WO2019037055A1 - 人 GRD4 基因的 shRNA 及其应用 - Google Patents

人 GRD4 基因的 shRNA 及其应用 Download PDF

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WO2019037055A1
WO2019037055A1 PCT/CN2017/098912 CN2017098912W WO2019037055A1 WO 2019037055 A1 WO2019037055 A1 WO 2019037055A1 CN 2017098912 W CN2017098912 W CN 2017098912W WO 2019037055 A1 WO2019037055 A1 WO 2019037055A1
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shrna
grd4
gene
expression
cells
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PCT/CN2017/098912
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毛吉炎
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深圳市博奥康生物科技有限公司
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Priority to PCT/CN2017/098912 priority Critical patent/WO2019037055A1/zh
Publication of WO2019037055A1 publication Critical patent/WO2019037055A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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

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  • 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 is directed to the synthesis of shRNA against the nucleotide sequence of the GRD4 gene on the surface of human T cells, and the shRNA can inhibit the expression of the human GRD4 gene after being transferred.
  • the anti-tumor immune mechanism of the body includes both cellular immunity and humoral immunity, but the effector cells that play a major role in cellular immune mechanisms are T cells, among which CD8+ T cells are tumor immunity. The most important effect is the execution of cells.
  • T cell activation requires dual signal recognition.
  • the tumor antigen first binds to the MHC molecule on the antigen presenting cell or target cell, and then binds to the T lymphocyte surface antigen recognition receptor (TCR) to provide the first signal for T cell activation; T cell
  • TCR T lymphocyte surface antigen recognition receptor
  • T cell The surface CD28 binds to the antigen presenting cell surface or the target cell surface B7 molecule to provide a second signal.
  • T cells After double signal recognition, T cells are fully activated, and the activated T cells first self-proliferate, producing a large number of antigen-specific T cells, which migrate to the tumor to kill. After 2 to 3 days of activated T cells, the expression of negative regulatory molecules such as GRD4 is up-regulated.
  • GRD4 is a transmembrane protein and belongs to the CD28 family.
  • the ligand of GRD4 is a member of the B7 family, which binds to CD27 molecules competitively with B7-1 and B7-2, inhibits the proliferation of activated T cells, and negatively regulates immunity.
  • the affinity of GRD4 and B7 molecules is more than 20 times that of CD28. Under physiological conditions, the immune over-amplification is prevented.
  • the binding of CD28 or GRD4 to CD80/86 exerts a counteraction on the function of immune cells, and plays a fine immune regulation role in normal humans. Maintain a dynamic balance between health and disease. However, it is often used by tumor cells to achieve immune escape. Therefore, the research on the role of GRD4 in tumor development can promote the development of tumor therapy. However, the lack of vectors specifically inhibiting GRD4 gene expression in the prior art makes the 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 designed a pair of GRD4-shRNA targeting the GRD4 gene based on the mRNA sequence of the human GRD4 gene in the GenBank database and the primer design principle of shRNA, and commissioned Shanghai Biotech to synthesize the G.
  • the oligonucleotide of the GRD4-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 transfects the pSilencer3.1-H1 hygro RNAi expression vector containing GRD4-shRNA into the Jurkat cell line, and the silencing efficiency of GRD4 mRNA is 83.0 ⁇ 3 ⁇ 4.
  • the GRD4-shRNA provided by the invention has high transduction efficiency and can efficiently and specifically inhibit Jurkat cells G
  • RD4 gene expression can be used as a powerful tool for the preparation of drugs for the treatment of diseases associated with abnormal expression of GRD4 gene.
  • FIG. 1 is a schematic diagram showing the results of fluorescent quantitative PCR detection of GRD4 gene expression of Jurkat cells transduced with GRD4-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'- ATGTACCCACCGCCATACT
  • the sense strand sequence ⁇ ij of GRD4-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 GRD4-shRNA expression vector was mixed and added to the electric shock cup, and electroporated 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 GRD4 gene after 48 h.
  • Example 4 Fluorescence quantitative PCR was used to detect the expression level of GRD4 gene.
  • Jurkat cells and normal Jurkat cells transduced with GRD4-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 adds 90 ⁇ M of RNase-Free dH20 to dilute cDNA, -20
  • ⁇ is the template, GAPDH is used as the internal reference, and the relative expression of GRD4 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 GRD4 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 GRD4 gene was significantly inhibited in Jurkat cells transduced with GRD4-shRNA expression vector, and the inhibitory efficiency of the interference fragment was 83.0 ⁇ 3 ⁇ 4 ⁇ 4.1%, which proved that the GRD4-shRNA expression vector used in this experiment carries shRNA. It can specifically inhibit the expression of the GRD4 gene, and the inhibitory effect is very remarkable.
  • the GRD4-shRNA provided by the invention has high transduction efficiency and can efficiently and specifically inhibit Jurkat cells G
  • RD4 gene expression can be used as a powerful tool for the preparation of drugs for the treatment of diseases associated with abnormal expression of GRD4 gene.

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Abstract

提供了一种人GRD4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。shRNA是根据人GRD4基因的核苷酸序列设计、合成并构建了shRNA表达载体，通过功能性实验验证，得到了抑制GRD4基因表达的shRNA。还提供了所述shRNA的应用。

Description

说明书 发明名称：人 GRD4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 GRD4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 GRD4基因的表达。
背景技术
[0002] 机体的抗肿瘤免疫机制包括细胞免疫和体液免疫两个方面， 但是以细胞免疫为 主， 在细胞免疫机制中起主要作用的效应细胞为 T细胞， 其中 CD8+T细胞是肿 瘤免疫中最主要的效应执行细胞。 T细胞活化需要双信号识别， 肿瘤抗原首先与 抗原提呈细胞或者靶细胞上的 MHC分子结合后， 与 T淋巴细胞表面抗原识别受 体 （TCR) 结合为 T细胞活化提供第一信号； T细胞表面的 CD28与抗原提呈细 胞表面或者靶细胞表面 B7分子结合提供第二信号。 双信号识别后， T细胞完全 活化， 活化的 T细胞首先自我增殖， 产生大量的抗原特异性 T细胞， 迁移到肿 瘤局部起到杀伤作用。 活化的 T细胞 2~3天后 GRD4等负调控分子幵始表达上 调。
技术问题
[0003] GRD4为跨膜蛋白， 属于 CD28家族成员。 GRD4的配体为 B7家族成员， 其 与 CD28分子竞争性地与 B7-1和 B7-2结合， 抑制活化的 T细胞增殖， 对免疫 起负调控作用。 GRD4与 B7分子的亲和力是 CD28的 20倍以上， 在生理情况下 ， 防止免疫过度放大， CD28或 GRD4与 CD80/86结合对免疫细胞的功能发挥反 作用， 在正常人体中起到精细的免疫调节作用， 维持着健康与疾病的动态平衡 。 但常常会被肿瘤细胞利用， 达到免疫逃逸的目的。 因此对 GRD4在肿瘤发生发 展中作用的研究可以很好地促进肿瘤治疗领域的发展， 但现有技术中也缺乏特 异抑制 GRD4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 GRD4基因的蛋白表达水平。
[0006] 本发明根据 GenBank数据库中人 GRD4基因的 mRNA序列以及 shRNA的引物设 计原则， 设计了 1对靶向 GRD4基因的 GRD4-shRNA， 并委托上海生工合成所述 G
RD4-shRNA。
[0007] 本发明将上述设计的 GRD4-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 GRD4-shRNA的 pSilencer3.1-H1 hygro RNAi表达载体转导进 Jurkat 细胞系， 其 GRD4 mRNA的被沉默效率为 83.0<¾。
发明的有益效果
有益效果
[0009] 本发明提供的 GRD4-shRNA具有转导效率高， 可高效、 特异地抑制 Jurkat细胞 G
RD4基因表达的优点， 可作为有力工具应用于制备治疗 GRD4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 GRD4-shRNA表达载体的 Jurkat细胞的荧光定量 PCR检测 GRD4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 GRD4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'- ATGTACCCACCGCCATACT
-3'， GRD4-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 GRD4-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 GRD4-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
GRD4-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 GRD4基因表达情 况。
[0020] 实施例四荧光定量 PCR检测 GRD4基因表达量。
[0021] 取正常 Jurkat细胞和转导 GRD4-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
Ιμί为模板， 以 GAPDH为内参， 实吋荧光定量 PCR (QPCR) 检测 GRD4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 GRD4基因相对表达量， 结果如图 1 所示。 结果显示转导 GRD4-shRNA表达载体的 Jurkat细胞， GRD4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 83.0<¾±4.1%， 从而证明本实验中 采用的 GRD4-shRNA表达载体携带 shRNA能特异抑制 GRD4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 GRD4-shRNA具有转导效率高， 可高效、 特异地抑制 Jurkat细胞 G
RD4基因表达的优点， 可作为有力工具应用于制备治疗 GRD4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 GRD4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 GRD4 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 GRD4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 GRD4基因表达异常相关疾病的药物 中的应用。
PCT/CN2017/098912 2017-08-24 2017-08-24 人 GRD4 基因的 shRNA 及其应用 WO2019037055A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016070136A1 (en) * 2014-10-31 2016-05-06 Massachusetts Institute Of Technology Delivery of biomolecules to immune cells
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
CN107058232A (zh) * 2017-04-12 2017-08-18 上海优卡迪生物医药科技有限公司 胆固醇转脂酶soat1被抑制的car‑t细胞及其制备方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016070136A1 (en) * 2014-10-31 2016-05-06 Massachusetts Institute Of Technology Delivery of biomolecules to immune cells
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
CN107058232A (zh) * 2017-04-12 2017-08-18 上海优卡迪生物医药科技有限公司 胆固醇转脂酶soat1被抑制的car‑t细胞及其制备方法和应用

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