WO2019033249A1 - 人 BTLA 基因的 shRNA 及应用 - Google Patents
人 BTLA 基因的 shRNA 及应用 Download PDFInfo
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- WO2019033249A1 WO2019033249A1 PCT/CN2017/097432 CN2017097432W WO2019033249A1 WO 2019033249 A1 WO2019033249 A1 WO 2019033249A1 CN 2017097432 W CN2017097432 W CN 2017097432W WO 2019033249 A1 WO2019033249 A1 WO 2019033249A1
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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 relates to a human BTLA gene, and more particularly to a shRNA (short hair clip R NA) of a human BTLA gene and use thereof.
- B/T lymphocyte attenuating factor is the third inhibitory molecule found in the CD28 family, and is mainly expressed on the cell surface of B cells, T cells, macrophages, and the like.
- BTLA has a similar structure to CTLA-4 and PD-1, but their expression characteristics are different.
- CTLA-4 and PD-1 are not expressed on resting T cells, and expression is gradually increased after activation, while BTLA is at rest. Constitutive expression on T cells, continued expression after activation. technical problem
- BTLA signaling is capable of inhibiting CD3 activated T cell proliferation and secretion of IL-2 and IFN- ⁇ . Research shows that B
- TLA plays an important role in maintaining peripheral immune tolerance and transplant immunity, and has a good clinical transformation prospect, which needs further study.
- One of the technical problems to be solved by the present invention is to provide a shRNA of a human BTLA gene.
- the second technical problem to be solved by the present invention is to provide a shRNA of human BTLA gene.
- the present invention provides a shRNA of a human BTLA gene, including:
- EQ ID NO: The sequence shown by 1 and the BTLA-RNAi consisting of the sequence shown in SEQ ID NO: 2.
- the shRNA provided by the invention has the advantages of high transduction efficiency, high and specific inhibition of BTLA gene expression in Jurkat cells, and can be used as a powerful tool for preparing a medicament for treating diseases related to abnormal expression of BTLA gene. Brief description of the drawing
- Figure 1 is a schematic diagram showing the results of Quantitative PCR detection of Jurkat cells after transduction of pLKO-BTLA vector.
- Jurkat cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences, Trizol was purchased from Invitrogen, and Endo-free Plasmid Mini Kit was purchased from Omega bio-tek.
- the complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
- the synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded BTLA-RNAi.
- Plasmid Mini Kit for endotoxin-free pLKO-BTLA vector extraction Plasmid Mini Kit for endotoxin-free pLKO-BTLA vector extraction.
- Example 3 The pLKO-BTLA vector transduced Jurkat cells.
- Jurkat cells cultured the growth state of the cell to take good 5,000,000, the cells were collected by centrifugation, then resuspended in 500 ⁇ L PBS is added and after mixing the cuvette 20 ⁇ ⁇ pLKO-BTLA vector, Invitrogen Neon electroporation application system Electrotransfer, 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 h.
- Example 4 Fluorescence quantitative PCR was used to detect the expression level of BTLA gene.
- Jurkat cells inoculated with Jurkat cells and transduced pLKO-BTLA vectors were separately cultured into cell culture flasks, and after 24 hours of culture, total RNA of each group was extracted with Trizol, and mRNA was reverse-transcribed into cDNA using PrimeScrip RT reagent Kit.
- the cDNA was diluted with 90 RNase-Free dH20 and stored at -20 °C.
- the shRNA provided by the present invention has the advantages of high transduction efficiency, and can efficiently and specifically inhibit the expression of BTLA gene in Jurkat cells, and can be used as a powerful tool for preparing a medicament for treating diseases related to abnormal expression of BTLA gene.
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Abstract
提供了一种人BTLA基因的shRNA及应用，该人BTLA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BTLA-RNAi。该人BTLA基因的shRNA可应用于制备治疗BTLA基因表达异常相关疾病的药物中。
Description
说明书 发明名称：人 BTLA基因的 shRNA及应用 技术领域
[0001] 本发明涉及一种人 BTLA基因， 特别是涉及一种人 BTLA基因的 shRNA (短发夹 R NA)及应用。
背景技术
[0002] B/T淋巴细胞弱化因子 （BTLA) 是 CD28家族发现的第 3个抑制性分子， 主要表 达在 B细胞、 T细胞、 巨噬细胞等细胞表面。 BTLA具有和 CTLA-4、 PD-1相似的 结构， 但它们的表达特点有所不同， CTLA-4和 PD-1在静止的 T细胞上不表达， 活化后表达逐步升高， 而 BTLA在静止 T细胞上组成性表达， 活化后继续表达。 技术问题
[0003] BTLA信号能够抑制 CD3活化的 T细胞增殖和 IL-2、 IFN-γ的分泌。 研究表明， B
TLA在维持外周免疫耐受和移植免疫中具有重要作用， 具有很好的临床转化前景 ， 有待进一步研究。
问题的解决方案
技术解决方案
[0004] 本发明要解决的技术问题之一是提供一种人 BTLA基因的 shRNA。
[0005] 本发明要解决的技术问题之二是提供一种人 BTLA基因的 shRNA
在制备治疗 BTLA基因表达异常相关疾病的药物。
[0006] 为解决上述技术问题， 本发明提供一种人 BTLA基因的 shRNA, 包括： 由编码 S
EQ ID NO: 1所示序列和编码 SEQ ID NO:2所示序列构成的 BTLA-RNAi。
发明的有益效果
有益效果
[0007] 本发明提供的 shRNA具有转导效率高， 可高效、 特异地抑制 Jurkat细胞 BTLA基 因表达的优点， 可作为有力工具应用于制备治疗 BTLA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
[0008] 图 1为转导 pLKO-BTLA载体后 Jurkat细胞的荧光定量 PCR检测结果 BTLA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
[0009] 下面结合附图与具体实施例对本发明做进一步的说明。
[0010] Jurkat细胞购自上海生命科学院细胞资源中心， 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。
[0011] 实施例一靶向 BTLA基因的 shRNA寡核苷酸序列的设计
[0012] (1) 根据人 BTLA基因特征设计沉默序列， 设计的原则： 19bp的特异性结合 B TLA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0。 将选取的片段进行 BLAST与人基因组比对， 确保特异性。
[0013] (2) 以 Age I-GN18-TT-loop-81NC-EcoR I形式设计一段 59bp序列 BTLA-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。 BTLA-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。
[0014] 实施例二 pLKO-BTLA载体的构建
[0015] 将合成的 shRNA寡核苷酸单链等量混合， 退火形成双链的 BTLA-RNAi。
[0016] 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：
[0017] 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;
[0018] 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-BTLA载体；
[0019] 3)转化；
[0020] 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-BTLA载体提取。
[0021] 实施例三 pLKO-BTLA载体转导 Jurkat细胞。
[0022] 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 μL PBS中， 与 20 μβ pLKO-BTLA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。
[0023] 实施例四荧光定量 PCR检测 BTLA基因表达量。
[0024] 分别接种 Jurkat细胞和转导 pLKO-BTLA载体的 Jurkat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。
[0025] 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 BTLA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， 禾 U 用 SYBR Primescript RT-PCR Kit检测各组细胞 BTLA基因相对表达量， 结果如图 1 所示。 结果显示转导 pLKO-BTLA载体的 Jurkat细胞， BTLA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 82.4%±7.6<¾， 从而证明本实验中采用的 p LKO-BTLA载体携带 shRNA能特异抑制 BTLA基因的表达， 且抑制效果非常显著 工业实用性
[0026] 本发明提供的 shRNA具有转导效率高， 可高效、 特异地抑制 Jurkat细胞 BTLA基 因表达的优点， 可作为有力工具应用于制备治疗 BTLA基因表达异常相关疾病的 药物。
Claims
[权利要求 1] 一种人 BTLA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 BTLA-RNAi。
[权利要求 2] 如权利要求 1所述的人 BTLA基因的 shRNA在制备治疗 BTLA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 BTLA基因的 shRN
A是 BTLA-RNAi。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2803525A1 (en) * | 2009-06-23 | 2011-01-13 | University Of Miami | Aptamer-targeted sirna to inhibit nonsense mediated decay |
| WO2012012518A2 (en) * | 2010-07-20 | 2012-01-26 | University Of Miami | Inhibition of nonsense mediated decay pathways |
| CN107034193A (zh) * | 2016-02-03 | 2017-08-11 | 北京马力喏生物科技有限公司 | 治疗b细胞白血病及b细胞淋巴瘤的治疗组合物 |
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- 2017-08-14 WO PCT/CN2017/097432 patent/WO2019033249A1/zh not_active Ceased
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2803525A1 (en) * | 2009-06-23 | 2011-01-13 | University Of Miami | Aptamer-targeted sirna to inhibit nonsense mediated decay |
| WO2012012518A2 (en) * | 2010-07-20 | 2012-01-26 | University Of Miami | Inhibition of nonsense mediated decay pathways |
| CN107034193A (zh) * | 2016-02-03 | 2017-08-11 | 北京马力喏生物科技有限公司 | 治疗b细胞白血病及b细胞淋巴瘤的治疗组合物 |
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