CN108728440A - Ctla-4基因的用途及相关药物 - Google Patents

Ctla-4基因的用途及相关药物 Download PDF

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CN108728440A
CN108728440A CN201710277551.6A CN201710277551A CN108728440A CN 108728440 A CN108728440 A CN 108728440A CN 201710277551 A CN201710277551 A CN 201710277551A CN 108728440 A CN108728440 A CN 108728440A
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ctla
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林菊芳
廖敬礼
袁纪军
李宗然
张秀
石钰
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Shanghai Jibi Biotechnology Co Ltd
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Abstract

本发明涉及生物技术领域，更具体地涉及CTLA‑4基因的用途及其相关药物。本发明设计了针对人CTLA‑4基因的134个RNAi靶点序列，构建相应的CTLA‑4 RNAi，使用转染载体将CTLA‑4 RNAi导入T细胞，能够降低T细胞中CTLA‑4基因的表达水平，显著抑制细胞中CTLA‑4的表达，促进T细胞活化。

Description

CTLA-4基因的用途及相关药物
技术领域
本发明涉及生物技术领域，更具体地涉及CTLA-4基因的用途及相关药物。
背景技术
细胞毒T淋巴细胞抗原4(CTLA-4)又名CD152，是由CTLA-4基因编码的一种跨膜蛋白质，表达于活化的T细胞，与T细胞表面的协同刺激分子受体(CD28)具有高度的同源性，能够结合抗原呈递细胞(APC)上的协同刺激分子B7，但不同于CD28，CTLA-4与B7分子结合后抑制T细胞活化。其机制一是与CD28竞争性的结合B7或者招募磷酸酶到CTLA-4的胞内结构域部分从而降解T细胞受体(TCR)和CD28的信号，二是降低CD80/CD86在APC的表达水平或者通过转胞吞作用将它们从APC上移除，这样就减少了CD28参与激活T细胞。此外CTLA-4还会介导树突细胞结合CD80/CD86并诱导色氨酸降解酶IDO的表达，从而导致TCR的抑制。伴随外源性抗原或肿瘤特异性靶抗原的刺激，T细胞被激活，CTLA-4表达水平上升抑制T细胞的免疫反应。因此阻断CTLA-4的免疫效应可刺激T细胞增殖，从而诱导或增强抗肿瘤免疫反应。目前CTLA-4抗体Ipilimumab已于2011年被FDA批准用于治疗黑色素瘤。
RNA干扰(RNA interference,RNAi)即用核苷酸组成的短的双链RNA(dsRNA)进行转录后基因沉默。它可高效、特异地剔除或关闭特定基因的表达，所以该技术已被广泛用于探索基因功能和传染性疾病及恶性肿瘤的基因治疗领域。已有很多针对不同基因的干扰RNA在体内和体外有效的抑制了肿瘤细胞的生长，这些目的基因包括：Bcl-2、survivin、EGFR、VEGF等。RNA干扰代替传统反义核酸进行转录后基因沉默，能高效地特异性抑制目的基因。与传统的方法相比，RNA干扰技术设计更简便、作用迅速、效果明显，其技术优势还在于能根据不同病情，设计个体化治疗方案。
发明内容
本发明的目的在于公开与人CTLA-4基因相关的治疗方法及药物，以RNA干扰(RNAi)为手段下调CTLA-4基因在T细胞中的表达。进而，防止CTLA-4与B7分子结合后抑制T细胞活化。
为实现上述目的及其他相关目的，本发明采用如下技术方案：
本发明的第一方面，提供了一种促进T细胞活化的方法，为降低T细胞中CTLA-4基因的表达，从而促进T细胞的活化。
优选地，所述方法包括但不限于特异性抑制CTLA-4基因的转录或翻译，或特异性抑制CTLA-4蛋白的表达或活性。
所述促进T细胞活化包括但不限于：刺激T细胞增殖和/或增强抗肿瘤免疫反应。
具体地，所述降低肿T细胞中CTLA-4基因的表达水平，可以采用各种化学、物理、生物的方法。包括但不限于：抑制CTLA-4基因的转录或翻译。抑制的方法可采用基因敲除，基因敲低使CTLA-4基因失活或活性降低；或者也可利用抗体、蛋白、小分子等与CTLA-4基因的表达产物结合使相关基因表达产物降低。
作为典型的代表，例如可以RNA干扰为手段，降低T细胞中CTLA-4基因的表达水平。
所述的促进T细胞活化的方法可以是体内或体外的。
采用本发明的方法体外处理后活化的T细胞可被用于治疗的或非治疗目的。
本发明的第二方面，提供了一种促进T细胞活化的分离的核酸分子，所述核酸分子包含：
a)双链RNA，所述双链RNA中含有能够在严紧条件下与CTLA-4基因杂交的核苷酸序列；或者
b)shRNA，所述shRNA中含有能够在严紧条件下与CTLA-4基因杂交的核苷酸序列。
进一步地，所述双链RNA包含第一链和第二链，所述第一链和所述第二链互补共同形成RNA二聚体，并且所述第一链的序列与CTLA-4基因中15-27个连续的核苷酸序列基本相同。较佳的，所述第一链的序列与CTLA-4基因中19-23个连续的核苷酸序列基本相同；更佳的，所述第一链的序列与CTLA-4基因中19、20或者21个连续的核苷酸序列基本相同。
优选地，所述CTLA-4基因来源于人。
更进一步地，所述双链RNA包含第一链和第二链，所述第一链和所述第二链互补共同形成RNA二聚体，并且所述第一链的序列与CTLA-4基因中的靶序列基本相同。
所述双链RNA第一链和第二链的长度均为15-27个核苷酸；较佳的，长度均为19-23个核苷酸；最佳的，长度均为19、20或者21个核苷酸。
优选地，所述CTLA-4基因中的靶序列选自如SEQ ID NO：1～134所示之任一序列或如SEQ ID NO：1～134所示之任一序列经过取代、缺失或添加一个或几个碱基序列且具有与如SEQ ID NO：1～134所示之任一序列相同活性的序列。
优选地，所述经过取代、缺失或添加一个或几个碱基序列具体是指在如SEQ IDNO：1～134所示之任一序列的5’或3’端增加或减少5个以下碱基，或在序列中突变少于2个碱基，或既有碱基的加减也有碱基突变。
进一步地，所述双链RNA为小干扰RNA(siRNA)。更进一步地，所述小干扰RNA第一链的序列如SEQ ID NO：135～268任一序列所示。
进一步地，所述shRNA包括正义链片段和反义链片段，以及连接所述正义链片段和反义链片段的茎环结构，所述正义链片段和所述反义链片段的序列互补，并且所述正义链片段的序列与CTLA-4基因中15-27个连续的核苷酸序列基本相同。所述shRNA经加工后可成为小干扰RNA(siRNA)进而起到特异性沉默T细胞中内源CTLA-4基因表达的作用。
更进一步地，所述shRNA包括正义链片段和反义链片段，以及连接所述正义链片段和反义链片段的茎环结构，所述正义链片段和所述反义链片段的序列互补，并且所述正义链片段的序列与CTLA-4基因中的靶序列基本相同。
较佳的，所述正义链片段与CTLA-4基因中19-23个连续的核苷酸序列基本相同；更佳的，所述正义链片段与CTLA-4基因中19、20或者21个连续的核苷酸序列基本相同。
进一步的，所述shRNA的茎环结构的序列可选自以下任一：UUCAAGAGA、AUG、CCC、UUCG、CCACC、CUCGAG、AAGCUU和CCACACC。
更进一步的，所述shRNA的序列如SEQ ID NO:269～402任一序列所示。
shRNA经酶切加工后可成为siRNA，进而起到特异性沉默T细胞内源性CTLA-4基因表达的作用。
所述双链RNA的第一链或所述shRNA的正义链片段与CTLA-4基因中的靶序列基本相同，所述CTLA-4基因的靶序列即为siRNA用于特异性沉默CTLA-4基因表达时，被所述siRNA识别并沉默的mRNA片段所对应的CTLA-4基因的片段。
较佳的，所述CTLA-4基因中的靶序列含有SEQ ID NO:1-134之任一序列。
进一步的，所述CTLA-4基因来源于人。
本发明的第三方面，提供一种CTLA-4基因干扰核酸构建体，含有编码本发明所述分离的核酸分子中的shRNA的基因片段，能表达所述shRNA。
所述的CTLA-4基因干扰核酸构建体可以是将编码前述CTLA-4shRNA的基因片段克隆入已知载体获得。
进一步地，所述CTLA-4基因干扰核酸构建体选自病毒载体或质粒载体。
更进一步地，所述CTLA-4基因干扰核酸构建体选自慢病毒载体、腺病毒载体、逆转录病毒载体、AAV。
本发明的CTLA-4基因干扰慢病毒载体是将编码前述CTLA-4基因shRNA的DNA片段克隆入已知载体获得，所述已知载体多为慢病毒载体，所述CTLA-4基因干扰慢病毒载体经过病毒包装成为有感染力的病毒颗粒后，感染T细胞，进而转录出本发明所述shRNA，通过酶切加工等步骤，最终获得所述siRNA，用于特异性沉默CTLA-4基因的表达。
进一步的，所述CTLA-4基因干扰慢病毒载体还含有启动子序列和/或编码T细胞中可被检测的标记物的核苷酸序列；较优的，所述可被检测的标记物如绿色荧光蛋白(GFP)。
进一步的，所述慢病毒载体可以选自：pLKO.1-puro、pLKO.1-CMV-tGFP、pLKO.1-puro-CMV-tGFP、pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、pLKO.1-puro-CMV-TagCFP、pLKO.1-puro-CMV-TagYFP、pLKO.1-puro-CMV-TagRFP、pLKO.1-puro-CMV-TagFP635、pLKO.1-puro-UbC-TurboGFP、pLKO.1-puro-UbC-TagFP635、pLKO-puro-IPTG-1xLacO、pLKO-puro-IPTG-3xLacO、pLP1、pLP2、pLP/VSV-G、pENTR/U6、pLenti6/BLOCK-iT-DEST、pLenti6-GW/U6-laminshrna、pcDNA1.2/V5-GW/lacZ、pLenti6.2/N-Lumio/V5-DEST、pGCSIL-GFP或pLenti6.2/N-Lumio/V5-GW/lacZ中的任一。
本发明实施例具体列举了以pGCSIL-GFP为载体构建的人CTLA-4基因干扰慢病毒载体，命名为pGCSIL-GFP-CTLA-4-siRNA。
本发明的CTLA-4基因siRNA可用于敲低T细胞的CTLA-4基因，进一步地可以用作T细胞活化促进剂。CTLA-4基因干扰慢病毒载体则可用于制备所述CTLA-4基因siRNA。当用作T细胞活化促进剂时，是将安全有效量的所述核酸分子施用于免疫细胞。具体剂量还应考虑具体情况，这些都是熟练医师技能范围之内的。
本发明的第四方面，公开了一种CTLA-4基因干扰慢病毒，由前述CTLA-4基因干扰慢病毒载体在慢病毒包装质粒、细胞系的辅助下，包装成慢病毒。该慢病毒可感染T细胞并产生针对CTLA-4基因的小分子干扰RNA，从而抑制CTLA-4的表达。该CTLA-4基因干扰慢病毒可用于制备T细胞活化促进剂。
本发明的第五方面，提供了前述分离的核酸分子、CTLA-4基因干扰核酸构建体、和/或所述的CTLA-4基因干扰慢病毒在制备T细胞活化促进剂中的用途。
优选地，所述T细胞活化促进剂，其有效物质含有以下任一项：(1)前述的分离的核酸分子；(2)CTLA-4基因干扰核酸构建体；(3)CTLA-4基因干扰慢病毒。
优选地，所述T细胞活化促进剂作用于T细胞，降低T细胞中CTLA-4基因的表达，获得活化的T细胞。所述活化的T细胞可通过CART，DC，CIK或DC-CIK等免疫治疗手段制备肿瘤治疗药物。
本发明的第六方面，公开了一种用于促进T细胞活化的促进剂，其有效物质含有以下之任一项：(1)前述的分离的核酸分子；(2)CTLA-4基因干扰核酸构建体；(3)CTLA-4基因干扰慢病毒。
进一步的，所述促进剂含有1～99wt％有效物质以及药学上可接受的载体、稀释剂或赋形剂。
在制备这些促进剂时，通常将活性成分与赋形剂混合，或用赋形剂稀释，或包在可以胶囊或药囊形式存在的载体中。当赋形剂起稀释剂作用时，它可以是固体、半固体或液体材料作为赋形剂、载体或活性成分的介质。因此，组合物可以是片剂、丸剂、粉剂、溶液剂、糖浆剂、灭菌注射溶液等。合适的赋形剂的例子包括：乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水、等。制剂还可包括:湿润剂、乳化剂、防腐剂(如羟基苯甲酸甲酯和丙酯)、甜味剂等。
本发明的第七方面，公开了一种促进T细胞活性的试剂盒，所述试剂盒包括：存在于容器中的以下之任一项：(1)前述的分离的核酸分子；(2)CTLA-4基因干扰核酸构建体；(3)CTLA-4基因干扰慢病毒。
本发明的第八方面，提供了一种T细胞，所述T细胞中含有以下之任一项：(1)前述的分离的核酸分子；(2)CTLA-4基因干扰核酸构建体；(3)CTLA-4基因干扰慢病毒。
所述T细胞可通过CAR-T技术用于治疗肿瘤或自身免疫疾病。
与现有技术相比，本发明具有如下有益效果：
本发明设计了针对人CTLA-4基因的134个RNAi靶点序列，构建相应的CTLA-4RNAi，所述CTLA-4RNAi能够显著下调CTLA-4基因在mRNA水平和蛋白水平的表达。使用转染载体将针对CTLA-4基因的RNAi序列高效导入T细胞，降低T细胞中CTLA-4基因的表达水平，显著抑制细胞中CTLA-4的表达，促进T细胞活化。
附图说明
图1：pGCSIL-GFP质粒DNA图谱。
图2：通过免疫印迹的方法(western blot)确定此293T细胞能够稳定表达CTLA-4基因。
图3：CTLA-4的蛋白表达水平明显下调。
具体实施方式
本发明的发明人首先制备了能够稳定表达CTLA-4的293T细胞株；并设计了一组针对人CTLA-4基因的小分子干扰RNA(siRNA)序列。选取人CTLA-4mRNA编码区序列作为siRNA的靶位点，根据靶位点中连续的10-30(优选15-27，更优选19-23)个碱基序列设计siRNA靶点序列。通过转染载体将小分子干扰RNA(siRNA)序列导入能够稳定表达CTLA-4的293T细胞株。细胞实验证明，上述siRNA序列能够特异性沉默人肿瘤细胞中内源CTLA-4基因的表达。
发明人发现，采用RNAi方法可以有效下调人CTLA-4基因的转录水平，并进一步可以有效抑制CTLA-4蛋白水平的表达。
下面结合实施例进一步阐述本发明。应理解，实施例仅用于说明本发明，而非限制本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照常规条件，如[美]Sambrook.J等著；黄培堂等译。分子克隆试验指南，第三版。北京：科学出版社2002中所述的条件或者制造商建议的条件进行或配置。
实施例1针对人CTLA-4基因的RNAi慢病毒的制备
1.筛选针对人CTLA-4基因的有效的siRNA靶点
从Genbank调取CTLA-4(Gene ID:1493)基因信息；设计针对CTLA-4基因的有效的siRNA靶点。表1列出了其中134条针对CTLA-4基因的有效siRNA靶点序列。
表1 靶向于人CTLA-4基因的siRNA靶点序列
2.慢病毒载体的制备
分别针对siRNA靶点合成两端含Age I和EcoR I酶切位点粘端的双链DNA Oligo序列(表2)；以Age I和EcoR I限制性内切酶作用于pGCSIL-GFP载体(上海吉凯基因化学技术有限公司提供，图1)，使其线性化，琼脂糖凝胶电泳鉴定酶切片段。
通过T4DNA连接酶将双酶切线性化(酶切体系如表4所示，37℃，反应1h)的载体DNA和纯化好的双链DNA Oligo连接，在适当的缓冲体系(连接体系如表5所示)中于16℃连接过夜，回收连接产物。将连接产物转化氯化钙制备的新鲜的大肠杆菌感受态细胞(转化操作参考：分子克隆实验指南第二版55-56页)。在连接转化产物长出菌克隆表面沾一下，溶于10μlLB培养基，混匀取1μl作为模板；在以慢病毒载体中RNAi序列的上下游，设计通用PCR引物，上游引物序列：5’-CCTATTTCCCATGATTCCTTCATA-3’(SEQ ID NO:671)；下游引物序列：5’-GTAATACGGTTATCCACGCG-3’(SEQ ID NO:672)，进行PCR鉴定实验(PCR反应体系如表6-1，反应条件如表6-2)。对PCR鉴定阳性的克隆进行测序和比对分析，比对正确的克隆即为构建成功的分别针对不同siRNA的表达RNAi的载体，分别命名为pGCSIL-GFP-CTLA-4-siRNA1～～pGCSIL-GFP-CTLA-4-siRNA134。
表2 两端含Age I和EcoR I酶切位点粘端的双链DNA Oligo
构建pGCSIL-GFP-Scr-siRNA阴性对照质粒，阴性对照siRNA靶序列为5’-TTCTCCGAACGTGTCACGT-3’(SEQ ID NO：673)。构建pGCSIL-GFP-Scr-siRNA阴性对照质粒时，针对Scr siRNA靶点合成两端含Age I和EcoR I酶切位点粘端的双链DNAOligo序列(表3)，其余构建方法、鉴定方法及条件均同pGCSIL-GFP-CTLA-4-siRNA1～～pGCSIL-GFP-CTLA-4-siRNA134。
表3 两端含Age I和EcoR I酶切位点粘端的双链DNA Oligo
表4 pGCSIL-GFP质粒酶切反应体系
试剂 体积(μl)
pGCSIL-GFP质粒(1μg/μl) 2.0
10×buffer 5.0
100×BSA 0.5
Age I(10U/μl) 1.0
EcoR I(10U/μl) 1.0
dd H2O 40.5
Total 50.0
表5 载体DNA和双链双链DNA Oligo连接反应体系
试剂 阳性对照(μl) 自连对照(μl) 连接组(μl)
线性化的载体DNA(100ng/μl) 1.0 1.0 1.0
退火的双链DNA Oligo(100ng/μl) 1.0 - 1.0
10×T4噬菌体DNA连接酶缓冲液 1.0 1.0 1.0
T4噬菌体DNA连接酶 1.0 1.0 1.0
dd H2O 16.0 17.0 16.0
Total 20.0 20.0 20.0
表6-1 PCR反应体系
试剂 体积(μl)
10×buffer 2.0
dNTPs(2.5mM) 0.8
上游引物 0.4
下游引物 0.4
Taq聚合酶 0.2
模板 1.0
ddH2O 15.2
Total 20.0
表6-2 PCR反应体系程序设定
3.包装CTLA-4-siRNA慢病毒
以Qiagen公司的质粒抽提试剂盒分别提取上述构建成功的RNAi质粒，配制成100ng/μl储存液。
转染前24h，用胰蛋白酶消化对数生长期的人胚肾细胞293T细胞，重悬在含10％胎牛血清的DMEM完全培养基中，调整细胞密度为1.5×105细胞/ml，接种于6孔板，37℃，5％CO2培养箱内培养。待细胞密度达70％-80％时即可用于转染。转染前2h，吸出原有培养基，加入1.5ml新鲜的完全培养基。按照Sigma-aldrich公司的MISSION Lentiviral Packaging Mix试剂盒的说明，向一灭菌离心管中加入Packing Mix(PVM)20μl，PEI 12μl，无血清DMEM培养基400μl，取20μl上述抽提的质粒DNA，加至上述PVM/PEI/DMEM混合液。
将上述转染混和物在室温下孵育15min，转移至人胚肾细胞293T细胞的培养基中，37℃，5％CO2培养箱内培养16h。弃去含有转染混和物的培养介质，PBS溶液洗涤，加入完全培养基2ml，继续培养48h。收集细胞上清液，Centricon Plus-20离心超滤装置(Millipore)纯化和浓缩慢病毒，步骤如下：(1)4℃，4000g离心10min，除去细胞碎片；(2)0.45μm滤器过滤上清液于40ml超速离心管中；(3)4000g离心，10-15min，至需要的病毒浓缩体积；(4)离心结束后，将过滤杯和下面的滤过液收集杯分开，将过滤杯倒扣在样品收集杯上，离心2min离心力不超过1000g；(5)把离心杯从样品收集杯上移开，样品收集杯中的即为病毒浓缩液。将病毒浓缩液分装后于-80摄氏度保存。
实施例2实时荧光定量RT-PCR法检测CTLA-4基因的沉默效率
(一)构建稳定表达CTLA-4的293T
具体方法包括如下步骤：
处于对数生长期的293T细胞进行胰酶消化，制成细胞悬液(细胞数约为5×104/ml)每孔500ul接种于24孔板中，培养至细胞融合度达到约20％。根据侵染复数值(MOI＝10)，加入适宜量的稳定表达CTLA-4同时表达嘌呤霉素抗性基因(嘌呤霉素N-乙酰转移酶(PAC))的慢病毒，16小时后换液，48小时后在培养基中加入2ug/ml的嘌呤霉素，继续培养3天，通过免疫印迹的方法(western blot)确定此293T细胞能够稳定表达CTLA-4基因，结果如图2所示。
(二)实时荧光定量RT-PCR法检测CTLA-4基因的沉默效率
2.1细胞转染
过表达CTLA-4的293T细胞培养在DMEM+10％FBS培养基中，用0.25％Trypsin-EDTA消化，调整细胞数量为50000cell/mL，12孔板中，每孔1ml，37℃，5％CO2培养箱孵育过夜。
采用Invitrogen3000作为转染试剂载体，将小分子干扰RNA(siRNA)序列导入能够稳定表达CTLA-4的293T细胞株，，所述小干扰RNA第一链的序列如SEQID NO：135～268任一序列所示。具体地，分别配置A液和B液：A液为siRNA(CTLA-4)或siRNA(Scr-siRNA)2ug/well，加入Opti-MEM 60ul/well，混合均匀；B液为Invitrogen3000，3ul/well，加入Opti-MEM 60ul/well，混合均匀；然后将A液和B液混合均匀，室温孵育5min，加入12孔板中，37℃，5％CO2培养箱孵育48h后检测敲减效率。
2.2总RNA的抽提
48小时后收集细胞(80％细胞密度)，2000rpm离心5min，去上清，细胞沉淀中加入1mL Trizol，充分混匀后室温静置5min，然后转移至新的1.5mL EP管中，每管加入200μL氯仿，用手上下颠倒EP管15s，室温静置10min。4℃、12800rpm，离心15min。吸取上层液体至新的1.5mL EP管，加入等体积预冷的异丙醇，混匀后4℃静置10min。4℃、12800rpm离心12min后，弃上清。加入1mL、75％乙醇(用DEPC水新鲜配制)，洗涤沉淀。4℃、11800rpm离心5min，弃去大部分上清。4℃、11800rpm再次离心5min，弃去上清，室温干燥。待RNA沉淀基本透明时，加入RNase-free水(加入体积视RNA沉淀量而定)至完全溶解，nanodrop 2000/2000C分光光度计分析测定所抽提RNA的浓度及质量。
2.3反转录获得cDNA
将1μL Oligo dT(0.5μg/μL)和2.0μg Total RNA加入到PCR小管中，补充RNase-Free H2O至10μL；混匀后离心，70℃温浴10min；之后立即置于冰水混合物中冰浴，使OligodT和模板退火。在上述混合物中，按表7的比例配制反应体系(冰上进行)，混匀，短暂离心,上述体系在42℃水浴反应1h，然后在70℃水浴10min使RT酶失活，将得到的RT产物--cDNA置于-20℃保存备用。
表7 逆转录反应体系
试剂 体积(μl)
5×RT buffer 4.0
10mM dNTPs 2.0
RNasin 0.4
M-MLV-RTase 1.0
DEPC H2O 2.6
Total 10.0
注：dNTPs是dATP,dCTP,dGTP,dTTP的混合，浓度为10mM。
3.4Real-time PCR检测
采用TP800型Real time PCR仪(TAKARA)进行实时定量检测。CTLA-4基因的引物如下：上游引物：5’-TTGCAGCAGTTAGTTCGGGG-3’(SEQ ID NO：676)和下游引物5’-GCTCTGTTGGGGGCATTTTC-3’(SEQ ID NO：677)。
以管家基因GAPDH为内参，引物序列如下：上游引物：5’-TGACTTCAACAGCGACACCCA-3’(SEQ ID NO：678)和下游引物5’-CACCCTGTTGCTGTAGCCAAA-3’(SEQ ID NO：679)。按表8中的比例配置反应体系。
表8 Real-time PCR反应体系
试剂 体积(μl)
SYBR premix ex taq: 6.0
上游引物(2.5μM)： 0.5
下游引物(2.5μM)： 0.5
cDNA 1.0
ddH2O 4.0
Total 12.0
设定程序为两步法Real-time PCR：预变性95℃，15s；之后每一步变性95℃，5s；退火延伸60℃，30s；共进行45个循环。每次在延伸阶段读取吸光值。PCR结束后，95℃变性1min，然后冷却至55℃，使DNA双链充分结合。从55℃开始到95℃，每一步增加0.5℃，保持4s，同时读取吸光值，制作熔解曲线。采用2-ΔΔCt分析法计算转染了CTLA-4siRNA之后的293T细胞内的CTLA-4mRNA的表达丰度，计算沉默效率。Scr-siRNA的细胞作为对照。实验结果如表9所示：
表9 CTLA-4siRNA沉默效率
注：+代表敲减效率小于10％，++代表敲减效率在10-30％，+++代表敲减效率在30％-50％，++++代表敲减效率在50％-70％，+++++代表敲减效率在70％及以上。
实施例3蛋白质印迹Western Blot法检测CTLA-4基因的沉默效率
293T过表达CTLA-4细胞株转染siRNA后48小时收取细胞PBS洗涤两次，加入适量预冷的2×Lysis Buffer裂解，配方如下：1M Tris-HCl(pH6.8)100mM；巯基乙醇2％；甘油20％；SDS 4％。细胞刮刮下细胞转移入EP管中，冰上裂解10-15min后超声破碎细胞(200W共4次，每次5s，间隔2s)。4℃、12000g，离心15min，取上清BCA法测定蛋白浓度(BCA ProteinAssay Kit)，调整每个样品蛋白浓度为2μg/μL。上SDS-PAGE胶，浓缩胶80mA，20min；分离胶120mA，1h。电泳结束后，使用转移电泳装置，在4℃、300mA恒流条件下电转150min，将蛋白转移到PVDF膜上。用封闭液(含5％脱脂牛奶的TBST溶液)室温封闭PVDF膜1h或4℃过夜。一抗选用可以识别CTLA-4的鼠源抗体孵育：封闭液稀释抗体，然后与封闭好的PVDF膜室温孵育2h或4℃过夜，并用TBST洗膜4次，每次8min。二抗使用羊抗鼠的抗体孵育：用封闭液稀释相应的二抗，室温下孵育PVDF膜1.5h，并用TBST洗膜4次，每次8min。采用Thermo公司PierceTMECL Western Blotting Substrate试剂盒进行显影。
实验结果如图3表明，CTLA-4的蛋白表达水平明显下调，与实施例2的实验结果基本相符。
此外，采用图1所示的载体作为转染载体包装慢病毒，详见实施例1。在细胞转染时，过表达CTLA-4的293T细胞培养在DMEM+10％FBS培养基中，用0.25％Trypsin-EDTA消化，调整细胞数量为50000cell/mL，12孔板中，每孔1ml，37℃，5％CO2培养箱孵育过夜。根据侵染复数(MOI＝1)值，加入适宜量的实施例1制备的病毒，培养24h后更换培养基。参照上述方法检测敲减效率。细胞实验证明，上述siRNA序列能够特异性沉默人肿瘤细胞中内源CTLA-4基因的表达。与表9和图3的实验结果一致。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
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<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 8
tgatgtgggt caaggaatt 19
<210> 9
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 9
gcaacggaac ccagattta 19
<210> 10
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 10
gtatgcatct ccaggcaaa 19
<210> 11
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 11
gctaaaggtt gtattgcat 19
<210> 12
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 12
ggagcgaggg agaagacta 19
<210> 13
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 13
acacaaggtc atttgctaa 19
<210> 14
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 14
ggactgaggg ccatggaca 19
<210> 15
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 15
tgagaaacca ttatgaaga 19
<210> 16
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 16
gaaagcatca cttgggatt 19
<210> 17
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 17
gagcgaggga gaagactat 19
<210> 18
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 18
gggaattcat ctctcttta 19
<210> 19
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 19
agacagagct gggatgttt 19
<210> 20
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 20
cagctttgtg tgtgagtat 19
<210> 21
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 21
gaacccagat ttatgtaat 19
<210> 22
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 22
atataaagtt ggatgcgga 19
<210> 23
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 23
aagcatcact tgggattaa 19
<210> 24
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 24
ggcacaaggc tcagctgaa 19
<210> 25
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 25
caacctacat gatggggaa 19
<210> 26
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 26
ccattatgaa gaagagagt 19
<210> 27
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 27
ggaattaagt tagggaatg 19
<210> 28
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 28
agggaatggc acagcccaa 19
<210> 29
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 29
ggagaagact atattgtac 19
<210> 30
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 30
aggcaaagcc actgaggtc 19
<210> 31
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 31
acccaaatta cgtgtacta 19
<210> 32
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 32
ggttaatgcc atggacaga 19
<210> 33
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 33
aagcaatgca cgtggccca 19
<210> 34
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 34
cagtggaaat caagtgaac 19
<210> 35
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 35
tctgcaaggt ggagctcat 19
<210> 36
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 36
cgccatacta cctgggcat 19
<210> 37
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 37
tgaagaagag agtccatat 19
<210> 38
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 38
gaagaagaga gtccatatt 19
<210> 39
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 39
ttccaagagc tgaggcaat 19
<210> 40
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 40
gagaagacta tattgtaca 19
<210> 41
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 41
cagtgatgct aaaggttgt 19
<210> 42
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 42
cactaataca ccaggtaga 19
<210> 43
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 43
gtggaaatca agtgaacct 19
<210> 44
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 44
gccagaatgt gaaaagcaa 19
<210> 45
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 45
ttaaagcaaa ggagtagaa 19
<210> 46
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 46
gcgagggaga agactatat 19
<210> 47
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 47
ctaggaagct ccagttcga 19
<210> 48
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 48
gaaggcagca ggtggcaga 19
<210> 49
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 49
gggcataggc aacggaacc 19
<210> 50
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 50
gccttggatt tcagcggca 19
<210> 51
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 51
gctcagctga acctggcta 19
<210> 52
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 52
cttctcttca tccctgtct 19
<210> 53
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 53
cctgtcttct gcaaagcaa 19
<210> 54
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 54
ggcatcgcca gctttgtgt 19
<210> 55
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 55
ccagctttgt gtgtgagta 19
<210> 56
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 56
ctttgtgtgt gagtatgca 19
<210> 57
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 57
gtgtgagtat gcatctcca 19
<210> 58
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 58
gagtatgcat ctccaggca 19
<210> 59
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 59
ctccaggcaa agccactga 19
<210> 60
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 60
ctgacagcca ggtgactga 19
<210> 61
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 61
ccaggtgact gaagtctgt 19
<210> 62
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 62
ggaatgagtt gaccttcct 19
<210> 63
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 63
ccttcctaga tgattccat 19
<210> 64
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 64
ctagatgatt ccatctgca 19
<210> 65
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 65
gattccatct gcacgggca 19
<210> 66
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 66
cacctccagt ggaaatcaa 19
<210> 67
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 67
ccagtggaaa tcaagtgaa 19
<210> 68
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 68
gaacctcact atccaagga 19
<210> 69
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 69
cgggactcta catctgcaa 19
<210> 70
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 70
gactctacat ctgcaaggt 19
<210> 71
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 71
gcaaggtgga gctcatgta 19
<210> 72
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 72
gcataggcaa cggaaccca 19
<210> 73
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 73
ggaacccaga tttatgtaa 19
<210> 74
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 74
cccagattta tgtaattga 19
<210> 75
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 75
cagattctga cttcctcct 19
<210> 76
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 76
gattctgact tcctcctct 19
<210> 77
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 77
ctgacttcct cctctggat 19
<210> 78
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 78
gatccttgca gcagttagt 19
<210> 79
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 79
gctttctcct cacagctgt 19
<210> 80
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 80
cccatcaatt gagaaacca 19
<210> 81
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 81
gaaaccatta tgaagaaga 19
<210> 82
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 82
gtccatattt caatttcca 19
<210> 83
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 83
caagagctga ggcaattct 19
<210> 84
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 84
ggatgcggaa cccaaatta 19
<210> 85
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 85
cggaacccaa attacgtgt 19
<210> 86
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 86
ggaacccaaa ttacgtgta 19
<210> 87
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 87
caaattacgt gtactacaa 19
<210> 88
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 88
ggagtagaaa gacagagct 19
<210> 89
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 89
ctgggatgtt tctgtcaca 19
<210> 90
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 90
ggatgtttct gtcacatca 19
<210> 91
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 91
cacatcagct ccactttca 19
<210> 92
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 92
cagctccact ttcagtgaa 19
<210> 93
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 93
gtgaaagcat cacttggga 19
<210> 94
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 94
ggatgcagca ttatgatgt 19
<210> 95
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 95
gcagcattat gatgtgggt 19
<210> 96
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 96
gcattatgat gtgggtcaa 19
<210> 97
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 97
caaggaatta agttaggga 19
<210> 98
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 98
gggaatggca cagcccaaa 19
<210> 99
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 99
gaatggcaca gcccaaaga 19
<210> 100
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 100
gcacagccca aagaaggaa 19
<210> 101
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 101
cacagcccaa agaaggaaa 19
<210> 102
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 102
gaagactata ttgtacaca 19
<210> 103
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 103
ctatattgta cacacctta 19
<210> 104
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 104
cacaccttat atttacgta 19
<210> 105
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 105
gagacgttta tagccgaaa 19
<210> 106
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 106
gattacatca aggcttcaa 19
<210> 107
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 107
gtgatgctaa aggttgtat 19
<210> 108
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 108
ctttccacct tgggtccca 19
<210> 109
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 109
gaggagctca ggacactaa 19
<210> 110
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 110
ggacactaat acaccaggt 19
<210> 111
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 111
gacactaata caccaggta 19
<210> 112
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 112
caaggtcatt tgctaacta 19
<210> 113
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 113
cttggaaact ggatgaggt 19
<210> 114
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 114
ggaaactgga tgaggtcat 19
<210> 115
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 115
gaaactggat gaggtcata 19
<210> 116
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 116
ggtcatagca gtgcttgat 19
<210> 117
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 117
cagtgcttga ttgcgtgga 19
<210> 118
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 118
gtgcttgatt gcgtggaat 19
<210> 119
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 119
gcgtggaatt gtgctgagt 19
<210> 120
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 120
cacagctggt ggtatctga 19
<210> 121
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 121
ctggtggtat ctgagttga 19
<210> 122
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 122
ggtggtatct gagttgact 19
<210> 123
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 123
gagttgactt gacagaaca 19
<210> 124
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 124
gttgacttga cagaacact 19
<210> 125
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 125
gcttactcca ggagaccca 19
<210> 126
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 126
cccacaggta tgaccttct 19
<210> 127
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 127
ccacaggtat gaccttcta 19
<210> 128
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 128
ccttctagga agctccagt 19
<210> 129
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 129
ccagttcgat gggcccaat 19
<210> 130
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 130
gatgggccca attcttaca 19
<210> 131
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 131
gcccaattct tacaaacat 19
<210> 132
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 132
ccaattctta caaacatgt 19
<210> 133
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 133
gttaatgcca tggacagaa 19
<210> 134
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶点序列
<400> 134
ggtgcatgaa ggtttctga 19
<210> 135
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 135
gcaaaggagu agaaagaca 19
<210> 136
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 136
aagcaaagga guagaaaga 19
<210> 137
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 137
gggucaagga auuaaguua 19
<210> 138
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 138
cuuacaaaca ugugguuaa 19
<210> 139
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 139
caacagagcc agaauguga 19
<210> 140
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 140
gagaaaccau uaugaagaa 19
<210> 141
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 141
aggaauuaag uuagggaau 19
<210> 142
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 142
ugaugugggu caaggaauu 19
<210> 143
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 143
gcaacggaac ccagauuua 19
<210> 144
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 144
guaugcaucu ccaggcaaa 19
<210> 145
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 145
gcuaaagguu guauugcau 19
<210> 146
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 146
ggagcgaggg agaagacua 19
<210> 147
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 147
acacaagguc auuugcuaa 19
<210> 148
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 148
ggacugaggg ccauggaca 19
<210> 149
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 149
ugagaaacca uuaugaaga 19
<210> 150
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 150
gaaagcauca cuugggauu 19
<210> 151
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 151
gagcgaggga gaagacuau 19
<210> 152
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 152
gggaauucau cucucuuua 19
<210> 153
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 153
agacagagcu gggauguuu 19
<210> 154
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 154
cagcuuugug ugugaguau 19
<210> 155
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 155
gaacccagau uuauguaau 19
<210> 156
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 156
auauaaaguu ggaugcgga 19
<210> 157
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 157
aagcaucacu ugggauuaa 19
<210> 158
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 158
ggcacaaggc ucagcugaa 19
<210> 159
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 159
caaccuacau gauggggaa 19
<210> 160
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 160
ccauuaugaa gaagagagu 19
<210> 161
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 161
ggaauuaagu uagggaaug 19
<210> 162
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 162
agggaauggc acagcccaa 19
<210> 163
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 163
ggagaagacu auauuguac 19
<210> 164
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 164
aggcaaagcc acugagguc 19
<210> 165
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 165
acccaaauua cguguacua 19
<210> 166
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 166
gguuaaugcc auggacaga 19
<210> 167
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 167
aagcaaugca cguggccca 19
<210> 168
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 168
caguggaaau caagugaac 19
<210> 169
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 169
ucugcaaggu ggagcucau 19
<210> 170
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 170
cgccauacua ccugggcau 19
<210> 171
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 171
ugaagaagag aguccauau 19
<210> 172
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 172
gaagaagaga guccauauu 19
<210> 173
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 173
uuccaagagc ugaggcaau 19
<210> 174
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 174
gagaagacua uauuguaca 19
<210> 175
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 175
cagugaugcu aaagguugu 19
<210> 176
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 176
cacuaauaca ccagguaga 19
<210> 177
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 177
guggaaauca agugaaccu 19
<210> 178
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 178
gccagaaugu gaaaagcaa 19
<210> 179
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 179
uuaaagcaaa ggaguagaa 19
<210> 180
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 180
gcgagggaga agacuauau 19
<210> 181
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 181
cuaggaagcu ccaguucga 19
<210> 182
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 182
gaaggcagca gguggcaga 19
<210> 183
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 183
gggcauaggc aacggaacc 19
<210> 184
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 184
gccuuggauu ucagcggca 19
<210> 185
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 185
gcucagcuga accuggcua 19
<210> 186
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 186
cuucucuuca ucccugucu 19
<210> 187
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 187
ccugucuucu gcaaagcaa 19
<210> 188
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 188
ggcaucgcca gcuuugugu 19
<210> 189
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 189
ccagcuuugu gugugagua 19
<210> 190
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 190
cuuugugugu gaguaugca 19
<210> 191
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 191
gugugaguau gcaucucca 19
<210> 192
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 192
gaguaugcau cuccaggca 19
<210> 193
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 193
cuccaggcaa agccacuga 19
<210> 194
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 194
cugacagcca ggugacuga 19
<210> 195
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 195
ccaggugacu gaagucugu 19
<210> 196
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 196
ggaaugaguu gaccuuccu 19
<210> 197
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 197
ccuuccuaga ugauuccau 19
<210> 198
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 198
cuagaugauu ccaucugca 19
<210> 199
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 199
gauuccaucu gcacgggca 19
<210> 200
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 200
caccuccagu ggaaaucaa 19
<210> 201
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 201
ccaguggaaa ucaagugaa 19
<210> 202
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 202
gaaccucacu auccaagga 19
<210> 203
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 203
cgggacucua caucugcaa 19
<210> 204
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 204
gacucuacau cugcaaggu 19
<210> 205
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 205
gcaaggugga gcucaugua 19
<210> 206
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 206
gcauaggcaa cggaaccca 19
<210> 207
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 207
ggaacccaga uuuauguaa 19
<210> 208
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 208
cccagauuua uguaauuga 19
<210> 209
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 209
cagauucuga cuuccuccu 19
<210> 210
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 210
gauucugacu uccuccucu 19
<210> 211
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 211
cugacuuccu ccucuggau 19
<210> 212
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 212
gauccuugca gcaguuagu 19
<210> 213
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 213
gcuuucuccu cacagcugu 19
<210> 214
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 214
cccaucaauu gagaaacca 19
<210> 215
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 215
gaaaccauua ugaagaaga 19
<210> 216
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 216
guccauauuu caauuucca 19
<210> 217
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 217
caagagcuga ggcaauucu 19
<210> 218
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 218
ggaugcggaa cccaaauua 19
<210> 219
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 219
cggaacccaa auuacgugu 19
<210> 220
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 220
ggaacccaaa uuacgugua 19
<210> 221
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 221
caaauuacgu guacuacaa 19
<210> 222
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 222
ggaguagaaa gacagagcu 19
<210> 223
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 223
cugggauguu ucugucaca 19
<210> 224
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 224
ggauguuucu gucacauca 19
<210> 225
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 225
cacaucagcu ccacuuuca 19
<210> 226
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 226
cagcuccacu uucagugaa 19
<210> 227
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 227
gugaaagcau cacuuggga 19
<210> 228
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 228
ggaugcagca uuaugaugu 19
<210> 229
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 229
gcagcauuau gaugugggu 19
<210> 230
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 230
gcauuaugau gugggucaa 19
<210> 231
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 231
caaggaauua aguuaggga 19
<210> 232
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 232
gggaauggca cagcccaaa 19
<210> 233
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 233
gaauggcaca gcccaaaga 19
<210> 234
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 234
gcacagccca aagaaggaa 19
<210> 235
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 235
cacagcccaa agaaggaaa 19
<210> 236
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 236
gaagacuaua uuguacaca 19
<210> 237
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 237
cuauauugua cacaccuua 19
<210> 238
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 238
cacaccuuau auuuacgua 19
<210> 239
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 239
gagacguuua uagccgaaa 19
<210> 240
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 240
gauuacauca aggcuucaa 19
<210> 241
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 241
gugaugcuaa agguuguau 19
<210> 242
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 242
cuuuccaccu uggguccca 19
<210> 243
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 243
gaggagcuca ggacacuaa 19
<210> 244
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 244
ggacacuaau acaccaggu 19
<210> 245
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 245
gacacuaaua caccaggua 19
<210> 246
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 246
caaggucauu ugcuaacua 19
<210> 247
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 247
cuuggaaacu ggaugaggu 19
<210> 248
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 248
ggaaacugga ugaggucau 19
<210> 249
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 249
gaaacuggau gaggucaua 19
<210> 250
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 250
ggucauagca gugcuugau 19
<210> 251
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 251
cagugcuuga uugcgugga 19
<210> 252
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 252
gugcuugauu gcguggaau 19
<210> 253
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 253
gcguggaauu gugcugagu 19
<210> 254
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 254
cacagcuggu gguaucuga 19
<210> 255
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 255
cuggugguau cugaguuga 19
<210> 256
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 256
ggugguaucu gaguugacu 19
<210> 257
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 257
gaguugacuu gacagaaca 19
<210> 258
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 258
guugacuuga cagaacacu 19
<210> 259
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 259
gcuuacucca ggagaccca 19
<210> 260
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 260
cccacaggua ugaccuucu 19
<210> 261
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 261
ccacagguau gaccuucua 19
<210> 262
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 262
ccuucuagga agcuccagu 19
<210> 263
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 263
ccaguucgau gggcccaau 19
<210> 264
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 264
gaugggccca auucuuaca 19
<210> 265
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 265
gcccaauucu uacaaacau 19
<210> 266
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 266
ccaauucuua caaacaugu 19
<210> 267
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 267
guuaaugcca uggacagaa 19
<210> 268
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA
<400> 268
ggugcaugaa gguuucuga 19
<210> 269
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 269
gcaaaggagu agaaagacau ucaagagaug ucuuucuacu ccuuugc 47
<210> 270
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 270
aagcaaagga guagaaagau ucaagagauc uuucuacucc uuugcuu 47
<210> 271
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 271
gggucaagga auuaaguuau ucaagagaua acuuaauucc uugaccc 47
<210> 272
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 272
cuuacaaaca ugugguuaau ucaagagauu aaccacaugu uuguaag 47
<210> 273
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 273
caacagagcc agaaugugau ucaagagauc acauucuggc ucuguug 47
<210> 274
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 274
gagaaaccau uaugaagaau ucaagagauu cuucauaaug guuucuc 47
<210> 275
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 275
aggaauuaag uuagggaauu ucaagagaau ucccuaacuu aauuccu 47
<210> 276
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 276
ugaugugggu caaggaauuu ucaagagaaa uuccuugacc cacauca 47
<210> 277
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 277
gcaacggaac ccagauuuau ucaagagaua aaucuggguu ccguugc 47
<210> 278
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 278
guaugcaucu ccaggcaaau ucaagagauu ugccuggaga ugcauac 47
<210> 279
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 279
gcuaaagguu guauugcauu ucaagagaau gcaauacaac cuuuagc 47
<210> 280
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 280
ggagcgaggg agaagacuau ucaagagaua gucuucuccc ucgcucc 47
<210> 281
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 281
acacaagguc auuugcuaau ucaagagauu agcaaaugac cuugugu 47
<210> 282
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 282
ggacugaggg ccauggacau ucaagagaug uccauggccc ucagucc 47
<210> 283
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 283
ugagaaacca uuaugaagau ucaagagauc uucauaaugg uuucuca 47
<210> 284
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 284
gaaagcauca cuugggauuu ucaagagaaa ucccaaguga ugcuuuc 47
<210> 285
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 285
gagcgaggga gaagacuauu ucaagagaau agucuucucc cucgcuc 47
<210> 286
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 286
gggaauucau cucucuuuau ucaagagaua aagagagaug aauuccc 47
<210> 287
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 287
agacagagcu gggauguuuu ucaagagaaa acaucccagc ucugucu 47
<210> 288
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 288
cagcuuugug ugugaguauu ucaagagaau acucacacac aaagcug 47
<210> 289
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 289
gaacccagau uuauguaauu ucaagagaau uacauaaauc uggguuc 47
<210> 290
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 290
auauaaaguu ggaugcggau ucaagagauc cgcauccaac uuuauau 47
<210> 291
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 291
aagcaucacu ugggauuaau ucaagagauu aaucccaagu gaugcuu 47
<210> 292
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 292
ggcacaaggc ucagcugaau ucaagagauu cagcugagcc uugugcc 47
<210> 293
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 293
caaccuacau gauggggaau ucaagagauu ccccaucaug uagguug 47
<210> 294
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 294
ccauuaugaa gaagagaguu ucaagagaac ucucuucuuc auaaugg 47
<210> 295
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 295
ggaauuaagu uagggaaugu ucaagagaca uucccuaacu uaauucc 47
<210> 296
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 296
agggaauggc acagcccaau ucaagagauu gggcugugcc auucccu 47
<210> 297
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 297
ggagaagacu auauuguacu ucaagagagu acaauauagu cuucucc 47
<210> 298
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 298
aggcaaagcc acugaggucu ucaagagaga ccucaguggc uuugccu 47
<210> 299
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 299
acccaaauua cguguacuau ucaagagaua guacacguaa uuugggu 47
<210> 300
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 300
gguuaaugcc auggacagau ucaagagauc uguccauggc auuaacc 47
<210> 301
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 301
aagcaaugca cguggcccau ucaagagaug ggccacgugc auugcuu 47
<210> 302
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 302
caguggaaau caagugaacu ucaagagagu ucacuugauu uccacug 47
<210> 303
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 303
ucugcaaggu ggagcucauu ucaagagaau gagcuccacc uugcaga 47
<210> 304
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 304
cgccauacua ccugggcauu ucaagagaau gcccagguag uauggcg 47
<210> 305
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 305
ugaagaagag aguccauauu ucaagagaau auggacucuc uucuuca 47
<210> 306
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 306
gaagaagaga guccauauuu ucaagagaaa uauggacucu cuucuuc 47
<210> 307
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 307
uuccaagagc ugaggcaauu ucaagagaau ugccucagcu cuuggaa 47
<210> 308
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 308
gagaagacua uauuguacau ucaagagaug uacaauauag ucuucuc 47
<210> 309
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 309
cagugaugcu aaagguuguu ucaagagaac aaccuuuagc aucacug 47
<210> 310
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 310
cacuaauaca ccagguagau ucaagagauc uaccuggugu auuagug 47
<210> 311
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 311
guggaaauca agugaaccuu ucaagagaag guucacuuga uuuccac 47
<210> 312
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 312
gccagaaugu gaaaagcaau ucaagagauu gcuuuucaca uucuggc 47
<210> 313
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 313
uuaaagcaaa ggaguagaau ucaagagauu cuacuccuuu gcuuuaa 47
<210> 314
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 314
gcgagggaga agacuauauu ucaagagaau auagucuucu cccucgc 47
<210> 315
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 315
cuaggaagcu ccaguucgau ucaagagauc gaacuggagc uuccuag 47
<210> 316
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 316
gaaggcagca gguggcagau ucaagagauc ugccaccugc ugccuuc 47
<210> 317
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 317
gggcauaggc aacggaaccu ucaagagagg uuccguugcc uaugccc 47
<210> 318
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 318
gccuuggauu ucagcggcau ucaagagaug ccgcugaaau ccaaggc 47
<210> 319
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 319
gcucagcuga accuggcuau ucaagagaua gccagguuca gcugagc 47
<210> 320
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 320
cuucucuuca ucccugucuu ucaagagaag acagggauga agagaag 47
<210> 321
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 321
ccugucuucu gcaaagcaau ucaagagauu gcuuugcaga agacagg 47
<210> 322
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 322
ggcaucgcca gcuuuguguu ucaagagaac acaaagcugg cgaugcc 47
<210> 323
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 323
ccagcuuugu gugugaguau ucaagagaua cucacacaca aagcugg 47
<210> 324
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 324
cuuugugugu gaguaugcau ucaagagaug cauacucaca cacaaag 47
<210> 325
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 325
gugugaguau gcaucuccau ucaagagaug gagaugcaua cucacac 47
<210> 326
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 326
gaguaugcau cuccaggcau ucaagagaug ccuggagaug cauacuc 47
<210> 327
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 327
cuccaggcaa agccacugau ucaagagauc aguggcuuug ccuggag 47
<210> 328
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 328
cugacagcca ggugacugau ucaagagauc agucaccugg cugucag 47
<210> 329
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 329
ccaggugacu gaagucuguu ucaagagaac agacuucagu caccugg 47
<210> 330
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 330
ggaaugaguu gaccuuccuu ucaagagaag gaaggucaac ucauucc 47
<210> 331
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 331
ccuuccuaga ugauuccauu ucaagagaau ggaaucaucu aggaagg 47
<210> 332
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 332
cuagaugauu ccaucugcau ucaagagaug cagauggaau caucuag 47
<210> 333
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 333
gauuccaucu gcacgggcau ucaagagaug cccgugcaga uggaauc 47
<210> 334
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 334
caccuccagu ggaaaucaau ucaagagauu gauuuccacu ggaggug 47
<210> 335
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 335
ccaguggaaa ucaagugaau ucaagagauu cacuugauuu ccacugg 47
<210> 336
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 336
gaaccucacu auccaaggau ucaagagauc cuuggauagu gagguuc 47
<210> 337
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 337
cgggacucua caucugcaau ucaagagauu gcagauguag agucccg 47
<210> 338
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 338
gacucuacau cugcaagguu ucaagagaac cuugcagaug uagaguc 47
<210> 339
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 339
gcaaggugga gcucauguau ucaagagaua caugagcucc accuugc 47
<210> 340
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 340
gcauaggcaa cggaacccau ucaagagaug gguuccguug ccuaugc 47
<210> 341
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 341
ggaacccaga uuuauguaau ucaagagauu acauaaaucu ggguucc 47
<210> 342
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 342
cccagauuua uguaauugau ucaagagauc aauuacauaa aucuggg 47
<210> 343
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 343
cagauucuga cuuccuccuu ucaagagaag gaggaaguca gaaucug 47
<210> 344
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 344
gauucugacu uccuccucuu ucaagagaag aggaggaagu cagaauc 47
<210> 345
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 345
cugacuuccu ccucuggauu ucaagagaau ccagaggagg aagucag 47
<210> 346
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 346
gauccuugca gcaguuaguu ucaagagaac uaacugcugc aaggauc 47
<210> 347
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 347
gcuuucuccu cacagcuguu ucaagagaac agcugugagg agaaagc 47
<210> 348
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 348
cccaucaauu gagaaaccau ucaagagaug guuucucaau ugauggg 47
<210> 349
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 349
gaaaccauua ugaagaagau ucaagagauc uucuucauaa ugguuuc 47
<210> 350
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 350
guccauauuu caauuuccau ucaagagaug gaaauugaaa uauggac 47
<210> 351
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 351
caagagcuga ggcaauucuu ucaagagaag aauugccuca gcucuug 47
<210> 352
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 352
ggaugcggaa cccaaauuau ucaagagaua auuuggguuc cgcaucc 47
<210> 353
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 353
cggaacccaa auuacguguu ucaagagaac acguaauuug gguuccg 47
<210> 354
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 354
ggaacccaaa uuacguguau ucaagagaua cacguaauuu ggguucc 47
<210> 355
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 355
caaauuacgu guacuacaau ucaagagauu guaguacacg uaauuug 47
<210> 356
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 356
ggaguagaaa gacagagcuu ucaagagaag cucugucuuu cuacucc 47
<210> 357
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 357
cugggauguu ucugucacau ucaagagaug ugacagaaac aucccag 47
<210> 358
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 358
ggauguuucu gucacaucau ucaagagaug augugacaga aacaucc 47
<210> 359
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 359
cacaucagcu ccacuuucau ucaagagaug aaaguggagc ugaugug 47
<210> 360
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 360
cagcuccacu uucagugaau ucaagagauu cacugaaagu ggagcug 47
<210> 361
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 361
gugaaagcau cacuugggau ucaagagauc ccaagugaug cuuucac 47
<210> 362
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 362
ggaugcagca uuaugauguu ucaagagaac aucauaaugc ugcaucc 47
<210> 363
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 363
gcagcauuau gauguggguu ucaagagaac ccacaucaua augcugc 47
<210> 364
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 364
gcauuaugau gugggucaau ucaagagauu gacccacauc auaaugc 47
<210> 365
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 365
caaggaauua aguuagggau ucaagagauc ccuaacuuaa uuccuug 47
<210> 366
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 366
gggaauggca cagcccaaau ucaagagauu ugggcugugc cauuccc 47
<210> 367
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 367
gaauggcaca gcccaaagau ucaagagauc uuugggcugu gccauuc 47
<210> 368
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 368
gcacagccca aagaaggaau ucaagagauu ccuucuuugg gcugugc 47
<210> 369
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 369
cacagcccaa agaaggaaau ucaagagauu uccuucuuug ggcugug 47
<210> 370
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 370
gaagacuaua uuguacacau ucaagagaug uguacaauau agucuuc 47
<210> 371
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 371
cuauauugua cacaccuuau ucaagagaua agguguguac aauauag 47
<210> 372
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 372
cacaccuuau auuuacguau ucaagagaua cguaaauaua aggugug 47
<210> 373
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 373
gagacguuua uagccgaaau ucaagagauu ucggcuauaa acgucuc 47
<210> 374
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 374
gauuacauca aggcuucaau ucaagagauu gaagccuuga uguaauc 47
<210> 375
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 375
gugaugcuaa agguuguauu ucaagagaau acaaccuuua gcaucac 47
<210> 376
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 376
cuuuccaccu ugggucccau ucaagagaug ggacccaagg uggaaag 47
<210> 377
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 377
gaggagcuca ggacacuaau ucaagagauu aguguccuga gcuccuc 47
<210> 378
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 378
ggacacuaau acaccagguu ucaagagaac cugguguauu agugucc 47
<210> 379
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 379
gacacuaaua caccagguau ucaagagaua ccugguguau uaguguc 47
<210> 380
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 380
caaggucauu ugcuaacuau ucaagagaua guuagcaaau gaccuug 47
<210> 381
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 381
cuuggaaacu ggaugagguu ucaagagaac cucauccagu uuccaag 47
<210> 382
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 382
ggaaacugga ugaggucauu ucaagagaau gaccucaucc aguuucc 47
<210> 383
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 383
gaaacuggau gaggucauau ucaagagaua ugaccucauc caguuuc 47
<210> 384
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 384
ggucauagca gugcuugauu ucaagagaau caagcacugc uaugacc 47
<210> 385
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 385
cagugcuuga uugcguggau ucaagagauc cacgcaauca agcacug 47
<210> 386
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 386
gugcuugauu gcguggaauu ucaagagaau uccacgcaau caagcac 47
<210> 387
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 387
gcguggaauu gugcugaguu ucaagagaac ucagcacaau uccacgc 47
<210> 388
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 388
cacagcuggu gguaucugau ucaagagauc agauaccacc agcugug 47
<210> 389
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 389
cuggugguau cugaguugau ucaagagauc aacucagaua ccaccag 47
<210> 390
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 390
ggugguaucu gaguugacuu ucaagagaag ucaacucaga uaccacc 47
<210> 391
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 391
guugacuuga cagaacacuu ucaagagaag uguucuguca agucaac 47
<210> 392
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 392
guugacuuga cagaacacuu ucaagagaag uguucuguca agucaac 47
<210> 393
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 393
gcuuacucca ggagacccau ucaagagaug ggucuccugg aguaagc 47
<210> 394
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 394
cccacaggua ugaccuucuu ucaagagaag aaggucauac cuguggg 47
<210> 395
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 395
ccacagguau gaccuucuau ucaagagaua gaaggucaua ccugugg 47
<210> 396
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 396
ccuucuagga agcuccaguu ucaagagaac uggagcuucc uagaagg 47
<210> 397
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 397
ccaguucgau gggcccaauu ucaagagaau ugggcccauc gaacugg 47
<210> 398
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 398
gaugggccca auucuuacau ucaagagaug uaagaauugg gcccauc 47
<210> 399
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 399
gcccaauucu uacaaacauu ucaagagaau guuuguaaga auugggc 47
<210> 400
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 400
ccaauucuua caaacauguu ucaagagaac auguuuguaa gaauugg 47
<210> 401
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 401
guuaaugcca uggacagaau ucaagagauu cuguccaugg cauuaac 47
<210> 402
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA
<400> 402
ggugcaugaa gguuucugau ucaagagauc agaaaccuuc augcacc 47
<210> 403
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 403
ccgggcaaag gagtagaaag acattcaaga gatgtctttc tactcctttg ctttttg 57
<210> 404
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 404
aattcaaaaa gcaaaggagt agaaagacat ctcttgaatg tctttctact cctttgc 57
<210> 405
<211> 53
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 405
aagcaaagga gtagaaagat tcaagagatc tttctactcc tttgcttttt ttg 53
<210> 406
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 406
aattcaaaaa aagcaaagga gtagaaagat ctcttgaatc tttctactcc tttgctt 57
<210> 407
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 407
ccgggggtca aggaattaag ttattcaaga gataacttaa ttccttgacc ctttttg 57
<210> 408
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 408
aattcaaaaa gggtcaagga attaagttat ctcttgaata acttaattcc ttgaccc 57
<210> 409
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 409
ccggcttaca aacatgtggt taattcaaga gattaaccac atgtttgtaa gtttttg 57
<210> 410
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 410
aattcaaaaa cttacaaaca tgtggttaat ctcttgaatt aaccacatgt ttgtaag 57
<210> 411
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 411
ccggcaacag agccagaatg tgattcaaga gatcacattc tggctctgtt gtttttg 57
<210> 412
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 412
aattcaaaaa caacagagcc agaatgtgat ctcttgaatc acattctggc tctgttg 57
<210> 413
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 413
ccgggagaaa ccattatgaa gaattcaaga gattcttcat aatggtttct ctttttg 57
<210> 414
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 414
aattcaaaaa gagaaaccat tatgaagaat ctcttgaatt cttcataatg gtttctc 57
<210> 415
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 415
ccggaggaat taagttaggg aatttcaaga gaattcccta acttaattcc ttttttg 57
<210> 416
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 416
aattcaaaaa aggaattaag ttagggaatt ctcttgaaat tccctaactt aattcct 57
<210> 417
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 417
ccggtgatgt gggtcaagga attttcaaga gaaattcctt gacccacatc atttttg 57
<210> 418
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 418
aattcaaaaa tgatgtgggt caaggaattt ctcttgaaaa ttccttgacc cacatca 57
<210> 419
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 419
ccgggcaacg gaacccagat ttattcaaga gataaatctg ggttccgttg ctttttg 57
<210> 420
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 420
aattcaaaaa gcaacggaac ccagatttat ctcttgaata aatctgggtt ccgttgc 57
<210> 421
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 421
ccgggtatgc atctccaggc aaattcaaga gatttgcctg gagatgcata ctttttg 57
<210> 422
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 422
aattcaaaaa gtatgcatct ccaggcaaat ctcttgaatt tgcctggaga tgcatac 57
<210> 423
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 423
ccgggctaaa ggttgtattg catttcaaga gaatgcaata caacctttag ctttttg 57
<210> 424
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 424
aattcaaaaa gctaaaggtt gtattgcatt ctcttgaaat gcaatacaac ctttagc 57
<210> 425
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 425
ccggggagcg agggagaaga ctattcaaga gatagtcttc tccctcgctc ctttttg 57
<210> 426
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 426
aattcaaaaa ggagcgaggg agaagactat ctcttgaata gtcttctccc tcgctcc 57
<210> 427
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 427
ccggacacaa ggtcatttgc taattcaaga gattagcaaa tgaccttgtg ttttttg 57
<210> 428
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 428
aattcaaaaa acacaaggtc atttgctaat ctcttgaatt agcaaatgac cttgtgt 57
<210> 429
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 429
ccggggactg agggccatgg acattcaaga gatgtccatg gccctcagtc ctttttg 57
<210> 430
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 430
aattcaaaaa ggactgaggg ccatggacat ctcttgaatg tccatggccc tcagtcc 57
<210> 431
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 431
ccggtgagaa accattatga agattcaaga gatcttcata atggtttctc atttttg 57
<210> 432
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 432
aattcaaaaa tgagaaacca ttatgaagat ctcttgaatc ttcataatgg tttctca 57
<210> 433
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 433
ccgggaaagc atcacttggg attttcaaga gaaatcccaa gtgatgcttt ctttttg 57
<210> 434
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 434
aattcaaaaa gaaagcatca cttgggattt ctcttgaaaa tcccaagtga tgctttc 57
<210> 435
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 435
ccgggagcga gggagaagac tatttcaaga gaatagtctt ctccctcgct ctttttg 57
<210> 436
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 436
aattcaaaaa gagcgaggga gaagactatt ctcttgaaat agtcttctcc ctcgctc 57
<210> 437
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 437
ccgggggaat tcatctctct ttattcaaga gataaagaga gatgaattcc ctttttg 57
<210> 438
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 438
aattcaaaaa gggaattcat ctctctttat ctcttgaata aagagagatg aattccc 57
<210> 439
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 439
ccggagacag agctgggatg tttttcaaga gaaaacatcc cagctctgtc ttttttg 57
<210> 440
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 440
aattcaaaaa agacagagct gggatgtttt ctcttgaaaa acatcccagc tctgtct 57
<210> 441
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 441
ccggcagctt tgtgtgtgag tatttcaaga gaatactcac acacaaagct gtttttg 57
<210> 442
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 442
aattcaaaaa cagctttgtg tgtgagtatt ctcttgaaat actcacacac aaagctg 57
<210> 443
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 443
ccgggaaccc agatttatgt aatttcaaga gaattacata aatctgggtt ctttttg 57
<210> 444
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 444
aattcaaaaa gaacccagat ttatgtaatt ctcttgaaat tacataaatc tgggttc 57
<210> 445
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 445
ccggatataa agttggatgc ggattcaaga gatccgcatc caactttata ttttttg 57
<210> 446
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 446
aattcaaaaa atataaagtt ggatgcggat ctcttgaatc cgcatccaac tttatat 57
<210> 447
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 447
ccggaagcat cacttgggat taattcaaga gattaatccc aagtgatgct ttttttg 57
<210> 448
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 448
aattcaaaaa aagcatcact tgggattaat ctcttgaatt aatcccaagt gatgctt 57
<210> 449
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 449
ccggggcaca aggctcagct gaattcaaga gattcagctg agccttgtgc ctttttg 57
<210> 450
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 450
aattcaaaaa ggcacaaggc tcagctgaat ctcttgaatt cagctgagcc ttgtgcc 57
<210> 451
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 451
ccggcaacct acatgatggg gaattcaaga gattccccat catgtaggtt gtttttg 57
<210> 452
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 452
aattcaaaaa caacctacat gatggggaat ctcttgaatt ccccatcatg taggttg 57
<210> 453
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 453
ccggccatta tgaagaagag agtttcaaga gaactctctt cttcataatg gtttttg 57
<210> 454
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 454
aattcaaaaa ccattatgaa gaagagagtt ctcttgaaac tctcttcttc ataatgg 57
<210> 455
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 455
ccggggaatt aagttaggga atgttcaaga gacattccct aacttaattc ctttttg 57
<210> 456
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 456
aattcaaaaa ggaattaagt tagggaatgt ctcttgaaca ttccctaact taattcc 57
<210> 457
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 457
ccggagggaa tggcacagcc caattcaaga gattgggctg tgccattccc ttttttg 57
<210> 458
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 458
aattcaaaaa agggaatggc acagcccaat ctcttgaatt gggctgtgcc attccct 57
<210> 459
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 459
ccggggagaa gactatattg tacttcaaga gagtacaata tagtcttctc ctttttg 57
<210> 460
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 460
aattcaaaaa ggagaagact atattgtact ctcttgaagt acaatatagt cttctcc 57
<210> 461
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 461
ccggaggcaa agccactgag gtcttcaaga gagacctcag tggctttgcc ttttttg 57
<210> 462
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 462
aattcaaaaa aggcaaagcc actgaggtct ctcttgaaga cctcagtggc tttgcct 57
<210> 463
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 463
ccggacccaa attacgtgta ctattcaaga gatagtacac gtaatttggg ttttttg 57
<210> 464
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 464
aattcaaaaa acccaaatta cgtgtactat ctcttgaata gtacacgtaa tttgggt 57
<210> 465
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 465
ccggggttaa tgccatggac agattcaaga gatctgtcca tggcattaac ctttttg 57
<210> 466
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 466
aattcaaaaa ggttaatgcc atggacagat ctcttgaatc tgtccatggc attaacc 57
<210> 467
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 467
ccggaagcaa tgcacgtggc ccattcaaga gatgggccac gtgcattgct ttttttg 57
<210> 468
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 468
aattcaaaaa aagcaatgca cgtggcccat ctcttgaatg ggccacgtgc attgctt 57
<210> 469
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 469
ccggcagtgg aaatcaagtg aacttcaaga gagttcactt gatttccact gtttttg 57
<210> 470
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 470
aattcaaaaa cagtggaaat caagtgaact ctcttgaagt tcacttgatt tccactg 57
<210> 471
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 471
ccggtctgca aggtggagct catttcaaga gaatgagctc caccttgcag atttttg 57
<210> 472
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 472
aattcaaaaa tctgcaaggt ggagctcatt ctcttgaaat gagctccacc ttgcaga 57
<210> 473
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 473
ccggcgccat actacctggg catttcaaga gaatgcccag gtagtatggc gtttttg 57
<210> 474
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 474
aattcaaaaa cgccatacta cctgggcatt ctcttgaaat gcccaggtag tatggcg 57
<210> 475
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 475
ccggtgaaga agagagtcca tatttcaaga gaatatggac tctcttcttc atttttg 57
<210> 476
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 476
aattcaaaaa tgaagaagag agtccatatt ctcttgaaat atggactctc ttcttca 57
<210> 477
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 477
ccgggaagaa gagagtccat attttcaaga gaaatatgga ctctcttctt ctttttg 57
<210> 478
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 478
aattcaaaaa gaagaagaga gtccatattt ctcttgaaaa tatggactct cttcttc 57
<210> 479
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 479
ccggttccaa gagctgaggc aatttcaaga gaattgcctc agctcttgga atttttg 57
<210> 480
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 480
aattcaaaaa ttccaagagc tgaggcaatt ctcttgaaat tgcctcagct cttggaa 57
<210> 481
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 481
ccgggagaag actatattgt acattcaaga gatgtacaat atagtcttct ctttttg 57
<210> 482
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 482
aattcaaaaa gagaagacta tattgtacat ctcttgaatg tacaatatag tcttctc 57
<210> 483
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 483
ccggcagtga tgctaaaggt tgtttcaaga gaacaacctt tagcatcact gtttttg 57
<210> 484
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 484
aattcaaaaa cagtgatgct aaaggttgtt ctcttgaaac aacctttagc atcactg 57
<210> 485
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 485
ccggcactaa tacaccaggt agattcaaga gatctacctg gtgtattagt gtttttg 57
<210> 486
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 486
aattcaaaaa cactaataca ccaggtagat ctcttgaatc tacctggtgt attagtg 57
<210> 487
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 487
ccgggtggaa atcaagtgaa cctttcaaga gaaggttcac ttgatttcca ctttttg 57
<210> 488
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 488
aattcaaaaa gtggaaatca agtgaacctt ctcttgaaag gttcacttga tttccac 57
<210> 489
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 489
ccgggccaga atgtgaaaag caattcaaga gattgctttt cacattctgg ctttttg 57
<210> 490
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 490
aattcaaaaa gccagaatgt gaaaagcaat ctcttgaatt gcttttcaca ttctggc 57
<210> 491
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 491
ccggttaaag caaaggagta gaattcaaga gattctactc ctttgcttta atttttg 57
<210> 492
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 492
aattcaaaaa ttaaagcaaa ggagtagaat ctcttgaatt ctactccttt gctttaa 57
<210> 493
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 493
ccgggcgagg gagaagacta tatttcaaga gaatatagtc ttctccctcg ctttttg 57
<210> 494
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 494
aattcaaaaa gcgagggaga agactatatt ctcttgaaat atagtcttct ccctcgc 57
<210> 495
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 495
ccggctagga agctccagtt cgattcaaga gatcgaactg gagcttccta gtttttg 57
<210> 496
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 496
aattcaaaaa ctaggaagct ccagttcgat ctcttgaatc gaactggagc ttcctag 57
<210> 497
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 497
ccgggaaggc agcaggtggc agattcaaga gatctgccac ctgctgcctt ctttttg 57
<210> 498
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 498
aattcaaaaa gaaggcagca ggtggcagat ctcttgaatc tgccacctgc tgccttc 57
<210> 499
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 499
ccgggggcat aggcaacgga accttcaaga gaggttccgt tgcctatgcc ctttttg 57
<210> 500
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 500
aattcaaaaa gggcataggc aacggaacct ctcttgaagg ttccgttgcc tatgccc 57
<210> 501
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 501
ccgggccttg gatttcagcg gcattcaaga gatgccgctg aaatccaagg ctttttg 57
<210> 502
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 502
aattcaaaaa gccttggatt tcagcggcat ctcttgaatg ccgctgaaat ccaaggc 57
<210> 503
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 503
ccgggctcag ctgaacctgg ctattcaaga gatagccagg ttcagctgag ctttttg 57
<210> 504
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 504
aattcaaaaa gctcagctga acctggctat ctcttgaata gccaggttca gctgagc 57
<210> 505
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 505
ccggcttctc ttcatccctg tctttcaaga gaagacaggg atgaagagaa gtttttg 57
<210> 506
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 506
aattcaaaaa cttctcttca tccctgtctt ctcttgaaag acagggatga agagaag 57
<210> 507
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 507
ccggcctgtc ttctgcaaag caattcaaga gattgctttg cagaagacag gtttttg 57
<210> 508
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 508
aattcaaaaa cctgtcttct gcaaagcaat ctcttgaatt gctttgcaga agacagg 57
<210> 509
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 509
ccggggcatc gccagctttg tgtttcaaga gaacacaaag ctggcgatgc ctttttg 57
<210> 510
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 510
aattcaaaaa ggcatcgcca gctttgtgtt ctcttgaaac acaaagctgg cgatgcc 57
<210> 511
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 511
ccggccagct ttgtgtgtga gtattcaaga gatactcaca cacaaagctg gtttttg 57
<210> 512
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 512
aattcaaaaa ccagctttgt gtgtgagtat ctcttgaata ctcacacaca aagctgg 57
<210> 513
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 513
ccggctttgt gtgtgagtat gcattcaaga gatgcatact cacacacaaa gtttttg 57
<210> 514
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 514
aattcaaaaa ctttgtgtgt gagtatgcat ctcttgaatg catactcaca cacaaag 57
<210> 515
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 515
ccgggtgtga gtatgcatct ccattcaaga gatggagatg catactcaca ctttttg 57
<210> 516
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 516
aattcaaaaa gtgtgagtat gcatctccat ctcttgaatg gagatgcata ctcacac 57
<210> 517
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 517
ccgggagtat gcatctccag gcattcaaga gatgcctgga gatgcatact ctttttg 57
<210> 518
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 518
aattcaaaaa gagtatgcat ctccaggcat ctcttgaatg cctggagatg catactc 57
<210> 519
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 519
ccggctccag gcaaagccac tgattcaaga gatcagtggc tttgcctgga gtttttg 57
<210> 520
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 520
aattcaaaaa ctccaggcaa agccactgat ctcttgaatc agtggctttg cctggag 57
<210> 521
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 521
ccggctgaca gccaggtgac tgattcaaga gatcagtcac ctggctgtca gtttttg 57
<210> 522
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 522
aattcaaaaa ctgacagcca ggtgactgat ctcttgaatc agtcacctgg ctgtcag 57
<210> 523
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 523
ccggccaggt gactgaagtc tgtttcaaga gaacagactt cagtcacctg gtttttg 57
<210> 524
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 524
aattcaaaaa ccaggtgact gaagtctgtt ctcttgaaac agacttcagt cacctgg 57
<210> 525
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 525
ccggggaatg agttgacctt cctttcaaga gaaggaaggt caactcattc ctttttg 57
<210> 526
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 526
aattcaaaaa ggaatgagtt gaccttcctt ctcttgaaag gaaggtcaac tcattcc 57
<210> 527
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 527
ccggccttcc tagatgattc catttcaaga gaatggaatc atctaggaag gtttttg 57
<210> 528
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 528
aattcaaaaa ccttcctaga tgattccatt ctcttgaaat ggaatcatct aggaagg 57
<210> 529
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 529
ccggctagat gattccatct gcattcaaga gatgcagatg gaatcatcta gtttttg 57
<210> 530
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 530
aattcaaaaa ctagatgatt ccatctgcat ctcttgaatg cagatggaat catctag 57
<210> 531
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 531
ccgggattcc atctgcacgg gcattcaaga gatgcccgtg cagatggaat ctttttg 57
<210> 532
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 532
aattcaaaaa gattccatct gcacgggcat ctcttgaatg cccgtgcaga tggaatc 57
<210> 533
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 533
ccggcacctc cagtggaaat caattcaaga gattgatttc cactggaggt gtttttg 57
<210> 534
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 534
aattcaaaaa cacctccagt ggaaatcaat ctcttgaatt gatttccact ggaggtg 57
<210> 535
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNAoligo
<400> 535
ccggccagtg gaaatcaagt gaattcaaga gattcacttg atttccactg gtttttg 57
<210> 536
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 536
aattcaaaaa ccagtggaaa tcaagtgaat ctcttgaatt cacttgattt ccactgg 57
<210> 537
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 537
ccgggaacct cactatccaa ggattcaaga gatccttgga tagtgaggtt ctttttg 57
<210> 538
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 538
aattcaaaaa gaacctcact atccaaggat ctcttgaatc cttggatagt gaggttc 57
<210> 539
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 539
ccggcgggac tctacatctg caattcaaga gattgcagat gtagagtccc gtttttg 57
<210> 540
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 540
aattcaaaaa cgggactcta catctgcaat ctcttgaatt gcagatgtag agtcccg 57
<210> 541
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 541
ccgggactct acatctgcaa ggtttcaaga gaaccttgca gatgtagagt ctttttg 57
<210> 542
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 542
aattcaaaaa gactctacat ctgcaaggtt ctcttgaaac cttgcagatg tagagtc 57
<210> 543
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 543
ccgggcaagg tggagctcat gtattcaaga gatacatgag ctccaccttg ctttttg 57
<210> 544
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 544
aattcaaaaa gcaaggtgga gctcatgtat ctcttgaata catgagctcc accttgc 57
<210> 545
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 545
ccgggcatag gcaacggaac ccattcaaga gatgggttcc gttgcctatg ctttttg 57
<210> 546
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 546
aattcaaaaa gcataggcaa cggaacccat ctcttgaatg ggttccgttg cctatgc 57
<210> 547
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 547
ccggggaacc cagatttatg taattcaaga gattacataa atctgggttc ctttttg 57
<210> 548
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 548
aattcaaaaa ggaacccaga tttatgtaat ctcttgaatt acataaatct gggttcc 57
<210> 549
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 549
ccggcccaga tttatgtaat tgattcaaga gatcaattac ataaatctgg gtttttg 57
<210> 550
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 550
aattcaaaaa cccagattta tgtaattgat ctcttgaatc aattacataa atctggg 57
<210> 551
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 551
ccggcagatt ctgacttcct cctttcaaga gaaggaggaa gtcagaatct gtttttg 57
<210> 552
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 552
aattcaaaaa cagattctga cttcctcctt ctcttgaaag gaggaagtca gaatctg 57
<210> 553
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 553
ccgggattct gacttcctcc tctttcaaga gaagaggagg aagtcagaat ctttttg 57
<210> 554
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 554
aattcaaaaa gattctgact tcctcctctt ctcttgaaag aggaggaagt cagaatc 57
<210> 555
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 555
ccggctgact tcctcctctg gatttcaaga gaatccagag gaggaagtca gtttttg 57
<210> 556
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 556
aattcaaaaa ctgacttcct cctctggatt ctcttgaaat ccagaggagg aagtcag 57
<210> 557
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 557
ccgggatcct tgcagcagtt agtttcaaga gaactaactg ctgcaaggat ctttttg 57
<210> 558
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 558
aattcaaaaa gatccttgca gcagttagtt ctcttgaaac taactgctgc aaggatc 57
<210> 559
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 559
ccgggctttc tcctcacagc tgtttcaaga gaacagctgt gaggagaaag ctttttg 57
<210> 560
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 560
aattcaaaaa gctttctcct cacagctgtt ctcttgaaac agctgtgagg agaaagc 57
<210> 561
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 561
ccggcccatc aattgagaaa ccattcaaga gatggtttct caattgatgg gtttttg 57
<210> 562
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 562
aattcaaaaa cccatcaatt gagaaaccat ctcttgaatg gtttctcaat tgatggg 57
<210> 563
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 563
ccgggaaacc attatgaaga agattcaaga gatcttcttc ataatggttt ctttttg 57
<210> 564
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 564
aattcaaaaa gaaaccatta tgaagaagat ctcttgaatc ttcttcataa tggtttc 57
<210> 565
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 565
ccgggtccat atttcaattt ccattcaaga gatggaaatt gaaatatgga ctttttg 57
<210> 566
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 566
aattcaaaaa gtccatattt caatttccat ctcttgaatg gaaattgaaa tatggac 57
<210> 567
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 567
ccggcaagag ctgaggcaat tctttcaaga gaagaattgc ctcagctctt gtttttg 57
<210> 568
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 568
aattcaaaaa caagagctga ggcaattctt ctcttgaaag aattgcctca gctcttg 57
<210> 569
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 569
ccggggatgc ggaacccaaa ttattcaaga gataatttgg gttccgcatc ctttttg 57
<210> 570
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 570
aattcaaaaa ggatgcggaa cccaaattat ctcttgaata atttgggttc cgcatcc 57
<210> 571
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 571
ccggcggaac ccaaattacg tgtttcaaga gaacacgtaa tttgggttcc gtttttg 57
<210> 572
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 572
aattcaaaaa cggaacccaa attacgtgtt ctcttgaaac acgtaatttg ggttccg 57
<210> 573
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 573
ccggggaacc caaattacgt gtattcaaga gatacacgta atttgggttc ctttttg 57
<210> 574
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 574
aattcaaaaa ggaacccaaa ttacgtgtat ctcttgaata cacgtaattt gggttcc 57
<210> 575
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 575
ccggcaaatt acgtgtacta caattcaaga gattgtagta cacgtaattt gtttttg 57
<210> 576
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 576
aattcaaaaa caaattacgt gtactacaat ctcttgaatt gtagtacacg taatttg 57
<210> 577
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 577
ccggggagta gaaagacaga gctttcaaga gaagctctgt ctttctactc ctttttg 57
<210> 578
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 578
aattcaaaaa ggagtagaaa gacagagctt ctcttgaaag ctctgtcttt ctactcc 57
<210> 579
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 579
ccggctggga tgtttctgtc acattcaaga gatgtgacag aaacatccca gtttttg 57
<210> 580
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 580
aattcaaaaa ctgggatgtt tctgtcacat ctcttgaatg tgacagaaac atcccag 57
<210> 581
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 581
ccggggatgt ttctgtcaca tcattcaaga gatgatgtga cagaaacatc ctttttg 57
<210> 582
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 582
aattcaaaaa ggatgtttct gtcacatcat ctcttgaatg atgtgacaga aacatcc 57
<210> 583
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 583
ccggcacatc agctccactt tcattcaaga gatgaaagtg gagctgatgt gtttttg 57
<210> 584
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 584
aattcaaaaa cacatcagct ccactttcat ctcttgaatg aaagtggagc tgatgtg 57
<210> 585
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 585
ccggcagctc cactttcagt gaattcaaga gattcactga aagtggagct gtttttg 57
<210> 586
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 586
aattcaaaaa cagctccact ttcagtgaat ctcttgaatt cactgaaagt ggagctg 57
<210> 587
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 587
ccgggtgaaa gcatcacttg ggattcaaga gatcccaagt gatgctttca ctttttg 57
<210> 588
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 588
aattcaaaaa gtgaaagcat cacttgggat ctcttgaatc ccaagtgatg ctttcac 57
<210> 589
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 589
ccggggatgc agcattatga tgtttcaaga gaacatcata atgctgcatc ctttttg 57
<210> 590
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 590
aattcaaaaa ggatgcagca ttatgatgtt ctcttgaaac atcataatgc tgcatcc 57
<210> 591
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 591
ccgggcagca ttatgatgtg ggtttcaaga gaacccacat cataatgctg ctttttg 57
<210> 592
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 592
aattcaaaaa gcagcattat gatgtgggtt ctcttgaaac ccacatcata atgctgc 57
<210> 593
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 593
ccgggcatta tgatgtgggt caattcaaga gattgaccca catcataatg ctttttg 57
<210> 594
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 594
aattcaaaaa gcattatgat gtgggtcaat ctcttgaatt gacccacatc ataatgc 57
<210> 595
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 595
ccggcaagga attaagttag ggattcaaga gatccctaac ttaattcctt gtttttg 57
<210> 596
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 596
aattcaaaaa caaggaatta agttagggat ctcttgaatc cctaacttaa ttccttg 57
<210> 597
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 597
ccgggggaat ggcacagccc aaattcaaga gatttgggct gtgccattcc ctttttg 57
<210> 598
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 598
aattcaaaaa gggaatggca cagcccaaat ctcttgaatt tgggctgtgc cattccc 57
<210> 599
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 599
ccgggaatgg cacagcccaa agattcaaga gatctttggg ctgtgccatt ctttttg 57
<210> 600
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 600
aattcaaaaa gaatggcaca gcccaaagat ctcttgaatc tttgggctgt gccattc 57
<210> 601
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 601
ccgggcacag cccaaagaag gaattcaaga gattccttct ttgggctgtg ctttttg 57
<210> 602
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 602
aattcaaaaa gcacagccca aagaaggaat ctcttgaatt ccttctttgg gctgtgc 57
<210> 603
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 603
ccggcacagc ccaaagaagg aaattcaaga gatttccttc tttgggctgt gtttttg 57
<210> 604
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 604
aattcaaaaa cacagcccaa agaaggaaat ctcttgaatt tccttctttg ggctgtg 57
<210> 605
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 605
ccgggaagac tatattgtac acattcaaga gatgtgtaca atatagtctt ctttttg 57
<210> 606
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 606
aattcaaaaa gaagactata ttgtacacat ctcttgaatg tgtacaatat agtcttc 57
<210> 607
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 607
ccggctatat tgtacacacc ttattcaaga gataaggtgt gtacaatata gtttttg 57
<210> 608
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 608
aattcaaaaa ctatattgta cacaccttat ctcttgaata aggtgtgtac aatatag 57
<210> 609
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 609
ccggcacacc ttatatttac gtattcaaga gatacgtaaa tataaggtgt gtttttg 57
<210> 610
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 610
aattcaaaaa cacaccttat atttacgtat ctcttgaata cgtaaatata aggtgtg 57
<210> 611
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 611
ccgggagacg tttatagccg aaattcaaga gatttcggct ataaacgtct ctttttg 57
<210> 612
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 612
aattcaaaaa gagacgttta tagccgaaat ctcttgaatt tcggctataa acgtctc 57
<210> 613
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 613
ccgggattac atcaaggctt caattcaaga gattgaagcc ttgatgtaat ctttttg 57
<210> 614
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 614
aattcaaaaa gattacatca aggcttcaat ctcttgaatt gaagccttga tgtaatc 57
<210> 615
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 615
ccgggtgatg ctaaaggttg tatttcaaga gaatacaacc tttagcatca ctttttg 57
<210> 616
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 616
aattcaaaaa gtgatgctaa aggttgtatt ctcttgaaat acaaccttta gcatcac 57
<210> 617
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 617
ccggctttcc accttgggtc ccattcaaga gatgggaccc aaggtggaaa gtttttg 57
<210> 618
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 618
aattcaaaaa ctttccacct tgggtcccat ctcttgaatg ggacccaagg tggaaag 57
<210> 619
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 619
ccgggaggag ctcaggacac taattcaaga gattagtgtc ctgagctcct ctttttg 57
<210> 620
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 620
aattcaaaaa gaggagctca ggacactaat ctcttgaatt agtgtcctga gctcctc 57
<210> 621
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 621
ccggggacac taatacacca ggtttcaaga gaacctggtg tattagtgtc ctttttg 57
<210> 622
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 622
aattcaaaaa ggacactaat acaccaggtt ctcttgaaac ctggtgtatt agtgtcc 57
<210> 623
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 623
ccgggacact aatacaccag gtattcaaga gatacctggt gtattagtgt ctttttg 57
<210> 624
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 624
aattcaaaaa gacactaata caccaggtat ctcttgaata cctggtgtat tagtgtc 57
<210> 625
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 625
ccggcaaggt catttgctaa ctattcaaga gatagttagc aaatgacctt gtttttg 57
<210> 626
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 626
aattcaaaaa caaggtcatt tgctaactat ctcttgaata gttagcaaat gaccttg 57
<210> 627
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 627
ccggcttgga aactggatga ggtttcaaga gaacctcatc cagtttccaa gtttttg 57
<210> 628
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 628
aattcaaaaa cttggaaact ggatgaggtt ctcttgaaac ctcatccagt ttccaag 57
<210> 629
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 629
ccggggaaac tggatgaggt catttcaaga gaatgacctc atccagtttc ctttttg 57
<210> 630
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 630
aattcaaaaa ggaaactgga tgaggtcatt ctcttgaaat gacctcatcc agtttcc 57
<210> 631
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 631
ccgggaaact ggatgaggtc atattcaaga gatatgacct catccagttt ctttttg 57
<210> 632
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 632
aattcaaaaa gaaactggat gaggtcatat ctcttgaata tgacctcatc cagtttc 57
<210> 633
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 633
ccggggtcat agcagtgctt gatttcaaga gaatcaagca ctgctatgac ctttttg 57
<210> 634
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 634
aattcaaaaa ggtcatagca gtgcttgatt ctcttgaaat caagcactgc tatgacc 57
<210> 635
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 635
ccggcagtgc ttgattgcgt ggattcaaga gatccacgca atcaagcact gtttttg 57
<210> 636
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 636
aattcaaaaa cagtgcttga ttgcgtggat ctcttgaatc cacgcaatca agcactg 57
<210> 637
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 637
ccgggtgctt gattgcgtgg aatttcaaga gaattccacg caatcaagca ctttttg 57
<210> 638
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 638
aattcaaaaa gtgcttgatt gcgtggaatt ctcttgaaat tccacgcaat caagcac 57
<210> 639
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 639
ccgggcgtgg aattgtgctg agtttcaaga gaactcagca caattccacg ctttttg 57
<210> 640
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 640
aattcaaaaa gcgtggaatt gtgctgagtt ctcttgaaac tcagcacaat tccacgc 57
<210> 641
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 641
ccggcacagc tggtggtatc tgattcaaga gatcagatac caccagctgt gtttttg 57
<210> 642
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 642
aattcaaaaa cacagctggt ggtatctgat ctcttgaatc agataccacc agctgtg 57
<210> 643
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 643
ccggctggtg gtatctgagt tgattcaaga gatcaactca gataccacca gtttttg 57
<210> 644
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 644
aattcaaaaa ctggtggtat ctgagttgat ctcttgaatc aactcagata ccaccag 57
<210> 645
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 645
ccggggtggt atctgagttg actttcaaga gaagtcaact cagataccac ctttttg 57
<210> 646
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 646
aattcaaaaa ggtggtatct gagttgactt ctcttgaaag tcaactcaga taccacc 57
<210> 647
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 647
ccgggagttg acttgacaga acattcaaga gatgttctgt caagtcaact ctttttg 57
<210> 648
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 648
aattcaaaaa gagttgactt gacagaacat ctcttgaatg ttctgtcaag tcaactc 57
<210> 649
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 649
ccgggttgac ttgacagaac actttcaaga gaagtgttct gtcaagtcaa ctttttg 57
<210> 650
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 650
aattcaaaaa gttgacttga cagaacactt ctcttgaaag tgttctgtca agtcaac 57
<210> 651
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 651
ccgggcttac tccaggagac ccattcaaga gatgggtctc ctggagtaag ctttttg 57
<210> 652
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 652
aattcaaaaa gcttactcca ggagacccat ctcttgaatg ggtctcctgg agtaagc 57
<210> 653
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 653
ccggcccaca ggtatgacct tctttcaaga gaagaaggtc atacctgtgg gtttttg 57
<210> 654
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 654
aattcaaaaa cccacaggta tgaccttctt ctcttgaaag aaggtcatac ctgtggg 57
<210> 655
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 655
ccggccacag gtatgacctt ctattcaaga gatagaaggt catacctgtg gtttttg 57
<210> 656
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 656
aattcaaaaa ccacaggtat gaccttctat ctcttgaata gaaggtcata cctgtgg 57
<210> 657
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 657
ccggccttct aggaagctcc agtttcaaga gaactggagc ttcctagaag gtttttg 57
<210> 658
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 658
aattcaaaaa ccttctagga agctccagtt ctcttgaaac tggagcttcc tagaagg 57
<210> 659
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 659
ccggccagtt cgatgggccc aatttcaaga gaattgggcc catcgaactg gtttttg 57
<210> 660
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 660
aattcaaaaa ccagttcgat gggcccaatt ctcttgaaat tgggcccatc gaactgg 57
<210> 661
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 661
ccgggatggg cccaattctt acattcaaga gatgtaagaa ttgggcccat ctttttg 57
<210> 662
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 662
aattcaaaaa gatgggccca attcttacat ctcttgaatg taagaattgg gcccatc 57
<210> 663
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 663
ccgggcccaa ttcttacaaa catttcaaga gaatgtttgt aagaattggg ctttttg 57
<210> 664
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 664
aattcaaaaa gcccaattct tacaaacatt ctcttgaaat gtttgtaaga attgggc 57
<210> 665
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 665
ccggccaatt cttacaaaca tgtttcaaga gaacatgttt gtaagaattg gtttttg 57
<210> 666
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 666
aattcaaaaa ccaattctta caaacatgtt ctcttgaaac atgtttgtaa gaattgg 57
<210> 667
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 667
ccgggttaat gccatggaca gaattcaaga gattctgtcc atggcattaa ctttttg 57
<210> 668
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 668
aattcaaaaa gttaatgcca tggacagaat ctcttgaatt ctgtccatgg cattaac 57
<210> 669
<211> 51
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 669
ccggggtgca tgaaggtttc tgattcaaga gatcagaaac cttcatgcac c 51
<210> 670
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 670
aattcaaaaa ggtgcatgaa ggtttctgat ctcttgaatc agaaaccttc atgcacc 57
<210> 671
<211> 24
<212> DNA
<213> Artificial
<220>
<223> 上游引物
<400> 671
cctatttccc atgattcctt cata 24
<210> 672
<211> 20
<212> DNA
<213> Artificial
<220>
<223> 下游引物
<400> 672
gtaatacggt tatccacgcg 20
<210> 673
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 阴性对照siRNA靶序列
<400> 673
ttctccgaac gtgtcacgt 19
<210> 674
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 674
ccggttctcc gaacgtgtca cgtttcaaga gaacgtgaca cgttcggaga atttttg 57
<210> 675
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 675
aattcaaaaa ttctccgaac gtgtcacgtt ctcttgaaac gtgacacgtt cggagaa 57
<210> 676
<211> 20
<212> DNA
<213> Artificial
<220>
<223> 上游引物
<400> 676
ttgcagcagt tagttcgggg 20
<210> 677
<211> 20
<212> DNA
<213> Artificial
<220>
<223> 下游引物
<400> 677
gctctgttgg gggcattttc 20
<210> 678
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 上游引物
<400> 678
tgacttcaac agcgacaccc a 21
<210> 679
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 下游引物
<400> 679
caccctgttg ctgtagccaa a 21

Claims (17)

1.一种分离的核酸分子，所述核酸分子包含：
a)双链RNA，所述双链RNA中含有能够在严紧条件下与CTLA-4基因杂交的核苷酸序列；或者
b)shRNA，所述shRNA中含有能够在严紧条件下与CTLA-4基因杂交的核苷酸序列。
2.根据权利要求1所述的分离的核酸分子，其特征在于，所述CTLA-4基因来源于人。
3.根据权利要求1所述的分离的核酸分子，其特征在于，所述双链RNA包含第一链和第二链，所述第一链和所述第二链互补共同形成RNA二聚体，并且所述第一链的序列与CTLA-4基因中的靶序列基本相同；所述shRNA包括正义链片段和反义链片段，以及连接所述正义链片段和反义链片段的茎环结构，所述正义链片段和所述反义链片段的序列互补，并且所述正义链片段的序列与CTLA-4基因中的靶序列基本相同。
4.根据权利要求1～3任一权利要求所述的分离的核酸分子，其特征在于，所述CTLA-4基因中的靶序列选自如SEQ ID NO：1～134所示之任一序列或如SEQ ID NO：1～134所示之任一序列经过取代、缺失或添加一个或几个碱基序列且具有与如SEQ ID NO：1～134所示之任一序列相同活性的序列。
5.根据权利要求1～3任一权利要求所述的分离的核酸分子，其特征在于，所述双链RNA为小干扰RNA，所述小干扰RNA第一链的序列如SEQ ID NO：135～268任一序列所示。
6.根据权利要求1～3任一权利要求所述的分离的核酸分子，其特征在于，所述shRNA的序列如SEQ ID NO:269～402任一序列所示。
7.一种CTLA-4基因干扰核酸构建体，含有编码如权利要求1～6任一权利要求所述分离的核酸分子中的shRNA的基因片段，能表达所述shRNA。
8.如权利要求7所述的CTLA-4基因干扰核酸构建体，其特征在于，所述CTLA-4基因干扰核酸构建体选自病毒载体或质粒载体。
9.如权利要求8所述的CTLA-4基因干扰核酸构建体，其特征在于，所述CTLA-4基因干扰核酸构建体选自慢病毒载体、腺病毒载体、逆转录病毒载体、AAV。
10.如权利要求9所述的CTLA-4基因干扰核酸构建体，其特征在于，所述慢病毒载体选自：pLKO.1-puro、pLKO.1-CMV-tGFP、pLKO.1-puro-CMV-tGFP、pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、pLKO.1-puro-CMV-TagCFP、pLKO.1-puro-CMV-TagYFP、pLKO.1-puro-CMV-TagRFP、pLKO.1-puro-CMV-TagFP635、pLKO.1-puro-UbC-TurboGFP、pLKO.1-puro-UbC-TagFP635、pLKO-puro-IPTG-1xLacO、pLKO-puro-IPTG-3xLacO、pLP1、pLP2、pLP/VSV-G、pENTR/U6、pLenti6/BLOCK-iT-DEST、pLenti6-GW/U6-laminshrna、pcDNA1.2/V5-GW/lacZ、pLenti6.2/N-Lumio/V5-DEST、pGCSIL-GFP或pLenti6.2/N-Lumio/V5-GW/lacZ中的任一。
11.一种CTLA-4基因干扰慢病毒，由权利要求7～10任一权利要求所述干扰核酸构建体在慢病毒包装质粒、细胞系的辅助下，经过病毒包装而成。
12.如权利要求1～6任一权利要求所述的分离的核酸分子、权利要求7～10任一权利要求所述CTLA-4基因干扰核酸构建体，和/或权利要求11所述的CTLA-4基因干扰慢病毒在制备T细胞活化促进剂中的用途。
13.根据权利要求12所述的用途，其特征在于，所述T细胞活化促进剂作用于T细胞，降低T细胞中CTLA-4基因的表达，获得活化的T细胞，所述活化的T细胞可通过CART，DC，CIK或DC-CIK免疫治疗手段制备肿瘤或自身免疫疾病治疗药物。
14.一种T细胞活化促进剂，其有效物质含有以下任一项所述有效成分以及药学上可接受的载体、稀释剂或赋形剂：
(1)如权利要求1-6任一权利要求所述的分离的核酸分子；(2)权利要求7～10任一权利要求所述CTLA-4基因干扰核酸构建体；(3)权利要求11所述的CTLA-4基因干扰慢病毒。
15.一种促进T细胞活化的试剂盒，所述试剂盒包括：存在于容器中的以下之任一项：
(1)如权利要求1-6任一权利要求所述的分离的核酸分子；(2)权利要求7～10任一权利要求所述CTLA-4基因干扰核酸构建体；(3)权利要求11所述的CTLA-4基因干扰慢病毒。
16.一种促进T细胞活化的方法，为降低T细胞中CTLA-4基因的表达，从而促进T细胞的活化。
17.一种T细胞，其特征在于，所述T细胞中含有以下之任以项：(1)如权利要求1-6任一权利要求所述的分离的核酸分子；(2)权利要求7～10任一权利要求所述CTLA-4基因干扰核酸构建体；(3)权利要求11所述的CTLA-4基因干扰慢病毒。
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