WO2018170709A1 - Vector for expressing human tnlg5a gene and application thereof - Google Patents
Vector for expressing human tnlg5a gene and application thereof Download PDFInfo
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- WO2018170709A1 WO2018170709A1 PCT/CN2017/077396 CN2017077396W WO2018170709A1 WO 2018170709 A1 WO2018170709 A1 WO 2018170709A1 CN 2017077396 W CN2017077396 W CN 2017077396W WO 2018170709 A1 WO2018170709 A1 WO 2018170709A1
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 19
- 239000013598 vector Substances 0.000 title claims abstract description 14
- 239000013604 expression vector Substances 0.000 claims abstract description 11
- 230000014509 gene expression Effects 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 6
- 230000002018 overexpression Effects 0.000 claims abstract description 6
- 238000010276 construction Methods 0.000 claims abstract description 4
- 239000002299 complementary DNA Substances 0.000 claims description 9
- 102000012410 DNA Ligases Human genes 0.000 claims description 6
- 108010061982 DNA Ligases Proteins 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 3
- 238000007796 conventional method Methods 0.000 claims description 3
- 239000013613 expression plasmid Substances 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 3
- 229930027917 kanamycin Natural products 0.000 claims description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 3
- 229960000318 kanamycin Drugs 0.000 claims description 3
- 229930182823 kanamycin A Natural products 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000012215 gene cloning Methods 0.000 claims description 2
- 230000029087 digestion Effects 0.000 claims 1
- 238000010367 cloning Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 20
- 239000013612 plasmid Substances 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
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Definitions
- the present invention belongs to the field of biotechnology, and relates to a method for constructing a human TNLG5A gene expression vector and an application thereof.
- ILA belongs to the TNFR superfamily and is mainly expressed in activated T cells and is an inducible T cell surface receptor;
- TNLG5A belongs to the TNF superfamily and is mainly expressed in concentrated antigen presenting cells (APC).
- APC concentrated antigen presenting cells
- IL A/TNLG5A is another important costimulatory molecule other than CD28/B7, which may or may not be dependent on CD28/
- the B7 pathway mediates the production of costimulatory signals that induce T cell activation, proliferation, and cytokine secretion.
- ILA and its ligand system have two-way signal transduction, which can transmit cells to T cells through TNLG5A, and can transmit signals to cells expressing ligands, which plays an important role in tumor immunotherapy, and needs to be done.
- a large number of studies can achieve clinical transformation, but the lack of recombinant vectors with high expression of TNLG5A gene in the prior art has hindered the progress of related research.
- the object of the present invention is to overcome the deficiencies in the prior art and to provide a method for constructing a human TNLG5A gene expression vector.
- the expression vector pEGFP-Cl/TNLG5A was constructed, which laid a foundation for the subsequent study of human TNLG5A gene function.
- a method for constructing a human TNLG5A gene overexpression vector comprising the steps of:
- RNA of Jurkat cells was extracted and reverse transcribed into cDNA, and primers TNLG5A-F and TNLG5A-R were designed.
- the cDNA was used as a template, and primers and PrimeStar high-fidelity DNA polymerase were used for PCR amplification according to a conventional method.
- the reaction conditions were : 98 ° C 2 min; 98 ° C 10 s, 58 ° C 10 s, 72 ° C 50 s, 30 cycles; 72 ° C 5 min.
- the PCR amplification products were identified by agarose gel electrophoresis.
- the purified PCR product and the eukaryotic expression vector pEGFP-Cl were digested with restriction enzymes Xho I and EcoR I, identified and recovered by 1% agarose gel electrophoresis;
- the target gene fragment was ligated with the same double-digested expression vector pEGFP-Cl; the reaction system was: 10XT4 DNA ligase Buffer 1 ⁇ , linearized pEGFP-Cl vector 1 ⁇ , PCR product 3 ⁇ , T4 DNA ligase 1 ⁇ , dH20 4 L, 4 °C ligation overnight; the ligation product was transformed into E.
- coli DH5ot competent cells ice bath for 30 min, heat shock at 42 °C for 90 s, immediately transferred to ice for 2 min, then added to 500 37
- the pre-warmed non-resistant LB medium was incubated at 37 °C for 1 h.
- the bacterial solution was uniformly coated on LB solid medium containing kanamycin, and placed in a 37 ° C incubator for overnight culture. Positive colonies were screened; 3 monoclonal colonies were randomly picked. After the colony PCR was positive, the bacterial liquid was sent to Shanghai Biotech for sequencing.
- the obtained recombinant eukaryotic expression plasmid was named pEGFP-Cl/TNLG5A.
- the present invention constructs the overexpression vector pEGFP-Cl/TNLG5A of the TNLG5A gene. Subsequent development of the TNLG5A gene will play an important role in TNLG5A-related drug research and development.
- 1 is the relative level of the TNLG5 A gene of 293T cells transfected with pEGFP-C 1/TNLG5 A vector.
- Jurkat cells and 293T cells were purchased from ATCC, Premix PrimeSTAR
- HS enzyme was purchased from Takara, RNeasy Mini Kit was purchased from QIAGEN, Endo-Free Plasmid
- Mini Kit II was purchased from Omega bio-tek.
- the purified PCR product and the eukaryotic expression vector pEGFP-Cl were digested with restriction enzymes Xho I and EcoR I, identified and recovered by 1% agarose gel electrophoresis;
- the target gene fragment was ligated with the same double-digested expression vector pEGFP-Cl; the reaction system was: 10XT4 DNA ligase Buffer 1 ⁇ , linearized pEGFP-Cl vector 1 ⁇ , PCR product 3 ⁇ , T4 DNA ligase 1 ⁇ , dH20 4 L, 4 °C ligation overnight; the ligation product was transformed into E.
- coli DH5ot competent cells ice bath for 30 min, heat shock at 42 °C for 90 s, immediately transferred to ice for 2 min, then added to 500 37
- the pre-warmed non-resistant LB medium was incubated at 37 °C for 1 h.
- the bacterial solution was uniformly coated on LB solid medium containing kanamycin, and placed in a 37 ° C incubator for overnight culture. Positive colonies were screened; 3 monoclonal colonies were randomly picked. After the colony PCR was positive, the bacterial liquid was sent to Shanghai Biotech for sequencing.
- the obtained recombinant eukaryotic expression plasmid was named pEGFP-Cl/TNLG5A.
- the primer design software Oligo 7.0 was used to design bows [0023] , CR production of the country
- GGGTCGG GA@CTTTGeCCeG
- Kit extracts total RNA from each group of cells, using PrimeScrip RT reagent
- Kit reverse transcribes mRNA into cDNA and stores at -20 °C.
- TNLG5A As a template, GAPDH was used as an internal reference, and the relative expression of TNLG5A was detected by real-time PCR. The reaction conditions were set: 95 ° C for 30 s, 1 cycle, 95 ° C for 10 s, and 54 ° C for 30 s for 40 cycles. The relative expression of TNLG5A gene in each group was detected by SYBR Primescript RT-PCR Kit. The results are shown in Figure 1. It can be seen that the expression level of TNLG5A gene in 293T cells transfected with pEGFP-Cl/TNLG5A plasmid is 170-fold higher than that of normal 293T cells, indicating that the pEGFP-Cl/TNLG5A plasmid can be specific, sustained and efficient. , stably promote high expression of TNLG5A gene.
- the present invention constructs the overexpression vector pEGFP-Cl/TNLG5A of the TNLG5A gene. Subsequent development of the TNLG5A gene will play an important role in TNLG5A-related drug research and development.
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Provided is a method for constructing a human TNLG5A gene expression vector, comprising the following steps: (1) cloning of a TNLG5A gene; and (2) construction of an overexpression vector pEGFP-Cl/TNLG5A.
Description
发明名称：一种用于表达人 TNLG5 A基因的载体及其应用 技术领域 Title of Invention: A Vector for Expressing Human TNLG5 A Gene and Application Thereof
[0001] 本发明属于生物技术领域， 涉及一种人 TNLG5A基因表达载体的构建方法及其 应用。 [0001] The present invention belongs to the field of biotechnology, and relates to a method for constructing a human TNLG5A gene expression vector and an application thereof.
背景技术 Background technique
[0002] ILA属于 TNFR超家族， 主要表达于活化的 T细胞中， 是一种可诱导的 T细胞表 面受体； TNLG5A属于 TNF超家族， 主要表达在集中抗原呈递细胞 （APC) 。 IL A/TNLG5A是 CD28/B7之外的另一重要的共刺激分子， 可依赖或不依赖于 CD28/ [0002] ILA belongs to the TNFR superfamily and is mainly expressed in activated T cells and is an inducible T cell surface receptor; TNLG5A belongs to the TNF superfamily and is mainly expressed in concentrated antigen presenting cells (APC). IL A/TNLG5A is another important costimulatory molecule other than CD28/B7, which may or may not be dependent on CD28/
B7途径而介导产生协同刺激信号， 诱导 T细胞的活化、 增殖与细胞因子的分泌。 技术问题 The B7 pathway mediates the production of costimulatory signals that induce T cell activation, proliferation, and cytokine secretion. technical problem
[0003] ILA及其配体系统存在双向信号传导， 既可通过 TNLG5A向 T细胞传递细胞， 又 可将信号传向表达配体的细胞， 其在肿瘤的免疫治疗中起重要的作用， 需做大 量研究方可实现临床转化， 但现有技术中缺乏 TNLG5A基因高表达的重组载体， 对相关研究的进展造成了一定的阻碍。 [0003] ILA and its ligand system have two-way signal transduction, which can transmit cells to T cells through TNLG5A, and can transmit signals to cells expressing ligands, which plays an important role in tumor immunotherapy, and needs to be done. A large number of studies can achieve clinical transformation, but the lack of recombinant vectors with high expression of TNLG5A gene in the prior art has hindered the progress of related research.
问题的解决方案 Problem solution
技术解决方案 Technical solution
[0004] 本发明的目的在于克服现有技术中的存在的缺陷， 提供一种人 TNLG5A基因表 达载体的构建方法。 该方法在克隆人 TNLG5A基因 cDNA序列的基础上， 构建过 表达载体 pEGFP-Cl/TNLG5A， 为后续研究人 TNLG5A基因功能奠定基础。 [0004] The object of the present invention is to overcome the deficiencies in the prior art and to provide a method for constructing a human TNLG5A gene expression vector. Based on the cDNA sequence of human TNLG5A gene, the expression vector pEGFP-Cl/TNLG5A was constructed, which laid a foundation for the subsequent study of human TNLG5A gene function.
[0005] 其具体技术方案为： [0005] The specific technical solution thereof is:
[0006] 一种人 TNLG5A基因过表达载体的构建方法， 包括以下步骤： [0006] A method for constructing a human TNLG5A gene overexpression vector, comprising the steps of:
[0007] (1) TNLG5A基因克隆 [0007] (1) TNLG5A gene cloning
[0008] 提取 Jurkat细胞总 RNA， 逆转录为 cDNA, 设计引物 TNLG5A-F、 TNLG5A-R ， 以 cDNA为模板， 使用引物和 PrimeStar高保真 DNA聚合酶， 按常规方法进行 PCR扩增； 反应条件为： 98°C 2 min； 98°C 10 s、 58°C 10 s、 72°C 50 s, 30个 循环； 72°C 5 min。 PCR扩增产物经琼脂糖凝胶电泳鉴定。
[0009] (2)过表达载体 pEGFP-Cl/TNLG5A的构建 [0008] The total RNA of Jurkat cells was extracted and reverse transcribed into cDNA, and primers TNLG5A-F and TNLG5A-R were designed. The cDNA was used as a template, and primers and PrimeStar high-fidelity DNA polymerase were used for PCR amplification according to a conventional method. The reaction conditions were : 98 ° C 2 min; 98 ° C 10 s, 58 ° C 10 s, 72 ° C 50 s, 30 cycles; 72 ° C 5 min. The PCR amplification products were identified by agarose gel electrophoresis. (2) Construction of overexpression vector pEGFP-Cl/TNLG5A
[0010] 利用限制酶 Xho I和 EcoR I对纯化后的 PCR扩增产物与真核表达载体 pEGFP-Cl 同吋进行双酶切， 经 1%的琼脂糖凝胶电泳鉴定并回收； 回收后的目的基因片段 和经同样双酶切的表达载体 pEGFP-Cl进行连接； 反应体系为： 10XT4 DNA连接 酶 Buffer 1 μί、 线性化的 pEGFP-Cl载体 1 μί、 PCR产物 3 μί、 T4 DNA连接酶 1 μί、 dH20 4 L、 4°C连接过夜； 将连接产物转化大肠杆菌 DH5ot感受态细胞中， 冰浴 30 min， 42°C热激 90 s， 立即移入冰中静置 2 min， 之后加入 500 37°C预温 的无抗性 LB培养基， 37°C摇床培养 lh， 最后将菌液均匀涂布含有卡那霉素的 LB 固体培养基上， 放入 37°C培养箱内过夜培养， 筛选阳性菌落； 随机挑取 3株单克 隆菌落， 菌落 PCR鉴定为阳性后将菌液送上海生工测序； 所得到的重组真核表达 质粒命名为 pEGFP-Cl/TNLG5A。 [0010] The purified PCR product and the eukaryotic expression vector pEGFP-Cl were digested with restriction enzymes Xho I and EcoR I, identified and recovered by 1% agarose gel electrophoresis; The target gene fragment was ligated with the same double-digested expression vector pEGFP-Cl; the reaction system was: 10XT4 DNA ligase Buffer 1 μί, linearized pEGFP-Cl vector 1 μί, PCR product 3 μί, T4 DNA ligase 1 Μί, dH20 4 L, 4 °C ligation overnight; the ligation product was transformed into E. coli DH5ot competent cells, ice bath for 30 min, heat shock at 42 °C for 90 s, immediately transferred to ice for 2 min, then added to 500 37 The pre-warmed non-resistant LB medium was incubated at 37 °C for 1 h. Finally, the bacterial solution was uniformly coated on LB solid medium containing kanamycin, and placed in a 37 ° C incubator for overnight culture. Positive colonies were screened; 3 monoclonal colonies were randomly picked. After the colony PCR was positive, the bacterial liquid was sent to Shanghai Biotech for sequencing. The obtained recombinant eukaryotic expression plasmid was named pEGFP-Cl/TNLG5A.
发明的有益效果 Advantageous effects of the invention
有益效果 Beneficial effect
[0011] 本发明通过构建 TNLG5A基因的过表达载体 pEGFP-Cl/TNLG5A。 后续幵展过 表达 TNLG5A基因在 TNLG5A相关的药物研究和幵发中将起重要作用。 [0011] The present invention constructs the overexpression vector pEGFP-Cl/TNLG5A of the TNLG5A gene. Subsequent development of the TNLG5A gene will play an important role in TNLG5A-related drug research and development.
对附图的简要说明 Brief description of the drawing
附图说明 DRAWINGS
[0012] 图 1为转染 pEGFP-C 1/TNLG5 A载体的 293T细胞的 TNLG5 A基因相对水平。 1 is the relative level of the TNLG5 A gene of 293T cells transfected with pEGFP-C 1/TNLG5 A 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
[0013] 下面结合附图与具体实施例对本发明做进一步的说明。 [0013] The present invention will be further described below in conjunction with the drawings and specific embodiments.
[0014] Jurkat细胞和 293T细胞购自 ATCC， Premix PrimeSTAR [0014] Jurkat cells and 293T cells were purchased from ATCC, Premix PrimeSTAR
HS酶购自 Takara公司， RNeasy Mini Kit购自 QIAGEN公司， Endo-Free Plasmid HS enzyme was purchased from Takara, RNeasy Mini Kit was purchased from QIAGEN, Endo-Free Plasmid
Mini Kit II购自 Omega bio-tek公司。 Mini Kit II was purchased from Omega bio-tek.
[0015] 实施例一 TNLG5A基因的克隆。 Example 1 Cloning of the TNLG5A gene.
[0016] 提取 Jurkat细胞总 RNA， 逆转录为 cDNA， 设计引物 （TNLG5A-F、 TNLG5A-R ) ， 以 cDNA为模板， 使用引物和 PrimeStar高保真 DNA聚合酶， 按常规方法进行
PCR扩增。 反应条件为： 98°C 2 min； 98。C 10 s、 58。C 10 s、 72。C 50 s， 30个循 环； 72°C 5 min。 PCR扩增产物经琼脂糖凝胶电泳鉴定正确。 TNLG5A-F的核苷 酸序列如 SEQ ID No: 1所示， TNLG5A-R的核苷酸序列如 SEQ ID No: 2所示 [0016] Extracting total RNA from Jurkat cells, reverse transcription into cDNA, designing primers (TNLG5A-F, TNLG5A-R), using cDNA as a template, using primers and PrimeStar high-fidelity DNA polymerase, according to conventional methods. PCR amplification. The reaction conditions were: 98 ° C 2 min; 98. C 10 s, 58. C 10 s, 72. C 50 s, 30 cycles; 72 ° C for 5 min. The PCR amplification products were identified by agarose gel electrophoresis. The nucleotide sequence of TNLG5A-F is shown in SEQ ID No: 1, and the nucleotide sequence of TNLG5A-R is shown in SEQ ID No: 2.
[0017] 实施例二 过表达载体 pEGFP-Cl/TNLG5A的构建 Example 2 Construction of overexpression vector pEGFP-Cl/TNLG5A
[0018] 利用限制酶 Xho I和 EcoR I对纯化后的 PCR扩增产物与真核表达载体 pEGFP-Cl 同吋进行双酶切， 经 1%的琼脂糖凝胶电泳鉴定并回收； 回收后的目的基因片段 和经同样双酶切的表达载体 pEGFP-Cl进行连接； 反应体系为： 10XT4 DNA连接 酶 Buffer 1 μί、 线性化的 pEGFP-Cl载体 1 μί、 PCR产物 3 μί、 T4 DNA连接酶 1 μί、 dH20 4 L、 4°C连接过夜； 将连接产物转化大肠杆菌 DH5ot感受态细胞中， 冰浴 30 min， 42°C热激 90 s， 立即移入冰中静置 2 min， 之后加入 500 37°C预温 的无抗性 LB培养基， 37°C摇床培养 lh， 最后将菌液均匀涂布含有卡那霉素的 LB 固体培养基上， 放入 37°C培养箱内过夜培养， 筛选阳性菌落； 随机挑取 3株单克 隆菌落， 菌落 PCR鉴定为阳性后将菌液送上海生工测序； 所得到的重组真核表达 质粒命名为 pEGFP-Cl/TNLG5A。 [0018] The purified PCR product and the eukaryotic expression vector pEGFP-Cl were digested with restriction enzymes Xho I and EcoR I, identified and recovered by 1% agarose gel electrophoresis; The target gene fragment was ligated with the same double-digested expression vector pEGFP-Cl; the reaction system was: 10XT4 DNA ligase Buffer 1 μί, linearized pEGFP-Cl vector 1 μί, PCR product 3 μί, T4 DNA ligase 1 Μί, dH20 4 L, 4 °C ligation overnight; the ligation product was transformed into E. coli DH5ot competent cells, ice bath for 30 min, heat shock at 42 °C for 90 s, immediately transferred to ice for 2 min, then added to 500 37 The pre-warmed non-resistant LB medium was incubated at 37 °C for 1 h. Finally, the bacterial solution was uniformly coated on LB solid medium containing kanamycin, and placed in a 37 ° C incubator for overnight culture. Positive colonies were screened; 3 monoclonal colonies were randomly picked. After the colony PCR was positive, the bacterial liquid was sent to Shanghai Biotech for sequencing. The obtained recombinant eukaryotic expression plasmid was named pEGFP-Cl/TNLG5A.
[0019] 实施例三 293T细胞的转导 Example 3 Transduction of 293T cells
[0020] 大量培养含 pEGFP-Cl/TNLG5A质粒的大肠杆菌， Endo-Free Plasmid Mini Kit II 提取 pEGFP-Cl/TNLG5A质粒。 取生长状态良好的 293T细胞接种到六孔中， 每孔 1000000个细胞， 培养 18 h后， 用 Lipofectamine 2000将 pEGFP-Cl/TNLG5A质粒转 导至 293T细胞中， 继续培养 48 h。 [0020] A large amount of Escherichia coli containing pEGFP-Cl/TNLG5A plasmid was cultured, and Endo-Free Plasmid Mini Kit II was used to extract pEGFP-Cl/TNLG5A plasmid. 293T cells with good growth were inoculated into six wells with 1000000 cells per well. After 18 h of culture, the pEGFP-Cl/TNLG5A plasmid was transfected into 293T cells with Lipofectamine 2000, and culture was continued for 48 h.
[0021] 实施例四荧光定量 PCR检测 TNLG5A基因表达量 [0021] Example 4 Fluorescence quantitative PCR detection of TNLG5A gene expression
[0022] 根据 GAPDH和 TNLG5A基因 mRNA序列， 利用引物设计软件 Oligo 7.0设计弓 |物 [0023]
，CR产猶 基国 [0022] According to the GAPDH and TNLG5A gene mRNA sequences, the primer design software Oligo 7.0 was used to design bows [0023] , CR production of the country
太 ( Bp ) Too (Bp)
： TCTGACTf GAACAOCGACACC : TCTGACTf GAACAOCGACACC
119 119
GGGTCGG:GA@CTTTGeCCeG GGGTCGG: GA@CTTTGeCCeG
T LG'5A, T LG'5A,
GTGTCGTGTTTGTAGCTCAG GTGTCGTGTTTGTAGCTCAG
[0024] 分别接种 293T细胞、 转导 pEGFP-Cl/TNLG5A质粒的 293T细胞至 6孔板。 细胞 密度达到 δΟ^^Ο^吋， 用 RNeasy Mini [2932] 293T cells transfected with 293T cells and transfected with pEGFP-Cl/TNLG5A plasmid into 6-well plates, respectively. 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， -20°C保存。 Kit reverse transcribes mRNA into cDNA and stores at -20 °C.
[0025] 取各组细胞的 cDNA 1 [0025] taking cDNA 1 of each group of cells
为模板， 以 GAPDH为内参， 荧光定量 PCR检测 TNLG5A相对表达量， 设置反 应条件： 95°C 30s， 1循环， 95°C 10s， 54°C 30s 40循环。 利用 SYBR Primescript RT-PCR Kit检测各组细胞 TNLG5A基因相对表达量， 结果如图 1所示。 可以看到 ， 转导 pEGFP-Cl/TNLG5A质粒的 293T细胞的 TNLG5A基因的表达量较正常 293T 细胞都有 170倍以上的升高， 说明本发明提供 pEGFP-Cl/TNLG5A质粒能特异、 持续、 高效、 稳定地促进 TNLG5A基因高表达。 As a template, GAPDH was used as an internal reference, and the relative expression of TNLG5A was detected by real-time PCR. The reaction conditions were set: 95 ° C for 30 s, 1 cycle, 95 ° C for 10 s, and 54 ° C for 30 s for 40 cycles. The relative expression of TNLG5A gene in each group was detected by SYBR Primescript RT-PCR Kit. The results are shown in Figure 1. It can be seen that the expression level of TNLG5A gene in 293T cells transfected with pEGFP-Cl/TNLG5A plasmid is 170-fold higher than that of normal 293T cells, indicating that the pEGFP-Cl/TNLG5A plasmid can be specific, sustained and efficient. , stably promote high expression of TNLG5A gene.
[0026] [0026]
[0027] [0027]
[0028] 。 [0028].
工业实用性 Industrial applicability
[0029] 本发明通过构建 TNLG5A基因的过表达载体 pEGFP-Cl/TNLG5A。 后续幵展过 表达 TNLG5A基因在 TNLG5A相关的药物研究和幵发中将起重要作用。
The present invention constructs the overexpression vector pEGFP-Cl/TNLG5A of the TNLG5A gene. Subsequent development of the TNLG5A gene will play an important role in TNLG5A-related drug research and development.
Claims
(1) TNLG5A基因克隆 (1) TNLG5A gene cloning
提取 Jurkat细胞总 RNA， 逆转录为 cDNA， 设计引物 TNLG5A-F、 TNLG5A-R, 以 cDNA为模板， 使用引物和 PrimeStar高 保真 DNA聚合酶， 按常规方法进行 PCR扩增； 反应条件为： 98°C 2 min； 98。C 10 s、 58。C 10 s、 72。C 50 s， 30个循环; 72。C 5 min。 PCR扩增产物经琼脂糖凝胶电泳鉴定。 The total RNA of Jurkat cells was extracted and reverse transcribed into cDNA. Primers TNLG5A-F and TNLG5A-R were designed. The cDNA was used as a template, and primers and PrimeStar high-fidelity DNA polymerase were used for PCR amplification according to the conventional method. The reaction conditions were: 98°. C 2 min; 98. C 10 s, 58. C 10 s, 72. C 50 s, 30 cycles; 72. C 5 min. The PCR amplification products were identified by agarose gel electrophoresis.
(2)过表达载体 pEGFP-Cl/TNLG5A的构建 (2) Construction of overexpression vector pEGFP-Cl/TNLG5A
利用限制酶 Xho I和 EcoR Use of restriction enzymes Xho I and EcoR
I对纯化后的 PCR扩增产物与真核表达载体 pEGFP-Cl同吋进行双酶切 ， 经 1%的琼脂糖凝胶电泳鉴定并回收； 回收后的目的基因片段和经 同样双酶切的表达载体 pEGFP-Cl进行连接； 反应体系为： 10xT4 DNA连接酶 Buffer 1 μί、 线性化的 pEGFP-Cl载体 1 μί、 PCR产物 3 μί、 T4 DNA连接酶 1μί、 dH20 4μί、 4°C连接过夜； 将连接产物转 化大肠杆菌 DH5ot感受态细胞中， 冰浴 30 min， 42°C热激 90 s， 立即移 入冰中静置 2 min， 之后加入 500 L 37°C预温的无抗性 LB培养基， 37 °C摇床培养 lh， 最后将菌液均匀涂布含有卡那霉素的 LB固体培养基 上， 放入 37°C培养箱内过夜培养， 筛选阳性菌落； 随机挑取 3株单克 隆菌落， 菌落 PCR鉴定为阳性后将菌液送上海生工测序； 所得到的重 组真核表达质粒命名为 pEGFP-Cl/TNLG5A。
I purified the PCR product and the eukaryotic expression vector pEGFP-Cl were digested and identified by 1% agarose gel electrophoresis; the recovered target gene fragment and the same double digestion The expression vector pEGFP-Cl was ligated; the reaction system was: 10xT4 DNA ligase Buffer 1 μί, linearized pEGFP-Cl vector 1 μί, PCR product 3 μί, T4 DNA ligase 1 μί, dH20 4 μί, 4 ° C overnight; The ligation product was transformed into E. coli DH5ot competent cells, incubated in ice bath for 30 min, heat shocked at 42 °C for 90 s, immediately transferred to ice for 2 min, and then added to 500 L 37 ° C pre-warmed non-resistant LB medium. After incubating at 37 °C for 1 h, finally, the bacterial solution was uniformly coated on LB solid medium containing kanamycin, placed in a 37 ° C incubator for overnight culture, and positive colonies were screened; 3 monoclonal clones were randomly picked. Colonies, colony PCR was positive, and the bacterial liquid was sent to Shanghai Biotech for sequencing; the obtained recombinant eukaryotic expression plasmid was named pEGFP-Cl/TNLG5A.
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