WO2019037049A1 - 人 ERBB2 基因的 shRNA 及应用 - Google Patents
人 ERBB2 基因的 shRNA 及应用 Download PDFInfo
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- WO2019037049A1 WO2019037049A1 PCT/CN2017/098906 CN2017098906W WO2019037049A1 WO 2019037049 A1 WO2019037049 A1 WO 2019037049A1 CN 2017098906 W CN2017098906 W CN 2017098906W WO 2019037049 A1 WO2019037049 A1 WO 2019037049A1
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- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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 relates to a human ERBB2 gene, and more particularly to a shRNA (short hairpin RNA) of a human ERBB2 gene and use thereof.
- shRNA short hairpin RNA
- ERBB2 is a 185 kDa cell membrane receptor encoded by the proto-oncogene ERBB2 and is a member of the epidermal growth factor receptor (EGFR) family.
- the family includes four members, ERBB1 (also known as EGFR, HERl), ERBB2 (also known as HER2), ERBB3 (HER3), and ERBB4 (HER4).
- ERBB2 is involved in the regulation of growth and development of normal tissues, promotes cell division and secretion of proteolytic enzymes, and enhances cell motility, but under pathological conditions, ERBB2 expression increases, thereby promoting tumor invasion and metastasis.
- ERBB2 has intracellular tyrosine kinase-like activity, plays a role in embryonic development and differentiation, is widely expressed in epithelial cells of human tissues, and is closely related to the survival and evolution of various tumors. . Therefore, the research on ERBB2 can greatly promote the development of tumor prevention and control, but the lack of vectors for specifically inhibiting the expression of ERBB2 gene in the prior art makes the related research not well developed.
- One of the technical problems to be solved by the present invention is to provide a shRNA of the human ERBB2 gene.
- the second technical problem to be solved by the present invention is to provide a shRNA of human ERBB2 gene.
- the present invention provides a shRNA of a human ERBB2 gene, including:
- sequence shown in SEQ ID NO: 1 and ERBB2-RNAi consisting of the sequence shown in SEQ ID NO: 2.
- the shRNA provided by the invention has high transduction efficiency and can efficiently and specifically inhibit the ERBB2 group of Raji cells. Due to the advantages of expression, it can be used as a powerful tool for the preparation of drugs for the treatment of diseases related to abnormal expression of ERBB2 gene.
- Figure 1 is a schematic diagram showing the results of Quantitative PCR detection of Raji cells after transduction of pLKO-ERBB2 vector.
- Kit was purchased from Omega bio-tek.
- the complete medium described below is a cell culture medium to which 10% fetal calf serum is added.
- the ERBB2-RNAi oligonucleotide chain was synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
- the synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded ERBB2-RNAi.
- Age I and EcoR I double-cleavage pLKO.l-puro vector, Fermentas restriction enzymes Age I and EcoR I double digestion, 50 reaction system is as follows: 5 ⁇ L 10x FastDigest Buffer, 2 ⁇ L Age I , 2 EcoR I, 2 g pLK0.1-puro plasmid, ddH20 supplemented to 20ul, 37 ° C reaction for 30 min; [0019] 2) ligation, 2 L annealing of phosphorylated double-stranded DNA oligos, 1 ligase buffer, 1 double-digested pLKO. l-puro plasmid, 1 ligase, 5 L ddH20, reaction at 16 ° C for 5 h, Obtaining the ligation product pLKO-ERBB2 vector;
- Plasmid Mini Kit for endotoxin-free pLKO-ERBB2 vector extraction Plasmid Mini Kit for endotoxin-free pLKO-ERBB2 vector extraction.
- Raji cells were cultured, and 5,000,000 cells with good growth state were taken, and the cells were collected by centrifugation, then resuspended in 500 ⁇ L ⁇ PBS, mixed with 20 pL KO-ERBB2 vector, and then added to an electric shock cup, and electrotransformed using Invitrogen Neon electrotransfer system.
- Electroporation procedure 2.1 KV, 25 ⁇ , pulse shock once; Transfer the cells to a 6 cm dish containing 5 mL DMEM complete medium, gently shake the dish to mix the cells, and detect the expression of TL6 gene after 48 hours.
- Example 4 Fluorescence quantitative PCR was used to detect the expression level of ERBB2 gene.
- Raji cells inoculated with Raji cells and transduced pLKO-ERBB2 vector were separately cultured into cell culture flasks, and after 24 h of culture, total RNA of each group was extracted with Trizol, and PrimeScrip RT reagent was used.
- Kit reverse-transcribes mRNA into cDNA, and is diluted with 90 RNase-Free dH20 and stored at -20 °C.
- ERBB2 For the template, GAPDH was used as the internal reference, and the relative expression of ERBB2 was detected by real-time quantitative PCR (QPCR). The reaction conditions were set: 95 ° C for 10 s, 1 cycle; 95 ° C for 5 s, 54 ° C for 30 s, for 40 cycles. The relative expression of ERBB2 gene in each group was detected by SYBR Primescript RT-PCR Kit. The results are shown in Figure 1. The results showed that the expression of ERBB2 gene was significantly inhibited in Raji cells transfected with pLKO-ERBB2 vector, and the inhibitory efficiency of the interference fragment on the target gene was 84.4% ⁇ 4.1%, which proved that the pLKO-ERBB2 vector used in this experiment can specifically inhibit shRNA. The expression of the ERBB2 gene is very significant.
- the shRNA provided by the invention has the advantages of high transduction efficiency, high-efficiency and specific inhibition of ERBB2 gene expression in Raji cells, and can be used as a powerful tool for preparing a medicament for treating ERBB2 gene expression-related diseases.
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Abstract
一种人ERBB2基因的shRNA，其编码序列的正义链和反义链分别如SEQ ID NO：1和SEQ ID NO：2所示。该人ERBB2基因的shRNA可用于制备治疗ERBB2基因表达异常相关疾病的药物。
Description
人 ERBB2基因的 shRNA及应用
技术领域
[0001] 本发明涉及一种人 ERBB2基因， 特别是涉及一种人 ERBB2基因的 shRNA (短发 夹 RNA)及应用。
背景技术
[0002] ERBB2是原癌基因 ERBB2编码的 185kDa的细胞膜受体， 为表皮生长因子受体（ EGFR)家族成员之一。 该家族包括 ERBB1 (又称 EGFR， HERl) 、 ERBB2 (又 称 HER2) 、 ERBB3 (HER3) 和 ERBB4 (HER4) 四个成员。
[0003] ERBB2参与了正常组织的生长与发育调节， 可以促进细胞分裂和蛋白水解酶的 分泌， 并增强细胞的运动能力， 但在病理状态下， ERBB2表达增多， 进而促进 肿瘤的侵袭和转移。
技术问题
[0004] ERBB2具有细胞内酪氨酸激酶样活性， 在胚胎发育形成及分化中有一定的作用 ， 在人类组织的上皮细胞中有广泛表达， 并且与多种肿瘤的生存和演进有密切 的关系。 因此对 ERBB2的研究可以大大促进肿瘤防治领域的发展， 但现有技术 中缺乏特异抑制 ERBB2基因表达的载体使得相关研究无法很好地幵展。
问题的解决方案
技术解决方案
[0005] 本发明要解决的技术问题之一是提供一种人 ERBB2基因的 shRNA。
[0006] 本发明要解决的技术问题之二是提供一种人 ERBB2基因的 shRNA
在制备治疗 ERBB2基因表达异常相关疾病的药物。
[0007] 为解决上述技术问题， 本发明提供一种人 ERBB2基因的 shRNA, 包括： 由编码
SEQ ID NO: 1所示序列和编码 SEQ ID NO:2所示序列构成的 ERBB2-RNAi。
发明的有益效果
有益效果
[0008] 本发明提供的 shRNA具有转导效率高， 可高效、 特异地抑制 Raji细胞 ERBB2基
因表达的优点， 可作为有力工具应用于制备治疗 ERBB2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
[0009] 图 1为转导 pLKO-ERBB2载体后 Raji细胞的荧光定量 PCR检测结果 ERBB2基因表 达结果示意图。
实施该发明的最佳实施例
本发明的最佳实施方式
[0010] 下面结合附图与具体实施例对本发明做进一步的说明。
[0011] Raji细胞购自 ATCC， Trizol购自 Invitrogen公司， Endo-free Plasmid Mini
Kit购自 Omega bio-tek。 下文所述完全培养基为加入了 10%胎牛血清的细胞培养 基。
[0012] 实施例一 shRNA序列设计
[0013] (1) 根据人 ERBB2基因特征设计沉默序列， 设计的原则： 19bp的特异性结合 E RBB2序列的 GC含量为 45<¾-55<¾， 退火温度 45°C-65°C， 同吋要求序列起始的第 一个碱基为 G。 将选取的片段进行 BLAST与人基因组比对， 确保特异性。
[0014] (2) 以 Age I-GN18-TT-loop-81NC-EcoR I形式设计一段 59bp序列 ERBB2-RNAi ， 81NC为 NG18的反向序列， 5'端为 Age I酶切位点， 3'端为 EcoR I酶切位点， 其 正义链序列如 SEQ ID No: 1所示， 反义链序列如 SEQ ID No:
2所示。 ERBB2-RNAi寡核苷酸链由上海生工生物工程技术服务有限公司合成。
[0015] 实施例二 pLKO-ERBB2载体的构建
[0016] 将合成的 shRNA寡核苷酸单链等量混合， 退火形成双链的 ERBB2-RNAi。
[0017] pLKO.l-puro载体 (Sigma Aldrich)全长 7086 bp, 选用限制性内切酶位点 Age
I和 EcoR I作为 DNA片段的插入位点， 应用不同 shRNA相对应 DNA片段的每种载 体构建具体步骤如下：
[0018] 1) Age l和 EcoR I双酶切 pLKO.l-puro载体， Fermentas公司限制性内切酶 Age I和 EcoR I双酶切， 50 反应体系如下： 5 μL 10x FastDigest Buffer, 2 μL Age I ， 2 EcoR I, 2 g pLK0.1-puro质粒， ddH20补至 20ul， 37°C反应 30 min;
[0019] 2) 连接， 2 L退火的磷酸化双链 DNA oligos , 1 连接酶 buffer, 1 双酶切 处理的 pLKO. l-puro质粒， 1 连接酶， 5 L ddH20， 16°C反应 5h， 获得连接产 物 pLKO-ERBB2载体；
[0020] 3) 转化；
[0021] 4) 挑取阳性克隆培养并进行测序验证插入序列完全正确， 然后用 Endo-free
Plasmid Mini Kit进行无内毒素的 pLKO-ERBB2载体提取。
[0022] 实施例三 pLKO-ERBB2载体转导 Raji细胞。
[0023] 培养 Raji细胞， 取生长状态良好的细胞 5000000个， 离心收集细胞， 然后重悬于 500 μL· PBS中， 与 20 pLKO-ERBB2载体混匀后加入电击杯， 应用 Invitrogen Neon电转系统进行电转， 电转程序： 2.1 KV， 25 μΐΌ , 脉冲电击一次； 将细胞 转移至含5 mL DMEM完全培养基的6 cm皿中， 轻轻晃动皿使细胞混匀， 48 h后 检测 TL6基因表达情况。
[0024] 实施例四荧光定量 PCR检测 ERBB2基因表达量。
[0025] 分别接种 Raji细胞和转导 pLKO-ERBB2载体的 Raji细胞细胞至细胞培养瓶， 培 养 24 h后， 用 Trizol提取各组细胞的总 RNA， 利用 PrimeScrip RT reagent
Kit将 mRNA逆转录为 cDNA， 加入 90 的 RNase-Free dH20稀释 cDNA， -20°C保 存。
[0026] 取各组细胞的 cDNA 1
为模板， 以 GAPDH为内参， 实吋荧光定量 PCR (QPCR) 检测 ERBB2相对表 达量， 设置反应条件： 95°C 10s， 1个循环； 95°C 5s， 54°C 30s， 共 40个循环， 利用 SYBR Primescript RT-PCR Kit检测各组细胞 ERBB2基因相对表达量， 结果如 图 1所示。 结果显示转导 pLKO-ERBB2载体的 Raji细胞， ERBB2基因表达明显受 到抑制， 干扰片段对目的基因的抑制效率达 84.4%±4.1 %， 从而证明本实验中采 用的 pLKO-ERBB2载体携带 shRNA能特异抑制 ERBB2基因的表达， 且抑制效果 非常显著。
[0027] 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
本发明提供的 shRNA具有转导效率高， 可高效、 特异地抑制 Raji细胞 ERBB2基 因表达的优点， 可作为有力工具应用于制备治疗 ERBB2基因表达异常相关疾病 的药物。
Claims
[权利要求 1] 一种人 ERBB2基因的 shRNA, 其特征在于,包括： 由编码 SEQ ID NO:
1所示序列和编码 SEQ ID NO: 2所示序列构成的 ERBB2-RNAi。
[权利要求 2] 如权利要求 1所述的人 ERBB2基因的 shRNA在制备治疗 ERBB2基因表 达异常相关疾病的药物中的应用。
[权利要求 3] 如权利要求 2所述的应用， 其特征在于： 所述的人 ERBB2基因的 shRN
A是 ERBB2-RNAi。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/098906 WO2019037049A1 (zh) | 2017-08-24 | 2017-08-24 | 人 ERBB2 基因的 shRNA 及应用 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/098906 WO2019037049A1 (zh) | 2017-08-24 | 2017-08-24 | 人 ERBB2 基因的 shRNA 及应用 |
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| WO2019037049A1 true WO2019037049A1 (zh) | 2019-02-28 |
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| PCT/CN2017/098906 Ceased WO2019037049A1 (zh) | 2017-08-24 | 2017-08-24 | 人 ERBB2 基因的 shRNA 及应用 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101918844A (zh) * | 2007-06-18 | 2010-12-15 | 米迪缪尼有限公司 | 表达epha2和erbb2的细胞的协同治疗 |
-
2017
- 2017-08-24 WO PCT/CN2017/098906 patent/WO2019037049A1/zh not_active Ceased
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101918844A (zh) * | 2007-06-18 | 2010-12-15 | 米迪缪尼有限公司 | 表达epha2和erbb2的细胞的协同治疗 |
Non-Patent Citations (2)
| Title |
|---|
| DATABASE Nucleotide 1 August 2016 (2016-08-01), TAL, M., XP055578017, Database accession no. AH002823 * |
| WEI BAO: "HER2-mediated upregulation of MMP-1 is involved in gastric cancer cell invasion", ARCHIEVES OF BIOCHEMISTRY AND BIOPHYSICS, 31 July 2010 (2010-07-31), XP027084046 * |
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