WO2018170711A1 - Human gp34 gene high-expression vector and application thereof - Google Patents
Human gp34 gene high-expression vector and application thereof Download PDFInfo
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- WO2018170711A1 WO2018170711A1 PCT/CN2017/077398 CN2017077398W WO2018170711A1 WO 2018170711 A1 WO2018170711 A1 WO 2018170711A1 CN 2017077398 W CN2017077398 W CN 2017077398W WO 2018170711 A1 WO2018170711 A1 WO 2018170711A1
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- 239000013604 expression vector Substances 0.000 title claims abstract description 13
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- 239000013598 vector Substances 0.000 claims abstract description 11
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Definitions
- the present invention belongs to the field of biotechnology, and relates to a method for constructing a human GP34 gene high expression vector and an application thereof.
- GP34 is a member of the TNF receptor superfamily and is a type I transmembrane glycoprotein.
- the expression profile of GP34 is restricted to the surface of activated CD4+ and CD8+ T cells, and is predominantly CD4+ T cells.
- the human GP34 ligand (GP34/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.
- GP34 can synergistically stimulate the activation of T cells, promote the production of high titer antibodies and class switching of B cells, mediate the infiltration of I MD16+ T cells into the inflammatory reaction site, and play an important role in the immunotherapy of tumors.
- the clinical transformation value is very large, and it requires a solid research before it can be put into practical use.
- the lack of the plasmid of the recombinant GP34 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 GP34 gene expression vector.
- the over-expression vector pEGFP-Cl/GP34 was constructed, which laid a foundation for the subsequent study of human GP34 gene function.
- a method for constructing a human GP34 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/GP34.
- the present invention constructs the overexpression vector pEGFP-Cl/GP34 of the GP34 gene. Subsequent development of overexpression GP3
- the 4 gene will play an important role in GP34-related drug research and development.
- 1 is the relative level of GP34 gene of 293T cells transfected with pEGFP-C 1/GP34 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
- RNA of Jurkat cells was extracted, reverse-transcribed into cDNA, primers (GP34-F, GP34-R) were designed, and PCR was carried out according to a conventional method using c-DNA as a template, using 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
- the PCR amplification products were identified by agarose gel electrophoresis.
- the nucleotide sequence of GP34-F is shown in SEQ ID No: 1
- the nucleotide sequence of GP34-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/GP34.
- the primer design software Oligo 7.0 was used to design the bow.
- the present invention constructs the overexpression vector pEGFP-Cl/GP34 of the GP34 gene. Subsequent development of overexpression GP3
- the 4 gene will play an important role in GP34-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 GP34 gene expression vector, comprising the steps of: (1) cloning of a GP34 gene; and (2) construction of an overexpression vector pEGFP-C1/GP34.
Description
发明名称:人 GP34基因高表达载体及其应用 技术领域 Title: Human GP34 gene high expression vector and its application
[0001] 本发明属于生物技术领域, 涉及一种人 GP34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 GP34 gene high expression vector and an application thereof.
背景技术 Background technique
[0002] GP34是 TNF受体超家族成员之一, 为 I型跨膜糖蛋白。 GP34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 GP34配体 (GP34/ 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。 [0002] GP34 is a member of the TNF receptor superfamily and is a type I transmembrane glycoprotein. The expression profile of GP34 is restricted to the surface of activated CD4+ and CD8+ T cells, and is predominantly CD4+ T cells. The human GP34 ligand (GP34/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] GP34能协同刺激 T细胞的活化, 促进 B细胞产生高效价抗体和类别转换, 介导 I MD16+T细胞向炎性反应部位浸润, 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 乏重组 GP34基因的质粒, 对相关研究的进展造成了一定的阻碍。 [0003] GP34 can synergistically stimulate the activation of T cells, promote the production of high titer antibodies and class switching of B cells, mediate the infiltration of I MD16+ T cells into the inflammatory reaction site, and play an important role in the immunotherapy of tumors. The clinical transformation value is very large, and it requires a solid research before it can be put into practical use. However, the lack of the plasmid of the recombinant GP34 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 GP34基因表达 载体的构建方法。 该方法在克隆人 GP34基因 cDNA序列的基础上, 构建过表达载 体 pEGFP-Cl/GP34, 为后续研究人 GP34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 GP34 gene expression vector. Based on the cloning of human GP34 gene cDNA sequence, the over-expression vector pEGFP-Cl/GP34 was constructed, which laid a foundation for the subsequent study of human GP34 gene function.
[0005] 其具体技术方案为: [0005] The specific technical solution thereof is:
[0006] 一种人 GP34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 GP34 gene overexpression vector, comprising the steps of:
[0007] (1) GP34基因克隆 [0007] (1) GP34 gene cloning
[0008] 提取 Jurkat细胞总 RNA, 逆转录为 cDNA, 设计引物 GP34-F、 GP34-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, and designing primers GP34-F and GP34-R to The cDNA was used as a template, and PCR amplification was carried out according to a conventional method using primers and PrimeStar high-fidelity DNA polymerase; the reaction conditions were: 98 ° C for 2 min; 98 ° C for 10 s, 58 ° C for 10 s, 72 ° C for 50 s, 30 cycles; 72 ° C for 5 min. The PCR amplification products were identified by agarose gel electrophoresis.
[0009] (2)过表达载体 pEGFP-Cl/GP34的构建 (2) Construction of overexpression vector pEGFP-Cl/GP34
[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/GP34。 [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/GP34.
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 GP34基因的过表达载体 pEGFP-Cl/GP34。 后续幵展过表达 GP3 [0011] The present invention constructs the overexpression vector pEGFP-Cl/GP34 of the GP34 gene. Subsequent development of overexpression GP3
4基因在 GP34相关的药物研究和幵发中将起重要作用。 The 4 gene will play an important role in GP34-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/GP34载体的 293T细胞的 GP34基因相对水平。 1 is the relative level of GP34 gene of 293T cells transfected with pEGFP-C 1/GP34 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。
[0015] 实施例一 GP34基因的克隆 Mini Kit II was purchased from Omega Bio-tek. [0015] Example 1 cloning of the GP34 gene
[0016] 提取 Jurkat细胞总 RNA, 逆转录为 cDNA, 设计引物 (GP34-F、 GP34-R) , 以 c DNA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 [0016] Total RNA of Jurkat cells was extracted, reverse-transcribed into cDNA, primers (GP34-F, GP34-R) were designed, and PCR was carried out according to a conventional method using c-DNA as a template, using 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。 PCR扩增产物经琼脂糖凝胶电泳鉴定正确。 GP34-F的核苷酸序列如 SEQ ID No: 1所示, GP34-R的核苷酸序列如 SEQ ID No: 2所示。 Min. The PCR amplification products were identified by agarose gel electrophoresis. The nucleotide sequence of GP34-F is shown in SEQ ID No: 1, and the nucleotide sequence of GP34-R is shown in SEQ ID No: 2.
[0017] 实施例二 过表达载体 pEGFP-Cl/GP34的构建 Example 2 Construction of overexpression vector pEGFP-Cl/GP34
[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/GP34。 [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/GP34.
[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/GP34质粒的大肠杆菌, Endo-Free Plasmid Mini Kit II提 取 pEGFP-Cl/GP34质粒。 取生长状态良好的 293T细胞接种到六孔中, 每孔 10000 00个细胞, 培养 18 h后, 用 Lipofectamine 2000将 pEGFP-Cl/GP34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/GP34 plasmid was cultured, and Endo-Free Plasmid Mini Kit II was used to extract pEGFP-Cl/GP34 plasmid. 293T cells with good growth were inoculated into six wells with 10000 cells per well. After 18 h of culture, pEGFP-Cl/GP34 plasmid was transduced 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 GP34基因表达量 [0021] Example 4 Fluorescence quantitative PCR detection of GP34 gene expression
[0022] 根据 GAPDH和 GP34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 GP34 gene mRNA sequences, the primer design software Oligo 7.0 was used to design the bow.
[0023] 分别接种 293T细胞、 转导 pEGFP-Cl/GP34质粒的 293T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。 [2933] 293T cells transfected with 293T cells and transfected with pEGFP-Cl/GP34 plasmid were separately plated into 6-well plates. The cell density reached δΟ^^Ο^吋, and the total RNA of each group was extracted with RNeasy Mini Kit. The mRNA was reverse-transcribed into cDNA using PrimeScrip RT reagent Kit and stored at -20 °C.
[0024] 取各组细胞的 cDNA 1 为模板, 以 GAPDH为内参, 荧光定量 PCR检测 GP34 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 GP34基因相对表达量, 结果如图 1 所示。 可以看到, 转导 pEGFP-Cl/GP34质粒的 293T细胞的 GP34基因的表达量较 正常 293T细胞都有 190倍以上的升高, 说明本发明提供 pEGFP-Cl/GP34质粒能特 异、 持续、 高效、 稳定地促进 GP34基因高表达。 [0024] Take cDNA 1 of each group as a template, use GAPDH as an internal reference, and detect the relative expression of GP34 by real-time PCR. Set the reaction conditions: 95 ° C 30 s, 1 cycle, 95 ° C 10 s, 54 ° C 30 s 40 cycles . The relative expression of GP34 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 GP34 gene of 293T cells transfected with pEGFP-Cl/GP34 plasmid is 190-fold higher than that of normal 293T cells, indicating that the pEGFP-Cl/GP34 plasmid can be specific, sustained and efficient. , stably promote high expression of GP34 gene.
[0025] 以上所述, 仅为本发明较佳的具体实施方式, 本发明的保护范围不限于此, 任 何熟悉本技术领域的技术人员在本发明披露的技术范围内, 还可以做出若干简 单推演或替换, 均落入本发明的保护范围内。 The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto. Any person skilled in the art can make some simple within the technical scope disclosed by the present invention. Deduction or replacement is within the scope of the present invention.
工业实用性 Industrial applicability
[0026] 本发明通过构建 GP34基因的过表达载体 pEGFP-Cl/GP34。 后续幵展过表达 GP3 The present invention constructs the overexpression vector pEGFP-Cl/GP34 of the GP34 gene. Subsequent development of overexpression GP3
4基因在 GP34相关的药物研究和幵发中将起重要作用。
The 4 gene will play an important role in GP34-related drug research and development.
Claims
(1) GP34基因克隆 (1) GP34 gene cloning
提取 Jurkat细胞总 RNA, 逆转录为 cDNA, 设计引物 GP34-F、 GP34-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 GP34-F and GP34-R were designed. Using cDNA as a template, 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 product was identified by agarose gel electrophoresis.
(2)过表达载体 pEGFP-Cl/GP34的构建 (2) Construction of overexpression vector pEGFP-Cl/GP34
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/GP34。
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/GP34.
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