CN100344331C - 用于预防或治疗丙型肝炎的靶向小干扰rna制剂及其制备方法 - Google Patents

用于预防或治疗丙型肝炎的靶向小干扰rna制剂及其制备方法 Download PDF

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CN100344331C
CN100344331C CNB2004100515625A CN200410051562A CN100344331C CN 100344331 C CN100344331 C CN 100344331C CN B2004100515625 A CNB2004100515625 A CN B2004100515625A CN 200410051562 A CN200410051562 A CN 200410051562A CN 100344331 C CN100344331 C CN 100344331C
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李宝健
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Tuopu Gene Tech Co Ltd Guangzhou
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Abstract

本发明公开了一种用于预防或治疗丙型肝炎的靶向小干扰RNA制剂及其制备方法,由小干扰RNA、肝细胞靶向的核酸导入载体组成;所述肝细胞靶向的核酸导入载体由肝细胞靶向分子和/或核酸结合分子组成;通过靶向肝细胞的载体建立核酸高效转染肝细胞的体系,在体外细胞水平筛选到的能够有效抑制丙型肝炎病毒复制和感染的siRNA,通过体内系统或局部给药将siRNA高效导入肝细胞,筛选出在体内能够有效抑制病毒复制和感染的siRNA及其体内给药技术。本发明还提供了用于靶向肝组织的给药技术,可用于蛋白类、核酸类药物的体外或体内给药以及靶向肝细胞的体内或体外药物筛选方法,尤其是核酸类药物。

Description

用于预防或治疗丙型肝炎的靶向小干扰RNA制剂及其制备方法
技术领域
本发明涉及一种用于预防或治疗丙型肝炎的靶向小干扰RNA制剂,本发明还涉及小干扰RNA制剂的制备方法。
背景技术
HCV急性感染后约75%发展为慢性肝炎,其中约20%最终结局为肝硬化、肝细胞肿瘤或肝功能衰竭。世界范围内大约有1.7亿患者,我国感染人数大约为5000万,因HCV感染是亚临床症状,实际人数可能更多。HCV感染已经成为严重威胁人类健康的社会公共卫生问题之一。迄今尚无特异有效的防治方法。
HCV为单股正链RNA病毒,属黄病毒科肝炎病毒属。HCV基因组变异较大,分为基因型、亚型、分离株、准种四个层次。世界范围内HCV要有六个基因型,80多个亚基因型,流行情况与地理和人群分布相关。我国主要为II/1b,III/2a型。HCV基因组全长约9.6Kb,含有单一开放读码框(ORF),两侧为非编码区(UTR)。5’UTR含有内部核糖体进入位点(IRES),非常保守;3’UTR与复制起始有关。开放读码框编码大约3010-3033个氨基酸的多聚蛋白前体。该蛋白前体在宿主信号肽酶和病毒蛋白酶的共同作用下生成10个成熟蛋白或酶,按顺序依次为Core-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B。
结构蛋白包括核心蛋白Core和两个囊膜蛋白E1,E2,它们是病毒囊膜与核蛋白体的主要成分。位于结构蛋白下游的是约7KD的膜相关蛋白,命名为p7,功能不详。NS2-NS5B蛋白是病毒基因组复制所必须的非结构区蛋白。NS2蛋白和部分NS3蛋白具有独特的蛋白酶活性,选择性水解NS2/3连接区。NS3蛋白是多功能的,N端180个氨基酸具有丝氨酸蛋白水解酶活性,切割NS3/4A,NS4A/4B,NS4B/NS5A,NS5A/NS5B连接区;C末端451个氨基酸具有ATP依赖的解旋酶活性,是复制所必须的区域。NS4A蛋白作为NS3蛋白的共作用分子,NS4B蛋白是膜蛋白。NS5A蛋白在细胞中以磷酸化形式存在,可以结合NS5B蛋白并调节其功能。NS5B蛋白具有RNA依赖的RNA聚合酶活性,负责HCV基因组RNA的复制。
目前,对HCV感染的治疗仅限于单独使用I FN-a,或者与广谱抗病毒药物利巴韦林联合使用。然而治疗的效果不令人满意,而且有一定的副作用。
RNAi技术(RNA干涉技术),简言之,就是当把21-23个核苷酸长的RNA片段(dsRNA)导入细胞或组织中后,后者会使目的mRNA被切割为21-23核苷酸长的片段,从而降解mRNA,阻碍或阻止了由mRNA翻译成相应蛋白质的过程。举例说明,根据病毒复制相关的mRNA序列,设计21-23个核苷酸长的RNA片段(dsRNA),当病毒侵入生物体后,把dsRNA导入生物体,引起病毒复制相关的mRNA的降解,从而阻断了病毒的复制,达到清除病毒的作用。
RNAi发现仅短短5年的时间,由于其高效性和高度特异性,RNAi成为颇为理想的细胞水平基因敲除工具,并迅速成为后基因组时代功能基因组学研究中不可或缺的重要手段。通过采取siRNA在哺乳动物细胞中诱导了强有力的特异性RNA干扰作用,这一突破性研究进展开创了人类疾病治疗的新天地,在SARS、AIDS等病毒感染性疾病治疗中的应用研究非常活跃和深入,并取得了令人鼓舞的成果。在AIDS的治疗上,RNA干扰可能成为对抗HIV病毒感染、复制的最有利方式。人们针对HIV生活周期的不同阶段设计了多种siRNA,针对HIV病毒gag、tat、rev、nef等基因的siRNA可用以控制病毒复制,针对宿主细胞的表面HIV受体基因的siRNA可用以控制病毒进入宿主细胞内,抑制病毒的感染过程。RNAi技术除用于抗HIV研究以外,还用于抗其他多种病毒的探索。它们包括乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、呼吸道合胞病毒(RSV)、流感病毒(influenzavirus)、脊髓灰质炎病毒(poliovirus)等。
2002年12月20日,Small RNA & RNAi被Science杂志评为年度十大科技成就之首,Nature杂志亦将Small RNA评为年度重大科技成果之一。RNA研究的突破性进展,是生物医学领域近20年来,可与人类基因组计划(human genomicsprogram,HGP)相提并论的最重大成果之一。RNAi最主要的功能在于可以调节和关闭基因的表达,进而调控细胞的各种高级生命活动。人们对小RNA分子的深入研究必将大大推进基因功能的研究,更为各种病毒性疾病和肿瘤等疾病的根治,开辟新的治疗途径。具有极其重要的理论和实际意义,它必将对生物学和医学的发展产生深远的影响。
病毒载体主要来源于鼠和人类的DNA、RNA病毒,包括逆转录病毒、腺病毒(Ad)、腺相关病毒(AAV)、慢病毒、单纯疱疹病毒(HSV)、牛痘病毒、杆状病毒、EB病毒(EBV)、水疱性口炎病毒(VSV)和人巨细胞病毒(CMV)等,并且其中一些已经进入了临床试验阶段。其他载体,如细菌载体和人工载体则还处于研究阶段。
经过改造的病毒载体,转染效率较高,但在于制备复杂,有免疫原性,体内不能反复应用,安全性存在隐患以及非导向性,在临床应用上存在一些困难。非病毒载体是核酸药物,尤其是反义核酸、质粒DNA以及siRNA核酸药物的重要途径。通常利用亲水或疏水的多价阳离子聚合物来凝聚重组质粒或反义寡核苷酸,形成微粒,被细胞内吞。尽管非病毒载体转染效率仍低于病毒性载体,但非病毒载体由于具有低毒、低免疫反应、靶向性和易于组装等优点。
脂质体是将药物包封于类脂双分子层形成的单层或多层球形微粒,类似细胞结构,有生物膜的特性和功能。它可以包裹水溶性和脂溶性两种类型的药物。应用于转基因脂质体DNA复合物分为阳性、中性脂质体、阴性脂质体以及pH-敏感的脂质体。还不断有新型的脂质体被推出,如双四铵化合物等。脂质体经聚乙二醇(PEG)修饰后,可降低脂质体-DNA复合物颗粒之间的相互聚集,降低了与血液成分的相互作用,使脂质体DNA复合物颗粒由大颗粒变成小颗粒,同时不影响脂质体与细胞的亲和力。RNA/DNA寡核苷链嵌合分子曾被用于修复DNA基因中的单个碱基突变。
阳离子多聚物可浓缩富含阴离子的DNA,然后粘附到细胞表面的硫酸粘多糖上,被细胞内吞进入细胞内,从而使质粒表达。阳离子多聚物种类很多,如聚乙烯亚胺(polyethylenimine,PEI),聚丙烯亚胺树突状物(polypropyleniminedendrimers),聚酰胺树突状物(polyamidoamine dendrimer),多聚赖氨酸、多聚组氨酸、多聚精氨酸、鱼精蛋白、聚氨基葡萄糖(chitosan)等以及上述聚合物的聚乙二醇修饰物。其中研究较早的是多聚赖氨酸,后来发现聚乙烯亚胺性能更佳。PEI可抑制溶酶体,在吞噬泡酸性环境中质子化、正电荷增加,对DNA提供更大的保护作用,有利于核酸逃离吞噬泡。由于DNA与阳离子多聚物形成的转移复合物是通过内吞进入细胞,易被溶酶体降解,因此加入吞噬泡释放剂有利于提高转染效率。最常用的吞噬泡释放剂是氯喹和两性分子肽。
多肽基因释放系统中最早使用的是二油酰蜂毒肽(dioleoylmelittin),与质粒DNA形成环形颗粒,可有效地转染真核细胞,建立了多肽转基因载体的方法。碱性氨基酸多肽长度16~18氨基酸足以转移DNA至细胞内。阳离子多聚赖氨酸或精氨酸肽链压缩DNA分子,最少需6到8个阳离子氨基酸残基;添加半胱氨酸和组氨酸后可以增加转染效率,如Cys-His-(Lys)(6)-His-Cys寡肽等。多肽载体是配体区、结合DNA的阳离子区、核定位信号区、两性分子功能区,是四位一体的合成短肽。如THALWHT寡肽可特异结合气道上皮,共聚焦显微镜发现标记的THALWHT寡肽与细胞结合后,被吞入细胞内;包含CTHALWHTC序列和DNA结合部分的合成多肽,能有效地将基因转移到气道上皮细胞内。添加吞噬泡释放剂、阳离子聚合物以及病毒的表面蛋白颗粒有助于提高转染效率。多肽载体的优势在于可用多肽合成仪合成,便于体内应用和基因工程法生产,克服了配体寡肽与多聚赖氨酸或PEI偶联制备的复杂性、不均一性以及系统误差大的缺陷。多肽载体是非病毒载体小型化的趋势。不足之处在于阳离子负荷相对偏少。
发明内容
本发明的目的在于提供一种用于预防或治疗丙型肝炎的靶向小干扰RNA制剂,本发明的另一个目的在于提供制备小干扰RNA制剂的方法。
本发明用于预防或治疗丙型肝炎的小干扰RNA制剂由小干扰RNA、肝细胞靶向的核酸导入载体组成;所述肝细胞靶向的核酸导入载体由肝细胞靶向分子和/或核酸结合分子组成。
本发明所述的小干扰RNA可以是由作用于一个靶序列的单一小干扰RNA组成,也可以由作用于一个基因的多个靶序列或多个基因上的靶序列的多个小干扰RNA组成;所述的靶序列是丙型肝炎病毒的RNA基因组序列,或发病宿主自身的内源基因的序列如人肝细胞的丙型肝炎病毒的受体CD81或Fas基因,可以由至少由序列表中的一个序列组成。
本发明所述的小干扰RNA可以是化学合成的21-23碱基对的双链RNA;也可以是载体或表达框架表达的21-23碱基对的双链RNA,所述载体或表达框架为RNA聚合酶III启动子和RNA聚合酶III终止子调控小干扰RNA在哺乳动物细胞中的表达,RNA聚合酶III启动子包括人源或鼠源的U6启动子和人H1启动子。
本发明所述肝细胞靶向分子为乙肝病毒的S基因区表达的大蛋白或中蛋白或小蛋白或部分S基因序列表达的蛋白;所述的核酸结合分子是富含碱性氨基酸和两性氨基酸分子,序列特征为KKALLALALHHLALLAHHLALALKKA或CCKKHHHKKHKKHGGLLALALHHLALLAHHLALAL,核酸结合分子可以是线性分子也可以是分枝型的分子(序列特征见图1),肝细胞靶向分子和核酸结合分子可以分别制备也可以通过2-10个GG或其它氨基酸连接成融合蛋白而成;可以是聚乙二醇(PEG)修饰的聚乙烯亚胺(PEI)。
本发明所述的肝细胞靶向分子或核酸结合分子可以是通过化学方法合成的,也可以在酵母细胞或哺乳动物细胞或昆虫细胞表达生产体系中表达。
本发明所述的在酵母细胞中表达生产体系包括酵母细胞和酵母表达载体,其中酵母细胞可以是毕赤酵母细胞、酿酒酵母细胞,酵母表达载体是pICZα或pYES2/CT或其它酵母表达载体;哺乳动物细胞表达生产体系包括人的CHO细胞和哺乳动物细胞外源基因表达载体;所述的昆虫细胞表达生产体系包括昆虫细胞和杆状病毒表达载体。
本发明用于预防或治疗丙型肝炎的小干扰RNA制剂的制备方法,重组生产的靶向蛋白经超速离心或柱纯化后,透析后冻干,溶于pH5-7PBS溶液中与siRNA以30-100∶1的比例混合,使用2-4mm的电激杯,在220V和500-1500uF的条件下处理,使siRNA与靶向蛋白形成复合物,可用于靶向肝细胞的siRNA的体内和体外转染。
本发明用于预防或治疗丙型肝炎的小干扰RNA制剂的制备方法,靶向蛋白与核酸结合分子的融合分子经超速离心或柱纯化后,透析后冻干;溶于pH5-7PBS溶液中与siRNA以30-150∶1的比例混合,使siRNA与融合分子形成复合物,可用于靶向肝细胞的siRNA的体内和体外转染。
本发明用于预防或治疗丙型肝炎的小干扰RNA制剂的制备方法,化学合成的分枝型核酸结合分子与siRNA按10∶1的比例混合,静置一定时间后,形成复合物,再与靶向蛋白混合,最终形成靶向蛋白/分枝型核酸结合分子/siRNA复合物,可用于靶向肝细胞的siRNA的体内和体外转染。
本发明用于预防或治疗丙型肝炎的小干扰RNA制剂的制备方法,取适量乳糖化或半乳糖化蛋白溶于二甲基亚砜(DMSO)加入TEA,在氮的保护下搅拌1分钟,PEG(PEG3400-N-hydroxy succinimide)以2-5∶1的重量比例加入,在室温下搅拌4小时,后使有机溶剂挥发完,获得乳糖化或半乳糖化蛋白螯合的PEG;制备好的乳糖化或半乳糖化蛋白螯合的PEG溶于DMSO,加入4-8倍体积的TEA,搅拌均匀,并在搅拌的条件下加入PEI,加入PEI质量是PEG的20-40倍,搅拌条件下过夜反应;获得的乳糖化或半乳糖化蛋白螯合的PEG-PEI和PEI按1∶1的比例混合溶于5mM HEPES缓冲液,合成的siRNA也溶于pH值为8的5mM HEPES缓冲液;两者按2∶1的质量比例混合,振荡混匀30秒,获得靶向肝细胞的小干扰RNA制剂。
本发明用于预防或治疗丙型肝炎的小干扰RNA制剂可以和脂质体混合使用。
本发明用于预防或治疗丙型肝炎的小干扰RNA制剂可通过肝门静脉直接注射入肝组织中,或通过呼吸道喷雾或滴注使siRNA制剂经过肺部吸收后到达肝脏部位;也可通过静脉给药途径注射或滴注进行系统给药。
siRNA制剂在病毒感染前和病毒感染后导入体外培养细胞或体内,通过病毒基因组拷贝数降低筛选出能有效防治丙型肝炎病毒的小干扰RNA制剂;通过动物模型的肝脏损伤减轻筛选出能有效防治丙型肝炎病毒的小干扰RNA制剂;通过大剂量和重复导入小干扰RNA制剂到动物模型中筛选出不影响心脏、肝脏、脾脏、肾脏、淋巴结以及血液系统正常功能的小干扰RNA制剂。
本发明治疗病毒性丙型肝炎的靶向小干扰RNA制剂能够提高肝脏的药物浓度,提高药物丙型肝炎疾病的治疗效果,并没有明显的毒副作用。治疗病毒性丙型肝炎的靶向小干扰RNA制剂能够抑制HCV病毒复制和感染及对肝组织的保护作用。
附图说明
图1分枝型核酸结合分子的结构特征。
图2siRNA、靶向分子和核酸结合分子所形成的复合物能够有效介导siRNA高效转染肝细胞。A:靶向分子、核酸结合分子和荧光标记的siRNA组成的制剂转染肝细胞;B:荧光标记的siRNA转染肝细胞。
图3pCI-HCV-Luc重组质粒图。
图4HCV特异的siRNA敲低HCV-Luc融合基因的表达,有效抑制肝组织中萤火虫荧光素酶的表达。
具体实施方式
L蛋白由110个氨基酸组成,在L蛋白的N末端108-119位氨基酸是Pre-S1蛋白,在L蛋白中位于表面,起到与肝细胞表面受体特异性结合的作用,是HBV病毒感染肝细胞所必需的。从人乙肝病毒的基因组中克隆出编码L protein以及部分编码L protein的DNA序列,将克隆的序列连接入T载体中,经测序验证正确,将编码序列克隆进酵母表达载体或昆虫杆状病毒表达载体或哺乳动物细胞表达载体,再将表达载体转染入相应的酵母或昆虫或动物细胞中,生产重组的有L蛋白基因或L蛋白部分DNA序列编码的重组蛋白,重组蛋白经超速离心或层析柱纯化后,由于靶向分子(L protein)具有肝细胞的靶向性,与脂质体或核酸结合多肽连接后,表型出高效的肝靶向性特点,从而制备出靶向肝细胞或肝组织的高效siRNA制剂;或与结合有siRNA的脂质体或分枝型多肽混合,制备出靶向肝细胞或肝组织的高效siRNA制剂。
上述的肝靶向L蛋白或部分L蛋白序列也可通过化学方法合成,并采用类似的办法与siRNA的脂质体或分枝型多肽形成复合物,制备出靶向肝细胞或肝组织的高效siRNA制剂。本发明使用的L蛋白可以是野生型的HBV的L蛋白,也可以是突变的能够逃避免疫系统识别的重组L蛋白。
设计了针对丙型肝炎病毒的RNA基因组,设计了175条siRNA,其中包括靶向5’非编码区、3’非编码区、核心蛋白C区、E1区、E2/NS1区、NS2a区、NS2b区、NS3区、NS4a区、NS4b区、NS5a区、NS5b区的siRNA。并采用化学方法和体外转录的合成了上述siRNA。采用本发明所制备的多肽与所设计的siRNA在室温下或在电激的条件下结合,在体外转染含有HCV复制元的肝细胞、也通过静脉注射或局部组织注射或经肺的系统给药方式导入到活体的肝组织中。肝细胞转染HCV复制元之前和之后分别对细胞转染由靶向分子、核酸结合蛋白和siRNA组成的制剂。
由靶向分子、核酸结合蛋白和siRNA组成的制剂中靶向分子是乙型肝炎病毒的表面大抗原,具有很好的肝靶向性。所采用的核酸结合分子有两种:线性核酸结合多肽和分枝型的核酸结合多肽。线性的核酸结合多肽的序列特征为KKALLALALHHKKALLALALHHLALLAHHLALALKKAGG-NH2,能够有效结合siRNA分子,与肝靶向分子融合,该融合蛋白可通过酵母或昆虫杆状病毒或哺乳动物细胞表达生产,纯化后就制备成肝靶向性的siRNA高效转染制剂。分枝型的多肽能够高效结合siRNA分子,且能够通过非共价键与靶向蛋白连接,因此,本发明通过化学方法合成了分枝型的多肽,序列特征结构见图1,分枝型核酸结合多肽与siRNA结合后,再与制备的重组肝靶向蛋白混合,形成复合物,从而制备成肝靶向性的siRNA高效转染制剂。
通过荧光标记的siRNA转染细胞的效率来评价肝靶向的siRNA高效转染制剂。靶向分子、核酸结合分子和荧光标记的siRNA形成复合物,将复合物与肝细胞共培养,24小时或37小时后,使用PBS溶液洗涤细胞后,在显微镜下观察培养细胞的荧光状况,以荧光标记的siRNA为对照,从图2中可见在未结合靶向分子、核酸结合蛋白的siRNA转染的细胞中未能观察到荧光,而siRNA、靶向分子和核酸结合蛋白复合物转染细胞后,可观察到细胞有明显的荧光。
在成功将siRNA高效导入肝细胞后,我们设计了丙型肝炎病毒基因序列和萤火虫荧光素酶基因(Luc基因)的融合基因,并将融合基因克隆到哺乳动物表达载体pCI,够建成pCI-HCV-Luc重组质粒,该质粒转录出的mRNA含有HCV mRNA并且能够翻译出有活性的萤火虫荧光素酶,如果HCV特异的siRNA能够降解该融合基因的mRNA,就检测不到萤火虫荧光素酶。该质粒和HCV特异的siRNA通过与靶向蛋白/核酸结合蛋白形成复合物,并通过静脉注射导入鼠体内,从图3和图4中可知,HCV特异的siRNA能有效抑制肝组织中萤火虫荧光素酶的表达。
在建立了siRNA药物靶向肝器官的给药方法后,我们将进一步在体外和体内评价siRNA对丙型肝炎的防治作用。通过实时定量RT-PCR评价siRNA分子对降低细胞内病毒基因组拷贝数的抑制作用。从中发现siRNA双链分子能有效抑制丙型肝炎病毒的感染和复制。
设计了一系列试验评价HCV特异siRNA在动物体内的药效和安全性。将权力要求中的至少一条siRNA与靶向分子、核酸结合蛋白配制成siRNA体内导入制剂。配制好的siRNA在病毒攻击前后不同时间把不同剂量的siRNA制剂通过静脉或呼吸道或局部肝组织给药。siRNA处理时间为HCV复制元转染或HCV病毒攻击动物4-72小时前(每隔24-48小时给药一次)、HCV复制元转染或HCV病毒攻击动物(每隔24小时-48小时给药一次)。在动物模型体内导入HCV特异的siRNA/靶向分子/核酸结合蛋白的复合物,在24小时、48小时、72小时以及96小时分别通过实时定量RT-PCR评价siRNA分子对降低肝组织样品中病毒基因组拷贝数的抑制作用;通过肝组织的病理指标评价siRNA分子对肝的保护作用,结果表明本发明的siRNA及制备的siRNA制剂在体内能够有效抑制丙型肝炎病毒的复制与感染,并有效保护了肝组织。
这类siRNA物质组成和制剂制备方法不仅适用丙型肝炎的的防治,而且适用于其他肝脏类疾病的治疗,如肝癌等。
实施例1:靶向蛋白的制备。设计引物上游引物5’atgggaggtt ggtcttccaaacct’3和下游引物5’aatgtatacccaaagacaaaagaaaatt’3,以HBV DNA为模板,反应体系为:5μl 10×RT Buffer,10μl MgCl2,10μl dNTPmixture,1μl上游引物,1μl下游引物,HBV DNA 2ng,TaqDNA聚合酶1μl,加水至50μl。PCR反应条件为:94℃预变性5min,94℃30s,60℃30s,72℃2min,进行30cycle,72℃延伸10min。PCR产物用PCR purify Kit纯化,并与pMD18-T Vector连接,转化,挑取单克隆,鉴定阳性克隆,测序。测序验证正确的靶向蛋白的DNA克隆至酵母表达载体pICZα,经酶切验证正确后,将表达载体导入毕赤酵母细胞中。首先,用Sacl切酵母表达载体pICZαDNA 1小时以上,使之线性化。补水至300ul,加300ul酚∶氯仿∶异戊醇(25∶24∶1)颠倒混匀。离心12000rpm,5分。取上清,加750ml乙醇,30ul NaAc,-20℃,30分钟。离心,12000rpm,10分。80%乙醇清洗,吹干,溶于10ul水。
再准备对数生长期酵母菌:(1挑单菌落接种于5ml YPD液体培养基30℃培养过夜。(2)取过夜培养的菌液0.1-0.5ml接种于50ml新鲜YPD液体培养基,再30℃培养至OD600=1.3-1.5。(3)在4℃,1500g离心5分钟,用50ml 0℃无菌水重悬菌体。(4)离心同上,再重悬于25ml 0℃无菌水。(5)离心同上,重悬于20ml 0℃ 1M山梨醇溶液。(6)离心同上,再重悬于1ml 0℃ 1M山梨醇溶液,置于冰上(当天使用)。
最后通过电击方法将表达载体导入酵母细胞:(1)将制备的细胞80μl和5-90μg线性化DNA注入0℃电击池,混匀,置冰上5分钟。(2)据酵母脉冲参数处理细胞。(电压:1500V;电容:25μF;电阻:400Ω;次数:1次;时间:8.9s)。后即加1ml 0℃ 1M山梨醇溶液到电击池内,混匀。(3)再将池内液体转置1.5ml EP管,30℃培养1-2小时。(4)取100μl菌液涂布于含ZeocinTM 100μg/ml的YPDS平板,剩余菌液全涂布于另一含ZeocinTM 100μg/ml的YPDS平板,30℃培养3-4天。
挑取酵母转化子单菌落接种于2.5mlBMGY中,置于50ml锥形瓶中,在28-30℃,250-300rpm摇瓶培养至OD600=2-6(大约16-18个小时),酵母细胞达到对数生长期。3000rpm,室温下,离心菌体5分钟,去上清,菌体重悬于10-20mlBMMY中,至OD600=1。置于100ml锥形瓶中盖两层纱布,继续摇瓶培养。每隔24小时加入100%甲醇至终浓度0.5%,保持诱导。每次加甲醇之前收集一次培养物(即在培养的不同时间段24,48,72,96小时,分别取样),每次取培养物2ml,12000rpm离心3分钟,取上清贮于-20℃,SDS-PAGE分析重组蛋白的表达量和分子量。采用Invitrogen公司的纯化试剂盒进行纯化,其过程如下:
1树脂处理:
(1)取2毫升亲和树脂,1000转离心1分钟,去上清液;
(2)加入6毫升水混匀,1000转离心1分钟,去上清液;
(3)加入6毫升Binding buffer混匀,1000转离心1分钟,去上清液;
2蛋白纯化:
(1)8毫升左右的发酵液与树脂混合,室温放置1小时;
(2)1000转离心1分钟,去上清液;
(3)加入8毫升Wash buffer混匀,1000转离心1分钟,去上清液;
(4)加入2毫升Elution buffer混匀,1000转离心1分钟,收集上清液。
收集到得上清液冷冻干燥后得到纯化的蛋白。
实施例2:靶向分子和核酸结合分子所组成的融合蛋白的制备。设计上游引物5’atgggaggtt ggtcttccaa acct’3和下游引物5’aatgtatacccaaagacaaaagaaaatt’3,以HBVDNA为模板,反应体系为:5μl 10×RT Buffer,10μl MgCl2,10μl dNTPmixture,1μl上游引物,1μl下游引物,HBV DNA 2ng,TaqDNA聚合酶1μl,加水至50μl。PCR反应条件为:94℃预变性5min,94℃30s,60℃30s,72℃2min,进行30cycle,72℃延伸10min。PCR产物用PCR purify Kit纯化,并与pMD18-T Vector连接,转化,挑取单克隆,鉴定阳性克隆,测序。编码KKALLALALHHKKALLALALHHLALLAHHLALALKKAGG-NH2多肽的5’ggtggcgccaagaagttcgccctccaccacgccctcctcgccctccaccacctcgccctcgccctcctcgccaagaagcaccacctcgccctcgccctcctcgccaagaag3’,序列经人工合成后,接入靶向分子形成融合基因。测序验证正确的编码靶向蛋白和核酸结合蛋白的融合基因克隆至酵母表达载体pICZα,经酶切验证正确后,将表达载体导入毕赤酵母细胞中。重组的酵母表达载体转化酵母细胞的方法以及重组蛋白的纯化方法同实施例1。
实施例3:分枝型核酸结合多肽的制备。
分枝型多肽合成可在ABI431A固相自动肽合成仪上进行也可通过手工合成,序列结构见图1。采用标准的Fmoc方法,选用0.125mmolFmoc-MAP-Branch-PSC树脂,按照序列使肽链从C端逐个向N端延伸,各氨基酸的用量为0.5mmol,氨基酸∶树脂=4∶1,第一个氨基酸连接到树脂上用DMAP,氨基酸的活化用HOBt和DCC偶联后用20%哌啶溶液去除Fmoc保护基团。多肽合成后按照PE公司推荐的步骤,将冰浴后合成物加入处于冰浴条件,下的10ml切割液中采用切割液B其成份为结晶苯酚0.75mg EDT0.25ml,苯甲硫醚0.5ml去离子水0.5ml TFA10ml待切割混合液温度上升至室温后,室温条件下持续搅拌反应1.5小时,使肽链从树脂上裂解下来,同时去除多种保护基团.将反应混合液G4玻沙漏斗过滤以滤掉树脂,先后用1mlTFA 5 10mlDCM冲洗反应瓶、树脂和漏斗。滤液在常温下低压蒸发至1-2ml加入至少50ml预冷后的乙醚沉淀多肽,4度放置过夜,经G6玻璃漏斗过滤,真空抽干至少3小时,所得便是多肽粗产品-20℃储存备用。将MAP粗品溶解浓度为15mg/ml经0.45m纤维滤膜过滤后explore100型中压液相色谱仪上纯化,根据其溶解特性为脂溶性采用POROS-50-R1,填充料的纯化柱,流动相为:A(10%乙醇+0.1%TFA),(90%乙醇+0.1%TFA)。洗脱梯度为100%流动相A平衡柱子1.5个柱体积;100%A-100%B10个柱体积;100%B 0.5个柱体积,上样量为2ml,在主峰处收集多肽溶液。使用树脂柱脱盐,冷冻干燥后待用。
实施例4:靶向分子、核酸结合分子和siRNA所组成制剂的制备。
使用两种方法制备靶向蛋白/核酸结合蛋白/siRNA的复合制剂。
1.首先分枝型核酸结合分子与siRNA通过非共价键结合。适量的siRNA溶于不含RNA酶的无菌水中,轻轻混匀,因为体积有限,可采用高浓度的siRNA,一般siRNA为8-15μg/μL。取适量的siRNA与分枝型核酸结合分子按1∶4-8的比例混合,在室温下温育30分钟,以形成siRNA/多肽复合物。
然后,靶向分子溶于500ul的PBS溶液中,再加入适量的siRNA,完全溶解混匀后,使用Gene Pulser II electroprotion system(Bio-Rad公司提供)仪器,在220V和950uF的条件下电激,使靶向分子包裹siRNA。
最后,将上述制备的siRNA/核酸结合分子和靶向分子/siRNA两种复合物混匀,在室温下放置30分钟,通过两种复合物之间相互作用,制备成用于体内和体外靶向转染siRNA的靶向分子/核酸结合分子/siRNA的制剂。
2.使用实施例2制备的靶向分子/核酸结合分子的融合蛋白制备siRNA制剂(制剂由靶向分子、核酸结合分子和siRNA)。适量的siRNA溶于不含RNA酶的无菌水中,轻轻混匀,因为体积有限,可采用高浓度的siRNA,一般siRNA为8-15μg/μL。取适量的siRNA与靶向分子/核酸结合分子的融合蛋白按1∶10-15的比例混合,在室温下温育30分钟,以形成靶向分子/核酸结合分子/siRNA的复合物,从而制备成由靶向分子、核酸结合分子和siRNA组成的siRNA制剂。
实施例5.靶向分子/核酸结合分子高效介导siRNA转染肝细胞。
荧光标记的siRNA与靶向分子/核酸结合分子按照实施例4中所述的方法制备成复合物。在转染的前一天,4-5×104细胞接种在24孔板上,0.5mL含FBS和抗生素的细胞培养基,应能在24小时内使细胞密度达到最终密度的40-70%。在100μl的无血清的培养基中稀释由靶向分子、核酸结合分子、siRNA所形成的制剂,充分混合,加入到细胞培养基中,轻轻混匀。细胞在37℃温育24h-120h后,在荧光显微镜下观察细胞的荧光状况图2。
实施例6靶向蛋白/核酸结合蛋白/siRNA敲低肝组织中外源基因的表达。
设计了HCV core基因序列和萤火虫荧光素酶基因(Luc基因)的融合基因,并将融合基因克隆到哺乳动物表达载体pCI,够建成pCI-HCV-Luc重组质粒,该质粒转录出的mRNA含有HCV core基因的mRNA并且能够翻译出有活性的萤火虫荧光素酶,如果HCV core特异的siRNA能够降解该融合基因的mRNA,就检测不到萤火虫荧光素酶。按照实施例4中所述的方法制备由HCV core基因特异的siRNA、靶向分子和核酸结合分子组成的制剂,通过静脉注射导入到体内,3天后将动物安乐死,分离肝组织,提取总RNA,通过RT-PCR定量分析荧火虫荧光素酶mRNA的表达水平。从图3和图4中可知,HCV特异的siRNA能有效抑制萤火虫荧光素酶的表达。
实施例7:应用转基因鼠评价siRNA抑制丙型肝炎病毒感染与复制的的药效及安全性。
(一)建立丙型肝炎的动物模型。
可供使用的模型为:Alb-uPA转基因(白蛋白基因启动子调控的4个串联的鼠尿激酶基因,使尿激酶过量表达,导致血纤维蛋白元过少和加速肝细胞的死亡)SCID小鼠(严重联合免疫缺陷小鼠)植入分离的人肝细胞,肝源人白蛋白用于移植。有于人类似的肝HCV血毒症,可持续35天,是较为合适的用于筛选抗HCV药物的动物模型。
可供使用的模型也可为:选用健康成年树鼩,中缅树鼩种,真西亚种,雌雄各半,体重95-148g。树鼩经尾静脉接种HCV阳性血清0.5ml,隔日第二次接种。症状:出现一次精神差,食欲下降及稀便(约持续一周),接种2-3周血清出现HCV-RNA,可见肝组织汇管区淋巴细胞浸润,肝细胞脂肪变、嗜酸性变等组织学改变,可见阳性物质呈颗粒样分布于细胞浆中,可见肝细胞内线粒体、滑面内质网中毒性改变。
(二)实验动物的安排
尽量选择性别、年龄一致的纯种动物,对实验动物进行随机分组。将实施例4中制备的由HCV core基因特异的siRNA、靶向分子和核酸结合分子组成的制剂通过静脉注射或腹腔注射或肝脏直接注射到肝细胞中。
(三)HCV core基因特异的siRNA、靶向分子和核酸结合分子组成的制剂抑制HCV病毒复制和感染及对肝组织的保护作用。分别在不同时期(第1、4、7、10、14、21天)取肝组织细胞和血液样品提取RNA,进行RT-PCR检验。由于HCV的5′端的NS5b、NS5a、NS4b或NS4a区最保守,多根据它们来设计检测引物,检测HCV RNA的拷贝数,靶向蛋白/核酸结合蛋白/siRNA制剂能有效将HCV特异的siRNA导入到肝细胞中,并有效抑制了HCV病毒复制。
Organization Applicant
----------------------
    Street:广州经济技术开发区宝石路11号8楼805室
    City:广州
    State:广东
    Country:中国
    PostalCode:510730
    PhoneNumber:(0)13302207795
    FaxNumber:020-37617478
    EmailAddress:chengducd@21cn.com
<110>OrganizationName:广州拓谱基因技术有限公司
Application Project
-------------------
<120>Title:用于预防或治疗丙型肝炎的靶向小干扰RNA制剂及其制备方法
<130>AppFileReference:0
<140>CurrentAppNumber:
<141>CurrentFilingDate:____-__-__
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cacaacauug guaaauugat t                                                21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA1
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA1
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caacauuggu aaauugacut t                                                21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA2
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA2
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agaucuggga auacgauaat t                                                21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA3
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA3
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ccaugauggu ggugucaaut t                                                21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA4
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA4
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
acggagugga ugugguuaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA5
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA5
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cucuacggau aggaguuuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA6
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA6
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caggacuugc agucugucat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA7
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA7
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aggacuugca gucugucaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA8
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA8
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggacuugcag ucugucaaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA9
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA9
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cuugcagucu gucaaaggut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA10
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA10
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ugcagucugu caaaggugat t                                            21
<212>Type:DNA/RNA
<211>Length:21
SequenceName:HCV-siRNA11
SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA11
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ucacaacauu gguaaauugt t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA12
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA12
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gucacaacau ugguaaauut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA13
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA13
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agucacaaca uugguaaaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA14
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA14
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aagucacaac auugguaaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA15
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA15
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caagucacaa cauugguaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA16
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA16
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
guaacaugug aggguguuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA17
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA17
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gccuucaagu aacaugugat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA18
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA18
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggaggaacau gauguuauct t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA19
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA19
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
acacauugga ggaacaugat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA20
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA20
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agugagucau cagaaucaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA21
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA21
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aagugaguca ucagaaucat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA22
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA22
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gaagugaguc aucagaauct t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA23
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA23
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agaagugagu caucagaaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA24
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA24
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aagaagugag ucaucagaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA25
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA25
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gaagaaguga gucaucagat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA26
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA26
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agaagaagug agucaucagt t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA27
     SequenceDescription:
Custom Codon
-----------
Sequence Name:HCV-siRNA27
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gagaagaagu gagucaucat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA28
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA28
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggagaagaag ugagucauct t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA29
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA29
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
auggagaaga agugagucat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA30
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA30
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gaguaacuau ggagugaaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA31
     SequenceDescription:
Custom Codon
-------------
Sequence Name:HCV-siRNA31
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ccuggagagu aacuauggat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA32
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA32
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ucaccuggag aguaacuaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA33
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA33
Sequence
-------
<213>OrganismName:人工序列
<400>PreSequenceString:
cucaccugga gaguaacuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA34
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA34
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gaucucaccu ggagaguaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA35
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA35
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agccaccauu aaagaaggat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA36
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA36
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
uggagccacc auuaaagaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA37
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA37
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agaagaacac gaggaaggat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA38
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA38
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cgcagaagaa cacgaggaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA39
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA39
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gcgcagaaga acacgaggat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA40
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA40
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agaguaccag accuacaaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA41
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA41
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aagaguacca gaccuacaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA42
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA42
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caagaguacc agaccuacat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA43
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA43
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gucaagagua ccagaccuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA44
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA44
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caaggucaag aguaccagat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA45
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA45
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gacaagguca agaguaccat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA46
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA46
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gugacaaggu caagaguact t                                            21
<212>Type:DNA
<211>Length:21
     SequenceName:HCV-siRNA47
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA47
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggugacaagg ucaagaguat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA48
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA48
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ccucggcccu ggugauaaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA49
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA49
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cgcauguaag gaggaugaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA50
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA50
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
uuggugaugu caaagauuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA51
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA51
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggaguuuggu gaugucaaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA52
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA52
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cgcacuaaca ugcaugcaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA53
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA53
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cuuccgcacu aacaugcaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA54
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA54
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cgaccuuccg cacuaacaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA55
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA55
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gcgaccuucc gcacuaacat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA56
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA56
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cagcgaccuu ccgcacuaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA57
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA57
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggccauuugg acauaaugat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA58
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA58
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aaggccauuu ggacauaaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA59
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA59
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gaaggccauu uggacauaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequcnceName:HCV-siRNA60
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA60
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
guaagauggu uguaaaugut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA61
     SecuenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA61
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cacccgaaga gcccuucaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA62
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA62
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ccacccgaag agcccuucat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA63
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA63
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ccggcacuuu aguacucuut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA64
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA64
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
auaugcagcc ggcacuuuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA65
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA65
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggagucacaa accuguagat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA66
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA66
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggacaaccgu ccucuuucut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA67
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA67
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cguccucuuu cuccguggat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA68
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA68
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
guccucuuuc uccguggagt t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA69
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA69
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ccucuuucuc cguggaggut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA70
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA70
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gcaccaccgg agggacguat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA71
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA71
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ucgaaagagu ccaggacuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA72
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA72
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
uccgacucca cgcgggugat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA73
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA73
Sequence
-------
<213>OrganismName:人工序列
<400>PreSequenceString:
cgacuccacg cgggugaugt t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA74
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA74
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
acuccacgcg ggugauguut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA75
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA75
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agacaacugg cuagcugaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA76
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA76
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
uacccgcguu ggcacgagat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA77
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA77
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
acccgcguug gcacgagaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA78
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA78
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cccgcguugg cacgagaaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA79
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA79
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caggucuuga agucagucat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA80
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA80
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggacccacgu gccgacgcat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA81
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA81
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gcaggaagua gauugaccat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA82
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA82
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggaaguagau ugaccaggut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA83
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA83
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gcgaugccgg cgcccacgat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA84
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA84
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cgaugccggc gcccacgaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA85
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA85
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gaugccggcg cccacgaaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA86
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA86
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggcgcccacg aaagccgaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA87
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA87
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
augccaucaa ugaugcuaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA88
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA88
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ugccaucaau gaugcuauut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA89
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA89
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agcgcuuucu gcuugaacut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA90
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA90
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
acucuuccau uucaucgaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA91
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA91
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ucuuccauuu caucgaacut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA92
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-si RNA92
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
uccauuucau cgaacuccut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA93
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA93
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
uuucaucgaa cuccugguat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA94
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA94
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ucaucgaacu ccugguagat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA95
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA95
Sequence
---------
<213>OrganismName:人工序列
<400>PreSequenceString:
ucgaacuccu gguagagaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA96
     SequenceDescription:
Custom Codon
-----------
Sequence Name:HCV-siRNA96
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gccucccgga caagauaaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA97
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA97
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agauaauccu acccacaaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA98
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA98
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ugcaugccau gauguauuut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA99
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA99
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
augccaugau guauuuggut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA100
     SequenceDescription:
Custom Codon
-----------
Sequence Name:HCV-siRNA100
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gccaugaugu auuugguuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA101
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA101
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
guaugagaca cuuccacaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA102
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA102
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
uaugagacac uuccacauut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA103
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA103
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
augagacacu uccacauuut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA104
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA104
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ugagacacuu ccacauuugt t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA105
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA105
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gagacacuuc cacauuugat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA106
     SequenceDescription:
Custom Codon
---------------
Sequence Name:HCV-siRNA106
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agacacuucc acauuugaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA107
     SequenceDescription:
Custom Codon
------------
Sequence Namc:HCV-siRNA107
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cgcacaccgu ggcuugguat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA108
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA108
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gcacaccgug gcuugguaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA109
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA109
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
accguggcuu gguaugcuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA110
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA110
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agcggguuga uccaagaaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA111
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA111
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ccaaaucucc aggcauugat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA112
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA112
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gcccaaaucu ccaggcauut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA113
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA113
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
acgcccaaau cuccaggcat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA114
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA114
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
acccaacacu acucggcuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA115
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA115
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
uucgcgaccc aacacuacut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA116
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA116
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggccuuucgc gacccaacat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA117
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA117
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cuaucaggca guaccacaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA118
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA118
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agcacccuau caggcaguat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA119
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA119
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
acucgcaagc acccuaucat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA120
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA120
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ugaugcacgg ucuacgagat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA121
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA121
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caugaugcac ggucuacgat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA122
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA122
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gcucaugaug cacggucuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA123
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA123
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gauuugugcu caugaugcat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA124
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA124
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
auacccucgu ugccauagat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA125
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA125
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
acccaaauua cgcgaccuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA126
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA126
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ugaccuuacc caaauuacgt t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA127
     SequenceDescription:
Custom Codon
-----------
Sequence Name:HCV-siRNA127
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
augaccuuac ccaaauuact t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA128
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA128
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gaugaccuua cccaaauuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA129
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA129
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cgaugaccuu acccaaauut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA130
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA130
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ucgaugaccu uacccaaaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA131
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA131
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aucgaugacc uuacccaaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA132
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA132
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agaggaagau agagaaagat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA133
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA133
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ucaagaccuu agcccaguut t                                            21
<2i2>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA134
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA134
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caaucaagac cuuagcccat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA135
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA135
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ucacaaucaa gaccuuagct t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA136
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA136
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aucacaauca agaccuuagt t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA137
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA137
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caucacaauc aagaccuuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA138
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA138
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gcaucacaau caagaccuut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA139
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA139
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
guagcaucac aaucaagact t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA140
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA140
Scquence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aguagcauca caaucaagat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA141
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA141
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agaguagcau cacaaucaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA142
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA142
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aagaguagca ucacaaucat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA143
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA143
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caaagaguag caucacaautt                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA144
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA144
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gcaaagagua gcaucacaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA145
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA145
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ccagcaaaga guagcaucat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA146
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA146
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ucagagcggu ccuguugaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA147
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA147
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caguuuggag ggagucauut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA148
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA148
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ccaguuugga gggagucaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA149
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA149
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cccaguuugg agggagucat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA150
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA150
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aaccugugug cguagaacat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA151
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA151
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ugaaccugug ugcguagaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA152
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA152
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cugcgacgcg gguacaauat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA153
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA153
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
caauacacug ggccacacat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA154
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA154
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ugcuauucau ccauguacat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA155
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA155
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ggugcuauuc auccauguat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA156
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA156
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agccgcauuu gguguaagut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA157
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA157
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
guagucaacu agacaccuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA158
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA158
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gccaaagucu guauggguat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA159
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA159
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
guagugccaa agucuguaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA160
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA160
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ugaccuugaa gaugguaaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA161
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA161
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cugaccuuga agaugguaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA162
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA162
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
ccugaccuug aagaugguat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA163
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA163
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
uccugaccuu gaagauggut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA164
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA164
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
acauccugac cuugaagaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA165
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA165
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
uacauccuga ccuugaagat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA166
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA166
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
guacauccug accuugaagt t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA167
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA167
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cguacauccu gaccuugaat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA168
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA168
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
acguacaucc ugaccuugat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA169
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA169
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
uacucccauu ugauuacgat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA170
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA170
Sequence
-------
<213>OrganismName:人工序列
<400>PreSequenceString:
acauacuccc auuugauuat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA171
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA171
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
aacauacucc cauuugauut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA172
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA172
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
gaacauacuc ccauuugaut t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA173
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA173
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
agaacauacu cccauuugat t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA174
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA174
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
cagaacauac ucccauuugt t                                            21
<212>Type:DNA/RNA
<211>Length:21
     SequenceName:HCV-siRNA175
     SequenceDescription:
Custom Codon
------------
Sequence Name:HCV-siRNA175
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
CCKKHHHKKH KKHGGLLALA LHHLALLAHH LALAL                             35
<212>Type:PRT
<211>Length:35
     SequenceName:核酸结合分子1
     SequenceDescription:
Sequence
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
KKALLALALH HLALLAHHLA LALKKA                                       26
<212>Type:PRT
<211>Length:26
     SequenceName:核酸结合分子2
     SequenceDescription:

Claims (11)

1.一种用于预防或治疗丙型肝炎的小干扰RNA制剂,其特征在于由小干扰RNA、肝细胞靶向的核酸导入载体组成;所述肝细胞靶向的核酸导入载体由肝细胞靶向分子和/或核酸结合分子组成;所述的小干扰RNA至少由序列表中的一个序列组成;所述肝细胞靶向分子为乙肝病毒的S基因区表达的大蛋白或中蛋白或小蛋白或部分S基因序列表达的蛋白;所述的核酸结合分子是富含碱性氨基酸和两性氨基酸分子,序列特征为KKALLALALHHLALLAHHLALALKKA或CCKKHHHKKHKKHGGLLALALHHLALLAHHLALAL,核酸结合分子是线性分子或分枝型的分子,序列特征见图1。
2.根据权利要求1所述的用于预防或治疗丙型肝炎的小干扰RNA制剂,其特征在于所述的小干扰RNA是由作用于一个靶序列的单一小干扰RNA组成,或由作用于一个基因的多个靶序列或多个基因上的靶序列的多个小干扰RNA组成;所述的靶序列是丙型肝炎病毒的RNA基因组序列,或发病宿主自身的内源基因的序列。
3.根据权利要求2所述的用于预防或治疗丙型肝炎的小干扰RNA制剂,其特征在于所述的发病宿主自身的内源基因的序列是人肝细胞的丙型肝炎病毒的受体CD81或Fas基因。
4.根据权利要求1或2所述的用于预防或治疗丙型肝炎的小干扰RNA制剂,其特征在于所述的小干扰RNA是化学合成的21-23碱基对的双链RNA;或是载体或表达框架表达的21-23碱基对的双链RNA,所述载体或表达框架为RNA聚合酶III启动子和RNA聚合酶III终止子调控小干扰RNA在哺乳动物细胞中的表达;所述的RNA聚合酶III启动子包括人源或鼠源的U6启动子和人H1启动子。
5.根据权利要求1所述的用于预防或治疗丙型肝炎的小干扰RNA制剂,其特征在于肝细胞靶向分子和核酸结合分子分别制备;或者是通过2-10个GG或其它氨基酸连接成融合蛋白而成;或者是聚乙二醇修饰的聚乙烯亚胺。
6.根据权利要求5所述的用于预防或治疗丙型肝炎的小干扰RNA制剂,其特征在于:肝细胞靶向分子或核酸结合分子是通过化学方法合成的,或者是在酵母细胞或哺乳动物细胞或昆虫细胞表达生产体系中表达。
7.根据权利要求6所述的用于预防或治疗丙型肝炎的小干扰RNA制剂,其特征在于:所述的在酵母细胞中表达生产体系包括酵母细胞和酵母表达载体,其中酵母细胞是毕赤酵母细胞、酿酒酵母细胞,酵母表达载体是pICZα或pYES2/CT或其它酵母表达载体;哺乳动物细胞表达生产体系包括人的CHO细胞和哺乳动物细胞外源基因表达载体;所述的昆虫细胞表达生产体系包括昆虫细胞和杆状病毒表达载体。
8.权利要求1所述的用于预防或治疗丙型肝炎的小干扰RNA制剂的制备方法,其特征在于:重组生产的靶向蛋白经超速离心或柱纯化后,透析后冻干,溶于pH5-7PBS溶液中与siRNA以30-100∶1的比例混合,使用2-4mm的电激杯,在220V和500-1500uF的条件下处理,使siRNA与靶向蛋白形成复合物,用于靶向肝细胞的siRNA的体内和体外转染。
9.权利要求1所述的用于预防或治疗丙型肝炎的小干扰RNA制剂的制备方法,其特征在于:靶向蛋白与核酸结合分子的融合分子经超速离心或柱纯化后,透析后冻干,溶于pH为5-7的PBS溶液中与siRNA以30-150∶1的比例混合,使siRNA与融合分子形成复合物,用于靶向肝细胞的siRNA的体内和体外转染。
10.权利要求1所述的用于预防或治疗丙型肝炎的小干扰RNA制剂的制备方法,其特征在于:化学合成的分枝型核酸结合分子与siRNA按10∶1的比例混合,静置一定时间后,形成复合物,再与靶向蛋白混合,最终形成靶向蛋白/分枝型核酸结合分子/siRNA复合物,用于靶向肝细胞的siRNA的体内和体外转染。
11.权利要求1所述的用于预防或治疗丙型肝炎的小干扰RNA制剂的制备方法,其特征在于:取适量乳糖化或半乳糖化蛋白溶于二甲基亚砜加入TEA,在氮的保护下搅拌1分钟,PEG3400-N-hydroxy succinimide以2-5∶1的重量比例加入,在室温下搅拌4小时,后使有机溶剂挥发完,获得乳糖化或半乳糖化蛋白螯合的PEG;制备好的乳糖化或半乳糖化蛋白螯合的PEG溶于DMSO,加入4-8倍体积的TEA,搅拌均匀,并在搅拌的条件下加入PEI,加入PEI质量是PEG的20-40倍,搅拌条件下过夜反应;获得的乳糖化或半乳糖化蛋白螯合的PEG-PEI和PEI按1∶1的比例混合溶于5mM HEPES缓冲液,合成的siRNA也溶于pH值为8的5mMHEPES缓冲液;两者按2∶1的质量比例混合,振荡混匀30秒,获得靶向肝细胞的小干扰RNA制剂。
CNB2004100515625A 2004-09-22 2004-09-22 用于预防或治疗丙型肝炎的靶向小干扰rna制剂及其制备方法 Expired - Fee Related CN100344331C (zh)

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CN102827841A (zh) * 2005-09-12 2012-12-19 索马根尼科斯公司 利用小干扰rna抑制病毒基因表达
US8829109B2 (en) * 2007-11-07 2014-09-09 University Of Utah Research Foundation Cleavable modifications to reducible poly(amido ethylenimine)s to enhance nucleotide delivery
CN108888777A (zh) * 2018-06-19 2018-11-27 徐州医科大学 一种半乳糖-peg-肌醇-pei基因载体及其与基因组成的复合物和应用
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