CN1277926C - 针对乙肝病毒基因的siRNA序列及其用途 - Google Patents
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Abstract
本发明提供针对乙肝病毒基因的2条siRNA的cDNA靶序列，SEQ ID NO.1为anti-s siRNA1的核苷酸序列，SEQ ID NO.2为anti-s siRNA2的核苷酸序列。这2条siRNA分别和报告质粒pGFP-S共转染HepG2细胞后，可抑制报告基因绿荧光蛋白的表达；这2条siRNA转染HepG2.2.15细胞后，可有效抑制HBsAg的表达。因此，针对HBV(ayw亚型)S基因的2条siRNA，可抑制HBsAg的表达，可在制备HBV慢性感染的治疗药物中应用。
Description
技术领域
本发明属于生物技术，涉及基因工程技术领域和生物信息学领域，具体地涉及针对乙肝病毒(HBV)基因的siRNA靶序列的设计、合成及其在体内外抑制HBV的应用。
背景技术
RNA干扰(RNA interference，RNAi)现象的发现，被《SCIENCE》杂志和美国科学促进会评为2002年十大科学成就之一。RNA干扰是指同源双链RNA的导入引起目标基因的不表达或减效表达，在哺乳动物细胞中，21-23个核苷酸长度的双链RNA(double-stranded RNA，dsRNA)，可有效降解同源RNA，从而阻断蛋白质的表达，这些特定长度的dsRNA被称为siRNA(small interferingRNAs)。RNA干扰被称为RNA基础上的细胞“免疫”系统。
经证实RNA干扰技术具有以下特点：(1)特异性高：一个碱基的不同就可导致作用的显著降低；(2)高效：少量siRNA分子就能较彻底抑制细胞内相应基因的表达；(3)稳定：由于RNA干扰采用的是双链RNA，它比较稳定，不易被降解，因此RNA干扰不仅在体外，而且在动物体内亦具有较好的效果；(4)筛选周期短：针对某一基因的有效siRNA序列筛选的方法较简单，因此研发周期较短。
RNAi在HIV的体外实验中取得了较理想的结果。Rossi等将HIV-1编码rev的基因和与它同源的双链RNA共转染293细胞，rev基因的表达被显著抑制；同时将siRNA的抑制效应与反义RNA和核酶等进行了比较，发现只有siRNA组抑制了HIV rev-EGFP的表达，其抑制率达到90％，远优于反义RNA和核酶组，这一结果同样被Novina等学者所证实。Novina等用针对CD4的dsRNA能将细胞表面HIV病毒的受体表达减少75％，从而抵抗HIV病毒的感染。因此，针对病毒基因的siRNA是慢性病毒性感染的最佳治疗策略，具有广阔的应用前景。
慢性乙型肝炎是我国的高发疾病之一，是导致肝纤维化和肝癌的主要原因。目前治疗乙型肝炎的药物主要是以α干扰素为主的免疫调节剂和以拉米呋啶、阿德福韦为主的核苷类似物，但治疗效果仍不满意。HBV DNA的持续复制是导致乙型肝炎慢性化的主要原因。因此有效抑制HBV DNA复制，是治疗慢性乙型肝炎和控制HBV持续感染的关键。
HBV DNA的复制是一个DNA-RNA-DNA的过程，其中前基因组RNA含有病毒DNA上全部遗传信息，是子代病毒前基因组反转录的模板。如果能够特异性阻断HBV前基因组RNA的逆转录及3.5kb，2.4kb，2.1kb和0.9Kb四种未剪切mRNA的翻译表达，将有效抑制HBVcccDNA在肝细胞中的再生成和子病毒的产生。因为HBV前基因组RNA的逆转录及未剪切mRNA的翻译过程均发生在细胞浆，容易受到针对靶序列的siRNAs的攻击而特异性地降解，所以本专利利用RNA干扰技术特异性降解HBV RNAs，达到抗HBV的作用。
发明内容
本发明的目的是根据siRNA的设计原理，提供针对HBV(ayw亚型)S基因的siRNA的cDNA靶序列。根据cDNA靶序列，化学合成法合成siRNA。这2条siRNA分别和报告质粒pGFP-S共转染HepG2细胞后，可抑制报告基因绿荧光蛋白的表达；这2条siRNA转染HepG2.2.15细胞后，可有效抑制HBsAg的表达。因此，针对HBV(ayw亚型)S基因的siRNA，可抑制HBsAg的表达，可在制备治疗HBV慢性感染的药物中应用。
本发明提供针对HBV(ayw亚型)S基因的2条siRNA的目标cDNA靶序列为：
SEQ ID NO.1(anti-s siRNA1)
5’-AAACCTTCGGACGGAAATTGC-3’
SEQ ID NO.2(anti-s siRNA2)
5’-AATACCGCAGAGTCTAGACTC-3’
具体实施方式
本发明结合实施例作进一步说明。
实施例一 合成干扰RNA体外抑制HBV表面抗原融合基因的表达
1.siRNA的设计 根据siRNA的设计原理，从转录本AUG起始密码子开始，搜寻HBV ayw亚型的S基因中的AA序列，记录跟每个AA 3’端相邻的19nt作为候选的siRNA靶位点，选择GC含量在40-55％的siRNA序列，通过Genebank的数据库的Blast分析，将潜在的序列和相应的人基因组数据库进行比较，排除那些和其他编码序列/EST同源的序列，设计针对HBV(ayw亚型)S基因的2条siRNA的目标cDNA靶序列。序列如下：
(anti-s siRNA1)5’-AAACCTTCGGACGGAAATTGC-3’
(anti-s siRNA2)5’-AATACCGCAGAGTCTAGACTC-3’
2.siRNA的化学合成和双链退火 根据siRNA设计原则，分别合成anti-ssiRNA1和anti-s siRNA2的正义链和反义链如下。每条链的5’端为磷酸盐，3’端羟基化，有两个碱基外悬，通过化学合成而得。形成双链siRNA的退火处理：正义链和反义链各20μM，加入退火缓冲液(100μM KAc，30mM HEPES-KOH，pH7.4，2mM MgAc2)，90℃水浴1min，37℃水浴1h。
anti-s siRNA1：
5’-ACCUUCGGACGGAAAUUGCdTdT-3’(sense siRNA)
5’-GCAAUUUCCGUCCGAAGGUdTdT-3’(antisense siRNA)
anti-s siRNA2：
5’-UACCGCAGAGUCUAGACUCdTdT-3’(sense siRNA)
5’-GAGUCUAGACUCUGCGGUAdTdT-3’(antisense siRNA)
3.anti-s siRNA抑制报告质粒pGFP-S和siRNA共转染HepG2细胞的绿荧光蛋白表达和HBV S基因表达的实验
(1)报告质粒pGFP-S和siRNA共转染HepG2细胞 报告质粒pGFP-S是表达报告基因绿荧光蛋白和HBsAg融合蛋白的质粒。HepG2细胞按照1×105/孔接种24孔板，培养24小时后达到80％-90％融合率，共转染pGFP-S和siRNA，具体步骤参照Invitrogen公司Lipofectamine2000说明书。6小时后换10％FCS DMEM，同时设置只转染pGFP-S载体的阴性组，每组重复3孔。
(2)荧光显微镜观察细胞绿荧光蛋白表达 转染48小时后，在倒置荧光显微镜(DM IRB，Leica)下观察绿荧光蛋白在HepG2细胞中的表达情况。结果显示，与pGFP-S载体的阴性组相比，转染pGFP-S和anti-S siRNA的HepG2细胞的荧光强度减弱，荧光细胞数减少。
(3)流式细胞仪观察细胞绿荧光蛋白表达 转染48小时后，常规消化HepG2细胞，离心悬液，重悬于PBS，流式细胞仪检测表达荧光蛋白的阳性细胞数比例和平均荧光强度。结果显示，与pGFP-S载体的阴性组相比，转染pGFP-S和siRNA的HepG2的阳性细胞数比例降低，平均荧光强度降低。
(4)逆转录-实时荧光定量PCR法检测HBV S基因RNA的表达RNA准备：转染72小时后，收获HepG2细胞，Trizol法抽提RNA，方法参照Invitrogen公司说明书。逆转录，过程参照MBI操作说明书。
荧光染料法定量PCR，
上游引物：5′-CTCACAATACCGCAGAGTC-3′；
下游引物：5′-TAAACTGAGCCAGGAGAAA-3′；内参照GAPDH，上游引物：5′-ACAGTCAGCCGCATCTTCTTT-3′，下游引物：5′-GCAACAATATCCACTTTACCAGAG-3′。PCR条件为：95℃ 3min，再94℃ 30s，56℃ 30s，72℃ 45s，25个循环，荧光检测点选择72℃。同时做阳性对照(直接以pGFP-S质粒DNA为模板组)和阴性对照(不加模板组)。实时定量PCR在25循环定点检测显示，与阴性对照相比，anti-S siRNA1和anti-S siRNA2对S基因的抑制率均达到50％以上。
4.anti-s siRNA抑制对siRNA转染HepG2.2.15细胞的HBsAg和HbeAg表达的实验
(1)siRNA转染HepG2.2.15细胞 HepG2.2.15细胞按照1×104接种24孔板，培养24小时后达到30％-40％融合率，转染siRNA各0.84μg，具体步骤参见Invitrogen公司OLIGOFECTAMINE试剂说明书。设置无关对照siGFP组(FEBS Letters 543(2003)51-54)：sense RNA 5-GGCUACGUCCAGGAGCGCACC-3，anti-sense RNA 5-UGCGCUCCUGGACGUAGCCTT-3。6小时后换10％FCS DMEM。每组设3个复孔。
(2)放射免疫法检测HBsAg和HBeAg浓度变化转染HepG2.2.15细胞后第48h，分别收集细胞培养液，离心去除细胞碎片，上清进行放射免疫法检测，检测方法参见试剂盒说明。转染anti-S siRNA1和anti-S siRNA2后，HepG2.2.15细胞上清中的HBsAg和HBeAg的浓度较HepG2.2.15细胞低，anti-SsiRNA1和anti-S siRNA2对HBsAg和HBeAg的抑制率均达到50％以上。
本发明是结合最佳实施例进行描述的，然而在阅读了本发明的上述内容后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (2)
1.一种乙肝病毒基因的ayw亚型S基因的siRNA的cDNA靶序列，其核苷酸序列为：AAACCTTCGG ACGGAAATTG C。
2.权利要求1所述的cDNA靶序列在制备抑制乙肝病毒复制药物中的应用。
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