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WO2019036869A1 - 人 TL6 基因的 shRNA 及其应用 - Google Patents

人 TL6 基因的 shRNA 及其应用 Download PDF

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Publication number
WO2019036869A1
WO2019036869A1 PCT/CN2017/098367 CN2017098367W WO2019036869A1 WO 2019036869 A1 WO2019036869 A1 WO 2019036869A1 CN 2017098367 W CN2017098367 W CN 2017098367W WO 2019036869 A1 WO2019036869 A1 WO 2019036869A1
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Prior art keywords
shrna
gene
expression
human
sequence
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English (en)
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 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 TL6 gene on the surface of human T cells, which can inhibit the expression of the human TL6 gene after transfer.
  • TL6 is a ligand for the surface molecule AITR on thymus-derived CD4+ CD25+ Treg cells. research shows
  • AITR/TL6 has many important biological activities, including cell proliferation, differentiation and survival. technical problem
  • the AITR/TL6 system participates in the role of Treg cells in the regulation of immune regulation, plays an important role in the immunotherapy of tumors, and has a good clinical transformation prospect.
  • the lack of vectors for specifically inhibiting the expression of TL6 gene in the prior art makes related research. Can't develop well.
  • 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 designed a pair of TL6-shRNA targeting TL6 gene according to the mRNA sequence of human TL6 gene in GenBank database and the primer design principle of shRNA, and commissioned Shanghai Biotech to synthesize the TL6-shR.
  • the oligonucleotide of the TL6-shRNA designed above 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 transduced the pSilencer3.1-H1 hygro RNAi expression vector containing TL6-shRNA into the Jurkat cell line, and the silencing efficiency of TL6 mRNA was 73.9%.
  • the TL6-shRNA provided by the invention has the advantages of high transduction efficiency, high-efficiency and specific inhibition of TL6 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 TL6 gene.
  • FIG. 1 is a schematic diagram showing the results of fluorescent quantitative PCR detection of TL6 gene expression by Jurkat cells transduced with TL6-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'- GCTCCCAATGCAAACTACA
  • the sense strand ⁇ ij of TL6-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 TL6-shRNA expression vector was mixed and added to the electric shock cup, and electroporated using the Invitrogen Neon electroporation 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. In the middle, the cells were gently shaken to mix the cells, and the expression of TL6 gene was detected 48 h later.
  • Example 4 Fluorescence quantitative PCR was used to detect the expression level of TL6 gene.
  • Jurkat cells were transfected with normal Jurkat cells and TL6-shRNA expression vector, total RNA was extracted from each group with RNeasy Mini Kit, mRNA was reverse transcribed into cDNA using PrimeScrip RT reagent Kit, and then 9 (L RNase) was added. -Free dH20 diluted cDNA, stored at -20 °C for later detection
  • ⁇ is the template, GAPDH is used as the internal reference, and the relative expression of TL6 is detected by real-time quantitative PCR (QPCR).
  • the reaction conditions are set: 95 ° C for 10 s, 1 cycle; 95 ° C 5 s, 60 ° C
  • the TL6-shRNA provided by the invention has the advantages of high transduction efficiency, high-efficiency and specific inhibition of TL6 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 TL6 gene.

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Abstract

一种人TL6基因的shRNA及其应用，其正义链序列SEQ ID NO：1所示，反义链序列如SEQ ID NO：2所示。本发明所述的shRNA是根据人TL6基因的核苷酸序列设计、合成并构建了shRNA表达载体。

Description

人 TL6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 TL6基因的核苷酸序列设计合成 shRNA, 所述 s hRNA转入后能抑制所述人 TL6基因的表达。
背景技术
[0002] TL6是胸腺来源的 CD4+ CD25+Treg细胞上的表面分子 AITR的配体。 研究表明
， AITR/TL6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。 技术问题
[0003] AITR/TL6系统参与 Treg细胞发挥免疫调节的作用， 在肿瘤的免疫治疗中起重要 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 TL6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 TL6基因的蛋白表达水平。
[0006] 本发明根据 GenBank数据库中人 TL6基因的 mRNA序列以及 shRNA的引物设计 原则， 设计了 1对靶向 TL6基因的 TL6-shRNA， 并委托上海生工合成所述 TL6-shR
NA。
[0007] 本发明将上述设计的 TL6-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 TL6-shRNA的 pSilencer3.1-Hl hygro RNAi表达载体转导进 Jurkat 细胞系， 其 TL6 mRNA的被沉默效率为 73.9%。
发明的有益效果
有益效果
[0009] 本发明提供的 TL6-shRNA具有转导效率高， 可高效、 特异地抑制 Jurkat细胞 TL6 基因表达的优点， 可作为有力工具应用于制备治疗 TL6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 TL6-shRNA表达载体的 Jurkat细胞的荧光定量 PCR检测 TL6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 TL6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'- GCTCCCAATGCAAACTACA
-3'， TL6-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 TL6-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 TL6-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
TL6-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 TL6基因表达情况。
[0020] 实施例四荧光定量 PCR检测 TL6基因表达量。
[0021] 取正常 Jurkat细胞和转导 TL6-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 9( L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
Ιμί为模板， 以 GAPDH为内参， 实吋荧光定量 PCR (QPCR) 检测 TL6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 TL6基因相对 表达量， 结果如图 1所示。 结果显示转导 TL6-shRNA表达载体的 Jurkat细胞， TL6 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 73.9<¾±7.4%， 从而 证明本实验中采用的 TL6-shRNA表达载体携带 shRNA能特异抑制 TL6基因的表达
， 且抑制效果非常显著。
工业实用性
[0023] 本发明提供的 TL6-shRNA具有转导效率高， 可高效、 特异地抑制 Jurkat细胞 TL6 基因表达的优点， 可作为有力工具应用于制备治疗 TL6基因表达异常相关疾病的 药物。

Claims

权利要求书
[权利要求 1] 一种抑制 TL6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 TL6 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 TL6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 TL6基因表达异常相关疾病的药物中 的应用。
PCT/CN2017/098367 2017-08-21 2017-08-21 人 TL6 基因的 shRNA 及其应用 Ceased WO2019036869A1 (zh)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104080909A (zh) * 2011-11-30 2014-10-01 中外制药株式会社 包含进入细胞内以形成免疫复合体的搬运体(载体)的药物

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104080909A (zh) * 2011-11-30 2014-10-01 中外制药株式会社 包含进入细胞内以形成免疫复合体的搬运体(载体)的药物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Identification of a novel activation-inducible protein of the tumor necrosis factor receptor superfamily and its ligand", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 10, 5 March 1999 (1999-03-05), pages 6056 - 6061, XP002147323, ISSN: 1083-351X *

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