WO2019037130A1 - 人 AMPAR 基因的 shRNA 及其应用 - Google Patents
人 AMPAR 基因的 shRNA 及其应用 Download PDFInfo
- Publication number
- WO2019037130A1 WO2019037130A1 PCT/CN2017/099174 CN2017099174W WO2019037130A1 WO 2019037130 A1 WO2019037130 A1 WO 2019037130A1 CN 2017099174 W CN2017099174 W CN 2017099174W WO 2019037130 A1 WO2019037130 A1 WO 2019037130A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ampar
- shrna
- gene
- expression
- human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- 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
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
Definitions
- AD Alzheimer's disease
- This disease is common in the elderly. It is a central nervous system degenerative degenerative disease with progressive cognitive impairment and memory impairment. The clinical manifestations are the deterioration of cognitive and memory functions, the progressive decline of daily living ability, and Various neuropsychiatric symptoms and behavioral disorders. More onset in the old age, latent onset, slow and irreversible, clinically based on intelligent damage.
- AMPAR glutamate a-amino-3-carbo-5-methyl-4-iso-propionate receptor
- a-amino-3-hy droxy-5-methyl-4 -isoxazole propionic acid receptor AMPAR is a key mechanism regulating central synaptic plasticity.
- AMPAR is one of the ionotropic glutamate receptors and mediates the central component of central excitatory postsynaptic currents and is essential for synaptic transmission.
- shRNA a small hairpin RNA
- RISC RNA-induced silencing complex
- the present invention constructs a shRNA, and the sense strand template sequence of the shRNA is SEQ NO.
- the antisense strand template sequence is shown in SEQ NO. 2 of the Sequence Listing.
- the present invention designed a pair of AMPAR-shRNA targeting AMPAR gene according to the mRNA sequence of human AMPAR gene in GenBank database and the primer design principle of shRNA, and commissioned Shanghai Biotech to synthesize the AMPAR-shRNA.
- the oligonucleotide of the AMPAR-shRNA designed above is routinely annealed and then double-stranded, digested and ligated to the pSilencer 3.1-H1 hygro RNAi expression vector, and the ligation product is transformed into Escherichia coli. Single colonies were picked for PCR and sequencing, and positive clones and plasmids were obtained.
- the present invention will contain AM-shRNA-containing pSilencer3.1-Hl hygro
- RNAi expression vector was transduced into the Jurkat cell line, and the silencing efficiency of AMPAR mRNA was 77.2 ⁇ 3 ⁇ 4.
- the AMPAR-shRNA provided by the present invention has the advantages of high transduction efficiency, high-efficiency and specific inhibition of the expression of the AMPAR gene of the Jurkat cell, and can be used as a powerful tool for preparing a medicament for treating an abnormal expression of AMPAR gene.
- FIG. 1 is a schematic diagram showing the results of fluorescent quantitative PCR detection of AMPAR gene expression by Jurkat cells transduced with AMPAR-shRNA expression vector.
- Jurkat cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences, and the RNeasy Mini Kit was purchased from Qiagen.
- the endotoxin-free plasmid extraction kit was purchased from Tiangen Biochemical (Beijing).
- the complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
- Example 1 Design of shRNA Oligonucleotide Sequences Targeting AMPAR Gene
- the "AA or NA" sequence was searched for and the 19-base sequence at the 3' end was recorded as a potential interference target site. It is ensured that the GC content of the target sequence should be around 30% to 60% and not on the 5' and 3' non-coding regions.
- NCBI BLAST confirmed that the selected sequences have no homology to other genes.
- the target sequence obtained in this example is 5'- GTGTCTGCAGGTCTGAGTA
- the sense strand sequence ⁇ ij of AMPAR-shRNA is shown in SEQ ID No: 1
- the antisense strand sequence is shown in SEQ ID No: 2.
- Jurkat cells were cultured, and 5,000,000 cells in good growth state were taken, and the cells were collected by centrifugation, and then resuspended in 500 L of PBS with 20 g.
- the AMPAR-shRNA expression vector was mixed and added to the electric shock cup.
- the Invitrogen Neon electroporation system was used for electroporation.
- the electroporation procedure was: 2.1 KV, 25 ⁇ , pulse shock once; the cells were transferred to a 6 cm dish containing 5 mL DMEM complete medium. Medium, gently shake the dish to mix the cells, and detect AMPAR gene expression 48 h later.
- Example 4 Fluorescence quantitative PCR was used to detect the AMPAR gene expression level.
- Jurkat cells were transfected with normal Jurkat cells and AMPAR-shRNA expression vector, and total RNA was extracted from each group using RNeasy Mini Kit, using PrimeScrip RT reagent.
- Kit reverse-transcribes mRNA into cDNA, and then cDNA is diluted with 90 ⁇ M of RNase-Free dH20 and stored at -20 °C for later detection.
- ⁇ is the template, GAPDH is used as the internal reference, and the relative expression of AMPAR is detected by real-time quantitative PCR (QPCR).
- the reaction conditions are set: 95°C for 10s, 1 cycle; 95°C for 5s, 60°C for 30s, for 40 cycles.
- the relative expression of AMPAR 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 AMPAR gene was significantly inhibited in Jurkat cells transduced with AMPAR-shRNA expression vector, and the inhibitory efficiency of the interference fragment on the target gene was 77.2% ⁇ 3.4%, which proved that the AMPAR-shRNA expression vector used in this experiment carries shRNA energy. It specifically inhibits the expression of the AMPAR gene, and the inhibitory effect is very remarkable.
- the AMPAR-shRNA provided by the present invention has the advantages of high transduction efficiency, high-efficiency and specific inhibition of the expression of the AMPAR gene of Jurkat cells, and can be used as a powerful tool for preparing a medicament for treating an abnormality in AMPAR gene expression.
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
提供了一种人AMPAR基因的shRNA及其应用。所述shRNA抑制AMPAR基因的表达，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。
Description
人 AMPAR基因的 shRNA及其应用 技术领域
[0001] 本发明属于基因工程技术领域， 涉及一种 shRNA表达序列的构建及应用。 具体 而言， 本发明针对人 T细胞表面的 AMPAR基因的核苷酸序列设计合成 shRNA, 所述 shRNA转入后能抑制所述人 AMPAR基因的表达。
背景技术
[0002] 阿尔茨海默病 （Alzheimer's disease , AD) ， 是引起老年性痴呆的最常见原因 。 此疾病常见于老年人， 是一种进行性认知障碍和记忆能力损害为主的中枢神 经系统退行性变性疾病， 临床表现为认知和记忆功能不断恶化， 日常生活能力 进行性减退， 并有各种神经精神症状和行为障碍。 多起病于老年期， 潜隐起病 ， 病程缓慢且不可逆， 临床上以智能损害为主。
[0003] 越来越多证据表明， 谷氨酸 a-氨基 -3-轻基 -5-甲基 -4-异挫丙酸受体 (a-amino-3-hy droxy-5-methyl-4-isoxazole propionic acid receptor, AMPAR)是调节中枢神经突触 可塑性的关键机制。 AMPAR是离子型谷氨酸受体之一， 介导中枢兴奋性突触后 电流的主要成分， 在突触传递过程中不可或缺。
技术问题
[0004] AMPAR的生理功能依赖受体数量、 亚基组成以及蛋白质磷酸化作用等因素的 调控 AMPAR功能紊乱， 导致神经元突触可塑性下降， 引起 AD认知障碍， 在 AD 的发病过程中起重要作用， 因此对 AMPAR在阿尔兹海默症中的作用及将其作为 一种治疗靶点的研究必不可少， 现有技术中也缺乏特异抑制 AMPAR基因表达的 载体使得相关研究无法很好地幵展。
[0005] shRNA即小发卡 RNA， 是一段外源性的具有茎环结构的 RNA序列， 能够在细胞 内被加工为 siRNA, siRNA进而与相关酶结合形成 RNA诱导沉默复合物 (RISC), 并结合到同源的 mRNA上并诱导其降解， 是一种很好的降低基因表达的方法。 问题的解决方案
技术解决方案
[0006] 本发明构建了一种 shRNA, 所述 shRNA的正义链模板序列如序列表 SEQ NO.
1所示， 其反义链模板序列如序列表 SEQ NO. 2所示。 所述 shRNA
能够降低人 AMPAR基因的蛋白表达水平。
[0007] 本发明根据 GenBank数据库中人 AMPAR基因的 mRNA序列以及 shRNA的引物设 计原则， 设计了 1对靶向 AMPAR基因的 AMPAR-shRNA, 并委托上海生工合成所 述 AMPAR-shRNA。
[0008] 本发明将上述设计的 AMPAR-shRNA的寡核苷酸常规退火后合成双链， 双酶切 后并连接到 pSilencer 3.1-H1 hygro RNAi表达载体上， 将连接产物转化大肠杆菌 。 挑取单菌落进行 PCR及测序鉴定， 得到了阳性的克隆和质粒。
[0009] 本发明将含有 AMPAR-shRNA的 pSilencer3.1-Hl hygro
RNAi表达载体转导进 Jurkat细胞系， 其 AMPAR mRNA的被沉默效率为 77.2<¾。 发明的有益效果
有益效果
[0010] 本发明提供的 AMPAR-shRNA具有转导效率高， 可高效、 特异地抑制 Jurkat细 胞 AMPAR基因表达的优点， 可作为有力工具应用于制备治疗 AMPAR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
[0011] 图 1为转导 AMPAR-shRNA表达载体的 Jurkat细胞的荧光定量 PCR检测 AMPAR 基因表达的结果示意图。
实施该发明的最佳实施例
本发明的最佳实施方式
[0012] 下面结合附图与具体实施例对本发明做进一步的说明。
[0013] Jurkat细胞购自上海生命科学院细胞资源中心， RNeasy Mini Kit购自 Qiagen公司
， 无内毒素质粒提取试剂盒购自天根生化 （北京） 。 下文所述完全培养基为加 入了 10%胎牛血清的细胞培养基。
[0014] 实施例一靶向 AMPAR基因的 shRNA寡核苷酸序列的设计
[0015] 从 mRNA的 AUG起始密码幵始， 寻找" AA或者 NA"二连序列， 并记下其 3'端的 1 9个碱基序列， 作为潜在的干扰靶位点。 确保靶序列的 GC含量应为 30%〜60%左 右， 并且不在 5'和 3'非编码区上。 NCBI BLAST确认挑选的序列与其它基因没有 同源性。 本实施例中获得的靶序列为 5'- GTGTCTGCAGGTCTGAGTA
-3'， AMPAR-shRNA的正义链序歹 ij如 SEQ ID No: 1所示， 反义链序列如 SEQ ID No:2所示。
[0016] 实施例二 AMPAR-shRNA表达载体的构建
[0017] 取等量 10 mmol/L的 DNA寡核苷酸单链片段混合， 在 TE缓冲液中 95°C加热 5min ， 缓慢降至室温。 用 BamHI、 Hindlll双酶切 pSilencer 3.1-H1 hygro表达载体， T4 连接酶将片段和载体连接。 然后将连接产物转化至大肠杆菌 ToplO中， 挑取单克 隆进行测序鉴定。
[0018] 选择测序正确的克隆接种到 5 mL培养基中， 培养过夜， 无内毒素提取质粒， 即 为 AMPAR-shRNA表达载体。
[0019] 实施例三 Jurkat细胞转导
[0020] 培养 Jurkat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 L PBS中， 与 20 g
AMPAR-shRNA表达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 μ¥Ό , 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 AMPAR基 因表达情况。
[0021] 实施例四荧光定量 PCR检测 AMPAR基因表达量。
[0022] 取正常 Jurkat细胞和转导 AMPAR-shRNA表达载体的 Jurkat细胞， 用 RNeasy Mini Kit提取各组细胞的总 RNA， 利用 PrimeScrip RT reagent
Kit将 mRNA逆转录为 cDNA， 然后加入 90μΙ^的 RNase-Free dH20稀释 cDNA， -20 °C保存， 以便后面检测使用。
[0023] 取各组细胞的 cDNA
Ιμί为模板， 以 GAPDH为内参， 实吋荧光定量 PCR (QPCR) 检测 AMPAR相对 表达量， 设置反应条件： 95°C 10s， 1个循环； 95°C 5s， 60°C 30s， 共 40个循环，
利用 SYBR Primescript RT-PCR Kit检测各组细胞 AMPAR基因相对表达量， 结果 如图 1所示。 结果显示转导 AMPAR-shRNA表达载体的 Jurkat细胞， AMPAR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 77.2%±3.4%， 从而证明 本实验中采用的 AMPAR-shRNA表达载体携带 shRNA能特异抑制 AMPAR基因的 表达， 且抑制效果非常显著。
[0024] 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
[0025] 本发明提供的 AMPAR-shRNA具有转导效率高， 可高效、 特异地抑制 Jurkat细 胞 AMPAR基因表达的优点， 可作为有力工具应用于制备治疗 AMPAR基因表达 异常相关疾病的药物。
Claims
[权利要求 1] 一种抑制 AMPAR基因表达的 shRNA, 其特征在于， 所述 shRNA的正 义链序列如 SEQ ID NO: 1所示， 所述 shRNA反义链序列如 SEQ ID NO:
2所示。
[权利要求 2] 权利要求 1所述的 shRNA在制备降低细胞 AMPAR mRNA试剂中的应 用。
[权利要求 3] 权利要求 1所述的 shRNA在制备抑制细胞 AMPAR蛋白表达试剂中的应 用。
[权利要求 4] 权利要求 1所述的 shRNA在制备 AMPAR基因表达异常相关疾病的药物 中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/099174 WO2019037130A1 (zh) | 2017-08-25 | 2017-08-25 | 人 AMPAR 基因的 shRNA 及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/099174 WO2019037130A1 (zh) | 2017-08-25 | 2017-08-25 | 人 AMPAR 基因的 shRNA 及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2019037130A1 true WO2019037130A1 (zh) | 2019-02-28 |
Family
ID=65439913
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2017/099174 Ceased WO2019037130A1 (zh) | 2017-08-25 | 2017-08-25 | 人 AMPAR 基因的 shRNA 及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2019037130A1 (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2338492A1 (en) * | 2009-12-24 | 2011-06-29 | Universidad del Pais Vasco | Methods and compositions for the treatment of alzheimer |
| WO2012047355A2 (en) * | 2010-07-16 | 2012-04-12 | The Research Foundation Of State University Of New York | Subunit-selective nucleic acid inhibitors of glutamate receptors |
-
2017
- 2017-08-25 WO PCT/CN2017/099174 patent/WO2019037130A1/zh not_active Ceased
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2338492A1 (en) * | 2009-12-24 | 2011-06-29 | Universidad del Pais Vasco | Methods and compositions for the treatment of alzheimer |
| WO2012047355A2 (en) * | 2010-07-16 | 2012-04-12 | The Research Foundation Of State University Of New York | Subunit-selective nucleic acid inhibitors of glutamate receptors |
Non-Patent Citations (4)
| Title |
|---|
| DATABASE Database Nucleotide 6 June 2016 (2016-06-06), ANONYMOUS: "PREDICTED: Homo sapiens calcium voltage-gated channel auxiliary subunit gamma 2 (CACNG2), transcript variant X1, mRNA.", XP055576961, retrieved from NCBI Genbank Database accession no. XM_017028531. 1 * |
| TRACY: "Acute knockdown of AMPA receptors reveals a trans-synaptic sig- nal for presynaptic maturation", EMBO J., vol. 30, no. 8, 4 March 2011 (2011-03-04) - 20 April 2011 (2011-04-20), pages 1577 - 1592, XP055576789, ISSN: 0261-4189, DOI: DOI10.1038/emboj.2011.59 * |
| XU: "The regulation of PTEN on expression and positioning of Ampa-glur2 in Rat primary Hippocainpal Neuron after stretch damage", CFDT- MEDICAL AND HEALTH SCIENCES, no. 2, 15 February 2011 (2011-02-15), ISSN: 1674-022X * |
| ZHENG: "Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice va- riant", J NEUROPHYSIOL., vol. 108, no. 1, 4 April 2012 (2012-04-04) - 1 July 2012 (2012-07-01), pages 101 - 111, XP055576799, ISSN: 0022-3077, DOI: doi.org/10.1152/jn.01097.2011 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2925866B1 (en) | Circular rna for inhibition of microrna | |
| WO2005047505A2 (en) | Methods and products for expression of micro rnas | |
| AU2021257890A1 (en) | Modulation of microRNAs against myotonic dystrophy type 1 and antagonists of microRNAs therefor | |
| WO2010029303A1 (en) | Gene silencing | |
| CN104726500A (zh) | MicroRNA26b-3p抑制剂在制备人脐带来源间充质干细胞中的应用 | |
| CN102433383A (zh) | 人stim1基因的用途及其相关药物 | |
| WO2019037130A1 (zh) | 人 AMPAR 基因的 shRNA 及其应用 | |
| WO2019037133A1 (zh) | 靶向沉默APP的shRNA | |
| CN102260673A (zh) | 靶向人血管紧张素原的rna干扰片段、表达载体与应用 | |
| CN101333529B (zh) | 抗HCMV UL122基因的siRNA序列及应用 | |
| WO2019037053A1 (zh) | 人 AITR 基因的 shRNA 及其应用 | |
| CN103710372B (zh) | 一种用于功能研究的miR-505高表达载体的构建方法 | |
| WO2019036869A1 (zh) | 人 TL6 基因的 shRNA 及其应用 | |
| CN102260672A (zh) | 抑制猪生长抑素受体2基因表达的siRNA | |
| CN106947778A (zh) | 靶向抑制整合素β1表达的小发夹RNA重组载体及其构建方法 | |
| CN102813926B (zh) | miR-7表达抑制剂在制备治疗系统性红斑狼疮药物中的应用 | |
| CN102229928B (zh) | 人rbbp6基因的小干扰rna及其应用 | |
| WO2019037055A1 (zh) | 人 GRD4 基因的 shRNA 及其应用 | |
| CN101333525A (zh) | 针对HCMV UL86基因的siRNA序列及应用 | |
| WO2019000145A1 (zh) | 一种ampar高表达的重组cho细胞的构建方法 | |
| CN103952413B (zh) | 靶向ifnar2基因的rna干扰表达载体构建及应用 | |
| CN113151275B (zh) | 一种抑制hsa_circ_0001610表达的shRNA及其表达载体 | |
| JP2022515211A (ja) | 合成マイクロｒｎａミミック | |
| WO2019000148A1 (zh) | 一种人ABCB6基因的siRNA及其应用 | |
| WO2018170764A1 (zh) | 一种用于 RNAi 的载体及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17922641 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 17922641 Country of ref document: EP Kind code of ref document: A1 |