WO2019037052A1 - Shrna for targeted silencing of wucam - Google Patents
Shrna for targeted silencing of wucam Download PDFInfo
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- 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 DNA recombination technology in bioengineering, and specifically relates to shRNA which specifically silences the target protein W UCAM.
- WUCAM is a newly discovered membrane protein molecule composed of immunoglobulin variable region (IgV), transmembrane region and immunoreceptor tyrosine-based inhibitor motif (ITIM). Expressed on Treg cells, memory T cells, activating T cells, and NK cell surfaces. The study found that: 1) WUCAM is highly constitutively expressed on Treg, and its expression is further up-regulated after activation, which is lowly expressed on memory T cells, while naive CD4+ CD25-T
- IgV immunoglobulin variable region
- ITIM immunoreceptor tyrosine-based inhibitor motif
- WUCAM-Fc fusion protein can promote DC secretion of IL-10 and reduce pro-inflammatory factor IL-12; 3) The ability of WUCAM-Fc fusion protein-treated mature DC to stimulate T cell proliferation is weakened , related to DC secretion of IL-10; 4) in vivo use of WUCAM protein can significantly reduce delayed hypersensitivity, its effect and CTLA-4
- WUCAM is an important immunomodulator and functions similarly to sputum, which inhibits the cytotoxicity of NK cells and the proliferation of CD4+ T cells and the production of cytokines.
- WUCAM is a substance that inhibits the action of T cells, and plays a role in inhibiting the excessive activation of T cells under normal conditions; however, this property also makes it easy to be utilized by tumor cells to achieve immune escape. Therefore, WU CAM is a potential target for tumor therapy and requires in-depth research. However, the lack of lentiviral vectors that specifically inhibit the expression of WUCAM gene in the prior art makes the research difficult to develop.
- the object of the present invention is to synthesize a specific base sequence capable of targeting silent WUCAM, and provide an effective means for further research on the role of WUCAM.
- the main construct required in the present invention is a silencing vector of WUCAM.
- the construction of the core silencing vector is divided into two major steps. The first step is to design a 19 bp shRNA core fragment based on the human WUCAM sequence.
- the synthesized fragment has appropriate restriction sites at both ends, and the target fragment is ligated to the pSUPER vector to form
- the core silencing vector after transformation, pick positive clones for PCR identification. The correct vector was identified, DNA sequencing was performed, and silencing efficiency was subsequently detected by immunoblotting.
- a 19 bp shRNA core fragment capable of targeting human WUCAM was designed and synthesized, and its sequence 'J is 5,-GGACGTACACTGGGAGAAT-3, (SEQ ID No: l).
- Construction of an expression vector that specifically silences WUCAM includes the following steps:
- the sequence of WUCAM is specifically identified based on the human WUCAM gene sequence using the corresponding software design.
- the second step the artificial synthesis of double-stranded shRNA fragments
- the linearized vector is recovered by using a recovery kit, and the recovered fragment is ligated with the shRNA fragment obtained by annealing, transformed, and the positive clone is identified, and the successfully constructed silencing vector is obtained by sequencing.
- the plasmid was extracted, and the total RNA was extracted after transfecting the cells. After reverse transcription into cDNA, the silencing efficiency was detected by quantitative PCR.
- the shRNA fragment synthesized in the present invention can specifically silence WUCAM.
- the experimental results show that the successful construction of the vector can specifically silence the WUCAM and reduce the expression of intracellular WUCAM. Therefore, the application of WUCAM silencing vector can study the function of WUCAM more elaborately and provide a theoretical basis for explaining related mechanisms.
- DRAWINGS 1 is a schematic diagram showing the results of quantitative PCR detection of WUCAM gene expression of Jurkat cells after transfection of pSUPER-WUCAM 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 complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
- a 59 bp sequence was designed in the form of Bgl II-GN18-TT-loop-81NC-Hind III, 81NC is the reverse complement of NG18, the Bgl II cleavage site at the 5' end and the Hind III cleavage site at the 3' end. Point, the obtained sequences are respectively SEQ ID NO:
- the synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded shRNA. Take 2 g
- the pSUPER vector, Bgl II and Hind III were digested, electrophoresed, and the linear pSUPER large fragment was recovered. Dilute the linearized pSUPER concentration to 100
- Ng/ ⁇ , and the double-stranded shRNA obtained by annealing were ligated overnight at 4 °C with T4 DNA ligase (NEB).
- the ligation product was transformed into ToplO competent cells and plated on LB plates with Kan resistance. Monoclonal shakes were picked on the transformation plates and cultured overnight before being sent to Shanghai Biotech for sequencing. The correct bacteria were sequenced for the extraction of endotoxin-free plasmids, and the pSUPER-WUCAM vector was obtained.
- Example 3 The pSUPER-WUCAM vector transduced Jurkat cells.
- Jurkat cells were cultured, and cells grown in good condition were inoculated into a six-well plate at 10 6 cells per well, and the cell density was about 60% after 18 h, using Lipofectamin.
- the extracted pSUPER-WUCAM vector 3 g was transduced into Jurkat cells. After 6 h, the medium was changed to fresh complete medium and cultured for 48 h.
- Example 4 Detection of WUCAM gene expression level
- Jurkat cells and Jurkat cells transduced with pSUPER-WUCAM vector were separately inoculated into 6-well plates. Cell density reaches ⁇ , with RNeasy Mini
- Kit extracts total RNA from each group of cells, using PrimeScrip RT reagent
- the shRNA fragment synthesized in the present invention can specifically silence WUCAM.
- the experimental results show that the successful construction of the vector can specifically silence the WUCAM and reduce the expression of intracellular WUCAM. Therefore, the application of WUCAM silencing vector can study the function of WUCAM more elaborately and provide a theoretical basis for explaining related mechanisms.
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Abstract
Description
靶向沉默 WUC AM的 shRNA Target Silence WUC AM shRNA
技术领域 Technical field
[0001] 本发明属于生物工程中的 DNA重组技术领域， 具体涉及特异性沉默靶标蛋白 W UCAM的 shRNA。 [0001] The present invention belongs to the field of DNA recombination technology in bioengineering, and specifically relates to shRNA which specifically silences the target protein W UCAM.
背景技术 Background technique
[0002] WUCAM是一种新发现的膜蛋白分子， 由免疫球蛋白可变区 （IgV) 、 跨膜区 以及受体酪氨酸抑制基序 (ITIM， immunoreceptor tyrosine-based inhibitor motif) 组成， 广泛表达于 Treg细胞、 记忆性 T细胞、 活化性 T细胞以及 NK细胞表面 。 研究发现： 1) WUCAM在 Treg上组成性高表达， 活化后表达进一步上调， 它 在记忆性 T细胞上低表达， 而 naive CD4+ CD25- T [0002] WUCAM is a newly discovered membrane protein molecule composed of immunoglobulin variable region (IgV), transmembrane region and immunoreceptor tyrosine-based inhibitor motif (ITIM). Expressed on Treg cells, memory T cells, activating T cells, and NK cell surfaces. The study found that: 1) WUCAM is highly constitutively expressed on Treg, and its expression is further up-regulated after activation, which is lowly expressed on memory T cells, while naive CD4+ CD25-T
则仅在活化后表达； 2) WUCAM-Fc融合蛋白可以促进 DC分泌 IL- 10， 而减少 促炎因子 IL-12; 3) WUCAM-Fc融合蛋白处理过的成熟 DC刺激 T细胞增殖的 能力减弱， 与 DC分泌 IL-10有关； 4) 体内使用 WUCAM蛋白可以显著减弱迟 发性超敏反应， 其效应与 CTLA-4 It is expressed only after activation; 2) WUCAM-Fc fusion protein can promote DC secretion of IL-10 and reduce pro-inflammatory factor IL-12; 3) The ability of WUCAM-Fc fusion protein-treated mature DC to stimulate T cell proliferation is weakened , related to DC secretion of IL-10; 4) in vivo use of WUCAM protein can significantly reduce delayed hypersensitivity, its effect and CTLA-4
蛋白的效果相当 [1]。 越来越多的证据表明： WUCAM是一种重要的免疫调节剂 ， 与 ΓΠΜ的功能相似， 它可以抑制 NK细胞的细胞毒作用和 CD4+ T细胞的活 化增殖以及细胞因子的产生。 The effect of the protein is quite [1]. There is increasing evidence that WUCAM is an important immunomodulator and functions similarly to sputum, which inhibits the cytotoxicity of NK cells and the proliferation of CD4+ T cells and the production of cytokines.
技术问题 technical problem
[0003] WUCAM是一种抑制 T细胞发挥作用的物质， 其在正常状态下发挥抑制 T细胞 过度活化的作用； 但该特性也使其容易被肿瘤细胞利用实现免疫逃逸。 因此 WU CAM是一个潜在的肿瘤治疗的靶点， 需要进行深入研究， 但现有技术中缺乏特 异抑制 WUCAM基因表达的慢病毒载体使得相关研究无法很好地幵展。 [0003] WUCAM is a substance that inhibits the action of T cells, and plays a role in inhibiting the excessive activation of T cells under normal conditions; however, this property also makes it easy to be utilized by tumor cells to achieve immune escape. Therefore, WU CAM is a potential target for tumor therapy and requires in-depth research. However, the lack of lentiviral vectors that specifically inhibit the expression of WUCAM gene in the prior art makes the research difficult to develop.
问题的解决方案 Problem solution
技术解决方案 Technical solution
[0004] 本发明的目的是合成特定的能够靶向沉默 WUCAM的特异碱基序列， 为更加深 入的研究 WUCAM的作用提供了有效的手段。 [0005] 本发明中所需要主要构建的是 WUCAM的沉默载体。 核心沉默载体构建分为两 个大的步骤， 首先是根据人 WUCAM序列设计出 19bp的 shRNA核心片段， 所合成 片段两端带有合适的酶切位点， 将目的片段连接到 pSUPER载体上， 形成核心的 沉默载体， 转化后， 挑取阳性克隆进行 PCR鉴定。 鉴定正确的载体， 进行 DNA 测序， 随后通过免疫印记实验进行沉默效率的检测。 [0004] The object of the present invention is to synthesize a specific base sequence capable of targeting silent WUCAM, and provide an effective means for further research on the role of WUCAM. [0005] The main construct required in the present invention is a silencing vector of WUCAM. The construction of the core silencing vector is divided into two major steps. The first step is to design a 19 bp shRNA core fragment based on the human WUCAM sequence. The synthesized fragment has appropriate restriction sites at both ends, and the target fragment is ligated to the pSUPER vector to form The core silencing vector, after transformation, pick positive clones for PCR identification. The correct vector was identified, DNA sequencing was performed, and silencing efficiency was subsequently detected by immunoblotting.
[0006] 本发明中设计合成了一个能够靶向人 WUCAM的 19bp的 shRNA核心片段， 其序 歹 'J为 5，- GGACGTACACTGGGAGAAT -3， (SEQ ID No: l) 。 In the present invention, a 19 bp shRNA core fragment capable of targeting human WUCAM was designed and synthesized, and its sequence 'J is 5,-GGACGTACACTGGGAGAAT-3, (SEQ ID No: l).
[0007] 特异性沉默 WUCAM的表达载体的构建包括以下步骤： [0007] Construction of an expression vector that specifically silences WUCAM includes the following steps:
[0008] 第一步， shRNA片段序列设计 [0008] First step, shRNA fragment sequence design
[0009] 根据人 WUCAM基因序列应用相应的软件设计特异性识别 WUCAM的序列。 [0009] The sequence of WUCAM is specifically identified based on the human WUCAM gene sequence using the corresponding software design.
[0010] 第二步， 双链 shRNA片段的人工合成 [0010] The second step, the artificial synthesis of double-stranded shRNA fragments
[0011] 将人工合成的 Sense (SEQ ID No: 2) 和 Antisense (SEQ ID No: 3) 通过变性、 退火形成双链 DNA。 [0011] The artificially synthesized Sense (SEQ ID No: 2) and Antisense (SEQ ID No: 3) were denatured and annealed to form double-stranded DNA.
[0012] 第三步， 沉默载体的构建 [0012] The third step, the construction of the silent carrier
[0013] 将沉默载体 pSUPER双酶切后,利用回收试剂盒回收线性化载体， 将回收片段与 退火所得的 shRNA片段进行连接、 转化、 鉴定阳性克隆、 测序得到构建成功的 沉默载体。 After double-clearing the silencing vector pSUPER, the linearized vector is recovered by using a recovery kit, and the recovered fragment is ligated with the shRNA fragment obtained by annealing, transformed, and the positive clone is identified, and the successfully constructed silencing vector is obtained by sequencing.
[0014] 第四步， 沉默靶蛋白效率的检测 [0014] The fourth step, detection of silencing target protein efficiency
[0015] 提取质粒， 转染细胞后提取总 RNA， 逆转录为 cDNA后利用定量 PCR检测沉默 效率。 [0015] The plasmid was extracted, and the total RNA was extracted after transfecting the cells. After reverse transcription into cDNA, the silencing efficiency was detected by quantitative PCR.
发明的有益效果 Advantageous effects of the invention
有益效果 Beneficial effect
[0016] 本发明中设计合成的 shRNA片段， 能够特异性的沉默 WUCAM。 实验结果表明 ， 构建成功的载体， 能够特异性的沉默 WUCAM, 降低细胞内 WUCAM的表达。 因此， WUCAM沉默载体的应用能够更为精细地研究 WUCAM的功能， 为解释相 关机制提供理论基础。 [0016] The shRNA fragment synthesized in the present invention can specifically silence WUCAM. The experimental results show that the successful construction of the vector can specifically silence the WUCAM and reduce the expression of intracellular WUCAM. Therefore, the application of WUCAM silencing vector can study the function of WUCAM more elaborately and provide a theoretical basis for explaining related mechanisms.
对附图的简要说明 Brief description of the drawing
附图说明 [0017] 图 1为转染 pSUPER-WUCAM载体后 Jurkat细胞的定量 PCR检测 WUCAM基因表 达情况结果示意图。 DRAWINGS 1 is a schematic diagram showing the results of quantitative PCR detection of WUCAM gene expression of Jurkat cells after transfection of pSUPER-WUCAM vector.
实施该发明的最佳实施例 BEST MODE FOR CARRYING OUT THE INVENTION
本发明的最佳实施方式 BEST MODE FOR CARRYING OUT THE INVENTION
[0018] 下面结合附图与具体实施例对本发明做进一步的说明。 [0018] The present invention will be further described below in conjunction with the drawings and specific embodiments.
[0019] 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 [0019] 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.
。 下文所述完全培养基为加入了 10%胎牛血清的细胞培养基。 . The complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
[0020] 实施例一 shRNA寡核苷酸序列的设计 Example 1 Design of shRNA Oligonucleotide Sequences
[0021] 在 GenBank査找到 WUCAM的 mRNA全序列， 经 BLAST同源性比对证实特异性 后应用 RNAstmcture 4.4软件对靶 mRNA序列的二级结构进行评估， 最后获得靶 核苷酸序列， 如 SEQ ID NO: 1所示。 [0021] The full sequence of the mRNA of WUCAM was found in GenBank, and the specificity was confirmed by BLAST homology alignment. The secondary structure of the target mRNA sequence was evaluated by RNAstmcture 4.4 software, and finally the target nucleotide sequence, such as SEQ ID, was obtained. NO: 1 is shown.
[0022] 以 Bgl II-GN18-TT-loop-81NC-Hind III形式设计 59bp序列， 81NC为 NG18的反向 互补， 5'端为 Bgl II酶切位点， 3'端为 Hind III酶切位点， 所获序列分别如 SEQ ID[0022] A 59 bp sequence was designed in the form of Bgl II-GN18-TT-loop-81NC-Hind III, 81NC is the reverse complement of NG18, the Bgl II cleavage site at the 5' end and the Hind III cleavage site at the 3' end. Point, the obtained sequences are respectively SEQ ID
NO: 2和 SEQ ID NO: 3所示。 NO: 2 and SEQ ID NO: 3.
[0023] 实施例二 shRNA慢病毒表达载体的构建 Example 2 Construction of shRNA Lentiviral Expression Vector
[0024] 将合成的 shRNA寡核苷酸单链等量混合， 退火形成双链的 shRNA。 取 2 g [0024] The synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded shRNA. Take 2 g
pSUPER载体， Bgl II和 Hind III双酶切， 电泳， 回收线性 pSUPER大片段。 将线 性化 pSUPER浓度稀释至 100 The pSUPER vector, Bgl II and Hind III were digested, electrophoresed, and the linear pSUPER large fragment was recovered. Dilute the linearized pSUPER concentration to 100
ng/μί, 与退火所得双链 shRNA用 T4DNA连接酶 （NEB) 4°C连接过夜。 将连接 产物转化 ToplO感受态细胞， 涂布在具有 Kan抗性的 LB平板上。 在转化平板上挑 取单克隆摇菌， 培养过夜后送至上海生工测序。 测序正确的菌用于无内毒素质 粒的提取， 获得 pSUPER-WUCAM载体。 Ng/μί, and the double-stranded shRNA obtained by annealing were ligated overnight at 4 °C with T4 DNA ligase (NEB). The ligation product was transformed into ToplO competent cells and plated on LB plates with Kan resistance. Monoclonal shakes were picked on the transformation plates and cultured overnight before being sent to Shanghai Biotech for sequencing. The correct bacteria were sequenced for the extraction of endotoxin-free plasmids, and the pSUPER-WUCAM vector was obtained.
[0025] 实施例三 pSUPER-WUCAM载体转导 Jurkat细胞。 Example 3 The pSUPER-WUCAM vector transduced Jurkat cells.
[0026] 培养 Jurkat细胞， 取生长状态良好的细胞接种到六孔板中， 每孔 10 6个细胞， 18 h后细胞密度约为 60%， 利用 Lipofectamin [0026] Jurkat cells were cultured, and cells grown in good condition were inoculated into a six-well plate at 10 6 cells per well, and the cell density was about 60% after 18 h, using Lipofectamin.
2000将提取的 pSUPER-WUCAM载体 3 g转导 Jurkat细胞。 6 h后， 将培养基换为 新鲜的完全培养基继续培养 48 h。 In 2000, the extracted pSUPER-WUCAM vector 3 g was transduced into Jurkat cells. After 6 h, the medium was changed to fresh complete medium and cultured for 48 h.
[0027] 实施例四 WUCAM基因表达水平检测 [0028] 分别接种 Jurkat细胞和转导了 pSUPER- WUCAM载体的 Jurkat细胞至 6孔板。 细 胞密度达到 δΟ^^Ο^吋， 用 RNeasy Mini [0027] Example 4 Detection of WUCAM gene expression level [0028] Jurkat cells and Jurkat cells transduced with pSUPER-WUCAM vector were separately inoculated into 6-well plates. Cell density reaches δΟ^^Ο^吋, with RNeasy Mini
Kit提取各组细胞的总 RNA， 利用 PrimeScrip RT reagent Kit extracts total RNA from each group of cells, using PrimeScrip RT reagent
Kit将 mRNA逆转录为 cDNA， 逆转录条件： 37°C， 15min; 85°C， 5s; 4°C， ∞。 反转录结束后， 加入 90μ1的 RNase-Free dH20稀释 cDNA， -20°C保存。 Kit reverse transcribes mRNA into cDNA, reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, ∞. After the reverse transcription was completed, the cDNA was diluted with 90 μl of RNase-Free dH20 and stored at -20 °C.
[0029] 取各组细胞的 cDNA 1 [0029] taking cDNA 1 of each group of cells
为模板， 以 GAPDH为内参， 实吋荧光定量 PCR (QPCR) 检测 WUCAM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 WUCAM基因相对表达量， 结 果如图 2所示。 结果显示转导了 pSUPER-WUCAM载体的 Jurkat细胞， WUCAM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 77.1%±5.5<¾， 从而证 明本实验中采用的 pSUPER-WUCAM载体能特异抑制 WUCAM基因的表达， 且抑 制效果非常显著。 For the template, GAPDH was used as the internal reference, and the relative expression of WUCAM was detected by real-time quantitative PCR (QPCR). The reaction conditions were set: 95 ° C for 10 s, 1 cycle; 95 ° C for 5 s, 60 ° C for 30 s, for 40 cycles. The relative expression of WUCAM gene in each group was detected by SYBR Primescript RT-PCR Kit. The results are shown in Figure 2. The results showed that the expression of WUCAM gene was significantly inhibited in Jurkat cells transduced with pSUPER-WUCAM vector. The inhibitory efficiency of the interference fragment on the target gene was 77.1%±5.5<3⁄4, which proved that the pSUPER-WUCAM vector used in this experiment can specifically inhibit The expression of the WUCAM gene is very significant.
工业实用性 Industrial applicability
[0030] 本发明中设计合成的 shRNA片段， 能够特异性的沉默 WUCAM。 实验结果表明 ， 构建成功的载体， 能够特异性的沉默 WUCAM, 降低细胞内 WUCAM的表达。 因此， WUCAM沉默载体的应用能够更为精细地研究 WUCAM的功能， 为解释相 关机制提供理论基础。 [0030] The shRNA fragment synthesized in the present invention can specifically silence WUCAM. The experimental results show that the successful construction of the vector can specifically silence the WUCAM and reduce the expression of intracellular WUCAM. Therefore, the application of WUCAM silencing vector can study the function of WUCAM more elaborately and provide a theoretical basis for explaining related mechanisms.
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| ZHANG, TAO ET AL.: "Increased Expression of TIGIT on CD4+ T Cells Ameliorates Immune-Mediated Bone Marrow Failure of Aplastic Anemia", JOURNAL OF CELLULAR BIOCHEMISTRY, vol. 115, no. 11, 10 September 2014 (2014-09-10), pages 1918 - 1927, XP055215805, ISSN: 1097-4644 * |
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