WO2019000145A1

WO2019000145A1 - Method for constructing recombinant cho cell highly expressing ampar

Info

Publication number: WO2019000145A1
Application number: PCT/CN2017/089925
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2017-06-26
Filing date: 2017-06-26
Publication date: 2019-01-03

Abstract

Provided is a method for constructing a recombinant CHO cell highly expressing AMPAR. The full-length AMPAR gene eukaryotic expression vector pCAG(m)-IRESblast-AMPAR is constructed using the eukaryotic expression vector pCAG(m)-IRESblast and is further used to transfect the CHO cells, and the blasticidin is used to screen same to obtain the engineered cell line CHO/AMPAR highly expressing the full-length AMPAR gene, and it is confirmed that the cell line can greatly increase the expression of the full-length AMPAR.

Description

一种 AMPAR高表达的重组 CHO细胞的构建方法技术领域 Method for constructing recombinant CHO cells with high AMPAR expression

[0001] 本发明属于重组细胞技术领域，涉及一种 AMPAR高表达的重组 CHO细胞的构建方法。 [0001] The present invention belongs to the field of recombinant cell technology, and relates to a method for constructing recombinant CHO cells with high AMPAR expression.

背景技术 Background technique

[0002] 阿尔茨海默病（Alzheimer's disease , AD) ，是引起老年性痴呆的最常见原因。此疾病常见于老年人，是一种进行性认知障碍和记忆能力损害为主的中枢神经***退行性变性疾病，临床表现为认知和记忆功能不断恶化，日常生活能力进行性减退，并有各种神经精神症状和行为障碍。多起病于老年期，潜隐起病，病程缓慢且不可逆，临床上以智能损害为主。 [0002] Alzheimer's disease (AD) is the most common cause of senile dementia. This disease is common in the elderly. It is a central nervous system degenerative degenerative disease with progressive cognitive impairment and memory impairment. The clinical manifestations are the deterioration of cognitive and memory functions, the progressive decline of daily living ability, and Various neuropsychiatric symptoms and behavioral disorders. More onset in the old age, latent onset, slow and irreversible, clinically based on intelligent damage.

[0003] 越来越多证据表明，谷氨酸 a-氨基 -3-轻基 -5-甲基 -4-异挫丙酸受体 (a-amino-3-hy droxy-5-methyl-4-isoxazole propionic acid receptor, AMPAR)是调节中枢神经突触可塑性的关键机制。 AMPAR是离子型谷氨酸受体之一，介导中枢兴奋性突触后电流的主要成分，在突触传递过程中不可或缺。 [0003] There is increasing evidence that glutamate a-amino-3-carbo-5-methyl-4-iso-propionate receptor (a-amino-3-hy droxy-5-methyl-4) -isoxazole propionic acid receptor (AMPAR) is a key mechanism regulating central synaptic plasticity. AMPAR is one of the ionotropic glutamate receptors and mediates the central component of central excitatory postsynaptic currents and is essential for synaptic transmission.

技术问题 technical problem

[0004] AMPAR的生理功能依赖受体数量、亚基组成以及蛋白质磷酸化作用等因素的调控 AMPAR功能紊乱，导致神经元突触可塑性下降，引起 AD认知障碍，在 AD 的发病过程中起重要作用，因此对 AMPAR在阿尔兹海默症中的作用及将其作为一种治疗靶点的研究必不可少，而现有技术中仍缺乏这些相关研究所需的高表达 AMPAR基因的细胞。 [0004] The physiological function of AMPAR depends on the number of receptors, subunit composition and protein phosphorylation to regulate AMPAR dysfunction, leading to decreased synaptic plasticity of neurons, causing AD cognitive impairment, which plays an important role in the pathogenesis of AD. The role, therefore, is essential for the role of AMPAR in Alzheimer's disease and its use as a therapeutic target, and the cells that express the AMPAR gene required for these related studies are still lacking in the prior art.

问题的解决方案 Problem solution

技术解决方案 Technical solution

[0005] 本发明的目的在于提供一种 AMPAR高表达的重组 CH0细胞的构建方法，通过以下技术方案来实现： [0005] The object of the present invention is to provide a method for constructing recombinant CH0 cells with high AMPAR expression, which is achieved by the following technical schemes:

[0006] 一种 AMPAR高表达的重组 CH0细胞的构建方法， AMPAR高表达的重组 CH0 细胞是以 CH0细胞为宿主细胞，转染宿主细胞的外源性表达载体的是包含 AMP AR全长基因的表达载体。 [0006] A method for constructing recombinant CH0 cells with high AMPAR expression, the recombinant CH0 cells highly expressed by AMPAR are CH0 cells as host cells, and the exogenous expression vector for transfecting host cells comprises AMP. An expression vector for the AR full-length gene.

[0007] 所述的 AMPAR全长基因的核苷酸序列如 SEQ ID No: 1所示。 [0007] The nucleotide sequence of the AMPAR full-length gene is shown in SEQ ID No: 1.

[0008] 所述的包含 AMPAR全长基因的表达载体是 pCAG(m)-IRESblast，该表达载体是通过 EcoR [0008] The expression vector comprising the full-length gene of AMPAR is pCAG(m)-IRESblast, and the expression vector is through EcoR

I与 Spel酶切位点将 AMPAR全长基因克隆入真核表达载体 pCAG(m)-IRESblast。发明的有益效果 The full-length AMPAR gene was cloned into the eukaryotic expression vector pCAG(m)-IRESblast by I and Spel restriction sites. Advantageous effects of the invention

有益效果 Beneficial effect

[0009] 本发明构建了人 AMPAR的真核表达载体 pCAG(m)-IRESblast，经过克隆测序证实序列同 NCBI数据库中的人 AMPAR序列一致；经过脂质体法转染 CHO细胞，杀稻瘟菌素抗性筛选获得能够稳定生长、存活的细胞株，经过定量 PCR检测，筛选出稳定、高表达 AMPAR的重组 CHO/AMPAR细胞株；本发明构建的包含 AMP AR基因全长的重组 CHO/AMPAR细胞株能够稳定表达 AMPAR, 具有完整的分子结构。 The present invention constructs the eukaryotic expression vector pCAG(m)-IRESblast of human AMPAR, which is confirmed by cloning and sequencing to be identical to the human AMPAR sequence in the NCBI database; transfected CHO cells by liposome method, blasticidal Resistant CHO/AMPAR cell line stably and highly expressed AMPAR was screened by restriction endonuclease screening, and recombinant CHO/AMPAR cells containing AMP AR gene full length were constructed according to the present invention. The strain is capable of stably expressing AMPAR and has a complete molecular structure.

[0010] 作为宿主细胞的 CHO细胞具有许多优势，包括细胞本身蛋白表达量相对较低，而对外源的重组基因则具有较高的扩增和表达能力，重组后的 CHO/AMPAR细胞表达的 AMPAR的相对表达量较 CHO空载明显升高，相对于其他表达产物， AMP AR占据优势表达量。 [0010] CHO cells as host cells have many advantages, including relatively low levels of protein expression in cells themselves, and higher amplification and expression ability of exogenous recombinant genes, AMPAR expressed by recombinant CHO/AMPAR cells. The relative expression level was significantly higher than that of CHO, and AMP AR occupied the dominant expression level compared with other expression products.

[0011] 通过定量 PCR检测技术，证明 AMPAR表达量相比 CHO明显升高，且其在细胞膜上的表达较 CHO细胞表达量明显提高。 [0011] By quantitative PCR detection technology, it was proved that the expression of AMPAR was significantly higher than that of CHO, and its expression on the cell membrane was significantly higher than that of CHO cells.

对附图的简要说明 Brief description of the drawing

附图说明 DRAWINGS

[0012] 图 1为杀稻瘟菌素筛选 CHO细胞后荧光定量 PCR检测 AMPAR表达水平结果示意图。 1 is a schematic diagram showing the results of fluorescent quantitative PCR detection of AMPAR expression levels after screening of CHO cells by blasticidin.

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

[0013] 本发明在构建 AMPAR全长基因的真核表达载体 pCAG(m)-IRESblast-AMPAR的基础上，转染 CHO细胞，获得稳定、高表达 AMPAR的重组 CHO/AMPAR细胞株，下面是对本发明实施例的解释而不是限定。 [0013] The present invention is based on the construction of the eukaryotic expression vector pCAG(m)-IRESblast-AMPAR of the AMPAR full-length gene, transfecting CHO cells to obtain a stable, high expression AMPAR-recombinant CHO/AMPAR cell line. The following is an explanation of the embodiments of the present invention and is not limiting.

[0014] 本实施例以 pCAG(m)-IRESblast为基础载体，具体选择 EcoR I与 Spel酶切位点作为连接外源基因的多克隆位点。 [0014] In this example, the pCAG(m)-IRESblast-based vector was used, and the EcoR I and Spel cleavage sites were specifically selected as the multiple cloning site for the foreign gene.

[0015] 实施例一 AMPAR基因的克隆 Example 1 Cloning of AMPAR Gene

[0016] 以 RGC5细胞的 cDNA为模板，采用 Premier Primer 6.0软件设计相应的特异性弓 | 物，并在两端分别加入 EcoR I和 Spe l酶切位点： AMPAR-F: 5'- GCGAATTCATGGGGCTCAACTGGAAAAATC -3'； AMPAR-R: 5'- AACTAGTTTATACGGGGGTGGTCCGG -3，。 [0016] Using the cDNA of RGC5 cells as a template, the corresponding specificity was designed by Premier Primer 6.0 software, and EcoR I and Spe l restriction sites were added at both ends: AMPAR-F: 5'-GCGAATTCATGGGGCTCAACTGGAAAAATC - 3'; AMPAR-R: 5'- AACTAGTTTATACGGGGGTGGTCCGG -3,.

[0017] 采用大连宝生物公司 PrimeSTAR酶进行扩增， PCR反应体系为：上游引物 4μί 、下游引物 4 L、 2xPremix PrimeSTAR 25 L、 cDNA: 1μΙ^、补加 ddH20至 50 L ； 98°C 2 min，再经过 30个循环作用 (98°C变性 10 s， 60°C退火 10 s， 72°C延伸 1 min) , 最后 72°C延伸 5 min，得到大量目的片段，对产物采用商品化 PCR纯化试剂盒将扩增产物纯化。 [0017] Amplification was carried out by using the PrimeSTAR enzyme of Dalian Bao Biotechnology Co., Ltd., and the PCR reaction system was: upstream primer 4 μί, downstream primer 4 L, 2×Premix PrimeSTAR 25 L, cDNA: 1 μΙ^, supplement ddH20 to 50 L; 98 ° C 2 min After 30 cycles (denaturation at 98 °C for 10 s, annealing at 60 °C for 10 s, extension at 72 °C for 1 min), and finally extension at 72 °C for 5 min, a large number of target fragments were obtained, and the product was purified by commercial PCR. The kit purifies the amplified product.

[0018] 实施例二重组表达载体构建 Example 2 Construction of Recombinant Expression Vector

[0019] 利用 EcoR I、 Spe I双酶切真核表达载体 pCAG(m)-IRESblast和 AMPAR基因纯化产物：酶切体系为： EcoR I l L、 Spe I 1.5 L、 DNA/质粒： 2 g、 lOxFastDigest Buffer 2 L、补加 ddH20至 20μί; 37°C反应 30 min，对酶切产物采用商品化 PCR 产物纯化试剂盒纯化。 [0019] using EcoR I, Spe I double-digested eukaryotic expression vector pCAG(m)-IRESblast and AMPAR gene purified product: The enzyme digestion system is: EcoR I l L, Spe I 1.5 L, DNA/plasmid: 2 g, lOxFastDigest Buffer 2 L, add ddH20 to 20μί; react at 37 °C for 30 min, and purify the product by using a commercial PCR product purification kit.

[0020] 将线性化载体与酶切后的 AMPAR扩增片段 4°C条件下， T4 DNA连接酶作用下过夜连接，反应体系为：酶切后的线性 pCAG(m)-IRESblast [0020] The linearized vector and the digested AMPAR amplified fragment were ligated overnight under the action of T4 DNA ligase at 4 ° C, and the reaction system was: linear pCAG(m)-IRESblast after digestion

2μί、酶切后的 AMPAR片段 5μί、 T4 DNA连接酶 1 μί、 10xT4 2μί, after digestion of AMPAR fragment 5μί, T4 DNA ligase 1 μί, 10xT4

DNA连接酶 Buffer Ιμί, 获得重组 pCAG(m)-IRESblast-AMPAR表达载体。 DNA ligase Buffer Ιμί, obtained recombinant pCAG(m)-IRESblast-AMPAR expression vector.

[0021] 将 5 重组 pCAG(m)-IRESblast-AMPAR质粒加入 5(VL Topl0感受态细胞中，冰浴 30 min， 42°C热激 60 s，冰浴 2 min，加入 50 无抗性 LB液体培养基， 37°C振摇 l h，取全部菌液，涂布至含有氨苄霉素抗性（终浓度 100 g/mL) LB固体培养基中， 37°C过夜培养，挑取单克隆在含有氨苄霉素抗性（终浓度 100 g/mL) LB液体培养基中扩大培养，并送上海生工进行测序，验证序列碱基，确保测序结果与基因库（GenBank) 中的一致。 [0022] 实施例三重组载体转导 CHO细胞 [0021] Add 5 recombinant pCAG(m)-IRESblast-AMPAR plasmid to 5 (VL Top10 competent cells, ice bath for 30 min, heat shock at 42 °C for 60 s, ice bath for 2 min, add 50 non-resistant LB liquid The medium was shaken at 37 ° C for 1 h, and all the bacterial liquid was taken and applied to LB solid medium containing ampicillin resistance (final concentration 100 g/mL), cultured overnight at 37 ° C, and the monoclonal was picked up. Ampicillin resistance (final concentration 100 g/mL) was expanded in LB liquid medium and sent to Shanghai Biotech for sequencing to verify the sequence bases, ensuring that the sequencing results were consistent with those in the GenBank. Example 3 Recombinant Vector Transduction of CHO Cells

[0023] 接种 CHO细胞于 6孔板中，每孔 10 ⁶个细胞， 18 [0023] CHO cells were seeded in 6-well plates at 10 ⁶ cells per well, 18

h后细胞密度约为 60<¾。取 pCAG(m)-IRESblast-AMPAR质粒 1 After h, the cell density is about 60 < 3⁄4. Take pCAG(m)-IRESblast-AMPAR Plasmid 1

g，应用 Lipofectamine 3000转导至 CHO细胞中，继续培养 24 h后，加入杀稻瘟菌素至终浓度为 5 g/mL，筛选细胞。 g, Lipofectamine 3000 was transduced into CHO cells, and after continuing to culture for 24 h, blasticidin was added to a final concentration of 5 g/mL, and the cells were selected.

[0024] 实施例四荧光定量 PCR检测 AMPAR基因表达量 [0024] Example 4 Fluorescence quantitative PCR detection of AMPAR gene expression

[0025] 分别接种 CHO细胞和 CHO/AMPAR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 [0025] CHO cells and CHO/AMPAR cells were seeded separately into 6-well plates. The cell density reached δΟ^^Ο^吋, and the total RNA of each group was extracted with RNeasy Mini Kit, using PrimeScrip RT reagent.

Kit将 mRNA逆转录为 cDNA， -20°C保存。 Kit reverse transcribes mRNA into cDNA and stores at -20 °C.

[0026] 取各组细胞的 cDNA 1 [0026] Take cDNA 1 of each group of cells

为模板，以 GAPDH为内参，实吋荧光定量 PCR检测 AMPAR相对表达量，设置反应条件： 95。C 30s， 1循环， 58。C 30s 40循环， 95。C 5s， 60°C lmin, 95。C 15s ，结果如图 1所示。可以看到， CHO/AMPAR细胞的 AMPAR基因表达水平较 CH 0细胞高 240倍，说明本发明提供的 AMPAR基因 cDNA序列成功***至 pCAG(m)- IRESblast载体中，能特异、高效地促进 AMPAR基因高表达。 As a template, GAPDH was used as an internal reference, and the relative expression of AMPAR was detected by real-time fluorescent quantitative PCR. The reaction conditions were set: 95. C 30s, 1 cycle, 58. C 30s 40 cycles, 95. C 5s, 60 ° C lmin, 95. C 15s , the results are shown in Figure 1. It can be seen that the AMPAR gene expression level of CHO/AMPAR cells is 240 times higher than that of CH 0 cells, indicating that the AMPAR gene cDNA sequence provided by the present invention is successfully inserted into the pCAG(m)-IRESblast vector, and the AMPAR gene can be specifically and efficiently promoted. High expression.

工业实用性 Industrial applicability

[0027] 本发明构建了人 AMPAR的真核表达载体 pCAG(m)-IRESblast，经过克隆测序证实序列同 NCBI数据库中的人 AMPAR序列一致；经过脂质体法转染 CHO细胞，杀稻瘟菌素抗性筛选获得能够稳定生长、存活的细胞株，经过定量 PCR检测，筛选出稳定、高表达 AMPAR的重组 CHO/AMPAR细胞株；本发明构建的包含 AMP AR基因全长的重组 CHO/AMPAR细胞株能够稳定表达 AMPAR, 具有完整的分子结构。 [0027] The present invention constructs the eukaryotic expression vector pCAG(m)-IRESblast of human AMPAR, which is confirmed by clone sequencing to be identical to the human AMPAR sequence in the NCBI database; transfected CHO cells by liposome method, blasticidal Resistant CHO/AMPAR cell line stably and highly expressed AMPAR was screened by restriction endonuclease screening, and recombinant CHO/AMPAR cells containing AMP AR gene full length were constructed according to the present invention. The strain is capable of stably expressing AMPAR and has a complete molecular structure.

[0028] 作为宿主细胞的 CHO细胞具有许多优势，包括细胞本身蛋白表达量相对较低，而对外源的重组基因则具有较高的扩增和表达能力，重组后的 CHO/AMPAR细胞表达的 AMPAR的相对表达量较 CHO空载明显升高，相对于其他表达产物， AMP AR占据优势表达量。 [0028] CHO cells as host cells have many advantages, including a relatively low amount of protein expression in the cell itself, and a higher amplification and expression ability of the foreign recombinant gene, and AMPAR expressed by the recombinant CHO/AMPAR cells. The relative expression level was significantly higher than that of CHO, and AMP AR occupied the dominant expression level compared with other expression products.

[0029] 通过定量 PCR检测技术，证明 AMPAR表达量相比 CHO明显升高，且其在细胞膜上的表达较 CHO细胞表达量明显提高。 [0029] By quantitative PCR detection technology, it was proved that the expression of AMPAR was significantly higher than that of CHO, and its expression on the cell membrane was significantly higher than that of CHO cells.

Claims

权利要求书 [权利要求 1] 一种 AMPAR高表达的重组 CHO细胞的构建方法，其特征在于： AMP AR高表达的重组 CHO细胞是以 CHO细胞为宿主细胞，转染宿主细胞的外源性表达载体的是包含 AMPAR全长基因的表达载体。 [权利要求 2] 根据权利要求 1所述的一种 AMPAR高表达的重组 CHO细胞的构建方法，其特征在于：所述的 AMPAR全长基因的核苷酸序列如 SEQ ID No: 1所示。 [权利要求 3] 根据权利要求 1所述的一种 AMPAR高表达的重组 CHO细胞的构建方法，其特征在于：所述的包含 AMPAR全长基因的表达载体是 pCAG( m)-IRESblast，该表达载体是通过 EcoR I和 Spe藤切位点将 AMPAR 全长基因克隆入真核表达载体 pCAG(m)-IRESblast。 [权利要求 4] 根据权利要求 1至 3任一权利要求所述的一种 AMPAR高表达的重组 CH 0细胞的构建方法，其特征在于：包括以下步骤： Claims [Claim 1] A method for constructing recombinant AMH cells with high expression of AMPAR, characterized in that: recombinant CHO cells with high expression of AMP AR are CHO cells as host cells, and the exogenous expression of the host cells is transfected. The vector is an expression vector comprising the AMPAR full-length gene. [Claim 2] The method for constructing a recombinant CHO cell with high expression of AMPAR according to claim 1, wherein the nucleotide sequence of the full-length AMPAR gene is shown in SEQ ID No: 1. [Claim 3] The method for constructing a AMPAR high expression recombinant CHO cell according to claim 1, wherein: the expression vector comprising the AMPAR full-length gene is pCAG(m)-IRESblast, the expression The vector was cloned into the eukaryotic expression vector pCAG(m)-IRESblast by the EcoR I and Spe cleavage sites. [Claim 4] A method for constructing recombinant AM 0 cells with high AMPAR expression according to any one of claims 1 to 3, comprising the steps of:

(1) AMPAR基因的克隆 (1) Cloning of the AMPAR gene

以 RGC5细胞的 cDNA为模板，采用 Premier Primer 6.0软件设计相应的特异性弓 I物，并在两端分别加入 EcoR I和 Spe I酶切位点： AMPAR-F ： 5， - GCGAATTC ATGGGGCTCAACTGGAAA AATC -3'； Using the cDNA of RGC5 cells as a template, the corresponding specific primers were designed using Premier Primer 6.0 software, and EcoR I and Spe I restriction sites were added at both ends: AMPAR-F : 5, - GCGAATTC ATGGGGCTCAACTGGAAA AATC -3 ';

AMPAR-R： 5'- AACTAGTTTATACGGGGGTGGTCCGG -3，。 AMPAR-R: 5'- AACTAGTTTATACGGGGGTGGTCCGG -3,.

采用大连宝生物公司 PrimeSTAR酶进行扩增， PCR反应体系为：上游引物 4μί、下游引物 4 L、 2xPremix PrimeSTAR 25 L、 cDNA: 1μΙ^、补加 ddH20至 50μί; 98°C 2 min, 再经过 30个循环作用 (98°C变性 10 s ， 60°C退火 10 s， 72°C延伸 l min)，最后 72°C延伸 5 min，得到大量目的片段，对产物采用商品化 PCR纯化试剂盒将扩增产物纯化。 Amplification was carried out by using the PrimeSTAR enzyme of Dalian Bao Biotech Co., Ltd. The PCR reaction system was: upstream primer 4 μί, downstream primer 4 L, 2x Premix PrimeSTAR 25 L, cDNA: 1 μΙ^, plus ddH20 to 50 μί; 98 °C 2 min, then 30 One cycle (denaturation at 98 °C for 10 s, annealing at 60 °C for 10 s, extension at 72 °C for 1 min), and finally extension at 72 °C for 5 min, a large number of target fragments were obtained, and the product was expanded using a commercial PCR purification kit. The product is purified.

(2) 重组表达载体构建 (2) Construction of recombinant expression vector

利用 EcoR I、 Spe I双酶切真核表达载体 pCAG(m)-IRESblast和 AMPAR 基因纯化产物：酶切体系为： EcoR I l L、 Spe l The EcoC I, Spe I double-digested eukaryotic expression vector pCAG(m)-IRESblast and AMPAR gene purified product: The enzyme digestion system is: EcoR I l L, Spe l

1.5 L、 DNA/质粒： 2 g、 lOxFastDigest Buffer 2μ^ 补加 ddH20至 20 μί; 37°C反应 30 min，对酶切产物采用商品化 PCR产物纯化试剂盒纯化。 1.5 L, DNA/plasmid: 2 g, lOxFastDigest Buffer 2μ^ plus ddH20 to 20 μί; 37 °C reaction for 30 min, commercial digestion product using pure PCR product purification kit Chemical.

将线性化载体与酶切后的 AMPAR扩增片段 4°C条件下， T4 DNA连接酶作用下过夜连接，反应体系为：酶切后的线性 pCAG(m)-IRESbla_St 2μί、酶切后的 AMPAR片段 5μί、 T4 DNA连接酶 1 μί、 10xT4 DNA 连接酶 Buffer Ιμί, 获得重组 pCAG(m)-IRESblast-AMPAR表达载体。将 5 重组 pCAG(m)-IRESblast-AMPAR质粒加入 5( L ToplO感受态细胞中，冰浴 30 min， 42°C热激 60 s，冰浴 2 min，加入 50 The linearized vector was ligated with the digested AMPAR amplified fragment at 4 ° C under the action of T4 DNA ligase overnight. The reaction system was: linear pCAG(m)-IRESbla _S t 2μί after digestion, after digestion The AMPAR fragment 5 μί, T4 DNA ligase 1 μί, 10×T4 DNA ligase Buffer Ιμί, obtained recombinant pCAG(m)-IRESblast-AMPAR expression vector. Add 5 recombinant pCAG(m)-IRESblast-AMPAR plasmid to 5 (L ToplO competent cells, ice bath for 30 min, heat shock at 42 °C for 60 s, ice bath for 2 min, add 50

无抗性 LB液体培养基， 37°C振摇 l h，取全部菌液，涂布至含有氨苄霉素抗性（终浓度 100 g/mL) LB固体培养基中， 37°C过夜培养，挑取单克隆在含有氨苄霉素抗性（终浓度 100 g/mL) LB液体培养基中扩大培养，并送上海生工进行测序，验证序列碱基，确保测序结果与基因库（GenBank) 中的一致。 Non-resistant LB liquid medium, shaken at 37 ° C for 1 h, take all the bacterial solution, apply to LB solid medium containing ampicillin resistance (final concentration 100 g / mL), culture overnight at 37 ° C, pick Monoclonal cultures were expanded in LB liquid medium containing ampicillin resistance (final concentration 100 g/mL) and sent to Shanghai Biotech for sequencing to verify sequence bases, ensuring sequencing results and gene banks (GenBank) Consistent.

(3) 重组载体转导 CHO细胞 (3) Recombinant vector transduction CHO cells

接种 CHO细胞于 6孔板中，每孔 10 ⁶个细胞， 18 h后细胞密度约为 60% 。取 pCAG(m)-IRESblast-AMPAR质粒 1 g，应用 Lipofectamine 3000 转导至 CHO细胞中，继续培养 24 h后，加入杀稻瘟菌素至终浓度为 5 筛选细胞。 CHO cells were seeded in 6-well plates at 10 ⁶ cells per well, and the cell density was approximately 60% after 18 h. 1 g of pCAG(m)-IRESblast-AMPAR plasmid was transfected into CHO cells using Lipofectamine 3000. After 24 hours of culture, blasticidin was added to a final concentration of 5 cells.

(4) 荧光定量 PCR检测 AMPAR基因表达量 (4) Fluorescence quantitative PCR detection of AMPAR gene expression

分别接种 CHO细胞和 CHO/AMPAR细胞至 6孔板。细胞密度达到 80<¾- 90%日寸，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。 CHO cells and CHO/AMPAR cells were inoculated separately into 6-well plates. The cell density reached 80<3⁄4-90% day, the total RNA of each group was extracted with RNeasy Mini Kit, and the mRNA was reverse transcribed into cDNA using the PrimeScrip RT reagent Kit. C save.

取各组细胞的 cDNA 1 Take cDNA 1 from each group of cells

为模板，以 GAPDH为内参，实吋荧光定量 PCR检测 AMPAR相对表达量，设置反应条件： 95。C 30s， 1循环， 58。C 30s 40循环， 95°C 5s, 60°C lmin, 95°C As a template, GAPDH was used as an internal reference, and the relative expression of AMPAR was detected by real-time fluorescent quantitative PCR. The reaction conditions were set: 95. C 30s, 1 cycle, 58. C 30s 40 cycles, 95°C 5s, 60°C lmin, 95°C

15s, 结果如图 1所示。可以看到， CHO/AMPAR细胞的 AMPAR基因表达水平较 CHO细胞高 240倍。 15s, the results are shown in Figure 1. It can be seen that the expression level of AMPAR gene in CHO/AMPAR cells is 240 times higher than that in CHO cells.