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WO2019000148A1 - 一种人ABCB6基因的siRNA及其应用 - Google Patents

一种人ABCB6基因的siRNA及其应用 Download PDF

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Publication number
WO2019000148A1
WO2019000148A1 PCT/CN2017/089928 CN2017089928W WO2019000148A1 WO 2019000148 A1 WO2019000148 A1 WO 2019000148A1 CN 2017089928 W CN2017089928 W CN 2017089928W WO 2019000148 A1 WO2019000148 A1 WO 2019000148A1
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sirna
abcb6 gene
gene
abcb6
sequence
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French (fr)
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毛吉炎
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Shenzhen Biocan Technologies Co Ltd
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Shenzhen Biocan Technologies Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention belongs to the field of molecular genetics, and particularly relates to an siRNA capable of inhibiting the expression of a human ABCB6 gene and an application thereof.
  • Adenosine triphosphate binding cassette transporter is a large class of transmembrane proteins that utilize the energy of hydrolyzing A TP to transport transmembrane transport of various endogenous and exogenous biomolecules in solute. Substrates include: sugars, amino acids, metal ions, peptides, proteins, cellular metabolites, and drugs. ABCB transporters are widely present in eukaryotic and prokaryotic organisms. To date, 49 ABC transgenic superfamily members have been identified in the human genome, which are divided into seven subfamilies of A-G.
  • ABCB1 is the first human ABC transporter to be discovered. Because multiple members are associated with multidrug resistance (MD R), the ABCB subfamily is also known as the ABC transporter MDR family. As one of them, A BCB6 is not only related to MDR, but also related to the pathogenesis of various tumors. However, the function and role of ABCB6 in it have not been clarified so far, and further research is needed.
  • RNA interference RNA interference
  • RNAi small interfering RNA
  • siABCB6 sequence is as follows: [0007] Justice Chain: 5'- GGCAUCUGGAUCAAGUUCA -3' (SEQ ID NO: 1)
  • Antisense strand 5,- UGAACUUGAUCCAGAUGCC -3, (SEQ ID NO: 2).
  • the present invention provides siABCB6 which has the advantages of high interference efficiency, high-efficiency and specific inhibition of ABCB6 gene expression, and can be used as a powerful tool for the preparation of a medicament for treating a disease associated with abnormal expression of ABCB6 gene.
  • 1 is a schematic diagram showing the results of quantitative PCR detection of ABCB6 gene expression levels after transfection of A375 cells with siABCB6.
  • A375 cells purchased from Biovector Plasmid Vector Culture Cell Gene Collection
  • DMEM complete medium containing 10% fetal bovine serum
  • 6-well plates were plated at a ratio of 150,000 cells/well at 37°C. C, 5 % C02 was cultured for 18 h.
  • Cell transfection was performed using the Lipofectamine 3000 Transfection Kit (Invitrogen) according to the product instructions.
  • RNA extraction Total RNA from normal and transfected siABCB6 ABCB6 cells was extracted using the QIAGEN RNeasy Mini Kit.
  • Reverse Transcription Reverse transcription was performed using FastQuant RT Super Mix.
  • Quantitative PCR was carried out, and the reaction system was 20 ⁇ , and 1 L cDNA was added as a template for each reaction.
  • the reaction procedure is: (1) 95 °C 30 s, (2) 95 °C 5s, (3) 60 °C 30s, (2)-(3), 40 cycles. With GAPDH as the internal reference, the results are shown in Figure 1.
  • the quantitative PCR primers used are shown in Table 1:
  • the mRNA expression level of ABCB6 gene in A375 cells transfected with siABCB6 was significantly decreased compared with normal A375 cells, indicating that siABCB6 of the present invention can efficiently and specifically inhibit the expression of ABCB6 gene.
  • the siABCB6 provided by the invention has high interference efficiency and can efficiently and specifically inhibit the ABCB6 gene expression.
  • the advantages of the drug can be used as a powerful tool for the preparation of drugs for the treatment of diseases related to abnormal expression of ABCB6 gene.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
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  • Medicinal Chemistry (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
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  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
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  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

提供一种高效特异性地抑制ABCB6基因的mRNA表达水平的RNA干扰片段，其可用于制备治疗ABCB6基因表达异常相关疾病的药物。

Description

说明书 发明名称：一种人 ABCB6基因的 siRNA及其应用 技术领域
[0001] 本发明属于分子遗传学领域， 尤其涉及一种能抑制人 ABCB6基因表达的 siRNA 及其应用。
背景技术
[0002] 腺苷三磷酸结合盒转运蛋白 （ABC转运蛋白） 是一大类跨膜蛋白， 利用水解 A TP的能量对溶质中多种内、 外源性生物分子进行跨膜转运， 其转运的底物包括 ： 糖、 氨基酸、 金属离子、 多肽、 蛋白质、 细胞代谢产物和药物等。 ABCB转运 蛋白广泛存在于真核和原核生物中， 在人类基因组中迄今已鉴定出 49个 ABC转 运蛋白超家族成员， 分为 A-G七个亚族。
技术问题
[0003] ABCB家族基因仅存在于脊椎动物基因组中， 人类基因组有 11个成员， 其中 AB CB1是第一个被发现的人类 ABC转运蛋白。 由于多个成员与肿瘤多药耐药 （MD R) 相关， 所以 ABCB亚族又被称为 ABC转运蛋白 MDR家族。 作为其中一员， A BCB6不仅与 MDR有关， 还与多种肿瘤发病相关， 但迄今 ABCB6在其中的功能 与作用尚未明确， 需要进一步深入研究。
[0004] RNA干扰（RNA interference, RNAi)现象最早是由 Jorgensen等在发现的， 它 是一种高效、 特异性强的基因阻断技术， 近年来发展迅速， 很快就成为功能基 因组研究的有力工具， 可高效、 特异地抑制人 ABCB6基因表达， 进而为其功能 研究打下基础。
问题的解决方案
技术解决方案
[0005] 本发明的目的在于提供一种基于 RNAi技术的针对人 ABCB6基因 mRNA的小分 子干扰 RNA (siRNA) 。
[0006] 从 GenBank获得人 ABCB6基因的 cDNA序列， 根据 siRNA靶序列的基本原则， 针对其设计了一条 19 nt的 siRNA: siABCB6序列如下： [0007] 正义链： 5'- GGCAUCUGGAUCAAGUUCA -3' (SEQ ID NO: 1)
[0008] 反义链： 5，- UGAACUUGAUCCAGAUGCC -3， （SEQ ID NO: 2) 。
发明的有益效果
有益效果
[0009] 本发明提供的 siABCB6具有干扰效率高， 可高效、 特异地抑制 ABCB6基因表 达的优点， 可作为有力工具应用于制备治疗 ABCB6基因表达异常相关疾病的药 物。
对附图的简要说明
附图说明
[0010] 图 1为 A375细胞转染 siABCB6后定量 PCR检测 ABCB6基因表达水平的结果示意 图。
实施该发明的最佳实施例
本发明的最佳实施方式
[0011] 下面结合附图与具体实施例对本发明做进一步的说明。 以下实施例中所使用的 技术， 包括 PCR扩增与检测、 细胞转染、 RNA提取等分子生物学技术， 以及细 胞培养、 检测技术等， 除非特别说明， 均为本领域内的技术人员已知的常规技 术； 所使用的仪器设备、 试剂和细胞系等， 除非是本说明书特别注明， 均为一 般本领域的研究和技术人员可以通过公共途径获得的。
[0012] 实施例一靶向 ABCB6基因的 siRNA寡核苷酸序列的设计
[0013] 在 GenBank査找到 ABCB6的 mRNA全序列， 从 ABCB6基因起始密码子 AUG下 游幵始， 搜索 AA序列， 其 3'端相邻 19 nt序列作为候选靶点， 从中选择 GC含量 在 40-50%的 siRNA序列， 经 BLAST同源性比对证实特异性后应用 RNA structure 4.4软件对靶 mRNA序列的二级结构进行评估， 最后获得靶核苷酸序列 siABCB6 ， 其正义链序列如 SEQ ID NO: 1， 反义链序列如 SEQ ID NO: 2所示。
[0014] 实施例二 siRNA转染 A375细胞。
[0015] A375细胞（购自 Biovector质粒载体菌种细胞基因保藏中心）， 用 DMEM完全培 养基 （含 10%胎牛血清） 培养， 按照 15万个 /孔的比例铺 6孔板， 于 37。C、 5 % C02培养 18 h。 用 Lipofectamine 3000转染试剂盒（Invitrogen)进行细胞转染， 方法按照产品说明。 转染吋， 用 100 pmol SiABCB6转染 A375细胞， siRNA与脂 质体比例为 lOO pmol: 10 μί。
[0016] 实施例三定量 PCR检测 ABCB6基因表达水平
[0017] 提取总 RNA: 使用 QIAGEN RNeasy Mini Kit提取正常和转染 siABCB6的 ABCB6 细胞的总 RNA。
[0018] 逆转录： 使用 FastQuant RT Super Mix (天根生化） 进行逆转录。
[0019] 定量 PCR: 使用 SYBR Premix Ex Taq (Tli RNaseH Plus) (大连宝生物）
进行定量 PCR， 检测， 反应体系为 20 μί， 每个反应加入 1 L cDNA作为模板。 反应程序为： (1)95 °C 30 s, (2)95 °C 5s, (3)60°C 30s, (2)-(3)， 40个循环。 同吋 以 GAPDH为内参， 结果如图 1所示， 使用的定量 PCR引物如表 1所示：
[0020] 表 1定量 PCR引物序列
Figure imgf000004_0001
如图 1所示， 转染 siABCB6后的 A375细胞， ABCB6基因的 mRNA表达水平与正 常 A375细胞相比显著下降， 说明本发明的 siABCB6能够高效特异性抑制 ABCB6 基因的表达。
[0022] 以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明， 不能认 定本发明的具体实施只局限于这些说明。 对于本发明所属技术领域的普通技术 人员来说， 在不脱离本发明构思的前提下， 还可以做出若干简单推演或替换， 都应当视为属于本发明的保护范围。
工业实用性
[0023] 本发明提供的 siABCB6具有干扰效率高， 可高效、 特异地抑制 ABCB6基因表 达的优点， 可作为有力工具应用于制备治疗 ABCB6基因表达异常相关疾病的药 物。

Claims

权利要求书
[权利要求 1] 一种 RNA干扰片段， 其特征在于， 所述 RNA干扰片段的序列如下:
正义链： 5，-GGCAUCUGGAUCAAGUUCA-3， （SEQIDNO: 1) 反义链： 5，-UGAACUUGAUCCAGAUGCC-3， （SEQIDNO: 2)
[权利要求 2] 权利要求 1中所述的 RNA干扰片段的应用，
其特征在于制备治疗 ABCB6基因表达异常相关疾病的药物。
PCT/CN2017/089928 2017-06-26 2017-06-26 一种人ABCB6基因的siRNA及其应用 Ceased WO2019000148A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116064527A (zh) * 2022-08-26 2023-05-05 广东省第二人民医院（广东省卫生应急医院） 抑制ABCB6基因表达的siRNA及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103849623A (zh) * 2012-11-28 2014-06-11 天津华大基因科技有限公司 Abcb6基因突变体及其应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103849623A (zh) * 2012-11-28 2014-06-11 天津华大基因科技有限公司 Abcb6基因突变体及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide [O] 28 March 2017 (2017-03-28), ALLIKMETS, R. ET AL., XP055558442, retrieved from ncbi Database accession no. NM_005689 *
ZHAO, SHIGUANG ET AL.: "Increased Expression of ABCB6 Enhances Protoporphyrin IX Accumulation and Photodynamic Effect in Human Glioma", ANNALS OF SURGICAL ONCOLOGY, vol. 20, no. 13, 12 June 2012 (2012-06-12), pages 4379 - 4388, XP055558440, ISSN: 1068-9265 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116064527A (zh) * 2022-08-26 2023-05-05 广东省第二人民医院（广东省卫生应急医院） 抑制ABCB6基因表达的siRNA及其应用

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