CN107523569A - Pdcd1基因的用途及其相关药物 - Google Patents
Pdcd1基因的用途及其相关药物 Download PDFInfo
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- CN107523569A CN107523569A CN201710470382.8A CN201710470382A CN107523569A CN 107523569 A CN107523569 A CN 107523569A CN 201710470382 A CN201710470382 A CN 201710470382A CN 107523569 A CN107523569 A CN 107523569A
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Abstract
本发明涉及生物技术领域,更具体地涉及人PDCD1基因的用途及其相关药物。本发明设计了针对人PDCD1基因的120个RNAi靶点序列,构建相应的PDCD1 RNAi载体,其中的RNAi载体pGCSIL‑GFP‑PDCD1‑siRNA能够显著下调PDCD1基因在mRNA水平和蛋白水平的表达。使用慢病毒(lentivirus,简写为Lv)作为基因操作工具携带RNAi载体pGCSIL‑GFP‑PDCD1‑siRNA能够靶向地将针对PDCD1基因的RNAi序列高效导入T细胞,降低T细胞中PDCD1基因的表达水平,显著抑制细胞中PDCD1的表达,提高T细胞活性。
Description
技术领域
本发明涉及生物技术领域,更具体地涉及人PDCD1基因的用途及其相关药物。
背景技术
PD-1(CD297、PDCD1)是B7-CD28家族的成员之一,在激活后的T细胞、B细胞、单核细胞和自然杀伤T细胞中表达(Keir,M.E.,M.J.Butte,G.J.Freeman,andA.H.Sharpe.2008.PD-1and its ligands in tolerance andimmunity.Annu.Rev.Immunol.26:677–704.)。PD-1有两种已知配体,分别为PD-L1(B7-H1)(Dong,H.,G.Zhu,K.Tamada,and L.Chen.1999.B7-H1,a third member of the B7family,costimulates T-cell proliferation and interleukin-10secretion.Nat.Med.5:1365–1369.)和PD-L2(B7-DC)(Latchman,Y.,C.R.Wood,T.Chernova,D.Chaudhary,M.Borde,I.Chernova,et al.2001.PD-L2is a second ligand for PD-1and inhibits T cellactivation.Nat.Immunol.2:261–268.)。这两种受体都在APCs细胞中表达。另外,PD-L1也在一些血液细胞中表达。当PD-L1或PD-L2与PD-1结合时,T细胞受体信号通路被抑制,抑凋亡因子表达下调,p15表达增加使细胞停滞在G1期,SKP2的表达下调。(Patsoukis,N.,J.Brown,V.Petkova,F.Liu,L.Li,and V.A.Boussiotis.2012.Selective effects of PD-1on Akt and Ras pathways regulate molecular components of the cell cycle andinhibit T cell proliferation.Sci.Signal.5:46.)PD-L1在非血液细胞的表达可以抑制免疫系统介导的组织损伤。
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、特异地阻断体内特定基因的表达,导致其降解,从而引起生物体内特异基因的沉默,使细胞表现出某种基因表型的缺失,是近年来新兴的一种常用的研究基因功能、寻找疾病治疗方法的实验室技术。研究表明,长度为21-23nt的双链RNA能够在转录和转录后水平特异性的引起RNAi(Tuschl T,Zamore PD,Sharp PA,Bartel DP.RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21to23nucleotide intervals.Cell 2000;101:25-33.)。
能够有效提高T细胞的活性,减少肿瘤细胞自身PD-L1对T细胞的抑制作用,增强T细胞对肿瘤细胞的杀伤力,是亟待解决的技术问题。
发明内容
本发明的目的在于公开与人PDCD1基因相关的治疗方法及药物,以RNA干扰(RNAi)为手段下调PDCD1基因在免疫细胞中的表达,进而阻断PD-1/PD-L1通路,减少肿瘤细胞自身PD-L1对T细胞的抑制作用,提高T细胞的活性,起到对肿瘤更有效的杀伤。
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,提供了一种提高T细胞活性的方法,为降低T细胞中PDCD1基因的表达,从而提高T细胞的活性。
优选地,所述方法包括但不限于特异性抑制PDCD1基因的转录或翻译,或特异性抑制PDCD1蛋白的表达或活性。
所述提高T细胞活性包括但不限于:增强T细胞对肿瘤细胞的杀伤力。
优选地,所述肿瘤细胞选自高表达PD1受体PD-L1的肿瘤细胞。
优选地,所述肿瘤选自胃癌、胸腺癌、卵巢癌、前列腺癌和肺癌。
具体地,所述降低肿T细胞中PDCD1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PDCD1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PDCD1基因失活或活性降低;或者也可利用抗体、蛋白、小分子等与PDCD1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PDCD1基因的表达水平。
所述的提高T细胞活性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性提高的T细胞可被用于治疗的或非治疗目的。
本发明的第二方面,提供了一种提高T细胞活性的分离的核酸分子,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PDCD1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PDCD1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PDCD1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PDCD1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PDCD1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PDCD1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PDCD1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PDCD1基因中的靶序列选自如SEQ ID NO:1~120所示之任一序列或如SEQ ID NO:1~120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:1~120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:1~120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:365~484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PDCD1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PDCD1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PDCD1基因中的靶序列基本相同。
较佳的,所述正义链片段与PDCD1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PDCD1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:489~608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PDCD1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PDCD1基因中的靶序列基本相同,所述PDCD1基因的靶序列即为siRNA用于特异性沉默PDCD1基因表达时,被所述siRNA识别并沉默的mRNA片段所对应的PDCD1基因的片段。
编码本发明所述shRNA的基因片段的干扰慢病毒载体含有SEQ ID NO:1-120中之任一序列及其互补序列。
所述双链RNA的第一链或所述shRNA的正义链片段与PDCD1基因中的靶序列基本相同,所述PDCD1基因的靶序列即为siRNA用于特异性沉默PDCD1基因表达时,被所述siRNA识别并沉默的mRNA片段所对应的PDCD1基因中的片段。
较佳的,所述PDCD1基因中的靶序列含有SEQ ID NO:1-120之任一序列。
进一步的,所述PDCD1基因来源于人。
本发明的第三方面,公开了一种PDCD1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。
所述的人PDCD1基因干扰核酸构建体可以是将编码前述人PDCD1基因shRNA的基因片段克隆入已知载体获得。
进一步地,所述PDCD1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PDCD1基因干扰核酸构建体选自PDCD1基因干扰慢病毒载体、腺病毒载体、逆转录病毒载体、AAV。
本发明的PDCD1基因干扰慢病毒载体是将编码前述PDCD1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PDCD1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,感染肿瘤细胞,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PDCD1基因的表达。
进一步的,所述PDCD1基因干扰慢病毒载体还含有启动子序列和/或编码肿瘤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PDCD1基因干扰慢病毒载体,命名为pGCSIL-GFP-PDCD1-siRNA。
本发明的PDCD1基因siRNA可用于敲低T胞的PDCD1,进一步地可以用作提高T细胞活性的促进剂。PDCD1基因干扰慢病毒载体则可用于制备所述PDCD1基因siRNA。当用作提高T细胞活性的促进剂时,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PDCD1基因干扰慢病毒,由前述PDCD1基因干扰慢病毒载体在慢病毒包装质粒、细胞系的辅助下,经过病毒包装而成。该慢病毒可感染肿瘤细胞并产生针对PDCD1基因的小分子干扰RNA,从而抑制PDCD1的表达。该PDCD1基因干扰慢病毒可用于制备提高T细胞活性的促进剂。
本发明的第五方面,提供了前述分离的核酸分子,PDCD1基因干扰核酸构建体或PDCD1基因干扰慢病毒在制备T细胞活性促进剂中的用途。
优选地,所述T细胞活性的促进剂,其有效物质含有前述的分离的核酸分子,和/或PDCD1基因干扰核酸构建体或PDCD1基因干扰慢病毒中的一种或多种的组合。
优选地,所述T细胞活性的促进剂用于作用于T细胞,降低T细胞中PDCD1基因的表达,获得具有较高的杀伤力的高活性T细胞。所述高活性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,公开了一种用于提高T细胞活性的促进剂,其有效物质含有前述的分离的核酸分子,和/或PDCD1基因干扰核酸构建体或PDCD1基因干扰慢病毒中的一种或多种的组合。
进一步的,所述促进剂含有1~99wt%所述双链RNA、shRNA、PDCD1基因干扰核酸构建体或PDCD1基因干扰慢病毒,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,公开了一种提高T细胞活性的试剂盒,所述试剂盒包括:存在于容器中的所述分离的核酸分子,和/或PDCD1基因干扰核酸构建体,和/或所述的PDCD1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细胞中含有所述分离的核酸分子,和/或PDCD1基因干扰核酸构建体,和/或所述的PDCD1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PDCD1基因的120个RNAi靶点序列,构建相应的PDCD1RNAi载体,其中的RNAi载体pGCSIL-GFP-PDCD1-siRNA能够显著下调PDCD1基因在mRNA水平和蛋白水平的表达。使用慢病毒(lentivirus,简写为Lv)作为基因操作工具携带RNAi载体pGCSIL-GFP-PDCD1-siRNA能够靶向地将针对PDCD1基因的RNAi序列高效导入T细胞,降低T细胞中PDCD1基因的表达水平,显著抑制细胞中PDCD1的表达,提高T细胞活性。
附图说明
图1:pGCSIL-GFP质粒DNA图谱。
图2:蛋白质印迹Western Blot法检测PDCD1基因的沉默效率图。
图3:慢病毒穿梭载体结构示意图。
图4:选取表达FMC63BBz及FMC63BBzshPD1的T细胞,并分析其表面PD1表达情况。
具体实施方式
本发明的发明人首先制备了能够稳定表达PDCD1的293T细胞株;并设计了一组针对人PDCD1基因的小分子干扰RNA(siRNA)序列、RNA干扰载体和RNA干扰慢病毒。选取人PDCD1mRNA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。通过基因克隆,构建表达上述siRNA的核酸构建体,包装表达上述siRNA的慢病毒。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PDCD1基因的表达。
发明人发现,采用RNAi方法可以有效下调人PDCD1基因的转录水平,并进一步可以有效抑制PDCD1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PDCD1基因RNAi慢病毒的制备
1.筛选针对人PDCD1基因的有效的siRNA靶点
从Genbank调取PDCD1(Gene ID:5133)基因信息;设计针对PDCD1基因的有效的siRNA靶点。表1列出了其中120条针对PDCD1基因的有效siRNA靶点序列。
表1靶向于人PDCD1基因的siRNA靶点序列
2.慢病毒载体的制备
分别针对siRNA靶点(以SEQ ID NO:1~120为例)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。
表2两端含Age I和EcoR I酶切位点粘端的双链DNA Oligo
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:363);下游引物序列:5’-GTAATACGGTTATCCACGCG-3’(SEQ ID NO:364),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针分别针对SEQ ID NO:1~120的表达RNAi的载体,分别命名为pGCSIL-GFP-PDCD1-siRNA1~~pGCSIL-GFP-PDCD1-siRNA120。
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:197)。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DNA Oligo序列(表3),其余构建方法、鉴定方法及条件均同pGCSIL-GFP-PDCD1-siRNA1~~pGCSIL-GFP-PDCD1-siRNA120。
表3两端含Age I和EcoR I酶切位点粘端的双链DNA Oligo
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。
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PDCD1-siRNA慢病毒
以Qiagen公司的质粒抽提试剂盒分别提取RNAi质粒pGCSIL-GFP-PDCD1-siRNA1~~pGCSIL-GFP-PDCD1-siRNA120的DNA,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,以含10%胎牛血清的DMEM完全培养基调整细胞密度为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。病毒浓缩液中含有的shRNA的第一链的序列,如SEQID NO:489~608任一序列所示。
对照慢病毒的包装过程同PDCD1-siRNA慢病毒,仅以pGCSIL-GFP-Scr-siRNA载体代替pGCSIL-GFP-PDCD1-siRNA载体。
实施例2实时荧光定量RT-PCR法检测PDCD1基因的沉默效率
首先,构建稳定表达PDCD1的293T,具体方法如下:
处于对数生长期的293T进行胰酶消化,制成细胞悬液(细胞数约为5×104/ml)接种于6孔板中,培养至细胞融合度达到约30%。根据侵染复数(MOI:1)值,加入适宜量的稳定表达PDCD1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,40小时后,在培养基中加入1ug/ml的嘌呤霉素,继续培养3天,通过免疫荧光印迹的方法确定此293T细胞能够稳定表达PDCD1基因。
处于对数生长期的稳定表达PDCD1的293T进行胰酶消化,制成细胞悬液(细胞数约为5×104/ml)接种于6孔板中,培养至细胞融合度达到约30%。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,待侵染时间达到5天后,收集细胞。根据Invitrogen公司的Trizol操作说明书,抽提总RNA。根据Promega公司的M-MLV操作说明书,将RNA逆转录获得cDNA(逆转录反应体系见表7,42℃反应1h,然后在70℃水浴锅中水浴10min使逆转录酶失活)。
采用TP800型Real time PCR仪(TAKARA)进行实时定量检测。PDCD1基因的引物如下:上游引物5’-GACATCCTACGGTCCCAAGGTC-3’(SEQ ID NO:485)和下游引物5’-TTCTGCCCTCCCAACACCC-3’(SEQ ID NO:486)。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:487)和下游引物5’-CACCCTGTTGCTGTAGCCAAA-3’(SEQ ID NO:488)。按表8中的比例配置反应体系。
表7逆转录反应体系
试剂 | 体积(μl) |
5×RT buffer | 4.0 |
10mM dNTPs | 2.0 |
RNasin | 0.5 |
M-MLV-RTase | 1.0 |
DEPC H2O | 3.5 |
Total | 11.0 |
表8Real-time PCR反应体系
试剂 | 体积(μl) |
SYBR premix ex taq: | 10.0 |
上游引物(2.5μM): | 0.5 |
下游引物(2.5μM): | 0.5 |
cDNA | 1.0 |
ddH2O | 8.0 |
Total | 20.0 |
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分析法计算侵染了PDCD1-siRNA慢病毒之后的293T细胞内的PDCD1mRNA的表达丰度,计算沉默效率。侵染对照病毒(Lv-Scr-siRNA)的细胞作为对照。实验结果如表9和图2所示:
表9
注:-代表没有敲减;+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PDCD1基因的沉默效率
处于对数生长期的稳定表达PDCD1的293T进行胰酶消化,制成细胞悬液(细胞数约为5×104/ml)接种于6孔板中,培养至细胞融合度达到约30%。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,待侵染时间达到5天后,收集细胞。接收细胞样品,PBS洗涤两次后,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 Protein AssayKit,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。由于293T所表达得PDCD1具有flag标签,因此一抗选用可以识别3×flag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2表明,PDCD1的蛋白表达水平明显下调,与实施例2的实验结果相符。
实施例4慢病毒感染T淋巴细胞制备PDCD1表达敲减的T细胞
感染实验按照本领域技术人员已知的常规方法进行。简述感染步骤如下:
1.外周血单核淋巴细胞(PBMC)的获得,通过血液单采系统获得>1x107的细胞。
2.抗人CD3/CD28抗体包被细胞培养皿。
用PBS稀释抗人CD3及抗人CD28抗体,终浓度为1ug/ml,向细胞培养皿中加入稀释后的抗体混合液,使其铺满培养皿,室温孵育2小时。2小时后用PBS洗一次,备用。
3.对T淋巴细胞进行激活处理
将分离的PBMC用T淋巴细胞培养液(TexMACS培养基+10%FBS+30IU/重组人IL-2)进行重悬,使终浓度为1*106个细胞/ml,并放入2步骤中处理过的培养皿中培养,培养条件为37℃+5%CO2,培养时间为24小时。
4.对激活的T淋巴细胞进行感染
1)培养板处理。
取1mg/ml CD3及0.5mg/ml的CD28抗体按1:1000体积比稀释至适量的PBS缓冲液中,并取retronectin试剂,按1:40体积比稀释至该PBS缓冲液中,混匀后均匀铺至细胞皿,室温孵育2小时。2小时后用PBS进行洗涤并待用。
2)慢病毒感染T淋巴细胞及T淋巴细胞维持
用完全培养基稀释已激活的T淋巴细胞,并按MOI=3加入实施例1中包被的慢病毒或对照慢病毒,并混匀。均匀铺在1)中所处理的培养皿中。
感染后监测细胞密度,使细胞维持在1*106个细胞/ml,5天后,使用实施例2中同样的实时荧光定量RT-PCR法检测PDCD1基因在T细胞中的沉默效率。结果发现,具有很好的沉默效率。
实施例5制备PD1敲减嵌合抗原受体T细胞
敲减PD1shRNA所对应的siRNA4的序列如SEQ ID NO.368所示。将所述敲减PD1shRNA插入U6启动子下游后,将U6-shRNA转录框插入含有抗CD19嵌合抗原受体的慢病毒穿梭载体(GV400和GV401,吉凯基因)中,置于EF1α启动子上游,示意图如图3所示。
将GV400-FMC63BBzshPD1与pCMV-VSVG、pCMV-gag/pol按照质量比1:1:1通过磷酸钙沉淀的转染293T细胞,经过48小时培养,将培养上清收集,经超速离心,重悬获得含有FMC63BBzshPD1慢病毒。
将人类PBMC用抗CD3和CD28抗体(OKT3和15E8克隆)刺激激活24小时,按照一定MOI将FMC63BBz(GV401载体)及FMC63BBzshPD1(GV401载体)慢病毒与PBMC混合继续培养8-10天后,用于实验检测。
将感染后8天的T细胞按照1:1比例与表达CD19肿瘤细胞Raji混合培养16小时,分别检测表达FMC63BBz及FMC63BBzshPD1表面PD1表达量,结果如下表10:
表10
Group | FMC63BBzT | FMC63BBzshPD1T |
Transduction Efficiency | 22.6% | 14.5% |
通过EGFP检测FMC63BBz及FMC63BBzshPD1感染效率,如表所示两种慢病毒均可以成功感染T细胞,其感染效率分别为22.6%和14.5%。
在上述细胞群中,选取表达FMC63BBz及FMC63BBzshPD1的T细胞,并分析其表面PD1表达情况,结果如表11和图4所示:
表11
Group | FMC63BBz | FMC63BBz+Raji | FMC63BBzshPD1 | FMC63BBzshPD1+Raji |
PD1MFI | 1636 | 4571 | 664 | 1672 |
经过计算表明,FMC63BBzshPD1细胞表面PD1表达降低59.4%,与Raji细胞混合培养组,其PD1表达降低63.4%。
实施例6LDH细胞裂解实验证明PDCD1敲除后的T细胞对PDL1高表达的肿瘤细胞杀伤力增强
为验证PDCD1敲除的T细胞对PDL1高表达的肿瘤的细胞杀伤作用,本研究采用PDCD1高表达的细胞作为靶细胞。试剂盒采用Cytotox96非放射性细胞毒性检测试剂盒(Promega),该方法原理为,用细胞内稳定表达且不分泌的乳酸脱氢酶(LDH),代替传统的放射性元素,当细胞发生凋亡时,LDH会释放到胞外,通过检测被LDH氧化的formazan含量来判断上清中酶含量,从而说明细胞凋亡水平。效应细胞分别采用PDCD1被敲低或未被敲低的T细胞,效靶比分别采用1:2、1:5、1:10、1:20、1:30。靶细胞数量为10000个/孔,各组设置3个附孔,检测时间为作用后4小时。
其中,各实验组及对照组设置如下:
实验组:不同效靶比的CAR-T细胞及不同种靶细胞;
对照组一:各靶细胞LDH最大释放组;
对照组二:各靶细胞LDH自发释放组;
对照组三:效应细胞自发释放组;
具体实验方法参加试剂盒说明书。细胞毒性计算公式为:
特异性裂解=(实验组-对照组2-对照组3)/(对照组1-对照组2)。
结果显示,PDCD1被敲低后的T细胞有更强的杀伤效果,而PDCD1未敲低的T细胞杀伤作用被抑制。
其中,将含有PDL1(CD274)读码框的慢病毒(含有筛选抗性Puromycin)转染Raji细胞24小时后,加入1ug/ml puromycin进行筛选1周后,通过FACS检测PDL1表达。
将表达实施例5中所述FMC63BBz及FMC63BBzshPD1的T细胞按照一定的E:T(10:1,3:1,1:1,0.3:1,0.1:1)与Raji细胞或Raji-PDL1细胞混合培养于含2%胎牛血清的RPMI1640培养基中4小时,取上清用
Cytotox96NonRadioactive Cytotoxicity Assay测定上清中LDH释放代表T细胞对肿瘤细胞的杀伤,比较E:T=10数据。
表12
经过计算表明FMC63BBzT在与Raji-PDL1混合后,其杀伤Emax(E:T=10时)下降了26%,而经过PD1敲减的FMC63BBzT与Raji-PDL1混合后,其杀伤Emax降低了11%。因此,通过PD1敲减可以缓解肿瘤细胞表达PDL1对T细胞杀伤的抑制。
实施例7细胞因子实验证明敲除后的T细胞对PDL1高表达的肿瘤细胞杀伤力增强
靶细胞为PDL1高表达的细胞,效应细胞为实施例4中提及的PDCD1敲减或未敲减的细胞,在CAR病毒感染后10天进行细胞因子分泌检测。
方法为:分别取1*105个靶细胞与效应细胞进行1:1混合,混合于100ul RPMI 1640+2%FBS培养基中,在37℃5%CO2培养箱中共孵育约16小时。16小时后200g离心5分钟,取上清液,检测上清液中细胞因子分泌水平。检测细胞因子含量用BD公司生产的HU TH1-TH2CBAKIT,其原理为,反应液中细胞因子可以与其对应Beads上的抗体结合,每种细胞因子所对应的Beads均带有不同强度的APC荧光标签,细胞因子与beads结合后,再用另一种PE荧光标记的抗体对结合于beads上的细胞因子进行标记,通过检测PE荧光强度来判断细胞因子含量,而通过区分APC荧光强度来区分不同细胞因子种类。本研究中检测IL-2、IFN-γ、TNF-α三种细胞因子分泌水平。具体检测方法参加该试剂盒说明书。检测结果通过FCAP Array v3软件进行分析。
结果表明,T细胞中PDCD1敲减后,IL-2、TNF-alpha、IFN-gamma等细胞因子均有很明显上升。表面PDCD1的敲减促进了T细胞对靶细胞的杀伤。
其中,将实施例5中表达FMC63BBz及FMC63BBzshPD1的T细胞,按照1:1比例与相同数量Raji细胞或Raji-PDL1细胞混合培养16小时检测IL2及TNFα细胞因子(BD CytometricBead Array)释放情况。
表13
IL2Fc及TNFαFc为细胞因子上升的倍数变化。经过计算表明FMC63BBzT在与Raji-PDL1混合后,IL2分泌下降了38%,TNFα下降了39%;而经过PD1敲减的FMC63BBzT与Raji-PDL1混合后,IL2分泌下降了7.5%,TNFα分泌下降了28%。此结果表明,通过敲减T细胞表面的PD1可以显著缓解由肿瘤细胞表达PDL1引起的对T细胞细胞因子分泌的抑制。
实施例8小鼠动物体内实验证明敲除后的T细胞对PDL1高表达的肿瘤细胞杀伤力增强
对NCG小鼠(购于南京大学模式生物所)进行皮下注射PDCD1高表达的人源性肿瘤细胞,待平均瘤体积达到160-180mm3时,对模式动物进行瘤内注射效应细胞。
本研究所用效应细胞为PDCD1被敲低的T细胞,对照组为PDCD1未敲低的T细胞。注射前,将效应细胞用PBS洗两次,用PBS重悬至3E7/ml及1E8/ml两种浓度,分别记为低剂量组及高剂量组。每只小鼠瘤内注射30ul效应细胞/PBS。
本研究检测内容包括:
小鼠体重及瘤体积/3天/次;
小鼠外周血细胞因子检测/7天/次;
瘤体积结果表明,从注射PDCD1被敲低的T细胞第四天起,高剂量组瘤体积开始有下降趋势,而正常T细胞组及PBS组无瘤体积无下降趋势。这说明PDCD1敲低后的T细胞具有增强的抑癌作用。
外周血中细胞因子分泌趋势表明,注射PDCD1被敲低的T细胞可以在外周血中明显检测到多种人源细胞因子分泌(IL-2、TNF-alpha、IFN-gamma),随着肿瘤体积减少,细胞因子分泌量也逐渐降低,而注射正常T细胞的小鼠外周血中细胞因子分泌并未明显上升,证明PDCD1的敲低对肿瘤细胞产生明显的激活效应。
实施例9 siRNA靶点的变体也可对PDCD1进行有效敲除
根据表1中列出的PDCD1基因的有效的siRNA靶点,在其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(以shRNA17为例,序列如下表),或既有碱基的加减也有碱基突变。之后按照实施例1的方法包装慢病毒,按照实施例2的方法对这些序列的敲减效果进行验证,结果表明这些序列仍旧可以起到对PDCD1的敲除效果。
CGGAGAGCTTCGTGCTAAACT | SEQ ID NO:17 | |
两端增加碱基 | CACATCGGAGAGCTTCGTGCTAAACTGTACC | SEQ ID NO:609 |
5'端增加碱基 | CACATCGGAGAGCTTCGTGCTAAACT | SEQ ID NO:610 |
3'端增加碱基 | CGGAGAGCTTCGTGCTAAACTGTACC | SEQ ID NO:611 |
5'端减少碱基 | CGGAGAGCTTCGTGCTAAA | SEQ ID NO:612 |
3'端减少碱基 | GAGAGCTTCGTGCTAAACT | SEQ ID NO:613 |
碱基突变 | CACATCGGAGAGCTTCGTGCTTAACTGTACC | SEQ ID NO:614 |
碱基突变 | CACATCGGTGTGCTTCGTGCTAAACTGTACC | SEQ ID NO:615 |
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。
SEQUENCE LISTING
<110> 上海吉凯基因化学技术有限公司
<120> PDCD1基因的用途及其相关药物
<130> PCNS
<160> 615
<170> PatentIn version 3.3
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ctagagaagt ttcagggaa 19
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cggagagctt cgtgctaaa 19
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ccaggatggt tcttagact 19
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ccaaggcgca gatcaaaga 19
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cctgtgttct ctgtggact 19
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gagtatgcca ccattgtct 19
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ctccttggaa cccattcct 19
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caggcctaga gaagtttca 19
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ccacaggact catgtctca 19
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tggcggccag gatggttctt a 21
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ggccaggatg gttcttagac t 21
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gggacaacgc caccttcacc t 21
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acgccacctt cacctgcagc t 21
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cagcttctcc aacacatcgg a 21
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atcggagagc ttcgtgctaa a 21
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cggagagctt cgtgctaaac t 21
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gagcttcgtg ctaaactggt a 21
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tcgtgctaaa ctggtaccgc a 21
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cgtgctaaac tggtaccgca t 21
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tgctaaactg gtaccgcatg a 21
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ccccagcaac cagacggaca a 21
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aggactgccg cttccgtgtc a 21
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tgccgcttcc gtgtcacaca a 21
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ccgcttccgt gtcacacaac t 21
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ccgtgtcaca caactgccca a 21
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acgggcgtga cttccacatg a 21
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gcgtgacttc cacatgagcg t 21
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ggcgcaatga cagcggcacc t 21
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atgacagcgg cacctacctc t 21
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ccccaaggcg cagatcaaag a 21
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ggcgcagatc aaagagagcc t 21
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agctcagggt gacagagaga a 21
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gtgacagaga gaagggcaga a 21
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agccggccag ttccaaaccc t 21
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cggccagttc caaaccctgg t 21
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aaccctggtg gttggtgtcg t 21
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gggcagcctg gtgctgctag t 21
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cgggccgcac gagggacaat a 21
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ccgcacgagg gacaatagga g 21
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<400> 41
cacgagggac aataggagcc a 21
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ggacaatagg agccaggcgc a 21
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agccgtgcct gtgttctctg t 21
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tgcctgtgtt ctctgtggac t 21
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gcctgtgttc tctgtggact a 21
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tggactatgg ggagctggat t 21
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ggactatggg gagctggatt t 21
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tggatttcca gtggcgagag a 21
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gatttccagt ggcgagagaa g 21
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ctgtgtccct gagcagacgg a 21
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gtgtccctga gcagacggag t 21
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ctgagcagac ggagtatgcc a 21
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agcagacgga gtatgccacc a 21
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gcagacggag tatgccacca t 21
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gacggagtat gccaccattg t 21
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cggagtatgc caccattgtc t 21
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accattgtct ttcctagcgg a 21
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ccattgtctt tcctagcgga a 21
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cattgtcttt cctagcggaa t 21
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ttcctagcgg aatgggcacc t 21
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cagccactga ggcctgagga t 21
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agccactgag gcctgaggat g 21
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ctgaggatgg acactgctct t 21
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caccagtgtt ctgcagaccc t 21
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cagtgttctg cagaccctcc a 21
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cgcatttcct caggagaagc a 21
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cccaccacag gactcatgtc t 21
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caccacagga ctcatgtctc a 21
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ggactcatgt ctcaatgccc a 21
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cccctacagg gagggccaga t 21
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gggccagatg cagtcactgc t 21
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tgcgtccagc tccctgaatc t 21
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ccctgaatct ctgctgctgc t 21
<210> 75
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atctctgctg ctgctgctgc t 21
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ccggagcctc ctgcctgaac t 21
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gggctggttg gagatggcct t 21
<210> 78
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ctggttggag atggccttgg a 21
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gttggagatg gccttggagc a 21
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cctggcagtg gcatcccgaa a 21
<210> 81
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ggcatcccga aacgccctgg a 21
<210> 82
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tgggcacagg agtgggaggt a 21
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<400> 83
gggcacagga gtgggaggta c 21
<210> 84
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<400> 84
ggcacaggag tgggaggtac a 21
<210> 85
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<400> 85
cccaggagtt atctgctccc t 21
<210> 86
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<212> DNA
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<400> 86
ccctgcaggc ctagagaagt t 21
<210> 87
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tgcaggccta gagaagtttc a 21
<210> 88
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gcctagagaa gtttcaggga a 21
<210> 89
<211> 21
<212> DNA
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<400> 89
cctagagaag tttcagggaa g 21
<210> 90
<211> 21
<212> DNA
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<400> 90
aagtttcagg gaaggtcaga a 21
<210> 91
<211> 21
<212> DNA
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<400> 91
tcagggaagg tcagaagagc t 21
<210> 92
<211> 21
<212> DNA
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<400> 92
cagggaaggt cagaagagct c 21
<210> 93
<211> 21
<212> DNA
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<400> 93
gggaaggtca gaagagctcc t 21
<210> 94
<211> 21
<212> DNA
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<400> 94
gtcagaagag ctcctggctg t 21
<210> 95
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 95
acctttacac atgcccaggc a 21
<210> 96
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 96
gggcagggaa gctgaggcag t 21
<210> 97
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 97
ggcagggaag ctgaggcagt a 21
<210> 98
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 98
agctgaggca gtaagcgggc a 21
<210> 99
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 99
ggcagtaagc gggcaggcag a 21
<210> 100
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 100
ggcagagctg gaggcctttc a 21
<210> 101
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 101
cccctcacac cactcgggag a 21
<210> 102
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 102
caccactcgg gagagggaca t 21
<210> 103
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 103
gggagaggga catcctacgg t 21
<210> 104
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 104
gagggacatc ctacggtccc a 21
<210> 105
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 105
agggacatcc tacggtccca a 21
<210> 106
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 106
ctgggtgttg ggagggcaga a 21
<210> 107
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 107
gggagggcag aagtgcaggc a 21
<210> 108
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 108
accctgggag ctctccttgg a 21
<210> 109
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 109
gggagctctc cttggaaccc a 21
<210> 110
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 110
ctctccttgg aacccattcc t 21
<210> 111
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 111
gggtgggaag gtacaggcgt t 21
<210> 112
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 112
cgccgtggcc tatccactcc t 21
<210> 113
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 113
gtggcctatc cactcctcac a 21
<210> 114
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 114
cactcctcac atccacacac t 21
<210> 115
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 115
tcctcacatc cacacactgc a 21
<210> 116
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 116
ggcacctgag tggctgggac a 21
<210> 117
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 117
ttccctgtgg ttctattata t 21
<210> 118
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 118
ttaaatatga gagcatgcta a 21
<210> 119
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 119
atatgagagc atgctaagga a 21
<210> 120
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 120
tatgagagca tgctaaggaa a 21
<210> 121
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 121
ccggctagag aagtttcagg gaattcaaga gattccctga aacttctcta gtttttg 57
<210> 122
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 122
aattcaaaaa ctagagaagt ttcagggaat ctcttgaatt ccctgaaact tctctag 57
<210> 123
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 123
ccggcggaga gcttcgtgct aaattcaaga gatttagcac gaagctctcc gtttttg 57
<210> 124
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 124
aattcaaaaa cggagagctt cgtgctaaat ctcttgaatt tagcacgaag ctctccg 57
<210> 125
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 125
ccggccagga tggttcttag actttcaaga gaagtctaag aaccatcctg gtttttg 57
<210> 126
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 126
aattcaaaaa ccaggatggt tcttagactt ctcttgaaag tctaagaacc atcctgg 57
<210> 127
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 127
ccgggcttcc gtgtcacaca actttcaaga gaagttgtgt gacacggaag ctttttg 57
<210> 128
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 128
aattcaaaaa gcttccgtgt cacacaactt ctcttgaaag ttgtgtgaca cggaagc 57
<210> 129
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 129
ccggccaagg cgcagatcaa agattcaaga gatctttgat ctgcgccttg gtttttg 57
<210> 130
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 130
aattcaaaaa ccaaggcgca gatcaaagat ctcttgaatc tttgatctgc gccttgg 57
<210> 131
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 131
ccggcctgtg ttctctgtgg actttcaaga gaagtccaca gagaacacag gtttttg 57
<210> 132
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 132
aattcaaaaa cctgtgttct ctgtggactt ctcttgaaag tccacagaga acacagg 57
<210> 133
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 133
ccgggagtat gccaccattg tctttcaaga gaagacaatg gtggcatact ctttttg 57
<210> 134
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 134
aattcaaaaa gagtatgcca ccattgtctt ctcttgaaag acaatggtgg catactc 57
<210> 135
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 135
ccggctcctt ggaacccatt cctttcaaga gaaggaatgg gttccaagga gtttttg 57
<210> 136
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 136
aattcaaaaa ctccttggaa cccattcctt ctcttgaaag gaatgggttc caaggag 57
<210> 137
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 137
ccggcaggcc tagagaagtt tcattcaaga gatgaaactt ctctaggcct gtttttg 57
<210> 138
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 138
aattcaaaaa caggcctaga gaagtttcat ctcttgaatg aaacttctct aggcctg 57
<210> 139
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 139
ccggccacag gactcatgtc tcattcaaga gatgagacat gagtcctgtg gtttttg 57
<210> 140
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 140
aattcaaaaa ccacaggact catgtctcat ctcttgaatg agacatgagt cctgtgg 57
<210> 141
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 141
ccggtggcgg ccaggatggt tcttattcaa gagataagaa ccatcctggc cgccattttt 60
g 61
<210> 142
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 142
aattcaaaaa tggcggccag gatggttctt atctcttgaa taagaaccat cctggccgcc 60
a 61
<210> 143
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 143
ccggggccag gatggttctt agactttcaa gagaagtcta agaaccatcc tggccttttt 60
g 61
<210> 144
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 144
aattcaaaaa ggccaggatg gttcttagac ttctcttgaa agtctaagaa ccatcctggc 60
c 61
<210> 145
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 145
ccgggggaca acgccacctt cacctttcaa gagaaggtga aggtggcgtt gtcccttttt 60
g 61
<210> 146
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 146
aattcaaaaa gggacaacgc caccttcacc ttctcttgaa aggtgaaggt ggcgttgtcc 60
c 61
<210> 147
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 147
ccggacgcca ccttcacctg cagctttcaa gagaagctgc aggtgaaggt ggcgtttttt 60
g 61
<210> 148
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 148
aattcaaaaa acgccacctt cacctgcagc ttctcttgaa agctgcaggt gaaggtggcg 60
t 61
<210> 149
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 149
ccggcagctt ctccaacaca tcggattcaa gagatccgat gtgttggaga agctgttttt 60
g 61
<210> 150
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 150
aattcaaaaa cagcttctcc aacacatcgg atctcttgaa tccgatgtgt tggagaagct 60
g 61
<210> 151
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 151
ccggatcgga gagcttcgtg ctaaattcaa gagatttagc acgaagctct ccgatttttt 60
g 61
<210> 152
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 152
aattcaaaaa atcggagagc ttcgtgctaa atctcttgaa tttagcacga agctctccga 60
t 61
<210> 153
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 153
ccggcggaga gcttcgtgct aaactttcaa gagaagttta gcacgaagct ctccgttttt 60
g 61
<210> 154
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 154
aattcaaaaa cggagagctt cgtgctaaac ttctcttgaa agtttagcac gaagctctcc 60
g 61
<210> 155
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 155
ccgggagctt cgtgctaaac tggtattcaa gagataccag tttagcacga agctcttttt 60
g 61
<210> 156
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 156
aattcaaaaa gagcttcgtg ctaaactggt atctcttgaa taccagttta gcacgaagct 60
c 61
<210> 157
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 157
ccggtcgtgc taaactggta ccgcattcaa gagatgcggt accagtttag cacgattttt 60
g 61
<210> 158
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 158
aattcaaaaa tcgtgctaaa ctggtaccgc atctcttgaa tgcggtacca gtttagcacg 60
a 61
<210> 159
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 159
ccggcgtgct aaactggtac cgcatttcaa gagaatgcgg taccagttta gcacgttttt 60
g 61
<210> 160
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 160
aattcaaaaa cgtgctaaac tggtaccgca ttctcttgaa atgcggtacc agtttagcac 60
g 61
<210> 161
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 161
ccggtgctaa actggtaccg catgattcaa gagatcatgc ggtaccagtt tagcattttt 60
g 61
<210> 162
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 162
aattcaaaaa tgctaaactg gtaccgcatg atctcttgaa tcatgcggta ccagtttagc 60
a 61
<210> 163
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 163
ccggccccag caaccagacg gacaattcaa gagattgtcc gtctggttgc tggggttttt 60
g 61
<210> 164
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 164
aattcaaaaa ccccagcaac cagacggaca atctcttgaa ttgtccgtct ggttgctggg 60
g 61
<210> 165
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 165
ccggaggact gccgcttccg tgtcattcaa gagatgacac ggaagcggca gtcctttttt 60
g 61
<210> 166
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 166
aattcaaaaa aggactgccg cttccgtgtc atctcttgaa tgacacggaa gcggcagtcc 60
t 61
<210> 167
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 167
ccggtgccgc ttccgtgtca cacaattcaa gagattgtgt gacacggaag cggcattttt 60
g 61
<210> 168
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 168
aattcaaaaa tgccgcttcc gtgtcacaca atctcttgaa ttgtgtgaca cggaagcggc 60
a 61
<210> 169
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 169
ccggccgctt ccgtgtcaca caactttcaa gagaagttgt gtgacacgga agcggttttt 60
g 61
<210> 170
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 170
aattcaaaaa ccgcttccgt gtcacacaac ttctcttgaa agttgtgtga cacggaagcg 60
g 61
<210> 171
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 171
ccggccgtgt cacacaactg cccaattcaa gagattgggc agttgtgtga cacggttttt 60
g 61
<210> 172
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 172
aattcaaaaa ccgtgtcaca caactgccca atctcttgaa ttgggcagtt gtgtgacacg 60
g 61
<210> 173
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 173
ccggacgggc gtgacttcca catgattcaa gagatcatgt ggaagtcacg cccgtttttt 60
g 61
<210> 174
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 174
aattcaaaaa acgggcgtga cttccacatg atctcttgaa tcatgtggaa gtcacgcccg 60
t 61
<210> 175
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 175
ccgggcgtga cttccacatg agcgtttcaa gagaacgctc atgtggaagt cacgcttttt 60
g 61
<210> 176
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 176
aattcaaaaa ggcgcaatga cagcggcacc ttctcttgaa acgctcatgt ggaagtcacg 60
c 61
<210> 177
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 177
ccggggcgca atgacagcgg cacctttcaa gagaaggtgc cgctgtcatt gcgccttttt 60
g 61
<210> 178
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 178
aattcaaaaa atgacagcgg cacctacctc ttctcttgaa aggtgccgct gtcattgcgc 60
c 61
<210> 179
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 179
ccggatgaca gcggcaccta cctctttcaa gagaagaggt aggtgccgct gtcatttttt 60
g 61
<210> 180
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 180
aattcaaaaa atgacagcgg cacctacctc ttctcttgaa agaggtaggt gccgctgtca 60
t 61
<210> 181
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 181
ccggccccaa ggcgcagatc aaagattcaa gagatctttg atctgcgcct tggggttttt 60
g 61
<210> 182
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 182
aattcaaaaa ccccaaggcg cagatcaaag atctcttgaa tctttgatct gcgccttggg 60
g 61
<210> 183
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 183
ccggggcgca gatcaaagag agcctttcaa gagaaggctc tctttgatct gcgccttttt 60
g 61
<210> 184
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 184
aattcaaaaa ggcgcagatc aaagagagcc ttctcttgaa aggctctctt tgatctgcgc 60
c 61
<210> 185
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 185
ccggagctca gggtgacaga gagaattcaa gagattctct ctgtcaccct gagctttttt 60
g 61
<210> 186
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 186
aattcaaaaa agctcagggt gacagagaga atctcttgaa ttctctctgt caccctgagc 60
t 61
<210> 187
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 187
ccgggtgaca gagagaaggg cagaattcaa gagattctgc ccttctctct gtcacttttt 60
g 61
<210> 188
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 188
aattcaaaaa gtgacagaga gaagggcaga atctcttgaa ttctgccctt ctctctgtca 60
c 61
<210> 189
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 189
ccggagccgg ccagttccaa accctttcaa gagaagggtt tggaactggc cggctttttt 60
g 61
<210> 190
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 190
aattcaaaaa agccggccag ttccaaaccc ttctcttgaa agggtttgga actggccggc 60
t 61
<210> 191
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 191
ccggcggcca gttccaaacc ctggtttcaa gagaaccagg gtttggaact ggccgttttt 60
g 61
<210> 192
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 192
aattcaaaaa cggccagttc caaaccctgg ttctcttgaa accagggttt ggaactggcc 60
g 61
<210> 193
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 193
ccggaaccct ggtggttggt gtcgtttcaa gagaacgaca ccaaccacca gggttttttt 60
g 61
<210> 194
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 194
aattcaaaaa aaccctggtg gttggtgtcg ttctcttgaa acgacaccaa ccaccagggt 60
t 61
<210> 195
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 195
ccgggggcag cctggtgctg ctagtttcaa gagaactagc agcaccaggc tgcccttttt 60
g 61
<210> 196
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 196
aattcaaaaa gggcagcctg gtgctgctag ttctcttgaa actagcagca ccaggctgcc 60
c 61
<210> 197
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 197
ccggcgggcc gcacgaggga caatattcaa gagatattgt ccctcgtgcg gcccgttttt 60
g 61
<210> 198
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 198
aattcaaaaa cgggccgcac gagggacaat atctcttgaa tattgtccct cgtgcggccc 60
g 61
<210> 199
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 199
ccggccgcac gagggacaat aggagttcaa gagactccta ttgtccctcg tgcggttttt 60
g 61
<210> 200
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 200
aattcaaaaa ccgcacgagg gacaatagga gtctcttgaa ctcctattgt ccctcgtgcg 60
g 61
<210> 201
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 201
ccggcacgag ggacaatagg agccattcaa gagatggctc ctattgtccc tcgtgttttt 60
g 61
<210> 202
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 202
aattcaaaaa cacgagggac aataggagcc atctcttgaa tggctcctat tgtccctcgt 60
g 61
<210> 203
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 203
ccggggacaa taggagccag gcgcattcaa gagatgcgcc tggctcctat tgtccttttt 60
g 61
<210> 204
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 204
aattcaaaaa ggacaatagg agccaggcgc atctcttgaa tgcgcctggc tcctattgtc 60
c 61
<210> 205
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 205
ccggagccgt gcctgtgttc tctgtttcaa gagaacagag aacacaggca cggctttttt 60
g 61
<210> 206
<211> 61
<212> DNA
<213> Artificial
<220>
<223> AATTCAAAAAAGCCGTGCCTGTGTTCTCTGTTCTCTTGAAACAGAGAACACAGGCACGGCT
<400> 206
aattcaaaaa agccgtgcct gtgttctctg ttctcttgaa acagagaaca caggcacggc 60
t 61
<210> 207
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 207
ccggtgcctg tgttctctgt ggactttcaa gagaagtcca cagagaacac aggcattttt 60
g 61
<210> 208
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 208
aattcaaaaa tgcctgtgtt ctctgtggac ttctcttgaa agtccacaga gaacacaggc 60
a 61
<210> 209
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 209
ccgggcctgt gttctctgtg gactattcaa gagatagtcc acagagaaca caggcttttt 60
g 61
<210> 210
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 210
aattcaaaaa gcctgtgttc tctgtggact atctcttgaa tagtccacag agaacacagg 60
c 61
<210> 211
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 211
ccggtggact atggggagct ggattttcaa gagaaatcca gctccccata gtccattttt 60
g 61
<210> 212
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 212
aattcaaaaa tggactatgg ggagctggat ttctcttgaa aatccagctc cccatagtcc 60
a 61
<210> 213
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 213
ccggggacta tggggagctg gatttttcaa gagaaaatcc agctccccat agtccttttt 60
g 61
<210> 214
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 214
aattcaaaaa ggactatggg gagctggatt ttctcttgaa aaatccagct ccccatagtc 60
c 61
<210> 215
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 215
ccggtggatt tccagtggcg agagattcaa gagatctctc gccactggaa atccattttt 60
g 61
<210> 216
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 216
aattcaaaaa tggatttcca gtggcgagag atctcttgaa tctctcgcca ctggaaatcc 60
a 61
<210> 217
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 217
ccgggatttc cagtggcgag agaagttcaa gagacttctc tcgccactgg aaatcttttt 60
g 61
<210> 218
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 218
aattcaaaaa gatttccagt ggcgagagaa gtctcttgaa cttctctcgc cactggaaat 60
c 61
<210> 219
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 219
ccggctgtgt ccctgagcag acggattcaa gagatccgtc tgctcaggga cacagttttt 60
g 61
<210> 220
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 220
aattcaaaaa ctgtgtccct gagcagacgg atctcttgaa tccgtctgct cagggacaca 60
g 61
<210> 221
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 221
ccgggtgtcc ctgagcagac ggagtttcaa gagaactccg tctgctcagg gacacttttt 60
g 61
<210> 222
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 222
aattcaaaaa gtgtccctga gcagacggag ttctcttgaa actccgtctg ctcagggaca 60
c 61
<210> 223
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 223
ccggtgtccc tgagcagacg gagtattcaa gagatactcc gtctgctcag ggacattttt 60
g 61
<210> 224
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 224
aattcaaaaa tgtccctgag cagacggagt atctcttgaa tactccgtct gctcagggac 60
a 61
<210> 225
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 225
ccggctgagc agacggagta tgccattcaa gagatggcat actccgtctg ctcagttttt 60
g 61
<210> 226
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 226
aattcaaaaa ctgagcagac ggagtatgcc atctcttgaa tggcatactc cgtctgctca 60
g 61
<210> 227
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 227
ccggagcaga cggagtatgc caccattcaa gagatggtgg catactccgt ctgctttttt 60
g 61
<210> 228
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 228
aattcaaaaa agcagacgga gtatgccacc atctcttgaa tggtggcata ctccgtctgc 60
t 61
<210> 229
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 229
ccgggcagac ggagtatgcc accatttcaa gagaatggtg gcatactccg tctgcttttt 60
g 61
<210> 230
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 230
aattcaaaaa gcagacggag tatgccacca ttctcttgaa atggtggcat actccgtctg 60
c 61
<210> 231
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 231
ccgggacgga gtatgccacc attgtttcaa gagaacaatg gtggcatact ccgtcttttt 60
g 61
<210> 232
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 232
aattcaaaaa gacggagtat gccaccattg ttctcttgaa acaatggtgg catactccgt 60
c 61
<210> 233
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 233
ccggcggagt atgccaccat tgtctttcaa gagaagacaa tggtggcata ctccgttttt 60
g 61
<210> 234
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 234
aattcaaaaa cggagtatgc caccattgtc ttctcttgaa agacaatggt ggcatactcc 60
g 61
<210> 235
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 235
ccggaccatt gtctttccta gcggattcaa gagatccgct aggaaagaca atggtttttt 60
g 61
<210> 236
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 236
aattcaaaaa accattgtct ttcctagcgg atctcttgaa tccgctagga aagacaatgg 60
t 61
<210> 237
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 237
ccggccattg tctttcctag cggaattcaa gagattccgc taggaaagac aatggttttt 60
g 61
<210> 238
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 238
aattcaaaaa ccattgtctt tcctagcgga atctcttgaa ttccgctagg aaagacaatg 60
g 61
<210> 239
<211> 61
<212> DNA
<213> Artificial
<220>
<223> CCGGCATTGTCTTTCCTAGCGGAATTTCAAGAGAATTCCGCTAGGAAAGACAATGTTTTTG
<400> 239
ccggcattgt ctttcctagc ggaatttcaa gagaattccg ctaggaaaga caatgttttt 60
g 61
<210> 240
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 240
aattcaaaaa cattgtcttt cctagcggaa ttctcttgaa attccgctag gaaagacaat 60
g 61
<210> 241
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 241
ccggttccta gcggaatggg cacctttcaa gagaaggtgc ccattccgct aggaattttt 60
g 61
<210> 242
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 242
aattcaaaaa ttcctagcgg aatgggcacc ttctcttgaa aggtgcccat tccgctagga 60
a 61
<210> 243
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 243
ccggcagcca ctgaggcctg aggatttcaa gagaatcctc aggcctcagt ggctgttttt 60
g 61
<210> 244
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 244
aattcaaaaa cagccactga ggcctgagga ttctcttgaa atcctcaggc ctcagtggct 60
g 61
<210> 245
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 245
ccggagccac tgaggcctga ggatgttcaa gagacatcct caggcctcag tggctttttt 60
g 61
<210> 246
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 246
aattcaaaaa agccactgag gcctgaggat gtctcttgaa catcctcagg cctcagtggc 60
t 61
<210> 247
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 247
ccggctgagg atggacactg ctcttttcaa gagaaagagc agtgtccatc ctcagttttt 60
g 61
<210> 248
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 248
aattcaaaaa ctgaggatgg acactgctct ttctcttgaa aagagcagtg tccatcctca 60
g 61
<210> 249
<211> 59
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 249
ccggcaccag tgttctgcag accctttcaa gagattccct gaaacttctc tagtttttg 59
<210> 250
<211> 59
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 250
aattcaaaaa caccagtgtt ctgcagaccc ttctcttgaa ttccctgaaa cttctctag 59
<210> 251
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 251
ccggcagtgt tctgcagacc ctccattcaa gagatggagg gtctgcagaa cactgttttt 60
g 61
<210> 252
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 252
aattcaaaaa cagtgttctg cagaccctcc atctcttgaa tggagggtct gcagaacact 60
g 61
<210> 253
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 253
ccggcgcatt tcctcaggag aagcattcaa gagatgcttc tcctgaggaa atgcgttttt 60
g 61
<210> 254
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 254
aattcaaaaa cgcatttcct caggagaagc atctcttgaa tgcttctcct gaggaaatgc 60
g 61
<210> 255
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 255
ccggcccacc acaggactca tgtctttcaa gagaagacat gagtcctgtg gtgggttttt 60
g 61
<210> 256
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 256
aattcaaaaa cccaccacag gactcatgtc ttctcttgaa agacatgagt cctgtggtgg 60
g 61
<210> 257
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 257
ccggcaccac aggactcatg tctcattcaa gagatgagac atgagtcctg tggtgttttt 60
g 61
<210> 258
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 258
aattcaaaaa caccacagga ctcatgtctc atctcttgaa tgagacatga gtcctgtggt 60
g 61
<210> 259
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 259
ccggggactc atgtctcaat gcccattcaa gagatgggca ttgagacatg agtccttttt 60
g 61
<210> 260
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 260
aattcaaaaa ggactcatgt ctcaatgccc atctcttgaa tgggcattga gacatgagtc 60
c 61
<210> 261
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 261
ccggccccta cagggagggc cagatttcaa gagaatctgg ccctccctgt aggggttttt 60
g 61
<210> 262
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 262
aattcaaaaa cccctacagg gagggccaga ttctcttgaa atctggccct ccctgtaggg 60
g 61
<210> 263
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 263
ccgggggcca gatgcagtca ctgctttcaa gagaagcagt gactgcatct ggcccttttt 60
g 61
<210> 264
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 264
aattcaaaaa gggccagatg cagtcactgc ttctcttgaa agcagtgact gcatctggcc 60
c 61
<210> 265
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 265
ccggtgcgtc cagctccctg aatctttcaa gagaagattc agggagctgg acgcattttt 60
g 61
<210> 266
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 266
aattcaaaaa tgcgtccagc tccctgaatc ttctcttgaa agattcaggg agctggacgc 60
a 61
<210> 267
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 267
ccggccctga atctctgctg ctgctttcaa gagaagcagc agcagagatt cagggttttt 60
g 61
<210> 268
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 268
aattcaaaaa ccctgaatct ctgctgctgc ttctcttgaa agcagcagca gagattcagg 60
g 61
<210> 269
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 269
ccggatctct gctgctgctg ctgctttcaa gagaagcagc agcagcagca gagatttttt 60
g 61
<210> 270
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 270
aattcaaaaa atctctgctg ctgctgctgc ttctcttgaa agcagcagca gcagcagaga 60
t 61
<210> 271
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 271
ccggccggag cctcctgcct gaactttcaa gagaagttca ggcaggaggc tccggttttt 60
g 61
<210> 272
<211> 61
<212> DNA
<213> Artificial
<220>
<223> AATTCAAAAA CCGGAGCCTCCTGCCTGAACT TCTCTTGAA AGTTCAGGCAGGAGGCTCCGG
<400> 272
aattcaaaaa ccggagcctc ctgcctgaac ttctcttgaa agttcaggca ggaggctccg 60
g 61
<210> 273
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 273
ccgggggctg gttggagatg gccttttcaa gagaaaggcc atctccaacc agcccttttt 60
g 61
<210> 274
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 274
aattcaaaaa gggctggttg gagatggcct ttctcttgaa aaggccatct ccaaccagcc 60
c 61
<210> 275
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 275
ccggctggtt ggagatggcc ttggattcaa gagatccaag gccatctcca accagttttt 60
g 61
<210> 276
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 276
aattcaaaaa ctggttggag atggccttgg atctcttgaa tccaaggcca tctccaacca 60
g 61
<210> 277
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 277
ccgggttgga gatggccttg gagcattcaa gagatgctcc aaggccatct ccaacttttt 60
g 61
<210> 278
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 278
aattcaaaaa gttggagatg gccttggagc atctcttgaa tgctccaagg ccatctccaa 60
c 61
<210> 279
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 279
ccggcctggc agtggcatcc cgaaattcaa gagatttcgg gatgccactg ccaggttttt 60
g 61
<210> 280
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 280
aattcaaaaa cctggcagtg gcatcccgaa atctcttgaa tttcgggatg ccactgccag 60
g 61
<210> 281
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 281
ccggggcatc ccgaaacgcc ctggattcaa gagatccagg gcgtttcggg atgccttttt 60
g 61
<210> 282
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 282
aattcaaaaa ggcatcccga aacgccctgg atctcttgaa tccagggcgt ttcgggatgc 60
c 61
<210> 283
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 283
ccggtgggca caggagtggg aggtattcaa gagatacctc ccactcctgt gcccattttt 60
g 61
<210> 284
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 284
aattcaaaaa tgggcacagg agtgggaggt atctcttgaa tacctcccac tcctgtgccc 60
a 61
<210> 285
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 285
ccgggggcac aggagtggga ggtacttcaa gagagtacct cccactcctg tgcccttttt 60
g 61
<210> 286
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 286
aattcaaaaa gggcacagga gtgggaggta ctctcttgaa gtacctccca ctcctgtgcc 60
c 61
<210> 287
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 287
ccggggcaca ggagtgggag gtacattcaa gagatgtacc tcccactcct gtgccttttt 60
g 61
<210> 288
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 288
aattcaaaaa ggcacaggag tgggaggtac atctcttgaa tgtacctccc actcctgtgc 60
c 61
<210> 289
<211> 61
<212> DNA
<213> Artificial
<220>
<223> CCGG CCCAGGAGTTATCTGCTCCCT TTCAAGAGA AGGGAGCAGATAACTCCTGGG TTTTTG
<400> 289
ccggcccagg agttatctgc tccctttcaa gagaagggag cagataactc ctgggttttt 60
g 61
<210> 290
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 290
aattcaaaaa cccaggagtt atctgctccc ttctcttgaa agggagcaga taactcctgg 60
g 61
<210> 291
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 291
ccggccctgc aggcctagag aagttttcaa gagaaacttc tctaggcctg cagggttttt 60
g 61
<210> 292
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 292
aattcaaaaa ccctgcaggc ctagagaagt ttctcttgaa aacttctcta ggcctgcagg 60
g 61
<210> 293
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 293
ccggtgcagg cctagagaag tttcattcaa gagatgaaac ttctctaggc ctgcattttt 60
g 61
<210> 294
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 294
aattcaaaaa tgcaggccta gagaagtttc atctcttgaa tgaaacttct ctaggcctgc 60
a 61
<210> 295
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 295
ccgggcctag agaagtttca gggaattcaa gagattccct gaaacttctc taggcttttt 60
g 61
<210> 296
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 296
aattcaaaaa gcctagagaa gtttcaggga atctcttgaa ttccctgaaa cttctctagg 60
c 61
<210> 297
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 297
ccggcctaga gaagtttcag ggaagttcaa gagacttccc tgaaacttct ctaggttttt 60
g 61
<210> 298
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 298
aattcaaaaa cctagagaag tttcagggaa gtctcttgaa cttccctgaa acttctctag 60
g 61
<210> 299
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 299
ccggaagttt cagggaaggt cagaattcaa gagattctga ccttccctga aacttttttt 60
g 61
<210> 300
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 300
aattcaaaaa aagtttcagg gaaggtcaga atctcttgaa ttctgacctt ccctgaaact 60
t 61
<210> 301
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 301
ccggtcaggg aaggtcagaa gagctttcaa gagaagctct tctgaccttc cctgattttt 60
g 61
<210> 302
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 302
aattcaaaaa tcagggaagg tcagaagagc ttctcttgaa agctcttctg accttccctg 60
a 61
<210> 303
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 303
ccggcaggga aggtcagaag agctcttcaa gagagagctc ttctgacctt ccctgttttt 60
g 61
<210> 304
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 304
aattcaaaaa cagggaaggt cagaagagct ctctcttgaa gagctcttct gaccttccct 60
g 61
<210> 305
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 305
ccgggggaag gtcagaagag ctcctttcaa gagaaggagc tcttctgacc ttcccttttt 60
g 61
<210> 306
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 306
aattcaaaaa gggaaggtca gaagagctcc ttctcttgaa aggagctctt ctgaccttcc 60
c 61
<210> 307
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 307
ccgggtcaga agagctcctg gctgtttcaa gagaacagcc aggagctctt ctgacttttt 60
g 61
<210> 308
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 308
aattcaaaaa gtcagaagag ctcctggctg ttctcttgaa acagccagga gctcttctga 60
c 61
<210> 309
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 309
ccggaccttt acacatgccc aggcattcaa gagatgcctg ggcatgtgta aaggtttttt 60
g 61
<210> 310
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 310
aattcaaaaa acctttacac atgcccaggc atctcttgaa tgcctgggca tgtgtaaagg 60
t 61
<210> 311
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 311
ccgggggcag ggaagctgag gcagtttcaa gagaactgcc tcagcttccc tgcccttttt 60
g 61
<210> 312
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 312
aattcaaaaa gggcagggaa gctgaggcag ttctcttgaa actgcctcag cttccctgcc 60
c 61
<210> 313
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 313
ccggggcagg gaagctgagg cagtattcaa gagatactgc ctcagcttcc ctgccttttt 60
g 61
<210> 314
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 314
aattcaaaaa ggcagggaag ctgaggcagt atctcttgaa tactgcctca gcttccctgc 60
c 61
<210> 315
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 315
ccggagctga ggcagtaagc gggcattcaa gagatgcccg cttactgcct cagctttttt 60
g 61
<210> 316
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 316
aattcaaaaa agctgaggca gtaagcgggc atctcttgaa tgcccgctta ctgcctcagc 60
t 61
<210> 317
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 317
ccggggcagt aagcgggcag gcagattcaa gagatctgcc tgcccgctta ctgccttttt 60
g 61
<210> 318
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 318
aattcaaaaa ggcagtaagc gggcaggcag atctcttgaa tctgcctgcc cgcttactgc 60
c 61
<210> 319
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 319
ccggggcaga gctggaggcc tttcattcaa gagatgaaag gcctccagct ctgccttttt 60
g 61
<210> 320
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 320
aattcaaaaa ggcagagctg gaggcctttc atctcttgaa tgaaaggcct ccagctctgc 60
c 61
<210> 321
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 321
ccggcccctc acaccactcg ggagattcaa gagatctccc gagtggtgtg aggggttttt 60
g 61
<210> 322
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 322
aattcaaaaa cccctcacac cactcgggag atctcttgaa tctcccgagt ggtgtgaggg 60
g 61
<210> 323
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 323
ccggcaccac tcgggagagg gacatttcaa gagaatgtcc ctctcccgag tggtgttttt 60
g 61
<210> 324
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 324
aattcaaaaa caccactcgg gagagggaca ttctcttgaa atgtccctct cccgagtggt 60
g 61
<210> 325
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 325
ccgggggaga gggacatcct acggtttcaa gagaaccgta ggatgtccct ctcccttttt 60
g 61
<210> 326
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 326
aattcaaaaa gggagaggga catcctacgg ttctcttgaa accgtaggat gtccctctcc 60
c 61
<210> 327
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 327
ccgggaggga catcctacgg tcccattcaa gagatgggac cgtaggatgt ccctcttttt 60
g 61
<210> 328
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 328
aattcaaaaa gagggacatc ctacggtccc atctcttgaa tgggaccgta ggatgtccct 60
c 61
<210> 329
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 329
ccggagggac atcctacggt cccaattcaa gagattggga ccgtaggatg tccctttttt 60
g 61
<210> 330
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 330
aattcaaaaa agggacatcc tacggtccca atctcttgaa ttgggaccgt aggatgtccc 60
t 61
<210> 331
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 331
ccggctgggt gttgggaggg cagaattcaa gagattctgc cctcccaaca cccagttttt 60
g 61
<210> 332
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 332
aattcaaaaa ctgggtgttg ggagggcaga atctcttgaa ttctgccctc ccaacaccca 60
g 61
<210> 333
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 333
ccgggggagg gcagaagtgc aggcattcaa gagatgcctg cacttctgcc ctcccttttt 60
g 61
<210> 334
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 334
aattcaaaaa gggagggcag aagtgcaggc atctcttgaa tgcctgcact tctgccctcc 60
c 61
<210> 335
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 335
ccggaccctg ggagctctcc ttggattcaa gagatccaag gagagctccc agggtttttt 60
g 61
<210> 336
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 336
aattcaaaaa accctgggag ctctccttgg atctcttgaa tccaaggaga gctcccaggg 60
t 61
<210> 337
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 337
ccgggggagc tctccttgga acccattcaa gagatccaag gagagctccc agggtttttt 60
g 61
<210> 338
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 338
aattcaaaaa gggagctctc cttggaaccc atctcttgaa tccaaggaga gctcccaggg 60
t 61
<210> 339
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 339
ccggctctcc ttggaaccca ttcctttcaa gagaaggaat gggttccaag gagagttttt 60
g 61
<210> 340
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 340
aattcaaaaa ctctccttgg aacccattcc ttctcttgaa aggaatgggt tccaaggaga 60
g 61
<210> 341
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 341
ccgggggtgg gaaggtacag gcgttttcaa gagaaacgcc tgtaccttcc cacccttttt 60
g 61
<210> 342
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 342
aattcaaaaa gggtgggaag gtacaggcgt ttctcttgaa aacgcctgta ccttcccacc 60
c 61
<210> 343
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 343
ccggcgccgt ggcctatcca ctcctttcaa gagaaggagt ggataggcca cggcgttttt 60
g 61
<210> 344
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 344
aattcaaaaa cgccgtggcc tatccactcc ttctcttgaa aggagtggat aggccacggc 60
g 61
<210> 345
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 345
ccgggtggcc tatccactcc tcacattcaa gagatgtgag gagtggatag gccacttttt 60
g 61
<210> 346
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 346
aattcaaaaa gtggcctatc cactcctcac atctcttgaa tgtgaggagt ggataggcca 60
c 61
<210> 347
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 347
ccggcactcc tcacatccac acactttcaa gagaagtgtg tggatgtgag gagtgttttt 60
g 61
<210> 348
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 348
aattcaaaaa cactcctcac atccacacac ttctcttgaa agtgtgtgga tgtgaggagt 60
g 61
<210> 349
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 349
ccggtcctca catccacaca ctgcattcaa gagatgcagt gtgtggatgt gaggattttt 60
g 61
<210> 350
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 350
aattcaaaaa tcctcacatc cacacactgc atctcttgaa tgcagtgtgt ggatgtgagg 60
a 61
<210> 351
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 351
ccggggcacc tgagtggctg ggacattcaa gagatgtccc agccactcag gtgccttttt 60
g 61
<210> 352
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 352
aattcaaaaa ggcacctgag tggctgggac atctcttgaa tgtcccagcc actcaggtgc 60
c 61
<210> 353
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 353
ccggttccct gtggttctat tatatttcaa gagaatataa tagaaccaca gggaattttt 60
g 61
<210> 354
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 354
aattcaaaaa ttccctgtgg ttctattata ttctcttgaa atataataga accacaggga 60
a 61
<210> 355
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 355
ccggttaaat atgagagcat gctaattcaa gagattagca tgctctcata tttaattttt 60
g 61
<210> 356
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 356
aattcaaaaa ttaaatatga gagcatgcta atctcttgaa ttagcatgct ctcatattta 60
a 61
<210> 357
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 357
ccggatatga gagcatgcta aggaattcaa gagattcctt agcatgctct catatttttt 60
g 61
<210> 358
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 358
aattcaaaaa atatgagagc atgctaagga atctcttgaa ttccttagca tgctctcata 60
t 61
<210> 359
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 359
ccggtatgag agcatgctaa ggaaattcaa gagatttcct tagcatgctc tcatattttt 60
g 61
<210> 360
<211> 61
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 360
aattcaaaaa tatgagagca tgctaaggaa atctcttgaa tttccttagc atgctctcat 60
a 61
<210> 361
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 361
ccggttctcc gaacgtgtca cgtttcaaga gaacgtgaca cgttcggaga atttttg 57
<210> 362
<211> 57
<212> DNA
<213> Artificial
<220>
<223> 双链DNA Oligo
<400> 362
aattcaaaaa ttctccgaac gtgtcacgtt ctcttgaaac gtgacacgtt cggagaa 57
<210> 363
<211> 24
<212> DNA
<213> Artificial
<220>
<223> 上游引物
<400> 363
cctatttccc atgattcctt cata 24
<210> 364
<211> 20
<212> DNA
<213> Artificial
<220>
<223> 下游引物
<400> 364
gtaatacggt tatccacgcg 20
<210> 365
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA 1
<400> 365
cuagagaagu uucagggaa 19
<210> 366
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA 2
<400> 366
cggagagcuu cgugcuaaa 19
<210> 367
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA 3
<400> 367
ccaggauggu ucuuagacu 19
<210> 368
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA 4
<400> 368
gcuuccgugu cacacaacu 19
<210> 369
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA 5
<400> 369
ccaaggcgca gaucaaaga 19
<210> 370
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA 6
<400> 370
ccuguguucu cuguggacu 19
<210> 371
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA 7
<400> 371
gaguaugcca ccauugucu 19
<210> 372
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA 8
<400> 372
cuccuuggaa cccauuccu 19
<210> 373
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA 9
<400> 373
caggccuaga gaaguuuca 19
<210> 374
<211> 19
<212> RNA
<213> Artificial
<220>
<223> siRNA 10
<400> 374
ccacaggacu caugucuca 19
<210> 375
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 11
<400> 375
uggcggccag gaugguucuu a 21
<210> 376
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 12
<400> 376
ggccaggaug guucuuagac u 21
<210> 377
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 13
<400> 377
gggacaacgc caccuucacc u 21
<210> 378
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 14
<400> 378
acgccaccuu caccugcagc u 21
<210> 379
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 15
<400> 379
cagcuucucc aacacaucgg a 21
<210> 380
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 16
<400> 380
aucggagagc uucgugcuaa a 21
<210> 381
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 17
<400> 381
cggagagcuu cgugcuaaac u 21
<210> 382
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 18
<400> 382
gagcuucgug cuaaacuggu a 21
<210> 383
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 19
<400> 383
ucgugcuaaa cugguaccgc a 21
<210> 384
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 20
<400> 384
cgugcuaaac ugguaccgca u 21
<210> 385
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 21
<400> 385
ugcuaaacug guaccgcaug a 21
<210> 386
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 22
<400> 386
ccccagcaac cagacggaca a 21
<210> 387
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 23
<400> 387
aggacugccg cuuccguguc a 21
<210> 388
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 24
<400> 388
ugccgcuucc gugucacaca a 21
<210> 389
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 25
<400> 389
ccgcuuccgu gucacacaac u 21
<210> 390
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 26
<400> 390
ccgugucaca caacugccca a 21
<210> 391
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 27
<400> 391
acgggcguga cuuccacaug a 21
<210> 392
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 28
<400> 392
gcgugacuuc cacaugagcg u 21
<210> 393
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 29
<400> 393
ggcgcaauga cagcggcacc u 21
<210> 394
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 30
<400> 394
augacagcgg caccuaccuc u 21
<210> 395
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 31
<400> 395
ccccaaggcg cagaucaaag a 21
<210> 396
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 32
<400> 396
ggcgcagauc aaagagagcc u 21
<210> 397
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 33
<400> 397
agcucagggu gacagagaga a 21
<210> 398
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 34
<400> 398
gugacagaga gaagggcaga a 21
<210> 399
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 35
<400> 399
agccggccag uuccaaaccc u 21
<210> 400
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 36
<400> 400
cggccaguuc caaacccugg u 21
<210> 401
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 37
<400> 401
aacccuggug guuggugucg u 21
<210> 402
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 38
<400> 402
gggcagccug gugcugcuag u 21
<210> 403
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 39
<400> 403
cgggccgcac gagggacaau a 21
<210> 404
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 40
<400> 404
ccgcacgagg gacaauagga g 21
<210> 405
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 41
<400> 405
cacgagggac aauaggagcc a 21
<210> 406
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 42
<400> 406
ggacaauagg agccaggcgc a 21
<210> 407
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 43
<400> 407
agccgugccu guguucucug u 21
<210> 408
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 44
<400> 408
ugccuguguu cucuguggac u 21
<210> 409
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 45
<400> 409
gccuguguuc ucuguggacu a 21
<210> 410
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 46
<400> 410
uggacuaugg ggagcuggau u 21
<210> 411
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 47
<400> 411
ggacuauggg gagcuggauu u 21
<210> 412
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 48
<400> 412
uggauuucca guggcgagag a 21
<210> 413
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 49
<400> 413
gauuuccagu ggcgagagaa g 21
<210> 414
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 50
<400> 414
cugugucccu gagcagacgg a 21
<210> 415
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 51
<400> 415
gugucccuga gcagacggag u 21
<210> 416
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 52
<400> 416
ugucccugag cagacggagu a 21
<210> 417
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 53
<400> 417
cugagcagac ggaguaugcc a 21
<210> 418
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 54
<400> 418
agcagacgga guaugccacc a 21
<210> 419
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 55
<400> 419
gcagacggag uaugccacca u 21
<210> 420
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 56
<400> 420
gacggaguau gccaccauug u 21
<210> 421
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 57
<400> 421
cggaguaugc caccauuguc u 21
<210> 422
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 58
<400> 422
accauugucu uuccuagcgg a 21
<210> 423
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 59
<400> 423
ccauugucuu uccuagcgga a 21
<210> 424
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 60
<400> 424
cauugucuuu ccuagcggaa u 21
<210> 425
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 61
<400> 425
uuccuagcgg aaugggcacc u 21
<210> 426
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 62
<400> 426
cagccacuga ggccugagga u 21
<210> 427
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 63
<400> 427
agccacugag gccugaggau g 21
<210> 428
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 64
<400> 428
cugaggaugg acacugcucu u 21
<210> 429
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 65
<400> 429
caccaguguu cugcagaccc u 21
<210> 430
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 66
<400> 430
caguguucug cagacccucc a 21
<210> 431
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 67
<400> 431
cgcauuuccu caggagaagc a 21
<210> 432
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 68
<400> 432
cccaccacag gacucauguc u 21
<210> 433
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 69
<400> 433
caccacagga cucaugucuc a 21
<210> 434
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 70
<400> 434
ggacucaugu cucaaugccc a 21
<210> 435
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 71
<400> 435
ccccuacagg gagggccaga u 21
<210> 436
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 72
<400> 436
gggccagaug cagucacugc u 21
<210> 437
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 73
<400> 437
ugcguccagc ucccugaauc u 21
<210> 438
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 74
<400> 438
cccugaaucu cugcugcugc u 21
<210> 439
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 75
<400> 439
aucucugcug cugcugcugc u 21
<210> 440
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 76
<400> 440
ccggagccuc cugccugaac u 21
<210> 441
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 77
<400> 441
gggcugguug gagauggccu u 21
<210> 442
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 78
<400> 442
cugguuggag auggccuugg a 21
<210> 443
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 79
<400> 443
guuggagaug gccuuggagc a 21
<210> 444
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 80
<400> 444
ccuggcagug gcaucccgaa a 21
<210> 445
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 81
<400> 445
ggcaucccga aacgcccugg a 21
<210> 446
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 82
<400> 446
ugggcacagg agugggaggu a 21
<210> 447
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 83
<400> 447
gggcacagga gugggaggua c 21
<210> 448
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 84
<400> 448
ggcacaggag ugggagguac a 21
<210> 449
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 85
<400> 449
cccaggaguu aucugcuccc u 21
<210> 450
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 86
<400> 450
cccugcaggc cuagagaagu u 21
<210> 451
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 87
<400> 451
ugcaggccua gagaaguuuc a 21
<210> 452
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 88
<400> 452
gccuagagaa guuucaggga a 21
<210> 453
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 89
<400> 453
ccuagagaag uuucagggaa g 21
<210> 454
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 90
<400> 454
aaguuucagg gaaggucaga a 21
<210> 455
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 91
<400> 455
ucagggaagg ucagaagagc u 21
<210> 456
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 92
<400> 456
cagggaaggu cagaagagcu c 21
<210> 457
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 93
<400> 457
gggaagguca gaagagcucc u 21
<210> 458
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 94
<400> 458
gucagaagag cuccuggcug u 21
<210> 459
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 95
<400> 459
accuuuacac augcccaggc a 21
<210> 460
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 96
<400> 460
gggcagggaa gcugaggcag u 21
<210> 461
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 97
<400> 461
ggcagggaag cugaggcagu a 21
<210> 462
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 98
<400> 462
agcugaggca guaagcgggc a 21
<210> 463
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 99
<400> 463
ggcaguaagc gggcaggcag a 21
<210> 464
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 100
<400> 464
ggcagagcug gaggccuuuc a 21
<210> 465
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 101
<400> 465
ccccucacac cacucgggag a 21
<210> 466
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 102
<400> 466
caccacucgg gagagggaca u 21
<210> 467
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 103
<400> 467
gggagaggga cauccuacgg u 21
<210> 468
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 104
<400> 468
gagggacauc cuacgguccc a 21
<210> 469
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 105
<400> 469
agggacaucc uacgguccca a 21
<210> 470
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 106
<400> 470
cuggguguug ggagggcaga a 21
<210> 471
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 107
<400> 471
gggagggcag aagugcaggc a 21
<210> 472
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 108
<400> 472
acccugggag cucuccuugg a 21
<210> 473
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 109
<400> 473
gggagcucuc cuuggaaccc a 21
<210> 474
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 110
<400> 474
cucuccuugg aacccauucc u 21
<210> 475
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 111
<400> 475
gggugggaag guacaggcgu u 21
<210> 476
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 112
<400> 476
cgccguggcc uauccacucc u 21
<210> 477
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 113
<400> 477
guggccuauc cacuccucac a 21
<210> 478
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 114
<400> 478
cacuccucac auccacacac u 21
<210> 479
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 115
<400> 479
uccucacauc cacacacugc a 21
<210> 480
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 116
<400> 480
ggcaccugag uggcugggac a 21
<210> 481
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 117
<400> 481
uucccugugg uucuauuaua u 21
<210> 482
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 118
<400> 482
uuaaauauga gagcaugcua a 21
<210> 483
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 119
<400> 483
auaugagagc augcuaagga a 21
<210> 484
<211> 21
<212> RNA
<213> Artificial
<220>
<223> siRNA 120
<400> 484
uaugagagca ugcuaaggaa a 21
<210> 485
<211> 22
<212> DNA
<213> Artificial
<220>
<223> 上游引物
<400> 485
gacatcctac ggtcccaagg tc 22
<210> 486
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 下游引物
<400> 486
ttctgccctc ccaacaccc 19
<210> 487
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 上游引物
<400> 487
tgacttcaac agcgacaccc a 21
<210> 488
<211> 21
<212> DNA
<213> Artificial
<220>
<223> 下游引物
<400> 488
caccctgttg ctgtagccaa a 21
<210> 489
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA1
<400> 489
cuagagaagu uucagggaau ucaagagauu cccugaaacu ucucuag 47
<210> 490
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA2
<400> 490
cggagagcuu cgugcuaaau ucaagagauu uagcacgaag cucuccg 47
<210> 491
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA3
<400> 491
ccaggauggu ucuuagacuu ucaagagaag ucuaagaacc auccugg 47
<210> 492
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA4
<400> 492
gcuuccgugu cacacaacuu ucaagagaag uugugugaca cggaagc 47
<210> 493
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA5
<400> 493
ccaaggcgca gaucaaagau ucaagagauc uuugaucugc gccuugg 47
<210> 494
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA6
<400> 494
ccuguguucu cuguggacuu ucaagagaag uccacagaga acacagg 47
<210> 495
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA7
<400> 495
gaguaugcca ccauugucuu ucaagagaag acaauggugg cauacuc 47
<210> 496
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA8
<400> 496
cuccuuggaa cccauuccuu ucaagagaag gaauggguuc caaggag 47
<210> 497
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA9
<400> 497
caggccuaga gaaguuucau ucaagagaug aaacuucucu aggccug 47
<210> 498
<211> 47
<212> RNA
<213> Artificial
<220>
<223> shRNA10
<400> 498
ccacaggacu caugucucau ucaagagaug agacaugagu ccugugg 47
<210> 499
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA11
<400> 499
uggcggccag gaugguucuu auucaagaga uaagaaccau ccuggccgcc a 51
<210> 500
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA12
<400> 500
ggccaggaug guucuuagac uuucaagaga agucuaagaa ccauccuggc c 51
<210> 501
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA13
<400> 501
gggacaacgc caccuucacc uuucaagaga aggugaaggu ggcguugucc c 51
<210> 502
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA14
<400> 502
acgccaccuu caccugcagc uuucaagaga agcugcaggu gaagguggcg u 51
<210> 503
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA15
<400> 503
cagcuucucc aacacaucgg auucaagaga uccgaugugu uggagaagcu g 51
<210> 504
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA16
<400> 504
aucggagagc uucgugcuaa auucaagaga uuuagcacga agcucuccga u 51
<210> 505
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA17
<400> 505
cggagagcuu cgugcuaaac uuucaagaga aguuuagcac gaagcucucc g 51
<210> 506
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA18
<400> 506
gagcuucgug cuaaacuggu auucaagaga uaccaguuua gcacgaagcu c 51
<210> 507
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA19
<400> 507
ucgugcuaaa cugguaccgc auucaagaga ugcgguacca guuuagcacg a 51
<210> 508
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA20
<400> 508
cgugcuaaac ugguaccgca uuucaagaga augcgguacc aguuuagcac g 51
<210> 509
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA21
<400> 509
ugcuaaacug guaccgcaug auucaagaga ucaugcggua ccaguuuagc a 51
<210> 510
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA22
<400> 510
ccccagcaac cagacggaca auucaagaga uuguccgucu gguugcuggg g 51
<210> 511
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA23
<400> 511
aggacugccg cuuccguguc auucaagaga ugacacggaa gcggcagucc u 51
<210> 512
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA24
<400> 512
ugccgcuucc gugucacaca auucaagaga uugugugaca cggaagcggc a 51
<210> 513
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA25
<400> 513
ccgcuuccgu gucacacaac uuucaagaga aguuguguga cacggaagcg g 51
<210> 514
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA26
<400> 514
ccgugucaca caacugccca auucaagaga uugggcaguu gugugacacg g 51
<210> 515
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA27
<400> 515
acgggcguga cuuccacaug auucaagaga ucauguggaa gucacgcccg u 51
<210> 516
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA28
<400> 516
gcgugacuuc cacaugagcg uuucaagaga acgcucaugu ggaagucacg c 51
<210> 517
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA29
<400> 517
ggcgcaauga cagcggcacc uuucaagaga aggugccgcu gucauugcgc c 51
<210> 518
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA30
<400> 518
augacagcgg caccuaccuc uuucaagaga agagguaggu gccgcuguca u 51
<210> 519
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA31
<400> 519
ccccaaggcg cagaucaaag auucaagaga ucuuugaucu gcgccuuggg g 51
<210> 520
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA32
<400> 520
ggcgcagauc aaagagagcc uuucaagaga aggcucucuu ugaucugcgc c 51
<210> 521
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA33
<400> 521
agcucagggu gacagagaga auucaagaga uucucucugu cacccugagc u 51
<210> 522
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA34
<400> 522
gugacagaga gaagggcaga auucaagaga uucugcccuu cucucuguca c 51
<210> 523
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA35
<400> 523
agccggccag uuccaaaccc uuucaagaga aggguuugga acuggccggc u 51
<210> 524
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA36
<400> 524
cggccaguuc caaacccugg uuucaagaga accaggguuu ggaacuggcc g 51
<210> 525
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA37
<400> 525
aacccuggug guuggugucg uuucaagaga acgacaccaa ccaccagggu u 51
<210> 526
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA38
<400> 526
gggcagccug gugcugcuag uuucaagaga acuagcagca ccaggcugcc c 51
<210> 527
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA39
<400> 527
cgggccgcac gagggacaau auucaagaga uauugucccu cgugcggccc g 51
<210> 528
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA40
<400> 528
ccgcacgagg gacaauagga guucaagaga cuccuauugu cccucgugcg g 51
<210> 529
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA41
<400> 529
cacgagggac aauaggagcc auucaagaga uggcuccuau ugucccucgu g 51
<210> 530
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA42
<400> 530
ggacaauagg agccaggcgc auucaagaga ugcgccuggc uccuauuguc c 51
<210> 531
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA43
<400> 531
agccgugccu guguucucug uuucaagaga acagagaaca caggcacggc u 51
<210> 532
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA44
<400> 532
ugccuguguu cucuguggac uuucaagaga aguccacaga gaacacaggc a 51
<210> 533
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA45
<400> 533
gccuguguuc ucuguggacu auucaagaga uaguccacag agaacacagg c 51
<210> 534
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA46
<400> 534
uggacuaugg ggagcuggau uuucaagaga aauccagcuc cccauagucc a 51
<210> 535
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA47
<400> 535
ggacuauggg gagcuggauu uuucaagaga aaauccagcu ccccauaguc c 51
<210> 536
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA48
<400> 536
uggauuucca guggcgagag auucaagaga ucucucgcca cuggaaaucc a 51
<210> 537
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA49
<400> 537
gauuuccagu ggcgagagaa guucaagaga cuucucucgc cacuggaaau c 51
<210> 538
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA50
<400> 538
cugugucccu gagcagacgg auucaagaga uccgucugcu cagggacaca g 51
<210> 539
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA51
<400> 539
gugucccuga gcagacggag uuucaagaga acuccgucug cucagggaca c 51
<210> 540
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA52
<400> 540
ugucccugag cagacggagu auucaagaga uacuccgucu gcucagggac a 51
<210> 541
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA53
<400> 541
cugagcagac ggaguaugcc auucaagaga uggcauacuc cgucugcuca g 51
<210> 542
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA54
<400> 542
agcagacgga guaugccacc auucaagaga ugguggcaua cuccgucugc u 51
<210> 543
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA55
<400> 543
gcagacggag uaugccacca uuucaagaga augguggcau acuccgucug c 51
<210> 544
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA56
<400> 544
gacggaguau gccaccauug uuucaagaga acaauggugg cauacuccgu c 51
<210> 545
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA57
<400> 545
cggaguaugc caccauuguc uuucaagaga agacaauggu ggcauacucc g 51
<210> 546
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA58
<400> 546
accauugucu uuccuagcgg auucaagaga uccgcuagga aagacaaugg u 51
<210> 547
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA59
<400> 547
ccauugucuu uccuagcgga auucaagaga uuccgcuagg aaagacaaug g 51
<210> 548
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA60
<400> 548
cauugucuuu ccuagcggaa uuucaagaga auuccgcuag gaaagacaau g 51
<210> 549
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA61
<400> 549
uuccuagcgg aaugggcacc uuucaagaga aggugcccau uccgcuagga a 51
<210> 550
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA62
<400> 550
cagccacuga ggccugagga uuucaagaga auccucaggc cucaguggcu g 51
<210> 551
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA63
<400> 551
agccacugag gccugaggau guucaagaga cauccucagg ccucaguggc u 51
<210> 552
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA64
<400> 552
cugaggaugg acacugcucu uuucaagaga aagagcagug uccauccuca g 51
<210> 553
<211> 49
<212> RNA
<213> Artificial
<220>
<223> shRNA65
<400> 553
caccaguguu cugcagaccc uuucaagaga uucccugaaa cuucucuag 49
<210> 554
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA66
<400> 554
caguguucug cagacccucc auucaagaga uggagggucu gcagaacacu g 51
<210> 555
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA67
<400> 555
cgcauuuccu caggagaagc auucaagaga ugcuucuccu gaggaaaugc g 51
<210> 556
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA68
<400> 556
cccaccacag gacucauguc uuucaagaga agacaugagu ccuguggugg g 51
<210> 557
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA69
<400> 557
caccacagga cucaugucuc auucaagaga ugagacauga guccuguggu g 51
<210> 558
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA70
<400> 558
ggacucaugu cucaaugccc auucaagaga ugggcauuga gacaugaguc c 51
<210> 559
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA71
<400> 559
ccccuacagg gagggccaga uuucaagaga aucuggcccu cccuguaggg g 51
<210> 560
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA72
<400> 560
gggccagaug cagucacugc uuucaagaga agcagugacu gcaucuggcc c 51
<210> 561
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA73
<400> 561
ugcguccagc ucccugaauc uuucaagaga agauucaggg agcuggacgc a 51
<210> 562
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA74
<400> 562
cccugaaucu cugcugcugc uuucaagaga agcagcagca gagauucagg g 51
<210> 563
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA75
<400> 563
aucucugcug cugcugcugc uuucaagaga agcagcagca gcagcagaga u 51
<210> 564
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA76
<400> 564
ccggagccuc cugccugaac uuucaagaga aguucaggca ggaggcuccg g 51
<210> 565
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA77
<400> 565
gggcugguug gagauggccu uuucaagaga aaggccaucu ccaaccagcc c 51
<210> 566
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA78
<400> 566
cugguuggag auggccuugg auucaagaga uccaaggcca ucuccaacca g 51
<210> 567
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA79
<400> 567
guuggagaug gccuuggagc auucaagaga ugcuccaagg ccaucuccaa c 51
<210> 568
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA80
<400> 568
ccuggcagug gcaucccgaa auucaagaga uuucgggaug ccacugccag g 51
<210> 569
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA81
<400> 569
ggcaucccga aacgcccugg auucaagaga uccagggcgu uucgggaugc c 51
<210> 570
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA82
<400> 570
ugggcacagg agugggaggu auucaagaga uaccucccac uccugugccc a 51
<210> 571
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA83
<400> 571
gggcacagga gugggaggua cuucaagaga guaccuccca cuccugugcc c 51
<210> 572
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA84
<400> 572
ggcacaggag ugggagguac auucaagaga uguaccuccc acuccugugc c 51
<210> 573
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA85
<400> 573
cccaggaguu aucugcuccc uuucaagaga agggagcaga uaacuccugg g 51
<210> 574
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA87
<400> 574
ugcaggccua gagaaguuuc auucaagaga ugaaacuucu cuaggccugc a 51
<210> 575
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA87
<400> 575
ugcaggccua gagaaguuuc auucaagaga ugaaacuucu cuaggccugc a 51
<210> 576
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA88
<400> 576
gccuagagaa guuucaggga auucaagaga uucccugaaa cuucucuagg c 51
<210> 577
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA89
<400> 577
ccuagagaag uuucagggaa guucaagaga cuucccugaa acuucucuag g 51
<210> 578
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA90
<400> 578
aaguuucagg gaaggucaga auucaagaga uucugaccuu cccugaaacu u 51
<210> 579
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA91
<400> 579
ucagggaagg ucagaagagc uuucaagaga agcucuucug accuucccug a 51
<210> 580
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA92
<400> 580
cagggaaggu cagaagagcu cuucaagaga gagcucuucu gaccuucccu g 51
<210> 581
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA93
<400> 581
gggaagguca gaagagcucc uuucaagaga aggagcucuu cugaccuucc c 51
<210> 582
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA94
<400> 582
gucagaagag cuccuggcug uuucaagaga acagccagga gcucuucuga c 51
<210> 583
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA95
<400> 583
accuuuacac augcccaggc auucaagaga ugccugggca uguguaaagg u 51
<210> 584
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA96
<400> 584
gggcagggaa gcugaggcag uuucaagaga acugccucag cuucccugcc c 51
<210> 585
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA97
<400> 585
ggcagggaag cugaggcagu auucaagaga uacugccuca gcuucccugc c 51
<210> 586
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA98
<400> 586
agcugaggca guaagcgggc auucaagaga ugcccgcuua cugccucagc u 51
<210> 587
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA99
<400> 587
ggcaguaagc gggcaggcag auucaagaga ucugccugcc cgcuuacugc c 51
<210> 588
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA100
<400> 588
ggcagagcug gaggccuuuc auucaagaga ugaaaggccu ccagcucugc c 51
<210> 589
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA101
<400> 589
ccccucacac cacucgggag auucaagaga ucucccgagu ggugugaggg g 51
<210> 590
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA102
<400> 590
caccacucgg gagagggaca uuucaagaga augucccucu cccgaguggu g 51
<210> 591
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA103
<400> 591
gggagaggga cauccuacgg uuucaagaga accguaggau gucccucucc c 51
<210> 592
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA104
<400> 592
gagggacauc cuacgguccc auucaagaga ugggaccgua ggaugucccu c 51
<210> 593
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA105
<400> 593
agggacaucc uacgguccca auucaagaga uugggaccgu aggauguccc u 51
<210> 594
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA106
<400> 594
cuggguguug ggagggcaga auucaagaga uucugcccuc ccaacaccca g 51
<210> 595
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA107
<400> 595
gggagggcag aagugcaggc auucaagaga ugccugcacu ucugcccucc c 51
<210> 596
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA108
<400> 596
acccugggag cucuccuugg auucaagaga uccaaggaga gcucccaggg u 51
<210> 597
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA109
<400> 597
gggagcucuc cuuggaaccc auucaagaga uccaaggaga gcucccaggg u 51
<210> 598
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA110
<400> 598
cucuccuugg aacccauucc uuucaagaga aggaaugggu uccaaggaga g 51
<210> 599
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA111
<400> 599
gggugggaag guacaggcgu uuucaagaga aacgccugua ccuucccacc c 51
<210> 600
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA112
<400> 600
cgccguggcc uauccacucc uuucaagaga aggaguggau aggccacggc g 51
<210> 601
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA113
<400> 601
guggccuauc cacuccucac auucaagaga ugugaggagu ggauaggcca c 51
<210> 602
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA114
<400> 602
cacuccucac auccacacac uuucaagaga agugugugga ugugaggagu g 51
<210> 603
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA115
<400> 603
uccucacauc cacacacugc auucaagaga ugcagugugu ggaugugagg a 51
<210> 604
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA116
<400> 604
ggcaccugag uggcugggac auucaagaga ugucccagcc acucaggugc c 51
<210> 605
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA117
<400> 605
uucccugugg uucuauuaua uuucaagaga auauaauaga accacaggga a 51
<210> 606
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA118
<400> 606
uuaaauauga gagcaugcua auucaagaga uuagcaugcu cucauauuua a 51
<210> 607
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA119
<400> 607
auaugagagc augcuaagga auucaagaga uuccuuagca ugcucucaua u 51
<210> 608
<211> 51
<212> RNA
<213> Artificial
<220>
<223> shRNA120
<400> 608
uaugagagca ugcuaaggaa auucaagaga uuuccuuagc augcucucau a 51
<210> 609
<211> 31
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 609
cacatcggag agcttcgtgc taaactgtac c 31
<210> 610
<211> 26
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 610
cacatcggag agcttcgtgc taaact 26
<210> 611
<211> 26
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 611
cggagagctt cgtgctaaac tgtacc 26
<210> 612
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 612
cggagagctt cgtgctaaa 19
<210> 613
<211> 19
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 613
gagagcttcg tgctaaact 19
<210> 614
<211> 31
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 614
cacatcggag agcttcgtgc ttaactgtac c 31
<210> 615
<211> 31
<212> DNA
<213> Artificial
<220>
<223> 靶序列
<400> 615
cacatcggtg tgcttcgtgc taaactgtac c 31
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PDCD1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PDCD1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PDCD1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PDCD1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PDCD1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PDCD1基因中的靶序列选自如SEQ ID NO:1~120所示之任一序列或如SEQ ID NO:1~120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:1~120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:365~484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:489~608任一序列所示。
7.一种PDCD1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PDCD1基因干扰核酸构建体,其特征在于,所述PDCD1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PDCD1基因干扰核酸构建体,其特征在于,所述PDCD1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PDCD1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PDCD1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PDCD1基因干扰核酸构建体,和/或权利要求11所述的PDCD1基因干扰慢病毒在制备T细胞活性促进剂中的用途。
13.根据权利要求12所述的用途,其特征在于,所述T细胞活性的促进剂用于作用于T细胞,降低T细胞中PDCD1基因的表达,获得具有较高的杀伤力的高活性T细胞,所述高活性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细胞活性促进剂,其有效物质含有如权利要求1-6任一权利要求所述的分离的核酸分子,和/或权利要求7-10任一权利要求所述PDCD1基因干扰核酸构建体,和/或权利要求11所述的PDCD1基因干扰慢病毒,以及药学上可接受的载体、稀释剂或赋形剂。
15.一种提高T细胞活性的试剂盒,所述试剂盒包括:存在于容器中的权利要求1-6任一权利要求所述的分离的核酸分子,和/或权利要求7-10任一权利要求所述PDCD1基因干扰核酸构建体,和/或权利要求11所述的PDCD1基因干扰慢病毒。
16.一种提高T细胞活性的方法,为降低T细胞中PDCD1基因的表达,从而提高T细胞的活性。
17.一种T细胞,其特征在于,所述T细胞中含有如权利要求1-6任一权利要求所述的分离的核酸分子,和/或权利要求7-10任一权利要求所述PDCD1基因干扰核酸构建体,和/或权利要求11所述的PDCD1基因干扰慢病毒。
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