WO2019037050A1 - 一种 pta1 基因稳定高表达细胞系的建立及其应用 - Google Patents

一种 pta1 基因稳定高表达细胞系的建立及其应用 Download PDF

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WO2019037050A1
WO2019037050A1 PCT/CN2017/098907 CN2017098907W WO2019037050A1 WO 2019037050 A1 WO2019037050 A1 WO 2019037050A1 CN 2017098907 W CN2017098907 W CN 2017098907W WO 2019037050 A1 WO2019037050 A1 WO 2019037050A1
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pta1
gene
cell line
cell
expression vector
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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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Publication of WO2019037050A1 publication Critical patent/WO2019037050A1/zh

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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention belongs to the fields of bioengineering and medical technology, and particularly relates to a PTA1 gene stable high expression cell line and its application in drug screening.
  • PTA1 molecule is a widely expressed T cell, NK cell, NK T cell, activated vascular endothelial cell
  • Type I transmembrane glycoproteins on the surface of immune cells such as megakaryocyte/platelet lineage, monocytes, thymocytes, certain B cell subsets, partial hematopoietic stem cells, and mast cells.
  • PTA1 The interaction of PTA1 with its ligands participates in the activation, differentiation and cytotoxicity of CTL and NK cells, regulates the proliferation and differentiation of CD4+ T cells, mediates the adhesion of endothelial cells to other cells, and promotes platelet activation and aggregation.
  • a method for establishing a stable high expression cell line of PTA1 gene comprises the following steps:
  • the recombinant expression vector obtained in the step 1 is introduced into a host cell, and a cell line stably expressing the PTA1 gene is selected.
  • the eukaryotic expression vector of the present invention is pIRES2-EGFP, and its map is shown in FIG.
  • the host cell of the present invention is HeLa cell, HepG2 cell or MCF-7 cell.
  • the method of the present invention wherein the prepared PTA1 gene stably stabilizes a cell line.
  • the PTA1 gene stably expressing cell line according to the present invention is characterized in that the PTA1 gene stably expressing cell line is a HepG2 cell line in which the PTA1 gene is stably expressed high.
  • the present invention encodes a PTA1 gene protein coding frame cDNA sequence, and the primers are as follows:
  • PTA1 -Reverse 5'-GCCCGGGTTAAACTCTAGTCTTTGGTC -3'.
  • the PTA1 gene stably high expression cell line of the present invention provides an experimental technical platform for further exploration of the action of the PTA1 gene, and can be used in drug research and development related to abnormal expression of PTA1.
  • Figure 1 is a map of the pIRES2-EGFP vector
  • PCR primers were designed according to the cDNA sequence of the coding cassette of the PTA1 protein as follows: Forward primer: 5'-GGAATTCATGGATTATCCTACTTTAC -3'; ; Reverse primer: 5'-GCCCGGGTTAAACTCTAGTCTTTGGTC -3'.
  • Jurkat cells were taken and total RNA was extracted, reverse-transcribed into cDNA and used as a template, and PCR amplification was carried out using the above primers.
  • the PCR amplification product was purified and recovered by agarose gel electrophoresis.
  • the purified product was digested with restriction endonucleases Ec oR I and Xma l, and the digested product was subjected to agarose gel electrophoresis purification and recovery. , the cDNA sequence of the PTA1 protein coding frame was obtained.
  • the eukaryotic expression vector pIRES 2-EGFP was also digested with EcoR I and Xma I, and the digested product was purified and recovered by agarose gel electrophoresis.
  • the cDNA sequence of the PTA1 protein coding cassette amplified by the above amplification and the product of the eukaryotic expression vector PIRES2-EGFP were ligated with DNA ligase, and the product was transformed into competent E. coli JM107 and coated with kanamycin. On the LB plate, it was sent to Shanghai Biotech for sequencing after cultivation. Sequencing results indicate that insertion
  • the cDNA sequence of the PTAl protein coding frame is identical to the sequence recorded on GenBank.
  • the resulting recombinant expression vector was named pIRES2-EGFP-PTAl.
  • the recombinant Escherichia coli obtained in the above step 1 was extracted with a Plasmid Plus SV Miniprep (purchased from Promega) to obtain a high-purity recombinant expression vector pIRES2-EGFP-PTAl, and the recombinant expression vector pIRES2-EGFP-PTAl was treated with Lipofectamine 2000 ( Transfected HepG2 cells from Invitrogen and continued to culture the transfected HepG2 cells for more than 24 hours. With neomycin (concentration 1
  • the recombinant HepG2 cells were screened, and after multiple screening, a stable cell line with high expression of PTA1 gene was obtained.
  • the HepG2 cell line stably expressing the PTA1 gene was stored in liquid nitrogen.
  • HepG2 cells and HepG2 cells transduced with pIRES2-EGFP-PTAl 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
  • Kit reverse transcribes mRNA into cDNA and stores at -20 °C. Take cDNA 1 from each group of cells
  • the PTA1 gene stable high expression cell line of the present invention provides an experimental technical platform for further exploration of the action of the PTA1 gene, and can be used in drug research and development related to abnormal expression of PTA1.

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Abstract

提供了一种稳定表达血小板T细胞活化抗原1（PTA1）基因的细胞系及其构建方法，步骤包括：（1）将PTA1基因序列插入真核载体的多克隆位点，得到重组表达载体；（2）将重组表达载体导入宿主细胞中，筛选得到稳定表达PTA1基因的细胞系。

Description

一种 PTAl基因稳定高表达细胞系的建立及其应用
技术领域
[0001] 本发明属于生物工程领域和医药技术领域， 具体涉及一种 PTA1基因稳定高表 达细胞系及其在药物筛选中的应用。
背景技术
[0002] PTA1分子是一种广泛表达 T细胞、 NK细胞、 NK T细胞、 活化的血管内皮细胞
、 巨核 /血小板谱系、 单核细胞、 胸腺细胞、 某些 B细胞亚群、 部分造血干细胞和 肥大细胞等免疫细胞表面的 I型跨膜糖蛋白。
技术问题
[0003] PTA1与其配体的相互作用参与 CTL和 NK细胞的活化、 分化和细胞毒作用， 调 节 CD4+ T细胞的增殖和分化， 介导内皮细胞与其他细胞的黏附， 促进血小板的 活化与聚集， 参与造血调控， 介导肥大细胞的脱颗粒作用， 参与 NKT细胞和胸 腺细胞的抗凋亡作用、 树突状细胞的成熟、 免疫突触和神经突触的形成， 在肿 瘤的免疫治疗中具有重要潜力， 需进行深入的转化研究， 但现有技术中缺乏哺 乳动物细胞高效表达 PTA1基因的细胞系， 对相关研究的进展造成了一定的阻碍 问题的解决方案
技术解决方案
[0004] 一种 PTA1基因稳定高表达细胞系的建立方法， 包括以下步骤：
[0005] ①将 PTA1基因的编码序列插入真核表达载体的多克隆位点， 得到重组表达载 体；
[0006] ②将步骤 1得到的重组表达载体导入宿主细胞中， 筛选得到稳定表达 PTA1基因 的细胞系。
[0007] 本发明所述真核表达载体为 pIRES2-EGFP， 其图谱如图 1所示。
[0008] 本发明所述宿主细胞为 HeLa细胞、 HepG2细胞或 MCF-7细胞。
[0009] 本发明所述的方法， 其中制备的 PTA1基因稳定高表达细胞系。 [0010] 本发明所述的 PTA1基因稳定表达细胞系， 其特征在于， 所述 PTA1基因稳定表 达细胞系为 PTA1基因稳定高表达的 HepG2细胞系。
[0011] 本发明编码 PTA1基因蛋白编码框 cDNA序列， 引物如下：
[0012] PTAl-Forward: 5'- GGAATTCATGGATTATCCTACTTTAC -3'；
[0013] PTA1 -Reverse: 5'- GCCCGGGTTAAACTCTAGTCTTTGGTC -3'。
发明的有益效果
有益效果
[0014] 本发明的 PTA1基因稳定高表达细胞系为深入探索 PTA1基因的作用提供实验技 术平台， 可用于与 PTA1表达异常相关的药物研究和幵发中。
对附图的简要说明
附图说明
[0015] 图 1为 pIRES2-EGFP载体的图谱； 图 2
为转导 PIRES2-EGFP-PTA1载体的细胞的 PTA1基因表达水平结果示意图。
实施该发明的最佳实施例
本发明的最佳实施方式
[0016] 实施例 1重组真核表达载体 pIRES2-EGFP-PTAl的构建
[0017] 根据 PTA1蛋白编码框的 cDNA序列， 设计 PCR引物如下： 正向引物： 5'- GGAATTCATGGATTATCCTACTTTAC -3'； ； 反向引物： 5'- GCCCGGGTTAAACTCTAGTCTTTGGTC -3'。
[0018] 取 Jurkat细胞并提取总 RNA， 逆转录为 cDNA并以其为模板， 利用上述引物进行 PCR扩增。 将 PCR扩增产物进行琼脂糖凝胶电泳纯化和回收， 用限制性内切酶 Ec oR I和 Xma l对纯化回收后的产物进行酶切， 酶切产物再次进行琼脂糖凝胶电泳 纯化和回收， 得到 PTA1蛋白编码框的 cDNA序列。 同吋， 将真核表达载体 pIRES 2-EGFP也用 EcoR I和 Xma I进行双酶切， 酶切产物进行琼脂糖凝胶电泳纯化和回 收。 用 DNA连接酶连接上述扩增得到的 PTA1蛋白编码框的 cDNA序列和真核表 达载体 PIRES2-EGFP酶切后的产物， 连接产物转化感受态大肠杆菌 JM107 , 并涂 布于含有卡那霉素的 LB平板上， 培养后送上海生工测序。 测序结果表明， 插入 的 PTAl蛋白编码框的 cDNA序列与 GenBank上记录的序列完全一致。 将得到的重 组表达载体命名为 pIRES2-EGFP-PTAl。
[0019] 实施例二 PTA1基因稳定高表达 HepG2细胞系的建立
[0020] 将上述步骤一得到的重组大肠杆菌利用 Wizard Plus SV Miniprep (购自 Promega ) 提取质粒， 得到高纯度的重组表达载体 pIRES2-EGFP-PTAl， 将重组表达载体 pIRES2-EGFP-PTAl利用 Lipofectamine2000 (购自 Invitrogen) 转染 HepG2细胞， 继续培养转染后的 HepG2细胞 24小吋以上。 用新霉素 （浓度为 1
g/ml)对重组 HepG2细胞进行筛选， 经多次筛选后， 得到 PTA1基因稳定高表达 细胞系。 将 PTA1基因稳定高表达的 HepG2细胞株保存于液氮中。
[0021] 实施例三 荧光定量 PCR检测 PTA1基因表达量。
[0022] 分别接种 HepG2细胞和转导 pIRES2-EGFP-PTAl载体的 HepG2细胞至 6孔板。 细 胞密度达到 δΟ^^Ο^吋， 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， -20°C保存。 取各组细胞的 cDNA 1
μΐ为模板， 以 GAPDH为内参， 实吋荧光定量 PCR检测 PTA1相对表达量， 设置反 应条件： 95。C 30s， 1循环， 54°C 30s 40循环， 95。C 5s， 60°C lmin, 95。C 15s， 结 果如图 2所示。 可以看到， 转导 pIRES2-EGFP-PTAl载体的 HepG2细胞的 PTA1基 因表达量较 Jurkat细胞有 130倍以上的升高， 说明本发明提供的 PTA1基因 cDNA序 列成功插入至 PIRES2-EGFP表达载体中， 能特异、 持续、 高效、 稳定地促进 PTA 1基因高表达。
工业实用性
[0023] 本发明的 PTA1基因稳定高表达细胞系为深入探索 PTA1基因的作用提供实验技 术平台， 可用于与 PTA1表达异常相关的药物研究和幵发中。

Claims

权利要求书 一种 PTAl受体稳定高表达细胞系的建立方法， 包括以下步骤：
1)将 PTA1受体的编码基因插入真核表达载体的多克隆位点， 得到重 组表达载体；
2)将步骤 1得到的重组表达载体导入宿主细胞中， 筛选得到稳定表达 P TA1受体的细胞系。
权利要求 1所述的方法， 其中 PTA1受体的编码基因引物如下： PTAl-Forward: 5 ' -GGAATTCATGGATTATCCTACTTTAC-3 '； PTA 1 -Reverse： 5'-GCCCGGGTTAAACTCTAGTCTTTGGTC。
权利要求 1所述的方法， 其特征在于， 所述真核表达载体为 PIRES2-E GFP。
权利要求 1所述的方法， 其特征在于所述宿主细胞为 HeLa细胞、 Hep G2细胞或 MCF-7细胞。
权利要求 1-3中任一项所述的方法， 其中制备的 PTA1基因稳定高表达 细胞系。
权利要求 5所述的 PTA1基因稳定表达细胞系， 其特征在于， 所述 PTA 1基因稳定表达细胞系为 PTA1基因稳定高表达的 HepG2细胞系。
权利要求 4-5中任一项所述的 PTA1基因稳定高表达细胞系在制备治疗 PTA1基因表达异常相关疾病的药物中的用途。
PCT/CN2017/098907 2017-08-24 2017-08-24 一种 pta1 基因稳定高表达细胞系的建立及其应用 WO2019037050A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1387043A (zh) * 2002-06-11 2002-12-25 中国人民解放军第四军医大学 可溶性血小板t细胞活化抗原1(cd226)的定量检测方法
CN103555762A (zh) * 2013-11-15 2014-02-05 新乡医学院 Afp和gm-csf双基因共表达重组载体及其制备方法和应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1387043A (zh) * 2002-06-11 2002-12-25 中国人民解放军第四军医大学 可溶性血小板t细胞活化抗原1(cd226)的定量检测方法
CN103555762A (zh) * 2013-11-15 2014-02-05 新乡医学院 Afp和gm-csf双基因共表达重组载体及其制备方法和应用

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JIA, WEI ET AL.: "Construction and Expression of CD 226 (PTA1) Truncated Gene Eukaryotic Expression Vectors and Identification of the Epitopes Localization Recognized by CD 226 McAbs", CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY, vol. 23, no. 1, 31 January 2003 (2003-01-31), pages 9 - 13, XP055579123, ISSN: 0254-5101 *

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