WO2019037053A1 - 人 AITR 基因的 shRNA 及其应用 - Google Patents
人 AITR 基因的 shRNA 及其应用 Download PDFInfo
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- WO2019037053A1 WO2019037053A1 PCT/CN2017/098910 CN2017098910W WO2019037053A1 WO 2019037053 A1 WO2019037053 A1 WO 2019037053A1 CN 2017098910 W CN2017098910 W CN 2017098910W WO 2019037053 A1 WO2019037053 A1 WO 2019037053A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A—HUMAN NECESSITIES
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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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- 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 genetic engineering technology, and relates to the construction and application of a shRNA expression sequence. Specifically, the present invention contemplates the synthesis of shRNA against the nucleotide sequence of the AITR gene on the surface of human T cells, which can inhibit the expression of the human AITR gene after being transferred.
- AITR is the 18th member of the tumor necrosis factor receptor (TNFR) superfamily and is a thymus-derived C D4+
- AITR/AITRL A surface molecule on CD25+Treg cells whose ligand is AITRL. Studies have shown that AITR/AITRL has many important biological activities, including cell proliferation, differentiation and survival. Because AITR is mainly expressed on resting Treg cells, anti-AITR antibody (DTA-1) abolishes the immunosuppressive effects of Treg cells.
- AITR/AITRL system is involved in the regulation of Treg cells, and has a good clinical transformation prospect.
- lack of vectors for specifically inhibiting the expression of AITR gene in the prior art makes the related research not well developed.
- 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 as shown in the sequence listing SEQ NO.
- the antisense strand template sequence is shown in SEQ NO. 2 of the Sequence Listing.
- the present invention designs a pair of AITR-shRNA targeting the AITR gene according to the mRNA sequence of the human AITR gene in the GenBank database and the primer design principle of the shRNA, and commissions Shanghai Biotech to synthesize the AITR. -shRNA.
- the oligonucleotide of the above-designed AITR-shRNA is routinely annealed and then double-stranded, double-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 transduces the pSilencer3.1-H1 hygro RNAi expression vector containing AITR-shRNA into the Jurkat cell line, and the siRNA efficiency of AITR mRNA is 66.1 ⁇ 3 ⁇ 4.
- the AITR-shRNA provided by the invention has high transduction efficiency and can efficiently and specifically inhibit Jurkat cell AI.
- TR gene expression can be used as a powerful tool for the preparation of drugs for the treatment of AITR gene expression-related diseases.
- FIG. 1 is a schematic diagram showing the results of AITR gene expression by real-time PCR detection of Jurkat cells transduced with an AITR-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.
- 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. Ensure that the target sequence has a GC content of 30% to 60% and is 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'-CGCGCTGCTGCCGGGTTCA
- the sense strand sequence ⁇ ij of AITR-shRNA is shown as SEQ ID No: 1
- the antisense strand sequence is SEQ ID No: 2 Shown.
- 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 AITR-shRNA expression vector was mixed and added to the electric shock cup.
- the electroporation was performed using the Invitrogen Neon electrotransfer system.
- 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 the expression of AITR gene after 48 hours.
- Example 4 Fluorescence quantitative PCR was used to detect the amount of AITR gene expression.
- Jurkat cells transfected with normal Jurkat cells and AITR-shRNA expression vector were used to extract total RNA from each group of cells 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.
- GAPDH was used as an internal reference, and real-time quantitative PCR (QPCR) was used to detect the relative expression of AITR.
- the reaction conditions were set: 95 ° C for 10 s, 1 cycle; 95 ° C 5 s, 60 ° C
- the AITR-shRNA provided by the invention has high transduction efficiency and can efficiently and specifically inhibit Jurkat cell AI.
- TR gene expression can be used as a powerful tool for the preparation of drugs for the treatment of AITR gene expression-related diseases.
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Abstract
提供了一种抑制AITR基因表达的shRNA，其编码核酸序列如SEQ ID NOs：1和2所示。还提供了该shRNA在制备降低细胞AITR mRNA试剂中的应用，在制备抑制细胞AITR蛋白表达试剂中的应用及在制备AITR基因表达异常相关疾病的药物中的应用。
Description
人 AITR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 AITR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 AITR基因的表达。
背景技术
[0002] AITR是肿瘤坏死因子受体 （TNFR) 超家族中的第 18个成员， 是胸腺来源的 C D4+
CD25+Treg细胞上的一个表面分子， 其配体为 AITRL。 研究表明， AITR/AITRL 具有许多重要的生物学活性， 包括细胞的增殖、 分化和存活等。 因为 AITR主要 表达在静息性的 Treg细胞上， 故抗 AITR抗体 (DTA-1)可消除 Treg细胞的免疫抑制 作用。
技术问题
[0003] AITR/AITRL系统参与 Treg细胞发挥免疫调节的作用， 具有较好的临床转化前 景， 但现有技术中缺乏特异抑制 AITR基因表达的载体使得相关研究无法很好地 幵展。
[0004] 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
[0005] 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 AITR基因的蛋白表达水平。
[0006] 本发明根据 GenBank数据库中人 AITR基因的 mRNA序列以及 shRNA的引物设计 原则， 设计了 1对靶向 AITR基因的 AITR-shRNA， 并委托上海生工合成所述 AITR
-shRNA。
[0007] 本发明将上述设计的 AITR-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。
[0008] 本发明将含有 AITR-shRNA的 pSilencer3.1-H1 hygro RNAi表达载体转导进 Jurkat 细胞系， 其 AITR mRNA的被沉默效率为 66.1<¾。
发明的有益效果
有益效果
[0009] 本发明提供的 AITR-shRNA具有转导效率高， 可高效、 特异地抑制 Jurkat细胞 AI
TR基因表达的优点， 可作为有力工具应用于制备治疗 AITR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为转导 AITR-shRNA表达载体的 Jurkat细胞的荧光定量 PCR检测 AITR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
[0011] 下面结合附图与具体实施例对本发明做进一步的说明。
[0012] 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。
[0013] 实施例一靶向 AITR基因的 shRNA寡核苷酸序列的设计
[0014] 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'- CGCGCTGCTGCCGGGTTCA
-3'， AITR-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。
[0015] 实施例二 AITR-shRNA表达载体的构建
[0016] 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。
[0017] 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 AITR-shRNA表达载体。
[0018] 实施例三 Jurkat细胞转导
[0019] 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
AITR-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 AITR基因表达情 况。
[0020] 实施例四荧光定量 PCR检测 AITR基因表达量。
[0021] 取正常 Jurkat细胞和转导 AITR-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。
[0022] 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 AITR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 AITR基因相 对表达量， 结果如图 1所示。 结果显示转导 AITR-shRNA表达载体的 Jurkat细胞， AITR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 66.1<¾±7.2%， 从而证明本实验中采用的 AITR-shRNA表达载体携带 shRNA能特异抑制 AITR基 因的表达， 且抑制效果非常显著。
工业实用性
本发明提供的 AITR-shRNA具有转导效率高， 可高效、 特异地抑制 Jurkat细胞 AI
TR基因表达的优点， 可作为有力工具应用于制备治疗 AITR基因表达异常相关疾 病的药物。
Claims
[权利要求 1] 一种抑制 AITR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 AITR 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 AITR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 AITR基因表达异常相关疾病的药物中 的应用。
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1809589A (zh) * | 2003-05-23 | 2006-07-26 | Wyeth公司 | Gitr配体和gitr配体相关分子和抗体及其应用 |
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- 2017-08-24 WO PCT/CN2017/098910 patent/WO2019037053A1/zh not_active Ceased
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1809589A (zh) * | 2003-05-23 | 2006-07-26 | Wyeth公司 | Gitr配体和gitr配体相关分子和抗体及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| DATABASE abstract Nucleotide 6 June 2016 (2016-06-06), ANONYMOUS: "PREDICTED: Homo sapiens TNF receptor superfamily member 18 (TNFRSF18), transcript variant X1, mRNA.", XP055579061, retrieved from NCBI GenBank Database accession no. XM_017002722 * |
| RONCHETTI S. ET AL.: "Glucocorticoid-Induced TNFR family Related gene (GITR) enhances dendritic cell activity", IMMUNOLOGY LETTERS, vol. 135, no. 1-2, 30 March 2011 (2011-03-30), pages 24 - 33, XP055579070, ISSN: 0165-2478, DOI: 10.1016/j.imlet.2010.09.008 * |
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