CN114990116B - 抑制人巨细胞病毒即刻早期基因的siRNA及其应用 - Google Patents
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Abstract
本发明涉及一种生物医药技术领域,具体涉及一种抑制人巨细胞病毒即刻早期基因的siRNA及其应用。本发明所述的siRNA分子包括核苷酸序列正义链和反义链,其中正义链的序列如序列表SEQ ID NO:1所示,反义链的序列如序列表SEQ ID NO:2所示;将本发明所述的siRNA分子转入人脑胶质瘤细胞后能明显抑制人巨细胞病毒即刻早期基因及蛋白的表达,并且能够有效抑制ATF5表达,从而抑制胶质瘤细胞增殖能力。
Description
技术领域
本发明属于生物医药技术领域,具体涉及抑制人巨细胞病毒即刻早期基因的siRNA及其应用。
背景技术
RNA干扰(RNAi)是一种强大的基因沉默技术,是通过导入与内源性mRNA同源的双链RNA(double-stranded RNA,dsRNA),来诱发同源靶mRNA高效、特异性地降解,从而导致靶基因沉默的现象。siRNA是RNA干扰的中间产物,通常是长度为19-25个核苷酸的双链RNA,在每条链的3’端都有两个悬挂碱基。siRNA能够抑制靶基因的表达,属于转录水平后调控。siRNA作为一种有效的基因研究工具,它的广泛应用加快了功能基因组学的研究步伐,同时也推动了基因治疗等相关领域的研究。
人巨细胞病毒(human cytomegalovirus,HCMV)属β疱疹病毒亚科,为双链线状DNA病毒,是疱疹病毒科中最大的DNA病毒。有研究资料证实HCMV感染与肿瘤的发生发展有密切关系。比如,Cobbs等人研究发现在恶性胶质瘤中HCMV即刻早期蛋白阳性率达60%(参考文献1)。再如,Scheurer等人明确提出了通过使用优化后的免疫组化方法,在石蜡包埋的恶性胶质瘤组织中检测到即刻早期蛋白的表达率为100%,在低度胶质瘤中为82%;同时,他们在上述胶质瘤组织中使用原位杂交的方法检测到HCMV特异性的寡核苷酸链(参考文献2)。即刻早期基因(immediate early,ie)是人巨细胞病毒原发感染或复发激活感染后最先表达的基因。UL122(ie2)为最主要的ie基因,它编码分子量为86kDa(又称IE86或IE2)的即刻早期蛋白,这种蛋白在HCMV基因表达的后续调控过程中起着主导作用。其中,有资料显示,在胶质瘤细胞系U87MG中,IE86与转录激活因子5(activating transcription factor 5,ATF5)共定位于细胞核;此外,IE86在细胞内能与ATF5相互作用,提高ATF5的乙酰化水平,进而可能在已有癌变基础上进一步促进胶质瘤细胞增殖和侵袭(参考文献3)。因此,抑制UL122基因表达不仅可以深入研究HCMV感染与恶性神经胶质瘤发生发展的分子机制,更有望为HCMV感染甚至是恶性胶质瘤疾病提供新的治疗手段。
RNA干扰(siRNA)是机体内广泛存在的一种双链RNA介导的序列特异性基因沉默现象,它可与蛋白质结合形成RNA诱导沉默复合物(RNA-induced silencing complex,RISC),该复合物能够结合到同源的mRNA上并诱导其降解。目前,已有报道关于抑制UL122基因的siRNA。比如,文献4提供了一组抑制UL122基因的siRNA,该siRNA在人胚肾AD293细胞系中对UL122基因的沉默效率较高,但是对IE86蛋白的抑制率远达不到要求。
因此,亟需一种特异性强、沉默效率高的靶向HCMV UL122基因的siRNA。
参考文献:
1.Cobbs C S,Harkins L Samanta M,et al.Human cytomegalovirus infectionand expression in human malignant glioma[J].Cancer research,2002,62(12):3347-3350.
2.Scheurer ME,Bondy ML,et al.Detection of human cytomegalovirus indifferent histological types of gliomas[J].Acta Neuropathol.2008,116(1):79-86.
3.Hu M,Wang B,et al.Human cytomegalovirus immediate-early proteinpromotes survival of glioma cells through interacting and acetylating ATF5[J].Oncotarget.2017,8(19):32157-32170.
4.Duan,Tao,et al.Efficient inhibition of human cytomegalovirus UL122gene expression in cell by small interfering RNAs[J].Journal of BasicMicrobiology 2009,49:531–537.
发明内容
针对现有技术存在的沉默效率不高问题,本发明提供一种抑制人巨细胞病毒即刻早期基因的siRNA及其应用,该siRNA分子及组合物具有特异性强、对HCMV UL122抑制效率高的特点,同时能够抑制胶质瘤细胞生长。
第一方面,本发明涉及一种双链siRNA分子,所述的siRNA分子由正义链和反义链组成;
所述siRNA的正义链为5’-GAACUUAGGAGAAAGAUGAUG-3’(SEQ ID NO:1),其中,5’端第14位碱基为2’-甲氧基修饰碱基;
所述siRNA的反义链为5’-UCAUCUUUCUCCUAAGUUCAU-3’(SEQ ID NO:2),其中,5’端第9位碱基为2’-甲氧基修饰碱基。
第二方面,本发明涉及一种双链siRNA分子组合物,所述的组合物包括上述siRNA,还包括siRNA2;所述的siRNA2由正义链和反义链组成;
所述siRNA2的正义链为5’-GCUAAAAAGGAUGAACUUAGG-3’(SEQ ID NO:3),其中,5’第16位碱基为2’-甲氧基修饰碱基;
所述siRNA2的反义链为5’-UAAGUUCAUCCUUUUUAGCAC-3’(SEQ ID NO:4),其中,5’第7位碱基为2’-甲氧基修饰碱基。
第三方面,本发明涉及上述siRNA分子、上述siRNA分子组合物在制备降低胶质瘤细胞中人巨细胞病毒即刻早期基因表达试剂中的应用;其中,所述的人巨细胞病毒即刻早期基因是UL122。
第四方面,本发明涉及一种试剂盒,该试剂盒包含上述siRNA分子、上述siRNA分子组合物。
第五方面,本发明涉及上述siRNA分子、上述siRNA分子组合物和上述试剂盒在制备预防和/或治疗病毒感染的产品中的应用。其中,所述的病毒感染是人巨细胞病毒感染。
第六方面,本发明还涉及上述siRNA分子、上述siRNA分子组合物和上述试剂盒在制备预防和/或治疗人脑胶质瘤的产品中的应用。
与现有技术相比,本发明的有益效果有:本发明设计了针对UL122基因RNA的小干扰RNA序列siRNA和siRNA2,该siRNA及其与siRNA2形成的siRNA分子组合物能够显著下调UL122基因在胶质瘤细胞中mRNA水平和蛋白水平的表达,且能够降低IE86募集ATF5的能力,从而抑制胶质瘤U87MG细胞系的增殖能力,进而为胶质瘤的治疗提供了新的治疗途径。
附图说明
图1为siRNA对HCMV UL122基因沉默效果的检测图;
图2为siRNA对HCMV IE2蛋白的抑制效果的检测图;
图3为siRNA对ATF5基因沉默效果的检测图;
图4为siRNA对ATF5蛋白的抑制效果的检测图;
图5为siRNA对U87MG细胞增殖能力的抑制效果的检测图;
图6为siRNA对U87MG细胞凋亡能力的促进效果的检测图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施例,进一步阐明本发明,但下述实施例仅仅为本发明的优选实施例,并非全部。基于实施方式中的实施例,本领域技术人员在没有做出创造性劳动的前提下所获得其它实施例,都属于本发明的保护范围。下述实施例中的实验方法,如无特殊说明,均为常规方法,下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
一、实验材料
U87MG细胞系,购自ATCC。
二、实验方法
1、细胞培养
使用含有10%FBS的DMEM培养基U87MG细胞,培养条件为37℃、5%CO2。
2、病毒感染
(1)准备处于对数生长期的达到80-90%汇合度的U87MG细胞,加病毒前将其用含2%FBS培养基饥饿处理12小时,不加病毒组为对照组;
(2)从-86℃冰箱取出HCMV AD169病毒,流水冲至完全融化,加入饥饿处理后的细胞中,病毒感染复数MOI=1,轻摇混匀后放入含5%二氧化碳的37℃恒温培养箱中,培养2小时,每15min轻轻晃动培养瓶,使病毒充分分散;
(3)2小时后,弃去含有病毒的培养液,PBS洗2次,更换新鲜的含2%FBS的DMEM培养液维持;
(4)每数小时在倒置显微镜下观察细胞形态并拍照记录;
(5)于加病毒后的72小时,收集感染HCMV的U87MG细胞,进行下步实验。
3、细胞铺板和转染
(1)将细胞(5×105个/孔)接种到6孔细胞培养板中,培养24小时。按照脂质体转染试剂2000(购自于Invitrogen公司,货号11668-019)的说明书进行转染,实验分为未转染组、阴性对照组和实验组。其中阴性对照组siRNA为通用对照siRNA,即si-NC,与HCMV UL122基因的序列无同源性。按siRNA:脂质体2000体积之比为1:2将siRNA转染细胞;
(2)稀释相应比例的质粒DNA于250μL Opti-MEM中,轻轻振荡混匀;
(3)取相应体积的脂质体于250μL Opti-MEM中,振荡混匀,室温放置5min;
(4)将250μL稀释的脂质体加入到250μL稀释的DNA溶液中,轻轻振荡混匀,室温孵育20min,使脂质体复合物形成;
(5)将细胞培养板中的细胞更换成新鲜的含有10%FBS的DMEM培养液;
(6)将转染混合物500μL轻轻滴加到每个培养孔内,轻轻前后晃动孔板,混匀;
(7)直接放入5%CO2培养箱中,37℃培养8小时,除去转染混合物,加入含2mL含有10%胎牛血清的MEM培养基,培养36小时,进行以下实验。
4、荧光定量PCR检测HCMV UL122 mRNA水平
(1)提取总RNA:
a.于6孔细胞培养板中加入1mL Trizol(购自于Invitrogen公司,货号:15596018)溶液裂解,置于冰上孵育15分钟使细胞解离内容物释放;
b.取一个新的1.5mL EP管,将细胞裂解产物移其中,向产物中加入200μL氯仿(购自国药集团化学试剂有限公司,货号10006818),手动震荡15秒,冰上放置5分钟,4℃离心机12000×g离心15分钟,离心后液体分为三相:下层红色的酚-氯仿相、中间的蛋白层及上层的水相;
c.将上层的水相移入新的EP管中约为500μL,加入等体积的异丙醇(购自国药集团化学试剂有限公司,货号40064360),-20℃静置90min,4℃离心机12000×g离心15分钟,弃掉上清;
d.加入1mL DEPC水配制的75%乙醇洗涤RNA沉淀,然后使用4℃离心机10000×g离心5分钟,弃上清;
e.放置于无菌通风橱中干燥5-10分钟,加入30μL无RNase的去离子水反复吹打混匀,溶解所得的RNA沉淀;
f.紫外分光光度计测定OD260、OD280,调整至终浓度分装保存。
(2)反转录合成第一条链cDNA
按照BeyoRTTM II cDNA第一链合成试剂盒(购自上海碧云天生物技术有限公司,货号:D7168S)说明书进行以下操作:
a.取高压3次的EP管置于冰上预冷,将反应物按表1体系加入EP管中;
表1总RNA反转录反应体系1
b.将以上反应物混合均匀,65℃水浴10分钟,以确保RNA的二级结构变性;
c.将反应管从恒温电热水浴锅中取出后立即置于冰上;
d.将混合有模板和引物的反应管与表2中的反应体系相混合;
表2总RNA反转录反应体系2
e.将以上反应物混合均匀,瞬离,42℃水浴1小时;
f.将反应管从42℃取出置于70℃水浴5分钟,反应结束后将反应管置于冰上静置5分钟,置于-86℃低温冰箱中保存。
(3)实时荧光定量PCR:
设计HCMV UL122基因、ATF5基因、内参GAPDH和β-actin基因特异性扩增引物,交由上海生工生物工程股份有限公司合成,引物序列见表3;以反转录所得cDNA为模版,进行PCR反应,反应体系见表4。
表3引物序列表
表4实时荧光定量PCR体系
采用两步法PCR扩增程序:95℃预变性10s,反应循环为95℃变性5s,60℃退火延伸35s,共40个循环。反应结束后进行实验特异性分析:确认Real time PCR的扩增曲线和融解曲线。数据结果以每组样品的GAPDH和β-actin基因表达为内参照,采用2-ΔΔCT法进行相对定量分析,分析相对于正常细胞组,胶质瘤样本和细胞组中靶基因表达的变化倍数。最终计算公式:变化倍数=2-ΔCT;ΔCt=(Ct靶基因-Ct内参)处理组或ΔCt=(Ct靶基因-Ct内参)对照组,ΔCt处理组/ΔCt对照组即为相对表达量。其中Ct值为PCR过程中扩增产物的荧光信号达到设定的阈值时所经过的循环次数。最终进行统计学分析。
5、CCK-8检测胶质瘤细胞增殖能力
CCK-8试剂盒购自北京索莱宝公司,货号:CA1210。
(1)接种:取对数生长期的U87细胞,消化后用含10%血清的完全培养基重悬,计数,将细胞浓度调至105个/mL,每孔100μL接种于96孔板中,仅含培养基的孔作为对照,边孔中加入200μL PBS,防止蒸发,培养48小时;
(2)根据分组,转染siRNA,培养48小时;
(3)向培养板每孔加入10μLCCK-8溶液,置于培养箱内孵育2小时;
(4)用酶标仪测定在450nm处的吸光度,并计算细胞的增殖率。
5、流式细胞术检测胶质瘤细胞凋亡能力
PI/FITC-Annexin V staining kit购自Invitrogen,货号:V13242。
(1)收集所有细胞上清与脱落细胞置于入15mL离心管中;
(2)用0.25%胰酶消化细胞,10%MEM培养液终止消化,并吹打成单细胞悬液;
(3)收集培养液和细胞于15mL离心管,1500rpm离心10min,弃上清;
(4)用100μL 10%MEM培养液重悬细胞,移至1.5ml的EP管;
(5)每管加入20μL annexin和2μL PI工作液,避光室温孵育15分钟;
(6)用流式细胞仪检测5000个细胞红色荧光和橙色荧光信号,采用流式四象限散点图表示实验结果。每个样品需重复检测三次,后做统计学分析。
6、Western-blot检测蛋白表达
(1)细胞总蛋白提取
a.清洗:弃去旧培养液,用4℃预冷的PBS洗3次,弃去PBS;
b.裂解:向培养瓶中加入300μL含1μmol/L PMSF的RIPA裂解液,冰上静置10min,慢慢摇晃并用细胞刮刀将细胞刮至培养瓶一侧,将细胞裂解液移至离心管;
c.12000rpm离心8min,上清液即细胞总蛋白,分装后至于-20℃保存。
(2)SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE电泳)
a.连接凝胶电泳装置,按表5所示,分别配置浓缩胶和分离胶,然后灌胶;
表5聚丙烯酰胺凝胶的配制
b.处理蛋白样品:蛋白样品和5×SDS蛋白上样缓冲液按1∶4的比例混匀,置于沸水浴10min,使蛋白质变性,冷却备用;
c.上样:按顺序向加样槽中加入样品,每孔15-20μL,并在邻近孔中加入3μL彩色预染蛋白分子量Marker;
d.电泳:安装好电泳设备,接通电源,设定电压80V,时间40min,待样品电泳至分离胶上端并处于同一水平线时,更换电压为120V,待溴酚蓝标记电泳至玻璃板底端时停止电泳。
(3)转膜
a.平衡:电泳结束后,将凝胶置于预冷的电转液中平衡15min;
b.处理PVDF膜和滤纸:剪裁与凝胶相同大小的PVDF膜及6张滤纸,将PVDF膜置于甲醇中浸泡1min,再置于电转液中浸泡5min,将滤纸和海绵浸泡于电转液中;
c.安装电转装置;
d.电转:将电转仪置于冰上,按照正负极安装电转装置,电转槽中加入电转缓冲液,接通电源,设定电流为130mA。
(4)免疫反应检测蛋白表达
a.封闭:取出PVDF膜,用TBST洗膜3次,每次10min,然后将膜放入5%的脱脂奶粉中室温封闭2小时,封闭结束后,用TBST洗膜3次,每次10min;
b.孵育一抗:用5%脱脂奶粉按照抗体说明书要求稀释一抗,一抗包括anti-IE2(购自Santa Crus Biotechnology,货号:sc-69835)、anti-ATF5(购自Abcam,货号:ab60126)和anti-β-actin(购自Abcam,货号:ab179467);稀释后的一抗按0.1mL/cm2比例加入,将置于4℃过夜,第二天取出,置于摇床上复苏2小时,将PVDF膜取出,TBST洗膜3次,每次10min;
c.孵育二抗:按照一抗的种属,加入相应HRP标记的山羊抗兔IgG(购自Abcam,货号:6721)和山羊抗鼠IgG(购自Abcam,货号:205719),置于摇床室温孵育2小时,TBST洗膜3次,每次10min。
(5)化学发光及图像采集
PierceTM Fast Western Blot Kit购自Thermo Fisher Scientific,货号:35050
a.按1∶1混合试剂;
b.将PVDF膜取出,膜的下端轻触吸水纸,除去膜上多余液体,将其置于洁净的小皿中,蛋白面向上,并置于避光的暗盒,将配置好的发光检测液滴加到膜上,使膜被检测液完全覆盖,室温避光反应5min;
c.将膜置于蛋白凝胶成像仪中曝光并采集图像。
(6)凝胶图像分析
用Quantity One图像分析软件分析采集的图像,得到目的蛋白条带的灰度值与内参蛋白条带的灰度值相比,得到的比值为目标蛋白的相对表达量。
7、统计学分析
所有实验均为多组平行实验(≥3),实验所得数据采用Graphpad 6.0软件进行分析处理。计量资料使用表示,多组间相互比较采用单因素方差分析,两组之间比较采用独立样本t检验,p<0.05时认为存在的差异具有显著性。
实施例1一种siRNA分子
siRNA1:正义链为5’-GAACUUAGGAGAAAGAUGAUG-3’(SEQ ID NO:1),其中,5’端第14位碱基为2’-甲氧基修饰碱基;反义链为5’-UCAUCUUUCUCCUAAGUUCAU-3’(SEQ ID NO:2),其中,5’端第9位碱基为2’-甲氧基修饰碱基。
实施例2一种siRNA分子
siRNA2:正义链为5’-GCUAAAAAGGAUGAACUUAGG-3’(SEQ ID NO:3),其中,5’第16位碱基为2’-甲氧基修饰碱基;反义链为5’-UAAGUUCAUCCUUUUUAGCAC-3’(SEQ ID NO:4),其中,5’第7位碱基为2’-甲氧基修饰碱基。
实施例3一种siRNA分子组合物由实施例1所述的siRNA1和实施例2所述的siRNA2组成。
对比例1
参考文献(Duan,Tao,et al.Efficient inhibition of human cytomegalovirusUL122 gene expression in cell by small interfering RNAs[J].Journal of BasicMicrobiology 2009,49:531–537.)中所述的si122-1(nts 618–638)序列。
对比例2
对比例1所述参考文献中的si122-2(nts 1103–1123)序列。
对比例3
对比例1所述参考文献中的si122-3(nts 1414–1434)序列。
实验例1合成HCMV UL122基因的siRNA序列
根据Genebank中的HCMV UL122基因序列(ID:X92746.1),由上海生工公司合成以下两对经修饰的siRNA,具体序列如下:
siRNA1:正义链为5’-GAACUUAGGAGAAAGAUGAUG-3’(SEQ ID NO:1),其中,5’端第14位碱基为2’-甲氧基修饰碱基;反义链为5’-UCAUCUUUCUCCUAAGUUCAU-3’(SEQ ID NO:2),其中,5’端第9位碱基为2’-甲氧基修饰碱基。
siRNA2:正义链为5’-GCUAAAAAGGAUGAACUUAGG-3’(SEQ ID NO:3),其中,5’第16位碱基为2’-甲氧基修饰碱基;反义链为5’-UAAGUUCAUCCUUUUUAGCAC-3’(SEQ ID NO:4),其中,5’第7位碱基为2’-甲氧基修饰碱基。
另外,合成对比例1-3相应的siRNA序列。
实验例2 siRNA对HCMV UL122沉默效率的检测实验
将实施例1-3和对比例1-3所述的siRNA瞬时转染到经HCMV感染后的U87MG细胞中,48小时后检测UL122 mRNA和蛋白表达水平。
UL122 mRNA表达水平:根据图1所示,与siNC组相比,siRNA1和siRNA2对U87MG细胞的UL122 mRNA表达抑制率分别为84%、83%;siRNA1+siRNA2共转染的抑制效率更高,达到90%;远高于对比例1-3使用的siRNA。
IE2(或IE86)蛋白水平:根据图2所示,与siNC组相比,siRNA1和siRNA2对U87MG细胞的IE2蛋白表达抑制率分别为85%、87%;siRNA1+siRNA2共转染的抑制效率更高,达到92%。
以上数据表明本发明提供的siRNA(实施例1-3)能在mRNA和蛋白水平明显抑制HCMV UL122的表达。
实验例3 siRNA对ATF5表达的影响
将实施例1-3和对比例1-3所述的siRNA瞬时转染到经HCMV感染后的U87MG细胞中,48小时后检测ATF5 mRNA和蛋白表达水平。
ATF5 mRNA表达水平:根据图3所示,与siNC组相比,siRNA1和siRNA2对U87MG细胞的ATF5 mRNA表达抑制率分别为76%、75%;siRNA1+siRNA2共转染的抑制效率更高,达到82%;远高于对比例1-3使用的siRNA。
ATF5蛋白表达水平:根据图4所示,与siNC组相比,siRNA1和siRNA2对U87MG细胞的ATF5蛋白表达抑制率分别为64%、61%;siRNA1+siRNA2共转染的抑制效率更高,达到50%。
以上数据表明本发明提供的siRNA(实施例1-3)能在mRNA和蛋白水平明显抑制ATF5的表达。
实验例4 siRNA对U87MG细胞增殖能力的影响
将实施例1-3和对比例1-3所述的siRNA瞬时转染到经HCMV感染后的U87MG细胞中,48小时后使用CCK-8试剂盒检测U87MG细胞存活率。
根据图5所示,与siNC组相比,转染siRNA1和siRNA2后,U87MG细胞存活率仅为36%、30%;siRNA1+siRNA2共转染组的细胞存活率更低,约为23%。因此,本发明提供的siRNA对U87MG细胞增殖能力的抑制效果远优于对比例1-3使用的siRNA。
实验例5 siRNA对U87MG细胞凋亡能力的影响
将实施例1-3和对比例1-3所述的siRNA瞬时转染到经HCMV感染后的U87MG细胞中,48小时后检测U87MG细胞凋亡率。
根据图6所示,与siNC组相比,转染siRNA1和siRNA2后,U87MG细胞凋亡率为22%、25%;siRNA1+siRNA2共转染组的细胞凋亡率更高,约为29.5%。因此,本发明提供的siRNA对U87MG细胞凋亡能力的促进作用远优于对比例1-3使用的siRNA。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 青岛大学
<120> 抑制人巨细胞病毒即刻早期基因的siRNA及其应用
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Claims (3)
1.一种双链siRNA分子组合物,其特征在于:包括双链siRNA1分子以及siRNA2分子
所述的siRNA1分子由正义链和反义链组成;
所述siRNA1的正义链为5’-GAACUUAGGAGAAAGAUGAUG-3’ (SEQ ID NO:1),其中,5’端第14位碱基为2’-甲氧基修饰碱基;
所述siRNA1的反义链为5’-UCAUCUUUCUCCUAAGUUCAU-3’ (SEQ ID NO:2),其中,5’端第9位碱基为2’-甲氧基修饰碱基;
所述siRNA2分子由正义链和反义链组成;
所述siRNA2的正义链为5’-GCUAAAAAGGAUGAACUUAGG-3’ (SEQ ID NO:3),其中,5’第16位碱基为2’-甲氧基修饰碱基;
所述siRNA2的反义链为5’-UAAGUUCAUCCUUUUUAGCAC-3’ (SEQ ID NO:4),其中,5’第7位碱基为2’-甲氧基修饰碱基。
2.权利要求1所述的双链siRNA分子组合物在制备降低胶质瘤细胞中人巨细胞病毒即刻早期基因表达试剂中的应用;
所述的人巨细胞病毒即刻早期基因是UL122。
3.一种试剂盒,该试剂盒包含权利要求1所述的双链siRNA分子组合物。
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