa tccaaactcc cacaaagaac aaaaatcccc 7800
cagaggatct tctgttctta tatttcctgc aatggcgtcc ctgtcatatc ccacaatggc 7860
ctccctgcca tttggatatc ccttccatat cctgttgaaa ttactcccta atagtaagct 7920
gaaatctgcc cctctagttg tagtcttggg attatttcat ttacatgatg accttttaat 7980
atttgactag aattaaatca tctccccttg gtctttccat tcctgggcta actaccatca 8040
atctgagggc taacaataca agtagaaaaa gtatacattt gtcactgatc actgatcaat 8100
tattaatcaa tgatcactga taactataaa ctcaaaaaca aaatcatgtg gggattaaga 8160
gaaatgtatc agttttatgt tgtatttctg gtccctgata ctggctcagg taatgccact 8220
attgtcaaga agataccact tgtaaagtag atttaatttt cattatattt taccatatgc 8280
ttctccattc atgacatctc ttgagatgtt gtggtttata ctttcagttt ttctccagtc 8340
catccgcaaa tatcaggcat ctactgtgtt ccaagatatt aaagaaatca tcatgactta 8400
gcctcatcaa cagcattgct agatctggga tggaaaggaa gagtataatc ctggcagtca 8460
ggaagaaggc agcataaagt ataagtttct gcttccaaaa aaggtctctc atcagcctgt 8520
agggagtgtg tagggaaggg acagctgtcc ttgtagtagg gaagggtttt attcaggtcg 8580
tctgggctcc ataatatccc ttgtgtatct gcagtctcct ttgccatgga tcaacacaat 8640
aggaaatctt ccggcactga tggtttttcc aagggggagt tcttcctgga gcaaagcaaa 8700
tgaccaacca ggtttgagga cctgatttgt ttgacaattc cattttgtat tgtaaattac 8760
ttaattggca ttctactccc aatccatctt gtcatttgca tacagtggtt ttgggattga 8820
gttcagctat accaaaagtc tgaaccttct gcacttagaa caaggcaacc accaagcttc 8880
acttgcactg aggccgtgtc tccaatggaa atgaggcagc tggcttgcag gagcttccca 8940
actcagggaa gtagaactcc tgagtcacct ccatatgcaa atgatttcac agtaatgctg 9000
ttgaacttca cttcccatca cagcaaatgt gtggtaacat agcttcccca caggagttta 9060
ctcaccatgg tattttaaag gtgaaacatt tcaaaactga aatttgaaag aatttagttt 9120
tggattcact caattatcac tatcacttcg ggtgttattg cacctttctt gtttgtgagt 9180
ttaaatgcca gactctcagg ccactaactt tcaattaaaa gtgtttttct ttaatcgctg 9240
aacctaacag cagggaaaac gaaatgttca ttcagacttt cagaaccttc aatgagatta 9300
ggcagctgaa agatcaaagt gttgcatagt tgtcccgata aagctatttg gatcatatgg 9360
accaaatcga ctgctgtcat tccccaccaa ccccatctct ccccaaaatt cccagccctg 9420
tttaagtgtt ctctgtagca tttatctcta tctagtatat tgtgtagcat atcatatcat 9480
acttttctgt tttgtttatt gtctctctcc tcctagaata taaactccac aagcacaaag 9540
atttgggcct gttttataat attgttgcat ccccagggcc tgatatacag cagagtggtg 9600
gtacgaaaag agcacacaaa aaaatatttg ttgagtcaat gaatgaatga tttcctcaaa 9660
taggattagc ctaaaatttt ggaaacatga acagatttgg atatgtgaaa atttatttcc 9720
agactgttca tcaggaactg ttagcagctt ctaaagggta cactggagca gcagtagtaa 9780
aaggaggaag aggagcagct ctgctactgc tactatcgag tactactaca attagcactt 9840
gcttattctg tgtgttaggc cctgtactga acactctgtc taaattagtt catttcctcc 9900
tggaaatgac tctagggggt aagtgcttca tcatgtaaga tgagtatttt tcacattttg 9960
ttgtgtctga aatctgagtg tgtctttcaa tgatggaatc tttgattcca tgataagtgg 10020
tattattccc attttaagga tgaggaaact gaggtccaaa gaaattaagt aatttgccca 10080
aattcaccca gcctagaaaa tgataaagct agttctaaac ccaagcagat tagctctgaa 10140
gtctgggccc ttaataacca ctttttattg cctatatttg tacctctggt gtacgtatca 10200
agttatatgt tgacttcaaa actatcatga ccttttcttg gttttgattg tccaacatta 10260
gtatagtgtt ctgggtctgc aaaaattttg attactcatc tcatctgtaa aacattttga 10320
actcgtgtgt ttgtgcatgc acatttgtgt gtaattataa aaattttact ttctgttaat 10380
atataagttg tatcataaga aactgccgtt tttgaagagc aaaaaaaggt tgaatgttac 10440
cagttacatc tggttcaacc taatagacat ttgtacaaaa acagacattt taagaggttg 10500
aaataaaaat ttaataaaca atattttcag tttttactaa ttgtgatgct tcactatcat 10560
tagctaatat gtcaaggcat aatatacctt agggtgaact ttatcattaa caaaggtgga 10620
tggtgtcaat aatcttgagg tttgtgtttt tttatataac actgcgaggt ctaattaagt 10680
acttactgtt taccacctca tacagtggcc gataaaaagt gtcacttctg ctgtttcctc 10740
tgggttgtgc ttgaattatt agtattatct tcagtcctca gtttctttgt gggaaacttt 10800
ttaattagtt gtttaatttt gtaagatggt tagtttagtc aaaattagat aagagaattt 10860
gaaaatccgt agctacccca aagcaaccta cacataagaa ctattatttt tgtgttttga 10920
aatcataatt ttattgattt ccagtgtttc cactggtagt ggtttcattg atataggagt 10980
atcaaaacat cactcattat ttatttcagt ttcatttgat cctagccgtt ttgtattaac 11040
tctctgtgaa gaaattacct cacaaatcta ttgctgtcct tggtaaagga atggagaatt 11100
aaggctctag atcattagtg gttacactat agtattagaa gtaaaaaaaa gattatacca 11160
acaaaataag aacatgttaa tgtacttgta atgaataaac atgaataaag ctcttatgct 11220
atataggtgc actaaacaat ctactagaat tgtcagcaaa ctacgtatct taatcctgaa 11280
agggtcccaa accaatgatc taaaattgaa tcaaactttc ttccttgagc ataattactt 11340
aaatgattta ttaaaatagc cagcatttaa aagcttaaaa tgtaaatatc ataatgtggt 11400
atcctagata gcatcccaga acagaaaaag gatattaggg aaaaactgga ggaatggaat 11460
aaattatgca gtttagttat taataatgta ctaacgtcct tagttatgac gattgtacca 11520
tggtaatgta agatactaac aatagaggaa accgggtaag gagtatacag taactctata 11580
ctatctttgc aacttttttg taaatttaaa acttctaaaa taaagaacaa atttaaacat 11640
taaaaagtat caccaggaac atatatcact gtttacagat gaaatactat gtattttcat 11700
atctaatttc tgatcattga cttcaaatca gaaaagtgaa tgacacctca aaatcaggtt 11760
ttctgtttac tgaagtctaa gaaaagaaag cataccagct ggagagattc atgtttataa 11820
agacagattt ataacaacaa aaataaaata tccaagaata aatttaagaa gaagcacttt 11880
actgagaaac atatgaaaac ctgaacaaat ggagagggat attttgtatt tgaatagaaa 11940
gacttctggt ttaaagataa ttctctttaa attatttttt gtagaaattt aaggggtaca 12000
agagcagtgt tgtcacatgg atatattaca tagtggtgaa gtctggggtt ttagtgtaaa 12060
ttaatcttta cattttgttt gagcccaata aatgtaccaa catgattttt atagaaagat 12120
agtcattcct attaatccaa acttgtccca actttgaatt gaattgaggc agagctagca 12180
ggtgttcccc acggctgagg catctgaaca ttaagcatat ccctctgaga accagcctgc 12240
attgatactc tttctaatgt ggacagcatc aagctatgta cgtagttctg tgctcagcaa 12300
aagccctgac ttctttttgt ttatgtccta gccccataca acaaaatcaa ccaaagaatt 12360
ttggttgtgg atccagtcac ctctgaacat gaactgacat gtcaggctga gggctacccc 12420
aaggccgaag tcatctggac aagcagtgac catcaagtcc tgagtggtaa gaccaccacc 12480
accaattcca agagagagga gaagcttttc aatgtgacca gcacactgag aatcaacaca 12540
acaactaatg agattttcta ctgcactttt aggagattag atcctgagga aaaccataca 12600
gctgaattgg tcatcccagg taatattctg aatgtgtcca ttaaaatatg tctaacactg 12660
tcccctagca cctagcatga tgtctgccta tcatagtcat tcagtgattg ttgaataaat 12720
gaatgaatga ataacactat gtttacaaaa tatatcctaa ttcctcacct ccattcatcc 12780
aaaccatatt gttacttaat aaacattcag cagatattta tggaatatac cttttgttcc 12840
atgcattgta gtactcattg gatacacata gaataataag actcagttca cactcttcag 12900
gaaacagata aaaaactaag aaacaaacaa aaaacaggca atccaacacc atgtgggaaa 12960
tgctttcata gccgggaaac ctggggaata cctgagagga atactcaatt caggccttgt 13020
ttcaggaatc caaatcctgg cacatcagag ctgcttccct ctttccaggg tggcaggaaa 13080
taaatggaac atatttttct atcttatgcc aaacatgagg gaccctttct ccccggtgcc 13140
tctcccaagg tagtctacaa tatttcaact ctagcagtct gcttagtgca tagaacatga 13200
ggctgtgtgt ccctgggcaa attactagac ttctgtgtgc ttcactttcc ctgtaggatt 13260
ataatctact gagcaagctt attgtaaggg tcagattagc aacagtgtat gaaaatgatt 13320
tgagaccatt gcctgcacaa attcaactat ttttttttat ctcactactc tacagaagta 13380
ggtagggtgg gagacagagt ctgatgagag gctcagaatg tgaaagaaag tgaggcgagt 13440
gagcatgata tttaatataa acacaaagat attctgagaa gagctgctca ctgccccctc 13500
ccccaataca tgttgatagg aaaatgccac gtacttcagc aaaaacaact gaaaaattag 13560
atagaaaagt caatcaatag gaaaagataa tccaggacgg tgttgtgaac agaaagaggg 13620
ggaaaaaact ttagaaaatg atggggatgc tcttactggg gtacgagtcc tcaggtattg 13680
aactggcttt cagtaaaagc tagattagtg ggttcctgcc atttacaagc tgttttatga 13740
caacttactt gttgggtggc ctacagtaac tcacctaact gcactgagtc tgtttcctca 13800
tctgtaaatt ggggattttt ttttaaatac ctggcatgcc taactcataa agttgttctg 13860
aaactgaaat aaaacatacg tgaacaggca ttgtaaactg taagttacgg aaaaagctgg 13920
ctgttgttgt gtctttaaag tttcacctgg gtagtcaaag atggatcatg ggtctcagtg 13980
gagagctgag ccaggcagga gctgactaag ggtgagaggt gggagttagc agcctctgaa 14040
catctgtgta ccatgggacc ccctttcctc ctgcatggta ccccagacaa ggagcctagt 14100
aagagatact aatggcttgt tgtccagaga tgttcaaact gcagagaaag ataagacaac 14160
aagcattggc ctccaatcat gatgacagat aggaggaggt gggagctcct tagcagtgct 14220
ggttggcctt ccatgttcta ctgtgggcca tctctgccat gtactgtagg ctactagctt 14280
ctatattaaa gaatgcaaga ggggccagga gcggaggctc atgcctgtaa tctcagcact 14340
ttgggaggcc aaggtgggca gatcacttga ggtcaggagt ttgtgaccag cctggccaac 14400
atggtgaaac tctgccttta ctaaaaatat aaaaattagc tgggtgtggt ggtgtgcacc 14460
tgtaatccca gctactcggg agactgaggc acaagaattg cttgaacctg ggaggcggaa 14520
gttgcagtga gcccagattg cgccactgca ctccaccctg ggcaacagag aaagactctg 14580
cctcaaaaaa aaaaaaaaaa agcaagagga agtgaaataa tcaaggccgc catttaatag 14640
tgagcagcca ctccatgtgg tactgtgcaa gcacattata aatattagcc tcacaagaaa 14700
tgtattagca tttgtatttt gtacactggt taagtatctt gcccaagacc tcaaaactgg 14760
ttaagggcag cagaatttag ccccagcacc accttttcaa agcctgggct tctcacactt 14820
ctccatgctg ttcccatttt aacacaggta tctcgccatt ccagccactc aaactttggc 14880
atttaagaaa attatcctaa agctaaacta aacttcaagg atgaccattc tcctgacccc 14940
ttcccatcaa aattttatct ttagtcagtt tgttttcgtt ttgttttgtt tttcagaact 15000
acctctggca catcctccaa atgaaaggac tcacttggta attctgggag ccatcttatt 15060
atgccttggt gtagcactga cattcatctt ccgtttaaga aaaggtagta tttccttaat 15120
tgcagtggtc tccactgggg gtgaggaagg ggtgagaatt ggatcatggc tgcaaggaaa 15180
cccgacttaa cctctgcaag gtggtgcaaa ggcattccac tgttcaacag caattatatt 15240
gaagctgagt gggatcactg ggtgaagatg aagcgtaagg ggtgaggggc aggagaatgg 15300
gtatggatgg aggtagaaga tgcagtgtca tacagttttt ttctatcatg aaaataacca 15360
cagacttaca gaagagaaag agctaaaatg cccgtcattt tcagttgcat tttagtcttg 15420
cattagttgc aaccagctgg tttctgggta ccctaagtaa taaaaatagt tcctctgtag 15480
aactgtagta tgtttaccat agagtatttt gcaaaatttt tggtagagga tgttacataa 15540
tttgcatgtg ttcatttctc catttacctg tgggaacaat taaaatccag gaaaatgagt 15600
atattcaaat aatttcctcc catttaagat gagtcagagt aaataattcc tccaatactt 15660
agagaagtat accaagagat ccagtgatgg tatagagttg tctgatgtta aatagggaag 15720
tagaatatgg aaggggattc caatagtcgt tgaaaaattc cccataaccc cttacatggg 15780
ggaaagtagt gttaactgag agagtagaga taagctgttt ccaaaaatta tattcttaac 15840
aggactgaga tagccagaat ataaggatca agtttcaatg acagtaagat cctgagatgg 15900
agttgatttg cacaaagaaa taattgttgc cagcatgcat tttgaatatt tctctggaaa 15960
aaaagattag ttggcagtag aaatggatag aaatcaatag atattaaaat acctcagaat 16020
ttggttcatc tctgggaaaa gatgaaaaat aaaagtgtat actcctcaag aacatctagg 16080
atcaaaagca tgtgccctac actattgaat taattaacct cataagttgg gacctgtgga 16140
ataaggatgt ccaccagact tcctagggat tacaaatgtt tcacagaact tgaaatttaa 16200
acttgggtca ctgtatggga tgtagagctg tgctatatgg aaataaaaat gatttctttt 16260
tctcaaggga gaatgatgga tgtgaaaaaa tgtggcatcc aagatacaaa ctcaaagaag 16320
caaagtggta agaatatcag aaggaattgg gaagtaaaag tcaaaggaaa caaaaagcta 16380
aagcaataac aaagagaaat ccatcagtca taatctcctc tccttttaaa gaatgctggt 16440
tcccctttgc ctcacagcta acacaagaac tcctccaccg tctgaggagg tttaggagca 16500
gggaagggga aggagtcagc ttcatttgct aatcttctgt tgccctgcac cctagcagct 16560
ccttgcagca ggggacaagg atgacttagg tggatggata attaattgat tctaaaatat 16620
tgtgtgtcag tattgtaata ctatgttaat tgcaccatgc acggtatctc atttaatccc 16680
ccaccccttg ccattaccaa agagagagag agagagagag agagaaatac tagaatttat 16740
cctcatttta cagtagagaa aacagagggt caagaagata atgtaaagtg cccaagaaca 16800
cacagctgat cacaaaaatc aagcttgggg gccattagcc taaccacaga cccttactct 16860
taacccatct gcttcaatcc attttgctac aaatgtttac atttataagc agggcagaaa 16920
aacctcatcc aggttattga actaagaaga aagttatatt aaggtttcta atttttttaa 16980
tgtagttaga aaccaaactt aacaatgagc ccaagtttaa agcagtctaa ttaacctgga 17040
caagctcagg caagtttcat tctgtggccc atagcatcat ctgtgttgta aagctaagta 17100
gcaaatgttg tttgggtcat gctgggggac aagccatccc aatttgctca ggactgaggg 17160
gttttccagg atatcatgta aggataattg ggtacaaata taacctgctg ctttctctca 17220
tttcaaattt atcatttatc atatcagcaa ctatgagtta tgttttttat tagatttctt 17280
gttacttttt ccccagacca cttcccatga aattaatata ctattatcac tctccagata 17340
cacatttgga ggagacgtaa tccagcattg gaacttctga tcttcaagca gggattctca 17400
acctgtggtt taggggttca tcggggctga gcgtgacaag aggaaggaat gggcccgtgg 17460
gatgcaggca atgtgggact taaaaggccc aagcactgaa aatggaacct ggcgaaagca 17520
gaggaggaga atgaagaaag atggagtcaa acagggagcc tggagggaga ccttgatact 17580
ttcaaatgcc tgaggggctc atcgacgcct gtgacaggga gaaaggatac ttctgaacaa 17640
ggagcctcca agcaaatcat ccattgctca tcctaggaag acgggttgag aatccctaat 17700
ttgagggtca gttcctgcag aagtgccctt tgcctccact caatgcctca atttcttttc 17760
tgcatgactg agagtctcag tgttggaacg ggacagtatt tatgtatgag tttttcctat 17820
ttattttgag tctgtgaggt cttcttgtca tgtgagtgtg gttgtgaatg atttcttttg 17880
aagatatatt gtagtagatg ttacaatttt gtcgccaaac taaacttgct gcttaatgat 17940
ttgctcacat ctagtaaaac atggagtatt tgtaaggtgc ttggtctcct ctataactac 18000
aagtatacat tggaagcata aagatcaaac cgttggttgc ataggatgtc acctttattt 18060
aacccattaa tactctggtt gacctaatct tattctcaga cctcaagtgt ctgtgcagta 18120
tctgttccat ttaaatatca gctttacaat tatgtggtag cctacacaca taatctcatt 18180
tcatcgctgt aaccaccctg ttgtgataac cactattatt ttacccatcg tacagctgag 18240
gaagcaaaca gattaagtaa cttgcccaaa ccagtaaata gcagacctca gactgccacc 18300
cactgtcctt ttataataca atttacagct atattttact ttaagcaatt cttttattca 18360
aaaaccattt attaagtgcc cttgcaatat caatcgctgt gccaggcatt gaatctacag 18420
atgtgagcaa gacaaagtac ctgtcctcaa ggagctcata gtataatgag gagattaaca 18480
agaaaatgta ttattacaat ttagtccagt gtcatagcat aaggatgatg cgaggggaaa 18540
acccgagcag tgttgccaag aggaggaaat aggccaatgt ggtctgggac ggttggatat 18600
acttaaacat cttaataatc agagtaattt tcatttacaa agagaggtcg gtacttaaaa 18660
taaccctgaa aaataacact ggaattcctt ttctagcatt atatttattc ctgatttgcc 18720
tttgccatat aatctaatgc ttgtttatat agtgtctggt attgtttaac agttctgtct 18780
tttctattta aatgccacta aattttaaat tcataccttt ccatgattca aaattcaaaa 18840
gatcccatgg gagatggttg gaaaatctcc acttcatcct ccaagccatt caagtttcct 18900
ttccagaagc aactgctact gcctttcatt catatgttct tctaaagata gtctacattt 18960
ggaaatgtat gttaaaagca cgtattttta aaattttttt cctaaatagt aacacattgt 19020
atgtctgctg tgtactttgc tatttttatt tattttagtg tttcttatat agcagatgga 19080
atgaatttga agttcccagg gctgaggatc catgccttct ttgtttctaa gttatctttc 19140
ccatagcttt tcattatctt tcatatgatc cagtatatgt taaatatgtc ctacatatac 19200
atttagacaa ccaccatttg ttaagtattt gctctaggac agagtttgga tttgtttatg 19260
tttgctcaaa aggagaccca tgggctctcc agggtgcact gagtcaatct agtcctaaaa 19320
agcaatctta ttattaactc tgtatgacag aatcatgtct ggaacttttg ttttctgctt 19380
tctgtcaagt ataaacttca ctttgatgct gtacttgcaa aatcacattt tctttctgga 19440
aattccggca gtgtaccttg actgctagct accctgtgcc agaaaagcct cattcgttgt 19500
gcttgaaccc ttgaatgcca ccagctgtca tcactacaca gccctcctaa gaggcttcct 19560
ggaggtttcg agattcagat gccctgggag atcccagagt ttcctttccc tcttggccat 19620
attctggtgt caatgacaag gagtaccttg gctttgccac atgtcaaggc tgaagaaaca 19680
gtgtctccaa cagagctcct tgtgttatct gtttgtacat gtgcatttgt acagtaattg 19740
gtgtgacagt gttctttgtg tgaattacag gcaagaattg tggctgagca aggcacatag 19800
tctactcagt ctattcctaa gtcctaactc ctccttgtgg tgttggattt gtaaggcact 19860
ttatcccttt tgtctcatgt ttcatcgtaa atggcatagg cagagatgat acctaattct 19920
gcatttgatt gtcacttttt gtacctgcat taatttaata aaatattctt atttattttg 19980
ttacttggta caccagcatg tccattttct tgtttatttt gtgtttaata aaatgttcag 20040
tttaacatcc cagtggagaa agtta 20065
<210> 2
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
acgttggatg atttgagggt cagttcctgc 30
<210> 3
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
acgttggatg actgagactc tcagtcatgc 30
<210> 4
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
acgttggatg tccactggga tgttaaactg 30
<210> 5
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
acgttggatg ttgtcacttt ttgtacctgc 30
<210> 6
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
acgttggatg acagagctcc ttgtgttatc 30
<210> 7
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
acgttggatg tgctcagcca caattcttgc 30
<210> 8
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
acgttggatg gaggatccat gccttctttg 30
<210> 9
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
acgttggatg cttaacaaat ggtggttgtc 30
<210> 10
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
acgttggatg ttaagtccca cattgcctgc 30
<210> 11
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
acgttggatg tctcaacctg tggtttaggg 30
<210> 12
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
acgttggatg atacctagta gaacctgccc 30
<210> 13
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
acgttggatg gcattgacag gttggaaaag 30
<210> 14
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
acgttggatg atttgctgcc ttgggcagag 30
<210> 15
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
acgttggatg tcgggaagct gcgcagaact 30
<210> 16
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
acgttggatg aacaggtggg aaagatgaac 30
<210> 17
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
acgttggatg gttgctgatg ggaattgagg 30
<210> 18
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
acgttggatg actttggtga ctgtgacctc 30
<210> 19
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
acgttggatg catgtgtgtg tcatacacag 30
<210> 20
<211> 20065
<212> DNA
<213> PDL1基因(Homo sapiens)
<400> 20
ggcgcaacgc tgagcagctg gcgcgtcccg cgcggcccca gttctgcgca gcttcccgag 60
gctccgcacc agccgcgctt ctgtccgcct gcaggtaggg agcgttgttc ctccgcgggt 120
gcccacggcc cagtatctct ggctagctcg ctgggcactt taggacggag ggtctctaca 180
ccctttcttt gggatggaga gaggagaagg gaaagggaac gcgatggtct agggggcagt 240
agagccaatt acctgttggg gttaataaga acaggcaatg catctggcct tcctccaggc 300
gcgattcagt tttgctctaa aaataattta tacctctaaa aataaataag ataggtagta 360
taggataggt agtcattctt atgcgactgt gtgttcagaa tatagctctg atgctaggct 420
ggaggtctgg acacgggtcc aagtccaccg ccagctgctt gctagtaaca tgacttgtgt 480
aagttatccc agctgcagca tctaagtaag tctcttcctg cgctaagcag gtccaggatc 540
cctgaacgga atttatttgc tctgtccatt ctgagaaccc aaaggagtcc taaaagagga 600
atggaggagc ctaagaataa aaatagtata ataaaacatt tcttagacac attgaccttg 660
gcctatgtca aagttcagtc tgggtttgtc ttataacaca aggagtaaaa gtaccattgt 720
tctacctctt tttttaatac ttgaaaaaaa tttactgtgg atgcttttct atgaattaaa 780
taaccttcta aaaaatgttt tcattgctgc attcgattag attgggtaac taaatgaaat 840
taattcctca ctgttgggta taaaggttat ttacagtggt tctgtcttag ccattcactg 900
aactcattgc atatatatct ctggaatatt gctgattgtt tccttcaagt aaacttagaa 960
gtgtaactac ttagtcaaag agcctgaata ttttaaaggc cttttgaaga aaactgaaaa 1020
tgctttccag aaaggatgta tcagttgaca atgacagtcg tcaacagtat ttaaggagaa 1080
ctatgatact ctgaagaaaa acttagcctt tctcagtaaa agtaggtagg cagaggccac 1140
atgacagcag ttagagtgtg gtcttcaagg aagtcacaga aatactgtgg ggaattgaaa 1200
ccccatgtgg aaaatgtaca agagtgtctc agtgtgactg agaaggaggt tgggcatggg 1260
gtttcatgga gtttaataaa gtttggtcac ttagtagagg tttaataaat caactgtctt 1320
aatctttgat cctacttaag aatttttttt ttgtttttgt agagatgggg ctcttgttat 1380
gttgcccagg ctgttctcga actcctagcc tcaggcgatc ctccctcctc aggctccaga 1440
agtcctggga ttactggcgg gagccaccat gcaggcctct tgctcctact tttgagaaag 1500
gaagtttaac cggttttttt tgtctttttt tttttttttt tgagacagag tctcactctg 1560
ttgcccatgc tggagtgcag tggtgcaatc tcagctcact gcctcccggg ttcaagtgat 1620
tctcctgcct cagcctcccg agtagctggg actacaggca cctgccacca cgcccagcta 1680
atttttgtat ttttagtaga aatggggttt caccatattg gccaggctga tctcgaactc 1740
ctgacctcag gtgatccgcc tgcctcggcc tcccaaagtg ctgggattac aggcatgagc 1800
cactgctcct ggctgcttaa ctttttctct atctcatcct cctacccatc ctacccttgg 1860
aagatagaga agtagtatta gttccatagt gttatactgg gcttccccca gggacaaacc 1920
cacttcccca acctgaatga gccatcactt cttccccagt ttacatttca ttgctcttta 1980
aatgtctcca ttcggatatg ggaattcaca tatggtcata attcttacct gaagaagatg 2040
tcagtcttct tctcttagac caactgccct gatatgaggt ttagaggtta aagaacatgt 2100
gtgtatttac atgatctttg tattctgcct tttcgtccct cactaatgac agctgcaccc 2160
caaggaaatg gagctgtgga agagagggtt tgataagaaa ttaagtaaat attggatcta 2220
atccatcacc ctccaggaag cctttattac tcctaaaaat ttcaaccaaa ttcattaaag 2280
gacaagaact ccaccagagt aggccataaa cattggcaaa attagttgta atccatgact 2340
agatttaatg tccctttgtt ttattcccat atggttataa tgctttgctt ggcattaggg 2400
gtattttaag ttttcttctg cctagtaagt gaatttgtgt ttataataca ataatcataa 2460
aatatcacat taatatttta taactgtaca gttataaaat attttataag taatatttat 2520
attttataag taatatttta taactgtaca gttaactctg gcccaaggaa aagatagtct 2580
gatagatgct gcagccccat tttagcaaat gtgacctcac aggcctgaat gccatcgcta 2640
ttccacatct acaggataga cggaaaggaa agaaataaaa aaataggtac ctaacactgg 2700
caagaggatg atgactcatg ttatttcact taaccttttt atcttttaac atgaaggact 2760
catacaggtt gataagaaac cagtgacata aacagaccaa aaaatgatca gatctttcaa 2820
attagcaaaa aaataatatt ttttaaacaa tgggtgaaaa tacagtgtaa cagtaccaat 2880
tatcaacatg tgttgagaac cagaaaaatg ttctttttct ttgatcagca acactatttg 2940
ggaaaatcta tcctcagggc ctagcctggg gccctggcac acagtaggca ctcaacgaat 3000
atttgctgaa cacacaaata cttatgatat tttaaaaaat tggcaacaat ctgataccta 3060
acaatagagg gattaaatat tatggaactg ttaaataaga tgcttatgaa taccatgcag 3120
taagatgggc aatatttatg ccataagctt taatgaaaca aatgggtatt aaatgtatga 3180
taaggttata aattactttt taaaagatta cagggaaaaa aattgaaaga tatacactga 3240
aatgtttttt gctcacagtg gtgacaaggt ttctcagcac tggcactgtt gacgttttag 3300
gctgtatgtc tttgctgtgg gaggctggcc tgtgcactgc agggtgtttg gcagcactct 3360
tggcctctgc ccctagatag caatagcagt cctccctcaa ccagcccaat tttgacaacc 3420
aaaaatgttt ccaggcatca ccagatgctc cctgggtgag agtgatgaaa tagtagggga 3480
ttttcccctt cttttcttat tttctgtaat tccattatat tactttaata ataaagaaaa 3540
aaacataaaa aataaacgaa tgttattatt ctacgtcagt ttggatgttt ggactccatt 3600
ttggggttct ttccattata tcacttggtc tgctaaacat tctacggttt ggtaaggtga 3660
agtgattcat gaaattttgg ttttattttt ttcctgatac taaaaataaa acattctttc 3720
acttggaaat ttggacacag aacaccaaaa aaaatccata atctcatctc tctttttctg 3780
tcttttcctt ccttttttcc ctttaaaaac aataaagagt gaaacctacc tgttctccct 3840
ctaatttaat tcctaaatat aatcactgtc aatatcttgg acatttcctg tgtctaaaca 3900
cacacacaca cttttttttt tcagcaaaag tggatttctg ctacatgtag tgttctgcaa 3960
cttactttct atgtgtttac aaaatcagta catgtacata tgctgaattc agtccttaat 4020
ggtattatat tttgtgaata taccaaaatt tgtttaacca cttagacaat ctaggatatt 4080
ctcagtttgc tgttatgagc aatgctcttc ctttacatat acagacatat atatatatat 4140
gtgtgtgtgt gtgtttttgt tttagtagga tagatttcta ggagagggtg aaaggtctta 4200
tgacatccgc atttacgatt gtaataggaa gtatcaaagt gccccctaaa gaaaaaaatc 4260
ctcccattag tgggtaagaa agcctatttg ttcatatctt cacaaacact aaatattaga 4320
aatatttaca attgtggtca agctcataag tgaaaatggt atttcatatc ttatattttt 4380
tattgtgaga ttgaacatct ttcatatgtt tacatgtcac ctgtatttct tattctctga 4440
actatatgtt atgacctttc actttttttc ctcatgggtt atgtgtagtt tgtatagttg 4500
tcttattgat tgttaggagc tatttatata ttaggaacat taatctcctg tcttatatat 4560
acgtggcatc gattagttga tcatttgtga gttcatgtct gtatacaaag attggagagg 4620
cactaagagg gaaaacttac ctctttctta tcaaagtttg taaatatatg tataacagaa 4680
gagggagaaa atattaataa atgcacagat tggctgaaat agagtataaa tcttttactc 4740
ccctacttca acataaactg caaaaggaga gtgacttttc tttcactctg acttccgtat 4800
tcctcatgct taaaatagtg cctagcacag aagaggtgct caatcagtgt ttgctaaacg 4860
aaataattag tcacatttca agcaggatga ctaaatgaag aatagaatct aggcagatac 4920
tctggaagag tggctgtgag tcattcatat cttagtatga attagtcaaa tccaactctc 4980
tccccttccc actccccact gttagtagaa gaatctgttt attgagagaa tagatttata 5040
atttagaata agtgagaggg gcagaagagg agattttgaa ggatggcacc tgaaggagga 5100
ctagcatggc tgagacagtg aagtggaagc cttgaatagc taaagggtaa gatgaaagta 5160
tttagctgta gggggaaaaa gcattgacag gttggaaaag taaaagtcag attctccttg 5220
ctctgaaatt ttgtacaggg caggttctac taggtatgtt acaatgcaga aaaaacatga 5280
aataattgag aggaatttgg tgcaatatta tcttcttggc ttcttttgag tgggcagatt 5340
tttttcacgg cctgtaacta taataaattt gaaacttctc atcttttagt aacttttttc 5400
acttaagttt atgtggctgt gggcaatgga atgaagatat tgaacttcca attccctgtt 5460
gggtttccac aattacaagt caatcatgac tggttattag aagactattt cagttagaac 5520
caccaagtcc catattgtca tattgtatgt ttaattatta agtgaagcag tcttcttttc 5580
gtgttttcca taattagggc attccagaaa gatgaggata tttgctgtct ttatattcat 5640
gacctactgg catttgctga acggtaagac accaaatcct tccattaggt tctatatttt 5700
aaatatttta accatgagtt taaaactaaa atgatcattt aaaatgcatg caattttctt 5760
atagagagaa cattctattc tttcttctac tttacacaat ggcaaagtct tctttctact 5820
ttacgcaatg ataaagttac ctgtgtcatt ttgtaaaaat atagagaata tagacaaatt 5880
gaaagacaca aaataatcta ttacccattt cccagggtta actactgaaa atatctgggg 5940
aaatggcctg tatgtataca tttatttgtt tgctttcaac aaggccaaga tcctttgatc 6000
tttcagtctt ggttgctctg tgacatgcct ttcctgatga ggatacttta aggaagaatt 6060
gtaagataca tggaaaatgt caggctaaca cagtactggc atcaccctgt gctctttcct 6120
gaactccata ccaatgtact tcttgccaga aaactgatca aaagtttagg gaagtaaaaa 6180
gagatgactg ttagaatcta ccattccctc tatgtaggaa gcaaataggt gtcctgtcaa 6240
aggacattct ggggatgtct acatgaaacc aagtctccct ggttgtaagg actccatctc 6300
catataatat ttatacagta atatatgttt ataaattgtg ggggcaactt gtttagctaa 6360
ttttattatt ctgctattgg gacactgtgt ctcagcatga gatatagtgt cccaaaacat 6420
atttcaagcc cattggataa aatatgtgtt tagcaagttc ttaaatataa tgataacata 6480
accgaccaga taaagtgatt tataaacgct gtgccaattt tgtaaatgtt tcgaggaatt 6540
ttcccttttc tgaagattgt ccttctttct ttttagcatt tactgtcacg gttcccaagg 6600
acctatatgt ggtagagtat ggtagcaata tgacaattga atgcaaattc ccagtagaaa 6660
aacaattaga cctggctgca ctaattgtct attgggaaat ggaggataag aacattattc 6720
aatttgtgca tggagaggaa gacctgaagg ttcagcatag tagctacaga cagagggccc 6780
ggctgttgaa ggaccagctc tccctgggaa atgctgcact tcagatcaca gatgtgaaat 6840
tgcaggatgc aggggtgtac cgctgcatga tcagctatgg tggtgccgac tacaagcgaa 6900
ttactgtgaa agtcaatggt aagaattatt atagatgaga ggcctgatct ttattgaaaa 6960
catattccaa gtgttgaaga cttttcattc ttgtaagtcc atacttattt tcaaacagaa 7020
cagcatagtc tgttcattca ttcattcaat tcatgaattc attcacataa ttatccaatt 7080
tcttgagcac ctatttgata gtcactggaa atccagagac aaacaacaca gagccatgtt 7140
ctacagtatg tacagttttc caaaaagaat ttctagtctt tactttttta ttacaaatgg 7200
aatacgtata cttgcaaata attcagatac tgtggaagag atcaaatgaa ttgcaaaagt 7260
gtccctcctc ccttcaccac tatctcccat ggcatgcaga gagagtaacc attatttgtg 7320
tgtccctcca gaaatttttt tattcaacta ctattttttt attttattag gtccgtcagt 7380
tttccttttt tgagcctctc tatatcaaat gcaaataaat atattcagaa caaaccccac 7440
tgtaaggttc acattaaaaa agacttgaag tcaccctatg aagacaaaaa ataatcacat 7500
taagtgtgaa agaacctatt cttccagtac aggataagcc atacttactg ggcatatatt 7560
catcttgaaa atctatactg atgttgtctt ggggaattga aaaggaacta ggagtgttag 7620
ttcctcggta ttgacccaca gttatgttat caggtcactt gagttcaaag ttttgtgttg 7680
gcactagcta agtaaaggaa aacacctctg ctttcattgt tgagtttcac agaattgaga 7740
gctgaaagga tcccaggcag gagcagctaa tccaaactcc cacaaagaac aaaaatcccc 7800
cagaggatct tctgttctta tatttcctgc aatggcgtcc ctgtcatatc ccacaatggc 7860
ctccctgcca tttggatatc ccttccatat cctgttgaaa ttactcccta atagtaagct 7920
gaaatctgcc cctctagttg tagtcttggg attatttcat ttacatgatg accttttaat 7980
atttgactag aattaaatca tctccccttg gtctttccat tcctgggcta actaccatca 8040
atctgagggc taacaataca agtagaaaaa gtatacattt gtcactgatc actgatcaat 8100
tattaatcaa tgatcactga taactataaa ctcaaaaaca aaatcatgtg gggattaaga 8160
gaaatgtatc agttttatgt tgtatttctg gtccctgata ctggctcagg taatgccact 8220
attgtcaaga agataccact tgtaaagtag atttaatttt cattatattt taccatatgc 8280
ttctccattc atgacatctc ttgagatgtt gtggtttata ctttcagttt ttctccagtc 8340
catccgcaaa tatcaggcat ctactgtgtt ccaagatatt aaagaaatca tcatgactta 8400
gcctcatcaa cagcattgct agatctggga tggaaaggaa gagtataatc ctggcagtca 8460
ggaagaaggc agcataaagt ataagtttct gcttccaaaa aaggtctctc atcagcctgt 8520
agggagtgtg tagggaaggg acagctgtcc ttgtagtagg gaagggtttt attcaggtcg 8580
tctgggctcc ataatatccc ttgtgtatct gcagtctcct ttgccatgga tcaacacaat 8640
aggaaatctt ccggcactga tggtttttcc aagggggagt tcttcctgga gcaaagcaaa 8700
tgaccaacca ggtttgagga cctgatttgt ttgacaattc cattttgtat tgtaaattac 8760
ttaattggca ttctactccc aatccatctt gtcatttgca tacagtggtt ttgggattga 8820
gttcagctat accaaaagtc tgaaccttct gcacttagaa caaggcaacc accaagcttc 8880
acttgcactg aggccgtgtc tccaatggaa atgaggcagc tggcttgcag gagcttccca 8940
actcagggaa gtagaactcc tgagtcacct ccatatgcaa atgatttcac agtaatgctg 9000
ttgaacttca cttcccatca cagcaaatgt gtggtaacat agcttcccca caggagttta 9060
ctcaccatgg tattttaaag gtgaaacatt tcaaaactga aatttgaaag aatttagttt 9120
tggattcact caattatcac tatcacttcg ggtgttattg cacctttctt gtttgtgagt 9180
ttaaatgcca gactctcagg ccactaactt tcaattaaaa gtgtttttct ttaatcgctg 9240
aacctaacag cagggaaaac gaaatgttca ttcagacttt cagaaccttc aatgagatta 9300
ggcagctgaa agatcaaagt gttgcatagt tgtcccgata aagctatttg gatcatatgg 9360
accaaatcga ctgctgtcat tccccaccaa ccccatctct ccccaaaatt cccagccctg 9420
tttaagtgtt ctctgtagca tttatctcta tctagtatat tgtgtagcat atcatatcat 9480
acttttctgt tttgtttatt gtctctctcc tcctagaata taaactccac aagcacaaag 9540
atttgggcct gttttataat attgttgcat ccccagggcc tgatatacag cagagtggtg 9600
gtacgaaaag agcacacaaa aaaatatttg ttgagtcaat gaatgaatga tttcctcaaa 9660
taggattagc ctaaaatttt ggaaacatga acagatttgg atatgtgaaa atttatttcc 9720
agactgttca tcaggaactg ttagcagctt ctaaagggta cactggagca gcagtagtaa 9780
aaggaggaag aggagcagct ctgctactgc tactatcgag tactactaca attagcactt 9840
gcttattctg tgtgttaggc cctgtactga acactctgtc taaattagtt catttcctcc 9900
tggaaatgac tctagggggt aagtgcttca tcatgtaaga tgagtatttt tcacattttg 9960
ttgtgtctga aatctgagtg tgtctttcaa tgatggaatc tttgattcca tgataagtgg 10020
tattattccc attttaagga tgaggaaact gaggtccaaa gaaattaagt aatttgccca 10080
aattcaccca gcctagaaaa tgataaagct agttctaaac ccaagcagat tagctctgaa 10140
gtctgggccc ttaataacca ctttttattg cctatatttg tacctctggt gtacgtatca 10200
agttatatgt tgacttcaaa actatcatga ccttttcttg gttttgattg tccaacatta 10260
gtatagtgtt ctgggtctgc aaaaattttg attactcatc tcatctgtaa aacattttga 10320
actcgtgtgt ttgtgcatgc acatttgtgt gtaattataa aaattttact ttctgttaat 10380
atataagttg tatcataaga aactgccgtt tttgaagagc aaaaaaaggt tgaatgttac 10440
cagttacatc tggttcaacc taatagacat ttgtacaaaa acagacattt taagaggttg 10500
aaataaaaat ttaataaaca atattttcag tttttactaa ttgtgatgct tcactatcat 10560
tagctaatat gtcaaggcat aatatacctt agggtgaact ttatcattaa caaaggtgga 10620
tggtgtcaat aatcttgagg tttgtgtttt tttatataac actgcgaggt ctaattaagt 10680
acttactgtt taccacctca tacagtggcc gataaaaagt gtcacttctg ctgtttcctc 10740
tgggttgtgc ttgaattatt agtattatct tcagtcctca gtttctttgt gggaaacttt 10800
ttaattagtt gtttaatttt gtaagatggt tagtttagtc aaaattagat aagagaattt 10860
gaaaatccgt agctacccca aagcaaccta cacataagaa ctattatttt tgtgttttga 10920
aatcataatt ttattgattt ccagtgtttc cactggtagt ggtttcattg atataggagt 10980
atcaaaacat cactcattat ttatttcagt ttcatttgat cctagccgtt ttgtattaac 11040
tctctgtgaa gaaattacct cacaaatcta ttgctgtcct tggtaaagga atggagaatt 11100
aaggctctag atcattagtg gttacactat agtattagaa gtaaaaaaaa gattatacca 11160
acaaaataag aacatgttaa tgtacttgta atgaataaac atgaataaag ctcttatgct 11220
atataggtgc actaaacaat ctactagaat tgtcagcaaa ctacgtatct taatcctgaa 11280
agggtcccaa accaatgatc taaaattgaa tcaaactttc ttccttgagc ataattactt 11340
aaatgattta ttaaaatagc cagcatttaa aagcttaaaa tgtaaatatc ataatgtggt 11400
atcctagata gcatcccaga acagaaaaag gatattaggg aaaaactgga ggaatggaat 11460
aaattatgca gtttagttat taataatgta ctaacgtcct tagttatgac gattgtacca 11520
tggtaatgta agatactaac aatagaggaa accgggtaag gagtatacag taactctata 11580
ctatctttgc aacttttttg taaatttaaa acttctaaaa taaagaacaa atttaaacat 11640
taaaaagtat caccaggaac atatatcact gtttacagat gaaatactat gtattttcat 11700
atctaatttc tgatcattga cttcaaatca gaaaagtgaa tgacacctca aaatcaggtt 11760
ttctgtttac tgaagtctaa gaaaagaaag cataccagct ggagagattc atgtttataa 11820
agacagattt ataacaacaa aaataaaata tccaagaata aatttaagaa gaagcacttt 11880
actgagaaac atatgaaaac ctgaacaaat ggagagggat attttgtatt tgaatagaaa 11940
gacttctggt ttaaagataa ttctctttaa attatttttt gtagaaattt aaggggtaca 12000
agagcagtgt tgtcacatgg atatattaca tagtggtgaa gtctggggtt ttagtgtaaa 12060
ttaatcttta cattttgttt gagcccaata aatgtaccaa catgattttt atagaaagat 12120
agtcattcct attaatccaa acttgtccca actttgaatt gaattgaggc agagctagca 12180
ggtgttcccc acggctgagg catctgaaca ttaagcatat ccctctgaga accagcctgc 12240
attgatactc tttctaatgt ggacagcatc aagctatgta cgtagttctg tgctcagcaa 12300
aagccctgac ttctttttgt ttatgtccta gccccataca acaaaatcaa ccaaagaatt 12360
ttggttgtgg atccagtcac ctctgaacat gaactgacat gtcaggctga gggctacccc 12420
aaggccgaag tcatctggac aagcagtgac catcaagtcc tgagtggtaa gaccaccacc 12480
accaattcca agagagagga gaagcttttc aatgtgacca gcacactgag aatcaacaca 12540
acaactaatg agattttcta ctgcactttt aggagattag atcctgagga aaaccataca 12600
gctgaattgg tcatcccagg taatattctg aatgtgtcca ttaaaatatg tctaacactg 12660
tcccctagca cctagcatga tgtctgccta tcatagtcat tcagtgattg ttgaataaat 12720
gaatgaatga ataacactat gtttacaaaa tatatcctaa ttcctcacct ccattcatcc 12780
aaaccatatt gttacttaat aaacattcag cagatattta tggaatatac cttttgttcc 12840
atgcattgta gtactcattg gatacacata gaataataag actcagttca cactcttcag 12900
gaaacagata aaaaactaag aaacaaacaa aaaacaggca atccaacacc atgtgggaaa 12960
tgctttcata gccgggaaac ctggggaata cctgagagga atactcaatt caggccttgt 13020
ttcaggaatc caaatcctgg cacatcagag ctgcttccct ctttccaggg tggcaggaaa 13080
taaatggaac atatttttct atcttatgcc aaacatgagg gaccctttct ccccggtgcc 13140
tctcccaagg tagtctacaa tatttcaact ctagcagtct gcttagtgca tagaacatga 13200
ggctgtgtgt ccctgggcaa attactagac ttctgtgtgc ttcactttcc ctgtaggatt 13260
ataatctact gagcaagctt attgtaaggg tcagattagc aacagtgtat gaaaatgatt 13320
tgagaccatt gcctgcacaa attcaactat ttttttttat ctcactactc tacagaagta 13380
ggtagggtgg gagacagagt ctgatgagag gctcagaatg tgaaagaaag tgaggcgagt 13440
gagcatgata tttaatataa acacaaagat attctgagaa gagctgctca ctgccccctc 13500
ccccaataca tgttgatagg aaaatgccac gtacttcagc aaaaacaact gaaaaattag 13560
atagaaaagt caatcaatag gaaaagataa tccaggacgg tgttgtgaac agaaagaggg 13620
ggaaaaaact ttagaaaatg atggggatgc tcttactggg gtacgagtcc tcaggtattg 13680
aactggcttt cagtaaaagc tagattagtg ggttcctgcc atttacaagc tgttttatga 13740
caacttactt gttgggtggc ctacagtaac tcacctaact gcactgagtc tgtttcctca 13800
tctgtaaatt ggggattttt ttttaaatac ctggcatgcc taactcataa agttgttctg 13860
aaactgaaat aaaacatacg tgaacaggca ttgtaaactg taagttacgg aaaaagctgg 13920
ctgttgttgt gtctttaaag tttcacctgg gtagtcaaag atggatcatg ggtctcagtg 13980
gagagctgag ccaggcagga gctgactaag ggtgagaggt gggagttagc agcctctgaa 14040
catctgtgta ccatgggacc ccctttcctc ctgcatggta ccccagacaa ggagcctagt 14100
aagagatact aatggcttgt tgtccagaga tgttcaaact gcagagaaag ataagacaac 14160
aagcattggc ctccaatcat gatgacagat aggaggaggt gggagctcct tagcagtgct 14220
ggttggcctt ccatgttcta ctgtgggcca tctctgccat gtactgtagg ctactagctt 14280
ctatattaaa gaatgcaaga ggggccagga gcggaggctc atgcctgtaa tctcagcact 14340
ttgggaggcc aaggtgggca gatcacttga ggtcaggagt ttgtgaccag cctggccaac 14400
atggtgaaac tctgccttta ctaaaaatat aaaaattagc tgggtgtggt ggtgtgcacc 14460
tgtaatccca gctactcggg agactgaggc acaagaattg cttgaacctg ggaggcggaa 14520
gttgcagtga gcccagattg cgccactgca ctccaccctg ggcaacagag aaagactctg 14580
cctcaaaaaa aaaaaaaaaa agcaagagga agtgaaataa tcaaggccgc catttaatag 14640
tgagcagcca ctccatgtgg tactgtgcaa gcacattata aatattagcc tcacaagaaa 14700
tgtattagca tttgtatttt gtacactggt taagtatctt gcccaagacc tcaaaactgg 14760
ttaagggcag cagaatttag ccccagcacc accttttcaa agcctgggct tctcacactt 14820
ctccatgctg ttcccatttt aacacaggta tctcgccatt ccagccactc aaactttggc 14880
atttaagaaa attatcctaa agctaaacta aacttcaagg atgaccattc tcctgacccc 14940
ttcccatcaa aattttatct ttagtcagtt tgttttcgtt ttgttttgtt tttcagaact 15000
acctctggca catcctccaa atgaaaggac tcacttggta attctgggag ccatcttatt 15060
atgccttggt gtagcactga cattcatctt ccgtttaaga aaaggtagta tttccttaat 15120
tgcagtggtc tccactgggg gtgaggaagg ggtgagaatt ggatcatggc tgcaaggaaa 15180
cccgacttaa cctctgcaag gtggtgcaaa ggcattccac tgttcaacag caattatatt 15240
gaagctgagt gggatcactg ggtgaagatg aagcgtaagg ggtgaggggc aggagaatgg 15300
gtatggatgg aggtagaaga tgcagtgtca tacagttttt ttctatcatg aaaataacca 15360
cagacttaca gaagagaaag agctaaaatg cccgtcattt tcagttgcat tttagtcttg 15420
cattagttgc aaccagctgg tttctgggta ccctaagtaa taaaaatagt tcctctgtag 15480
aactgtagta tgtttaccat agagtatttt gcaaaatttt tggtagagga tgttacataa 15540
tttgcatgtg ttcatttctc catttacctg tgggaacaat taaaatccag gaaaatgagt 15600
atattcaaat aatttcctcc catttaagat gagtcagagt aaataattcc tccaatactt 15660
agagaagtat accaagagat ccagtgatgg tatagagttg tctgatgtta aatagggaag 15720
tagaatatgg aaggggattc caatagtcgt tgaaaaattc cccataaccc cttacatggg 15780
ggaaagtagt gttaactgag agagtagaga taagctgttt ccaaaaatta tattcttaac 15840
aggactgaga tagccagaat ataaggatca agtttcaatg acagtaagat cctgagatgg 15900
agttgatttg cacaaagaaa taattgttgc cagcatgcat tttgaatatt tctctggaaa 15960
aaaagattag ttggcagtag aaatggatag aaatcaatag atattaaaat acctcagaat 16020
ttggttcatc tctgggaaaa gatgaaaaat aaaagtgtat actcctcaag aacatctagg 16080
atcaaaagca tgtgccctac actattgaat taattaacct cataagttgg gacctgtgga 16140
ataaggatgt ccaccagact tcctagggat tacaaatgtt tcacagaact tgaaatttaa 16200
acttgggtca ctgtatggga tgtagagctg tgctatatgg aaataaaaat gatttctttt 16260
tctcaaggga gaatgatgga tgtgaaaaaa tgtggcatcc aagatacaaa ctcaaagaag 16320
caaagtggta agaatatcag aaggaattgg gaagtaaaag tcaaaggaaa caaaaagcta 16380
aagcaataac aaagagaaat ccatcagtca taatctcctc tccttttaaa gaatgctggt 16440
tcccctttgc ctcacagcta acacaagaac tcctccaccg tctgaggagg tttaggagca 16500
gggaagggga aggagtcagc ttcatttgct aatcttctgt tgccctgcac cctagcagct 16560
ccttgcagca ggggacaagg atgacttagg tggatggata attaattgat tctaaaatat 16620
tgtgtgtcag tattgtaata ctatgttaat tgcaccatgc acggtatctc atttaatccc 16680
ccaccccttg ccattaccaa agagagagag agagagagag agagaaatac tagaatttat 16740
cctcatttta cagtagagaa aacagagggt caagaagata atgtaaagtg cccaagaaca 16800
cacagctgat cacaaaaatc aagcttgggg gccattagcc taaccacaga cccttactct 16860
taacccatct gcttcaatcc attttgctac aaatgtttac atttataagc agggcagaaa 16920
aacctcatcc aggttattga actaagaaga aagttatatt aaggtttcta atttttttaa 16980
tgtagttaga aaccaaactt aacaatgagc ccaagtttaa agcagtctaa ttaacctgga 17040
caagctcagg caagtttcat tctgtggccc atagcatcat ctgtgttgta aagctaagta 17100
gcaaatgttg tttgggtcat gctgggggac aagccatccc aatttgctca ggactgaggg 17160
gttttccagg atatcatgta aggataattg ggtacaaata taacctgctg ctttctctca 17220
tttcaaattt atcatttatc atatcagcaa ctatgagtta tgttttttat tagatttctt 17280
gttacttttt ccccagacca cttcccatga aattaatata ctattatcac tctccagata 17340
cacatttgga ggagacgtaa tccagcattg gaacttctga tcttcaagca gggattctca 17400
acctgtggtt taggggttca tcggggctga gcgtgacaag aggaaggaat gggcccgtgg 17460
gatgcaggca atgtgggact taaaaggccc aagcactgaa aatggaacct ggcgaaagca 17520
gaggaggaga atgaagaaag atggagtcaa acagggagcc tggagggaga ccttgatact 17580
ttcaaatgcc tgaggggctc atcgacgcct gtgacaggga gaaaggatac ttctgaacaa 17640
ggagcctcca agcaaatcat ccattgctca tcctaggaag acgggttgag aatccctaat 17700
ttgagggtca gttcctgcag aagtgccctt tgcctccact caatgcctca atttgttttc 17760
tgcatgactg agagtctcag tgttggaacg ggacagtatt tatgtatgag tttttcctat 17820
ttattttgag tctgtgaggt cttcttgtca tgtgagtgtg gttgtgaatg atttcttttg 17880
aagatatatt gtagtagatg ttacaatttt gtcgccaaac taaacttgct gcttaatgat 17940
ttgctcacat ctagtaaaac atggagtatt tgtaaggtgc ttggtctcct ctataactac 18000
aagtatacat tggaagcata aagatcaaac cgttggttgc ataggatgtc acctttattt 18060
aacccattaa tactctggtt gacctaatct tattctcaga cctcaagtgt ctgtgcagta 18120
tctgttccat ttaaatatca gctttacaat tatgtggtag cctacacaca taatctcatt 18180
tcatcgctgt aaccaccctg ttgtgataac cactattatt ttacccatcg tacagctgag 18240
gaagcaaaca gattaagtaa cttgcccaaa ccagtaaata gcagacctca gactgccacc 18300
cactgtcctt ttataataca atttacagct atattttact ttaagcaatt cttttattca 18360
aaaaccattt attaagtgcc cttgcaatat caatcgctgt gccaggcatt gaatctacag 18420
atgtgagcaa gacaaagtac ctgtcctcaa ggagctcata gtataatgag gagattaaca 18480
agaaaatgta ttattacaat ttagtccagt gtcatagcat aaggatgatg cgaggggaaa 18540
acccgagcag tgttgccaag aggaggaaat aggccaatgt ggtctgggac ggttggatat 18600
acttaaacat cttaataatc agagtaattt tcatttacaa agagaggtcg gtacttaaaa 18660
taaccctgaa aaataacact ggaattcctt ttctagcatt atatttattc ctgatttgcc 18720
tttgccatat aatctaatgc ttgtttatat agtgtctggt attgtttaac agttctgtct 18780
tttctattta aatgccacta aattttaaat tcataccttt ccatgattca aaattcaaaa 18840
gatcccatgg gagatggttg gaaaatctcc acttcatcct ccaagccatt caagtttcct 18900
ttccagaagc aactgctact gcctttcatt catatgttct tctaaagata gtctacattt 18960
ggaaatgtat gttaaaagca cgtattttta aaattttttt cctaaatagt aacacattgt 19020
atgtctgctg tgtactttgc tatttttatt tattttagtg tttcttatat agcagatgga 19080
atgaatttga agttcccagg gctgaggatc catgccttct ttgtttctaa gttatctttc 19140
ccatagcttt tcattatctt tcatatgatc cagtatatgt taaatatgtc ctacatatac 19200
atttagacaa ccaccatttg ttaagtattt gctctaggac agagtttgga tttgtttatg 19260
tttgctcaaa aggagaccca tgggctctcc agggtgcact gagtcaatct agtcctaaaa 19320
agcaatctta ttattaactc tgtatgacag aatcatgtct ggaacttttg ttttctgctt 19380
tctgtcaagt ataaacttca ctttgatgct gtacttgcaa aatcacattt tctttctgga 19440
aattccggca gtgtaccttg actgctagct accctgtgcc agaaaagcct cattcgttgt 19500
gcttgaaccc ttgaatgcca ccagctgtca tcactacaca gccctcctaa gaggcttcct 19560
ggaggtttcg agattcagat gccctgggag atcccagagt ttcctttccc tcttggccat 19620
attctggtgt caatgacaag gagtaccttg gctttgccac atgtcaaggc tgaagaaaca 19680
gtgtctccaa cagagctcct tgtgttatct gtttgtacat gtgcatttgt acagtaattg 19740
gtgtgacagt gttctttgtg tgaattacag gcaagaattg tggctgagca aggcacatag 19800
tctactcagt ctattcctaa gtcctaactc ctccttgtgg tgttggattt gtaaggcact 19860
ttatcccttt tgtctcatgt ttcatcgtaa atggcatagg cagagatgat acctaattct 19920
gcatttgatt gtcacttttt gtacctgcat taatttaata aaatattctt atttattttg 19980
ttacttggta caccagcatg tccattttct tgtttatttt gtgtttaata aaatgttcag 20040
tttaacatcc cagtggagaa agtta 20065
Claims (3)
1.rs4143815基因型在制备诊断预测乳腺癌新辅助化疗疗效标记物的用途，所述rs4143815基因型为CC、CG或者GG，所述的新辅助化疗为接受紫杉醇联合顺铂的单周新辅助化疗方案；所述的新辅助化疗的疗效是指：基因型为CC和CG的患者与基因型为GG的患者相比更容易出现中性粒细胞减少，而不容易出现水肿的不良反应，基因型为CC+GG的患者与基因型为CG的患者相比，更容易出现谷草转氨酶升高；在预测病理完全缓解（pCR）方面，基因型为GC的患者比基因型为GG和CC的患者易于pCR；在生存方面，基因型为GG的患者生存劣于基因型为GC和CC 的患者；基因型为GG患者比基因型为GC和CC的患者复发风险较高。
2.根据权利要求1所述的用途，其特征在于：所述的乳腺癌为激素受体阳性。
3.rs4143815基因型在制备诊断预测乳腺癌新辅助化疗疗效的试剂中的用途，所述rs4143815基因型为CC、CG或者GG，所述的新辅助化疗为接受紫杉醇联合顺铂的单周新辅助化疗方案；所述的新辅助化疗的疗效是指：基因型为CC和CG的患者与基因型为GG的患者相比更容易出现中性粒细胞减少，而不容易出现水肿的不良反应，基因型为CC+GG的患者与基因型为CG的患者相比，更容易出现谷草转氨酶升高；在预测病理完全缓解（pCR）方面，基因型为GC的患者比基因型为GG和CC的患者易于pCR；在生存方面，基因型为GG的患者生存劣于基因型为GC和CC 的患者；基因型为GG患者比基因型为GC和CC的患者复发风险较高。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602746A (zh) * | 2013-11-26 | 2014-02-26 | 浙江省肿瘤医院 | 预测乳腺癌新辅助化疗疗效的生物标记物及其用途 |
| US9045545B1 (en) * | 2014-07-15 | 2015-06-02 | Kymab Limited | Precision medicine by targeting PD-L1 variants for treatment of cancer |
| US9139648B1 (en) * | 2014-07-15 | 2015-09-22 | Kymab Limited | Precision medicine by targeting human NAV1.9 variants for treatment of pain |
| CN106062004A (zh) * | 2013-12-17 | 2016-10-26 | 科马布有限公司 | 人靶标 |
| CN107530429A (zh) * | 2015-03-30 | 2018-01-02 | 斯特库比股份有限公司 | 特异性针对糖基化的pd‑l1的抗体及其使用方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103602746A (zh) * | 2013-11-26 | 2014-02-26 | 浙江省肿瘤医院 | 预测乳腺癌新辅助化疗疗效的生物标记物及其用途 |
| CN106062004A (zh) * | 2013-12-17 | 2016-10-26 | 科马布有限公司 | 人靶标 |
| US9045545B1 (en) * | 2014-07-15 | 2015-06-02 | Kymab Limited | Precision medicine by targeting PD-L1 variants for treatment of cancer |
| US9139648B1 (en) * | 2014-07-15 | 2015-09-22 | Kymab Limited | Precision medicine by targeting human NAV1.9 variants for treatment of pain |
| CN107530429A (zh) * | 2015-03-30 | 2018-01-02 | 斯特库比股份有限公司 | 特异性针对糖基化的pd‑l1的抗体及其使用方法 |
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