WO2018170765A1 - Imd16基因的rna干涉载体及其应用 - Google Patents

Imd16基因的rna干涉载体及其应用 Download PDF

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WO2018170765A1
WO2018170765A1 PCT/CN2017/077607 CN2017077607W WO2018170765A1 WO 2018170765 A1 WO2018170765 A1 WO 2018170765A1 CN 2017077607 W CN2017077607 W CN 2017077607W WO 2018170765 A1 WO2018170765 A1 WO 2018170765A1
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imd16
sequence
vector
gene
rna interference
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PCT/CN2017/077607
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English (en)
French (fr)
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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/CN2017/077607 priority Critical patent/WO2018170765A1/zh
Publication of WO2018170765A1 publication Critical patent/WO2018170765A1/zh

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  • RNA interference vector of IMD16 gene Inventive name: RNA interference vector of IMD16 gene and its application
  • the present invention belongs to the field of genetic engineering, and relates to an RNA interference vector and application thereof, and particularly relates to an RNA interference vector of I MD16 gene and application thereof.
  • IMD16 is a member of the TNF receptor superfamily and is a type I transmembrane glycoprotein.
  • the expression profile of IMD16 is restricted to the surface of activated CD4+ and CD8+ T cells, and is CD4+
  • the human IMD16 ligand (IMD16L/CD 134L) contains 183 amino acids (extracellular 139 amino acids transmembrane 21 amino acids, intracellular 23 amino acids) and belongs to the TNF family member, which is a scorpion-type transmembrane glycoprotein.
  • IMD 16/GP34 is an important co-stimulatory molecule that plays an important role in the body's immune response and various diseases. Its interaction can promote the activation, proliferation, migration, prolongation of life and promote hair growth of CD+4T cells. The formation of the center and the differentiation and maturation of DC.
  • IMD16 plays an important role in tumor immunotherapy, and a large amount of research is needed to achieve clinical transformation.
  • the lack of a vector that specifically inhibits the expression of IMD16 gene in the prior art makes the related research not well developed.
  • the object of the present invention is an RNA interference vector of the IMD16 gene and a construction method and application thereof.
  • an isolated polynucleotide is provided, the nucleotide sequence of which is set forth in SEQ ID NO: 1.
  • a recombinant vector comprising the nucleotide sequence set forth in SEQ ID NO: 2, wherein the recombinant vector is a pLVX-shRNA1 vector as a backbone vector, in a polyclonal enzyme IMD16- consisting of Bam HI restriction site + target nucleotide sequence + stem loop structure sequence + target nucleotide sequence complementary sequence + termination site sequence + EcoR I restriction site inserted at the cleavage site RNAi sequence.
  • RNA interference vector of the aforementioned IMD16 gene including the following steps:
  • IMD16-RNAi sequence According to the full sequence of IMD16 mRNA, design the interference fragment according to the design principle of RNAi fragment, NCBI BLSAT for homology comparison, confirm the specificity to obtain the target nucleotide sequence, obtain SEQ. ID NO: sequence shown by l, named IMD16-RNAi sequence;
  • RNA interference vector of IMD16 gene The synthesized IMD16-RNAi sequence and the extracted plasmid pLVX-shRNA1 were digested with restriction endonucleases Bam HI and EcoR I, electrophoresis and gelatinization. The vector was recovered, and the IMD16-RNAi sequence was ligated into the SJpLVX-shRNA1 expression vector by T4 DNA Ligase to obtain a ligation product; the ligation product was transformed into competent E. coli Stbl3 and uniformly coated onto an ampicillin-containing LB medium plate. The positive monoclonal colonies were picked and cultured and identified by PCR. The preliminary identification results indicated that the IMD16-RNAi sequence was inserted into the successful bacterial solution for sequencing and identification;
  • RNA interference vector of IMD16 gene Extraction of RNA interference vector of IMD16 gene: The sequencing result confirmed that the IMD16-RNAi sequence was inserted into a successful bacterial cell expansion culture, and a large amount of recombinant plasmid was extracted to obtain an RNA interference carrier of IMD16 gene.
  • the present invention utilizes RNAi technology to construct an RNA interference vector targeting IMD16 gene expression. After successful identification, the Jurkat cells are electroporated, and the inhibitory effect of IMD16 gene expression is verified from the mRNA level by using real-time fluorescent quantitative PCR technology. The IMD16-RNAi sequence provided by the present invention was successfully inserted into the pLVX-shRNA1 expression vector, and the inhibitory effect on IMD16 gene expression was remarkable.
  • a polynucleotide of SEQ ID NO: 1 or a vector comprising the polynucleotide of SEQ ID NO: 1 or an RNA interference vector of the aforementioned IMD16 gene is provided at IMD16 Use in drugs for diseases with abnormal gene expression.
  • RNA interference vector of the IMD16 gene provided by the invention has the advantages of high transfection efficiency, low dosage, high and specific inhibition of IMD16 gene expression in Jurkat cells, and can be used as a powerful tool for preparing and treating I MD16 gene expression abnormality. Drugs related to the disease.
  • FIG. 1 is a schematic diagram showing the results of real-time quantitative PCR detection of RNA interference vector cells transfected with IMD16 gene.
  • Jurkat cells were purchased from ATCC, RNAi vector pLVX-shRNAl was purchased from Clontech, RNeasyMi niKit was purchased from Qiagen, and endotoxin free plasmid extraction kit was purchased from Omega bio-tek.
  • the complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
  • the Ai fragment design principle designed the interference fragment, NCBI BLSAT for homology comparison, and confirmed the specificity to obtain the target nucleotide sequence.
  • the IMD16-RNAi sequence was designed as follows: Bam HI restriction site +19 nt target nucleotide sequence ⁇ ij + stem loop structure
  • TTCAAGAGA target sequence complementary sequence + RNA Polym polymerase transcriptional stop site (TTTTTT) + EcoR I restriction site six regions, the sequence of which is shown in SEQ ID NO: 1.
  • the designed IMD16-RNAi sequence chain was synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • the synthesized IMD16-RNAi sequence and the extracted plasmid pLVX-shRNA1 were digested with restriction endonucleases Bam HI and EcoR I, electrophoresed, and the gel was recovered, and the IMD16-RNAi sequence was further amplified by T4 DNA Ligase.
  • An RNA interference vector that forms the IMD16 gene is ligated into the vector pLVX-shRNA1.
  • the ligation product was transformed into competent E. coli Stbl3, plated on a plate containing ampicillin LB medium, and cultured at 37 ° C for 14 h. Pick up the grown colonies and send them to Shanghai Biotech for sequencing. The sequencing results are fully consistent with the designed sequence and can be used in subsequent experiments.
  • Jurkat cells were cultured, and 35,000 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.
  • RNA interference vector of IMD16 gene was mixed and added to the electric shock cup.
  • the electric rotation was performed by BTX ECM830 electro-rotation instrument.
  • the electroporation procedure was: 2.1 KV, 25 ⁇ , pulse electric shock once; the cells were transferred to 6 cm containing 5 mL DMEM complete medium. In the dish, the cells were gently shaken to mix the cells, and the expression of IMD16 gene was detected 48 h later.
  • 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 ⁇ l of RNase-Free dH20 and stored at -20 ° C for later detection. Take the cDNA of each group of cells
  • ⁇ is the template, with GAPDH as the internal reference, real-time quantitative PCR (QPCR) to detect the relative expression of IMD16, set the reaction conditions: 95 ° C 10s, 1 cycle; 95 ° C 5s, 54 ° C 30s, a total of 40 cycles
  • QPCR real-time quantitative PCR
  • RNA interference vector of the IMD16 gene provided by the invention has the advantages of high transfection efficiency, low dosage, high and specific inhibition of IMD16 gene expression in Jurkat cells, and can be used as a powerful tool for preparing and treating I MD16 gene expression abnormality. Drugs related to the disease.

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Abstract

提供了一种针对IMD16基因的RNAi序列，其核苷酸序列如SEQ ID NO:1所示。还提供了包含该RNAi序列的表达载体及其构建方法和应用。

Description

说明书 发明名称： IMD16基因的 RNA干涉载体及其应用 技术领域
[0001] 本发明属于基因工程领域， 涉及一种 RNA干涉载体及其应用， 尤其涉及一种 I MD16基因的 RNA干涉载体及其应用。
背景技术
[0002] IMD16是 TNF受体超家族成员之一， 为 I型跨膜糖蛋白。 IMD16的表达谱局限于 活化的 CD4+和 CD8+ T细胞表面， 且以 CD4+
T细胞为主。 人 IMD16配体 (IMD16L/ CD 134L)含 183个氨基酸 (胞外 139个氨基酸 跨膜 21个氨基酸， 胞内 23个氨基酸)， 属 TNF家庭成员， 为 Π型跨膜糖蛋白。 IMD 16/GP34是一对重要的协同刺激分子， 在机体的免疫应答和多种疾病中起重要作 用， 其相互作用能促进 CD+4T细胞的活化、 增殖、 迁移， 延长其寿命， 并促进 生发中心的形成和 DC的分化成熟。
技术问题
[0003] IMD16在肿瘤免疫治疗中可起重要作用， 需做大量研究方可实现临床转化， 但 现有技术中缺乏特异抑制 IMD16基因表达的载体使得相关研究无法很好地幵展。 问题的解决方案
技术解决方案
[0004] 本发明的目的是 IMD16基因的 RNA干涉载体及其构建方法和应用。
[0005] 在本发明的第一方面， 提供一种分离的多核苷酸， 其核苷酸序列如 SEQ ID ΝΟ:1所示。
[0006] 在本发明的另一方面， 提供一种重组载体， 含有如 SEQ ID NO:2所示的核苷酸 序列， 所述重组载体是以 pLVX-shRNAl载体为骨架载体， 在多克隆酶切位点处 插入顺序连接的由 Bam HI酶切位点 +靶核苷酸序列 +茎环结构序列 +靶核苷酸序列 互补序列 +终止位点序列 +EcoR I酶切位点组成的 IMD16-RNAi序列。
[0007] 在本发明的另一方面， 提供一种前述的 IMD16基因的 RNA干涉载体的制备方法 ， 包括如下步骤：
[0008] 1) IMD16-RNAi序列的设计： 根据 IMD16的 mRNA全序列， 按 RNAi片段设计 原则设计干涉片段， NCBI BLSAT进行同源性对比， 确认特异性后得到靶核苷酸 序列， 获得如 SEQ ID NO:l所示序列， 命名为 IMD16-RNAi序列；
[0009] 2) IMD16基因的 RNA干涉载体的构建和鉴定： 将合成的 IMD16-RNAi序列与提 取的质粒 pLVX-shRNAl， 用限制性内切酶 Bam HI、 EcoR I双酶切， 电泳、 切胶 回收载体， 再用 T4 DNA Ligase分别将 IMD16-RNAi序列连接 SJpLVX-shRNAl表 达载体中， 得到连接产物； 将连接产物转化到感受态大肠杆菌 Stbl3中， 均匀涂 布到含氨苄青霉素 LB培养基平板上， 挑取阳性单克隆菌落培养保存并进行 PCR 鉴定， 将初步鉴定结果说明 IMD16-RNAi序列插入成功的菌液进行测序鉴定；
[0010] 3) IMD16基因的 RNA干涉载体的抽提： 将测序结果证实 IMD16-RNAi序列插入 成功的菌液扩增培养， 进行大量抽提重组质粒， 得到 IMD16基因的 RNA干涉载 体。
[0011] 本发明利用 RNAi技术构建靶向 IMD16基因表达的 RNA干涉载体， 经鉴定构建 成功后， 电转 Jurkat细胞， 使用实吋荧光定量 PCR技术从 mRNA水平验证 IMD16 基因表达的抑制效果， 实验结果证明本发明提供的 IMD16-RNAi序列成功插入至 pLVX-shRNAl表达载体中， 且对 IMD16基因表达的抑制效果显著。
[0012] 在本发明的另一方面， 提供一种 SEQ ID ΝΟ:1所示的多核苷酸或含有 SEQ ID ΝΟ:1所示的多核苷酸的载体或前述 IMD16基因的 RNA干涉载体在 IMD16基因表 达异常相关疾病的药物中的用途。
发明的有益效果
有益效果
[0013] 本发明提供的 IMD16基因的 RNA干涉载体具有转染效率高， 用量少， 可高效、 特异地抑制 Jurkat细胞 IMD16基因表达的优点， 可作为有力工具应用于制备治疗 I MD16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
[0014] 图 1为转染 IMD16基因的 RNA干涉载体细胞的荧光定量 PCR检测结果示意图。 实施该发明的最佳实施例
本发明的最佳实施方式
[0015] 下面结合附图与具体实施例对本发明做进一步的说明。
[0016] Jurkat细胞购自 ATCC， RNAi载体 pLVX-shRNAl购自 Clontech公司， RNeasyMi niKit购自 Qiagen公司， 无内毒素质粒提取试剂盒购自 Omega bio-tek公司。 下文所 述完全培养基为加入了 10%胎牛血清的细胞培养基。
[0017] 实施例一 IMD16-RNAi序列的设计
[0018] 在 GenBank査找到 IMD16的 mRNA全序列， 根据 IMD16的 mRNA全序列， 按 RN
Ai片段设计原则设计干涉片段， NCBI BLSAT进行同源性对比， 确认特异性后得 到靶核苷酸序列。
[0019] 设计 IMD16-RNAi序列， 如下： Bam HI酶切位点 +19nt靶核苷酸序歹 ij+茎环结构
(TTCAAGAGA) +靶序列互补序列 +RNAPolym聚合酶转录中止位点 （TTTTTT ) +EcoR I酶切位点六个区域， 其序列如 SEQ ID NO: 1所示。 设计的 IMD16-RNAi 序列链由上海生工生物工程技术服务有限公司合成。
[0020] 实施例二 IMD16基因的 RNA干涉载体的构建。
[0021] 将合成的 IMD16-RNAi序列与提取的质粒 pLVX-shRNAl， 用限制性内切酶 Bam HI、 EcoR I双酶切， 电泳、 切胶回收载体， 再用 T4 DNA Ligase将 IMD16-RNAi序 列连接到载体 pLVX-shRNAl中， 形成 IMD16基因的 RNA干涉载体。 将连接产物 转化到感受态大肠杆菌 Stbl3中， 涂布到含氨苄青霉素 LB培养基的平板上， 于 37 °C培养 14 h。 挑取长出的菌落， 送至上海生工测序。 测序结果与所设计的序列完 全相符， 可用于后续实验。
[0022] 实施例三 Jurkat细胞的电转
[0023] 培养 Jurkat细胞， 取生长状态良好的细胞 3510000个， 离心收集细胞， 然后重悬 于 500 L PBS中， 与 20 g
IMD16基因的 RNA干涉载体混匀后加入电击杯， 应用 BTX ECM830电转仪进行电 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 IMD16基因表达情 况。 [0024] 实施例四荧光定量 PCR检测 IMD16基因表达量
[0025] 分别培养正常 Jurkat细胞和电转 IMD16基因的 RNA干涉载体的 Jurkat细胞 48 h， 禾1 J用 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， 以便后面 检测使用。 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 IMD16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， 利用 SYBR Prime script RT-PCR Kit检测各组细胞 IMD16基因相对表达量， 结果如 图 1所示。 结果显示转染 IMD16基因的 RNA干涉载体的 Jurkat细胞， IMD16基因表 达明显受到抑制， IMD16-RNAi序列对目的基因的抑制效率达 78.1<¾±6.7<¾， 从而 证明本实验中采用的 IMD16基因的 RNA干涉载体能特异抑制 IMD16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 IMD16基因的 RNA干涉载体具有转染效率高， 用量少， 可高效、 特异地抑制 Jurkat细胞 IMD16基因表达的优点， 可作为有力工具应用于制备治疗 I MD16基因表达异常相关疾病的药物。

Claims

权利要求书
[权利要求 1] 一种分离的多核苷酸， 其特征在于， 其核苷酸序列如 SEQ ID NO: 1所
[权利要求 2] 一种载体， 其特征在于， 它含有权利要求 1所述的多核苷酸。
[权利要求 3] 一种重组载体， 含有如 SEQ ID
NO:2所示的序列， 所述重组载体是以 pLVX-shRNAl为骨架载体， 在 重组位点处插入顺序连接的由 Bam HI酶切位点 +靶核苷酸序歹 ij+茎环 结构序列 +靶核苷酸序列互补序列 +终止位点序列 +EcoR I酶切位点组 成的 shRNA寡核苷酸序列。
[权利要求 4] 一种权利要求 3所述的 IMD16基因的 RNA干涉载体的制备方法， 其特 征在于， 包括如下步骤：
1) IMD16-RNAi序列的设计： 根据 IMD16的 mRNA全序列， 按 RNAi 片段设计原则设计干涉片段， NCBI BLSAT进行同源性对比， 确认特 异性后得到靶核苷酸序列， 获得如 SEQ ID
NO: 1所示序列， 命名为 IMD16-RNAi序列；
2) IMD16基因的 RNA干涉载体的构建和鉴定： 将合成的 IMD16-RNA i序列与提取的质粒 pLVX-shRNAl， 用限制性内切酶 Bam HI、 EcoR I 双酶切， 电泳、 切胶回收载体， 再用 T4 DNA
Ligase分别将 IMD16-RNAi序列连接 SJpLVX-shRNAl表达载体中， 得 到连接产物； 将连接产物转化到感受态大肠杆菌 Stbl3中， 均匀涂布 到含氨苄青霉素 LB培养基平板上， 挑取阳性单克隆菌落培养保存并 进行 PCR鉴定， 将初步鉴定结果说明 IMD16-RNAi序列插入成功的菌 液进行测序鉴定；
3) IMD16基因的 RNA干涉载体的抽提： 将测序结果证实 IMD16-RNA i序列插入成功的菌液扩增培养， 进行大量抽提重组质粒， 得到 IMD1 6基因的 RNA干涉载体。
[权利要求 5] 一种权利要求 1所述的多核苷酸或权利要求 2所述的重组载体或权利要 求 3所述的 IMD16基因的 RNA干涉载体在治疗 IMD16基因表达异常相 关疾病的应用。
PCT/CN2017/077607 2017-03-22 2017-03-22 Imd16基因的rna干涉载体及其应用 WO2018170765A1 (zh)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
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WO2009045397A1 (en) * 2007-10-02 2009-04-09 Stowers Institute For Medical Research Methods for treating polycystic kidney desease (pkd) or other cyst forming diseases
CN102427809A (zh) * 2009-03-16 2012-04-25 根梅迪卡治疗公司 用于治疗代谢性疾病的抗炎剂和抗氧化剂轭合物

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2005085443A2 (en) * 2004-03-01 2005-09-15 Massachusetts Institute Of Technology Rnai-based therapeutics for allergic rhinitis and asthma
WO2009045397A1 (en) * 2007-10-02 2009-04-09 Stowers Institute For Medical Research Methods for treating polycystic kidney desease (pkd) or other cyst forming diseases
CN102427809A (zh) * 2009-03-16 2012-04-25 根梅迪卡治疗公司 用于治疗代谢性疾病的抗炎剂和抗氧化剂轭合物

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