WO2018170710A1 - Human imd16 gene high-expression vector and application thereof - Google Patents
Human imd16 gene high-expression vector and application thereof Download PDFInfo
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Definitions
- the present invention belongs to the field of biotechnology, and relates to a method for constructing a human IMD16 gene high expression vector and an 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+
- IMD16L/CD 134L Human IMD16 ligand contained 183 amino acids in 1991 (extracellular 139 amino acids, transmembrane 21 amino acids, intracellular 23 amino acids), belonging to the TNF family, and is a scorpion-type transmembrane glycoprotein.
- IMD16/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+4 T cells. The formation of the center and the differentiation and maturation of DC.
- the object of the present invention is to overcome the deficiencies in the prior art and to provide a method for constructing a human IMD16 gene expression vector.
- the overexpression vector pEGFP-Cl/IMD16 was constructed to lay a foundation for the subsequent study of human IMD16 gene function.
- a method for constructing a human IMD16 gene overexpression vector comprising the steps of:
- 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/IMD16.
- the present invention constructs the overexpression vector pEGFP-Cl/IMD16 of the IMD16 gene. Subsequent development of overexpression I MD16 gene will play an important role in IMD16-related drug research and development.
- 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.
- Example 1 Cloning of the IMD16 gene [0016] Total RNA of Jurkat cells was extracted, reverse-transcribed into cDNA, primers (IMD16-F, IMD16-R) were designed, 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 nucleotide sequence of IMD16-F is shown in SEQ ID No: 1, and the nucleotide sequence of IMD16-R is shown in SEQ ID No: 2.
- 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/IMD16.
- Escherichia coli containing the PEGFP-C1/IMD16 plasmid was cultured in large amounts, and the pEGFP-Cl/IMD16 plasmid was extracted from Endo-Free Plasmid Mini Kit II. 293T cells with good growth were inoculated into six wells with 1000 000 cells per well. After 18 h of culture, Lipofectamine was used.
- the pEGFP-Cl/IMD16 plasmid was transduced into 293T cells in 2000 and cultured for 48 h.
- the primer design software Oligo 7.0 was used to design the bow.
- the RT reagent kit reverse-transcribes mRNA into cDNA and stores it at -20 °C.
- Kit detected the relative expression of IMD16 gene in each group, and the results are shown in Figure 1. It can be seen that the expression level of IMD 16 gene in 293T cells transfected with pEG FP-C1/IMD 16 plasmid is more than 160-fold higher than that of normal 293T cells, indicating that the present invention provides PEGFP-C1/IMD16 plasmid specificity, Continuous, efficient, and stable promotion of high expression of IMD16 gene.
- the present invention constructs the overexpression vector pEGFP-Cl/IMD16 of the IMD16 gene. Subsequent development of overexpression I MD16 gene will play an important role in IMD16-related drug research and development.
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Abstract
Provided is a method for constructing a human IMD16 gene expression vector, comprising the steps of: (1) cloning of an IMD16 gene; and (2) construction of an overexpression vector pEGFP-C1/IMD16.
Description
发明名称：人 IMD16基因高表达载体及其应用 技术领域 Title: Human IMD16 gene high expression vector and its application
[0001] 本发明属于生物技术领域， 涉及一种人 IMD16基因高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 IMD16 gene high expression vector and an application thereof.
背景技术 Background technique
[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+ [0002] 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+
T细胞为主。 人 IMD16配体 (IMD16L/ CD 134L)1991年含 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。 IMD16/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+4 T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。 T cells are predominant. Human IMD16 ligand (IMD16L/CD 134L) contained 183 amino acids in 1991 (extracellular 139 amino acids, transmembrane 21 amino acids, intracellular 23 amino acids), belonging to the TNF family, and is a scorpion-type transmembrane glycoprotein. IMD16/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+4 T cells. The formation of the center and the differentiation and maturation of DC.
技术问题 technical problem
[0003] IMD16在肿瘤的免疫治疗中起重要的作用， 其潜在的临床转化价值很大， 需进 行扎实的研究方可投入实际应用， 但现有技术中缺乏 IMD16基因表达载体， 对相 关研究的进展造成了一定的阻碍。 [0003] IMD16 plays an important role in the immunotherapy of tumors, and its potential clinical transformation value is very large. It requires a solid research to be put into practical application, but the prior art lacks the IMD16 gene expression vector for related research. Progress has caused some obstacles.
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 IMD16基因表达 载体的构建方法。 该方法在克隆人 IMD16基因 cDNA序列的基础上， 构建过表达 载体 pEGFP-Cl/IMD16， 为后续研究人 IMD16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 IMD16 gene expression vector. Based on the cDNA sequence of human IMD16 gene, the overexpression vector pEGFP-Cl/IMD16 was constructed to lay a foundation for the subsequent study of human IMD16 gene function.
[0005] 其具体技术方案为： [0005] The specific technical solution thereof is:
[0006] 一种人 IMD16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 IMD16 gene overexpression vector, comprising the steps of:
[0007] (1) IMD16基因克隆 [0007] (1) IMD16 gene cloning
[0008] 提取 Jurkat细胞总 RNA， 逆转录为 cDNA， 设计引物 IMD16-F、 IMD16-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。 [0008] Extracting total RNA from Jurkat cells, reverse transcription into cDNA, designing primers IMD16-F, IMD16-R, using cDNA as a template, using primers and PrimeStar high-fidelity DNA polymerase, performing PCR according to conventional methods. Amplification; 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.
[0009] (2)过表达载体 pEGFP-Cl/IMD16的构建 (2) Construction of overexpression vector pEGFP-Cl/IMD16
[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/IMD16。 [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/IMD16.
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 IMD16基因的过表达载体 pEGFP-Cl/IMD16。 后续幵展过表达 I MD16基因在 IMD16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/IMD16 of the IMD16 gene. Subsequent development of overexpression I MD16 gene will play an important role in IMD16-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/IMD 16载体的 293T细胞的 IMD 16基因相对水平。 1 is the relative level of IMD 16 gene of 293T cells transfected with pEGFP-C 1/IMD 16 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 IMD16基因的克隆
[0016] 提取 Jurkat细胞总 RNA， 逆转录为 cDNA， 设计引物 （IMD16-F、 IMD16-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。 IMD16-F的核 苷酸序列如 SEQ ID No: 1所示， IMD16-R的核苷酸序列如 SEQ ID No: 2所示 [0015] Example 1 Cloning of the IMD16 gene [0016] Total RNA of Jurkat cells was extracted, reverse-transcribed into cDNA, primers (IMD16-F, IMD16-R) were designed, 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 nucleotide sequence of IMD16-F is shown in SEQ ID No: 1, and the nucleotide sequence of IMD16-R is shown in SEQ ID No: 2.
[0017] 实施例二 过表达载体 pEGFP-Cl/IMD16的构建 Example 2 Construction of overexpression vector pEGFP-Cl/IMD16
[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/IMD16。 [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/IMD16.
[0019] 实施例三 293T细胞的转导 Example 3 Transduction of 293T cells
[0020] 大量培养含 PEGFP-C1/IMD16质粒的大肠杆菌， Endo-Free Plasmid Mini Kit II提 取 pEGFP-Cl/IMD16质粒。 取生长状态良好的 293T细胞接种到六孔中， 每孔 1000 000个细胞， 培养 18 h后， 用 Lipofectamine [0020] Escherichia coli containing the PEGFP-C1/IMD16 plasmid was cultured in large amounts, and the pEGFP-Cl/IMD16 plasmid was extracted from Endo-Free Plasmid Mini Kit II. 293T cells with good growth were inoculated into six wells with 1000 000 cells per well. After 18 h of culture, Lipofectamine was used.
2000将 pEGFP-Cl/IMD16质粒转导至 293T细胞中， 继续培养 48 h。 The pEGFP-Cl/IMD16 plasmid was transduced into 293T cells in 2000 and cultured for 48 h.
[0021] 实施例四荧光定量 PCR检测 IMD16基因表达量 [0021] Example 4 Fluorescence quantitative PCR detection of IMD16 gene expression
[0022] 根据 GAPDH和 IMD16基因 mRNA序列， 利用引物设计软件 Oligo 7.0设计弓 |物。 [0022] According to the GAPDH and IMD16 gene mRNA sequences, the primer design software Oligo 7.0 was used to design the bow.
[0023]
[0023]
[0024] 分别接种 293T细胞、 转导 pEGFP-Cl/IMD16质粒的 293T细胞至 6孔板。 细胞密 度达到 80<¾-90<¾吋， 用 RNeasy Mini Kit提取各组细胞的总 RNA， 利用 PrimeScrip [29324] 293T cells transfected with 293T cells and transfected with pEGFP-Cl/IMD16 plasmid were plated into 6-well plates, respectively. The cell density reached 80<3⁄4-90<3⁄4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。 The RT reagent kit reverse-transcribes mRNA into cDNA and stores it at -20 °C.
[0025] 取各组细胞的 cDNA 1 为模板， 以 GAPDH为内参， 荧光定量 PCR检测 IMD16 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 [0025] Take cDNA 1 of each group as a template, use GAPDH as an internal reference, and detect the relative expression of IMD16 by real-time PCR. Set the reaction conditions: 95 ° C 30 s, 1 cycle, 95 ° C 10 s, 54 ° C 30 s 40 cycles . Use SYBR Primescript RT-PCR
Kit检测各组细胞 IMD16基因相对表达量， 结果如图 1所示。 可以看到， 转导 pEG FP-C1/IMD 16质粒的 293T细胞的 IMD 16基因的表达量较正常 293T细胞都有 160倍 以上的升高， 说明本发明提供 PEGFP-C1/IMD16质粒能特异、 持续、 高效、 稳定 地促进 IMD16基因高表达。 Kit detected the relative expression of IMD16 gene in each group, and the results are shown in Figure 1. It can be seen that the expression level of IMD 16 gene in 293T cells transfected with pEG FP-C1/IMD 16 plasmid is more than 160-fold higher than that of normal 293T cells, indicating that the present invention provides PEGFP-C1/IMD16 plasmid specificity, Continuous, efficient, and stable promotion of high expression of IMD16 gene.
工业实用性 Industrial applicability
[0026] 本发明通过构建 IMD16基因的过表达载体 pEGFP-Cl/IMD16。 后续幵展过表达 I MD16基因在 IMD16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/IMD16 of the IMD16 gene. Subsequent development of overexpression I MD16 gene will play an important role in IMD16-related drug research and development.
Claims
(1) IMD16基因克隆 (1) IMD16 gene cloning
提取 Jurkat细胞总 RNA， 逆转录为 cDNA， 设计引物 IMD16-F、 IMD16-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 IMD16-F and IMD16-R were designed. Using cDNA as a template, PCR amplification was carried out by using conventional primers and PrimeStar high-fidelity DNA polymerase. 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 product was identified by agarose gel electrophoresis.
(2)过表达载体 pEGFP-Cl/IMD16的构建 (2) Construction of overexpression vector pEGFP-Cl/IMD16
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/IMD16。
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/IMD16.
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