WO2018170710A1 - 人imd16基因高表达载体及其应用 - Google Patents
人imd16基因高表达载体及其应用 Download PDFInfo
- Publication number
- WO2018170710A1 WO2018170710A1 PCT/CN2017/077397 CN2017077397W WO2018170710A1 WO 2018170710 A1 WO2018170710 A1 WO 2018170710A1 CN 2017077397 W CN2017077397 W CN 2017077397W WO 2018170710 A1 WO2018170710 A1 WO 2018170710A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- imd16
- pegfp
- expression vector
- gene
- pcr
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 17
- 239000013604 expression vector Substances 0.000 title claims abstract description 13
- 208000014136 immunodeficiency 16 Diseases 0.000 claims abstract description 50
- 239000013598 vector Substances 0.000 claims abstract description 12
- 230000014509 gene expression Effects 0.000 claims abstract description 10
- 230000002018 overexpression Effects 0.000 claims abstract description 9
- 238000000034 method Methods 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
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 239000007787 solid Substances 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
- 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
- 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 16
- 239000013612 plasmid Substances 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 101150076401 16 gene Proteins 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 238000011530 RNeasy Mini Kit Methods 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 2
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000003208 gene overexpression Methods 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 1
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.
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
提供了一种人IMD16基因表达载体的构建方法，步骤包括：（1）IMD16基因的克隆；（2）过表达载体pEGFP-C1/ IMD16的构建。
Description
发明名称：人 IMD16基因高表达载体及其应用 技术领域
[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。
背景技术
[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+
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。
技术问题
[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。
问题的解决方案
技术解决方案
[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。
[0005] 其具体技术方案为：
[0006] 一种人 IMD16基因过表达载体的构建方法， 包括以下步骤：
[0007] (1) IMD16基因克隆
[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。
[0009] (2)过表达载体 pEGFP-Cl/IMD16的构建
[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。
发明的有益效果
有益效果
[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。
对附图的简要说明
附图说明
[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。
实施该发明的最佳实施例
本发明的最佳实施方式
[0013] 下面结合附图与具体实施例对本发明做进一步的说明。
[0014] Jurkat细胞和 293T细胞购自 ATCC， Premix PrimeSTAR
HS酶购自 Takara公司， RNeasy Mini Kit购自 QIAGEN公司， Endo-Free Plasmid
Mini Kit II购自 Omega bio-tek公司。
[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
[0017] 实施例二 过表达载体 pEGFP-Cl/IMD16的构建
[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。
[0019] 实施例三 293T细胞的转导
[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
2000将 pEGFP-Cl/IMD16质粒转导至 293T细胞中， 继续培养 48 h。
[0021] 实施例四荧光定量 PCR检测 IMD16基因表达量
[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。
[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
RT reagent Kit将 mRNA逆转录为 cDNA， -20°C保存。
[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
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。
工业实用性
[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。
Claims
(1) IMD16基因克隆
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。
(2)过表达载体 pEGFP-Cl/IMD16的构建
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 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。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/077397 WO2018170710A1 (zh) | 2017-03-20 | 2017-03-20 | 人imd16基因高表达载体及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/077397 WO2018170710A1 (zh) | 2017-03-20 | 2017-03-20 | 人imd16基因高表达载体及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2018170710A1 true WO2018170710A1 (zh) | 2018-09-27 |
Family
ID=63583897
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2017/077397 WO2018170710A1 (zh) | 2017-03-20 | 2017-03-20 | 人imd16基因高表达载体及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2018170710A1 (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2718868A1 (fr) * | 2008-03-18 | 2009-09-24 | Centre National De La Recherche Scientifique | Polynucleotides et polypeptides chimeriques permettant la secretion d'un polypeptide d'interet en association avec des exosomes et leur utilisation pour la production de compositions immunogenes |
| CN102061310A (zh) * | 2010-11-24 | 2011-05-18 | 中国人民解放军第四军医大学 | 人fhl1c真核表达载体的构建及其应用 |
| CN102337297A (zh) * | 2011-10-21 | 2012-02-01 | 南京医科大学 | 一种mbr-FPGS高效表达载体及其构建方法和应用 |
-
2017
- 2017-03-20 WO PCT/CN2017/077397 patent/WO2018170710A1/zh active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2718868A1 (fr) * | 2008-03-18 | 2009-09-24 | Centre National De La Recherche Scientifique | Polynucleotides et polypeptides chimeriques permettant la secretion d'un polypeptide d'interet en association avec des exosomes et leur utilisation pour la production de compositions immunogenes |
| CN102061310A (zh) * | 2010-11-24 | 2011-05-18 | 中国人民解放军第四军医大学 | 人fhl1c真核表达载体的构建及其应用 |
| CN102337297A (zh) * | 2011-10-21 | 2012-02-01 | 南京医科大学 | 一种mbr-FPGS高效表达载体及其构建方法和应用 |
Non-Patent Citations (1)
| Title |
|---|
| LIU, HONGXIANG ET AL.,: "Advances in Research on TNF-TNFR Superfamily T Cell Costimulatory Molecules and Organ Transplantation", SURGERY FOREIGN MEDICAL SCIENCES, vol. 32, no. 6, 30 November 2005 (2005-11-30), pages 401 - 404 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109266650A (zh) | 一种诱导型启动子、其重组载体、转化体以及诱导基因表达的方法及其应用 | |
| WO2018170710A1 (zh) | 人imd16基因高表达载体及其应用 | |
| WO2018170711A1 (zh) | 人 gp34 基因高表达载体及其应用 | |
| CN103031285B (zh) | 冬虫夏草中国被毛孢尿苷-胞苷激酶、编码基因及其应用 | |
| CN102260673A (zh) | 靶向人血管紧张素原的rna干扰片段、表达载体与应用 | |
| WO2018170708A1 (zh) | 人ila基因表达载体的构建方法及其应用 | |
| WO2018170709A1 (zh) | 一种用于表达人 tnlg5a 基因的载体及其应用 | |
| CN115838713A (zh) | 一种蛋白酶及其在l-肌肽合成中的应用 | |
| WO2017214940A1 (zh) | 特异促进Cplx2基因高表达的慢病毒表达载体及其应用 | |
| WO2019237391A1 (zh) | CRISPR/Cas9 靶向敲除人 TXGP1 基因及其特异性 gRNA | |
| WO2019000145A1 (zh) | 一种ampar高表达的重组cho细胞的构建方法 | |
| WO2019037133A1 (zh) | 靶向沉默APP的shRNA | |
| WO2019000150A1 (zh) | 一种tl6高表达的重组cho细胞的构建方法 | |
| CN103031286B (zh) | 冬虫夏草中国被毛孢尿苷酸-胞苷酸激酶、编码基因及应用 | |
| WO2019000152A1 (zh) | 一种表达rhbdd1基因的cho细胞及其应用 | |
| WO2019037053A1 (zh) | 人 AITR 基因的 shRNA 及其应用 | |
| WO2019000144A1 (zh) | 一种表达aitr基因的cho细胞及其应用 | |
| WO2018170765A1 (zh) | Imd16基因的rna干涉载体及其应用 | |
| WO2018170762A1 (zh) | TNLG5A基因的RNAi表达载体、构建方法及其应用 | |
| WO2019036870A1 (zh) | 一种人arntl基因高表达载体的构建方法及应用 | |
| WO2017214937A1 (zh) | 一种促进 app 基因表达的慢病毒表达载体及其应用 | |
| WO2019037054A1 (zh) | 一种人 cvid1 基因高表达载体的构建方法及应用 | |
| WO2017214938A1 (zh) | 特异促进 bace1 基因高表达的慢病毒表达载体及应用 | |
| WO2019036869A1 (zh) | 人 TL6 基因的 shRNA 及其应用 | |
| WO2017214910A1 (zh) | 特异促进Tβ4基因高表达的慢病毒表达载体及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17901841 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 17901841 Country of ref document: EP Kind code of ref document: A1 |