CN104059916A - 乙肝病毒特异性的microRNA样siRNA序列及其用途 - Google Patents
乙肝病毒特异性的microRNA样siRNA序列及其用途 Download PDFInfo
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Abstract
本发明提供针对乙肝病毒S基因的1个microRNA样siRNA的序列即msiHBs的核苷酸序列,及其二级结构。这个msiRNA与报告HBV复制型质粒pHY106+wta共转染HepG2和Huh7细胞后,可抑制HBsAg和HBeAg的表达;msiHBs转染HepG2.2.15细胞后,也可有效抑制HBsAg的和HBeAg表达水平,和上清中HBV病毒载量。另外msiRNA还具有免疫刺激活性,可以通过Toll样受体通路活化天然免疫。因此,这个msiRNA,具有抑制HBV基因表达和复制及免疫刺激双重活性,可在制备HBV慢性感染的治疗药物中应用。
Description
技术领域
本发明属于基因工程技术领域,涉及乙肝病毒特异性的microRNA样siRNA序列及其用途。
背景技术
乙型肝炎病毒(HepatitisBvirus,HBV)感染是导致慢性肝炎、肝硬化和肝细胞肝癌的重要原因之一。我国现有慢性HBV感染者约9300万,对乙型肝炎的防治研究一直是医疗卫生科研的重要领域。迄今,对HBV感染仍缺乏长期有效的抗病毒药物。干扰素(IFN-α)和核苷(酸)类似物治疗虽然可以取得一定疗效,但是仍存在持续应答率较低,有一定副作用以及长期应用易产生耐药性等问题。研究表明,慢性乙肝患者免疫系统不健全、免疫细胞应答数量和质量的不足,加上病毒颗粒和抗原的耐受原作用等因素形成HBV特异性免疫耐受,这种免疫耐受是导致HBV持续感染和久治不愈的重要根源,仅仅抑制病毒复制难以清除HBV感染。多样性、强大的特异性免疫是控制HBV感染的关键因素。因此,新的抗HBV治疗策略应该是:一方面高效抑制HBV基因表达和复制以清除“耐受原”,另一方面诱导并增强天然免疫和HBV特异性免疫应答以达到控制甚至清除HBV感染的目的。
抗病毒和免疫调节的联合治疗是公认的研究方向,但是方案还不成熟。如何活化肝脏天然免疫,尽可能降低病毒抗原和病毒颗粒的水平,以恢复或诱导HBV特异性免疫应答,可能在控制HBV感染方面发挥重要作用。microRNA样siRNA(microRNAlikesiRNA,msiRNA)是在siRNA的信使链引入非配对的类似microRNA的尿嘧啶(uracil,U)环状突起,这种结构修饰既增加了siRNA序列中U的数目,又降低了siRNA双链的亲和力,因而既具有很强的免疫刺激活性,又保持高度的RNAi活性。msiRNA首先可以与内体中丰富的Toll样受体(Tolllikereceptor,TLR)7/8结合,触发IFN通路,活化免疫应答;然后随着内体的裂解,msiRNA被释放到胞浆后,一方面可以触发RIGI通路,进一步活化天然免疫;另一方面进入RNAi通路,发挥直接的抗病毒作用;再者,乙肝病毒RNA降解产物又可以通过TLR3、TLR7/8,以及PKR等通路进一步活化细胞天然免疫;最后,病毒抗原表达的阻断还可以部分解除抗原特异性免疫耐受。msiRNA的免疫刺激和抗病毒双重活性,一方面肝脏天然免疫,诱导增强毒特异性免疫应答;另一方面发挥直接的病毒抑制作用,在乙型肝炎病毒慢性感染个体内打破病毒特异性免疫耐受中发挥重要作用,有可能开发为全新的乙型肝炎治疗方案。
发明内容
本发明的目的在于提供乙肝病毒特异性的miRNA样siRNA序列及其用途,为乙型肝炎的治疗提供双活性的siRNA,为打破HBV特异性免疫耐受提供新的技术方法,在此基础上可开发为全新的乙型肝炎治疗方案。
序列1为HBVS基因特异性msiRNA的sense链序列:
5’-GAAUCCUCUUUUUACCGCAtt-3’,和
antisense:3’-ttCUUAGGAGUGUUAUGGCGU-5’。
进一步,乙肝病毒特异性的microRNA样siRNA的二级结构如下:
本发明提供针对乙肝病毒S基因的1个msiRNA的序列,即msiHBs的核苷酸序列。这个msiRNA与报告HBV复制型质粒pHY106+wta共转染HepG2和Huh7细胞后,可抑制HBsAg和HBeAg的表达;msiHBs转染HepG2.2.15细胞后,也可有效抑制HBsAg的和HBeAg表达水平,和上清中HBV病毒载量。另外msiRNA还具有免疫刺激活性,可以通过Toll样受体通路活化天然免疫。因此,这个msiRNA,具有抑制HBV基因表达和复制及免疫刺激双重活性,可在制备HBV慢性感染的治疗药物中应用。
附图说明
图1是本发明msiHBs结构示意图;
图2是msiHBs在Huh7细胞抑制HBsAg表达示意图;
图3是msiHBs在HepG2细胞抑制HBsAg和HBeAg表达示意图;
图4是msiHBs在HepG2.2.15细胞抑制HBsAg和HBeAg表达示意图;
图5是msiHBs在HepG2.2.15细胞抑制HBVDNA复制示意图。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
图1为乙肝病毒特异性的microRNA样siRNA的二级结构。
(1)msiHBs在Huh7细胞中抑制HBV抗原表达。
HBVS基因特异性msiRNA与报告HBV复制型质粒pHY106+wta共转染Huh7细胞,72h收集上清,ELISA检测HBsAg的表达。结果,msiHBs显著抑制HBsAg表达(图2)。
(2)msiHBs在HepG2细胞中抑制HBV抗原表达。
HBVS基因特异性msiRNA与报告HBV复制型质粒pHY106+wta共转染HepG2细胞,72h收集上清,ELISA检测HBsAg和HBeAg的表达。结果,msiHBs显著抑制HBsAg和HBeAg的表达(图3)。
(3)msiHBs在HepG2.2.15细胞抑制HBV表达和复制。
HBVS基因特异性msiRNA转染HepG2.2.15细胞,72h收集上清,ELISA和RealtimePCR分别检测培养上清中HBsAg和HBeAg的和HBVDNA的水平;结果,msiHBs显著抑制HBsAg和HBeAg的表达(图4)和HBVDNA复制(图5)。
Claims (2)
1.乙肝病毒特异性的microRNA样siRNA序列,其特征在于:
序列1为HBVS基因特异性msiRNA的sense链序列:
5’-GAAUCCUCUUUUUACCGCAtt-3’,和
antisense:3’-ttCUUAGGAGUGUUAUGGCGU-5’。
2.按照权利要求1所述乙肝病毒特异性的microRNA样siRNA序列,其特征在于:乙肝病毒特异性的microRNA样siRNA的二级结构如下:
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US10799524B2 (en) | 2017-10-20 | 2020-10-13 | Dicerna Pharmaceuticals, Inc. | Methods for treating hepatitis B infection |
CN113186224A (zh) * | 2021-04-29 | 2021-07-30 | 中国人民解放军陆军军医大学士官学校 | 具有抑制乙肝病毒复制活性的microRNA-27a及其应用 |
US11534453B2 (en) | 2015-08-07 | 2022-12-27 | Arrowhead Pharmaceuticals, Inc. | RNAi therapy for hepatitis B virus infection |
US11590156B2 (en) | 2016-08-04 | 2023-02-28 | Arrowhead Pharmaceuticals, Inc. | RNAi agents for hepatitis B virus infection |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11534453B2 (en) | 2015-08-07 | 2022-12-27 | Arrowhead Pharmaceuticals, Inc. | RNAi therapy for hepatitis B virus infection |
US11590156B2 (en) | 2016-08-04 | 2023-02-28 | Arrowhead Pharmaceuticals, Inc. | RNAi agents for hepatitis B virus infection |
US10799524B2 (en) | 2017-10-20 | 2020-10-13 | Dicerna Pharmaceuticals, Inc. | Methods for treating hepatitis B infection |
US11052105B2 (en) | 2017-10-20 | 2021-07-06 | Dicerna Pharmaceuticals, Inc. | Methods for treating hepatitis B infection |
US11052104B2 (en) | 2017-10-20 | 2021-07-06 | Dicerna Pharmaceuticals, Inc. | Methods for treating hepatitis B infection |
US11273173B2 (en) | 2017-10-20 | 2022-03-15 | Dicerna Pharmaceuticals, Inc. | Methods for treating hepatitis B infection |
CN113186224A (zh) * | 2021-04-29 | 2021-07-30 | 中国人民解放军陆军军医大学士官学校 | 具有抑制乙肝病毒复制活性的microRNA-27a及其应用 |
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