WO2019033249A1 - Sharn du gène btla humain et utilisation correspondante - Google Patents
Sharn du gène btla humain et utilisation correspondante Download PDFInfo
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- WO2019033249A1 WO2019033249A1 PCT/CN2017/097432 CN2017097432W WO2019033249A1 WO 2019033249 A1 WO2019033249 A1 WO 2019033249A1 CN 2017097432 W CN2017097432 W CN 2017097432W WO 2019033249 A1 WO2019033249 A1 WO 2019033249A1
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- btla
- shrna
- gene
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- btla gene
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Links
- 101150025010 Btla gene Proteins 0.000 title claims abstract description 14
- 108091027967 Small hairpin RNA Proteins 0.000 title claims abstract 6
- 230000014509 gene expression Effects 0.000 claims abstract description 17
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 claims abstract description 12
- 230000002159 abnormal effect Effects 0.000 claims abstract description 5
- 201000010099 disease Diseases 0.000 claims abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 5
- 239000003814 drug Substances 0.000 claims abstract description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims abstract description 3
- 238000002360 preparation method Methods 0.000 claims abstract description 3
- 239000004055 small Interfering RNA Substances 0.000 claims abstract 5
- 229940079593 drug Drugs 0.000 abstract 1
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- 230000005764 inhibitory process Effects 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000010361 transduction Methods 0.000 description 3
- 230000026683 transduction Effects 0.000 description 3
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 2
- 229940045513 CTLA4 antagonist Drugs 0.000 description 2
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
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- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
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- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
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- 230000008685 targeting Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
Definitions
- the present invention relates to a human BTLA gene, and more particularly to a shRNA (short hair clip R NA) of a human BTLA gene and use thereof.
- B/T lymphocyte attenuating factor is the third inhibitory molecule found in the CD28 family, and is mainly expressed on the cell surface of B cells, T cells, macrophages, and the like.
- BTLA has a similar structure to CTLA-4 and PD-1, but their expression characteristics are different.
- CTLA-4 and PD-1 are not expressed on resting T cells, and expression is gradually increased after activation, while BTLA is at rest. Constitutive expression on T cells, continued expression after activation. technical problem
- BTLA signaling is capable of inhibiting CD3 activated T cell proliferation and secretion of IL-2 and IFN- ⁇ . Research shows that B
- TLA plays an important role in maintaining peripheral immune tolerance and transplant immunity, and has a good clinical transformation prospect, which needs further study.
- One of the technical problems to be solved by the present invention is to provide a shRNA of a human BTLA gene.
- the second technical problem to be solved by the present invention is to provide a shRNA of human BTLA gene.
- the present invention provides a shRNA of a human BTLA gene, including:
- EQ ID NO: The sequence shown by 1 and the BTLA-RNAi consisting of the sequence shown in SEQ ID NO: 2.
- the shRNA provided by the invention has the advantages of high transduction efficiency, high and specific inhibition of BTLA gene expression in Jurkat cells, and can be used as a powerful tool for preparing a medicament for treating diseases related to abnormal expression of BTLA gene. Brief description of the drawing
- Figure 1 is a schematic diagram showing the results of Quantitative PCR detection of Jurkat cells after transduction of pLKO-BTLA vector.
- Jurkat cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences, Trizol was purchased from Invitrogen, and Endo-free Plasmid Mini Kit was purchased from Omega bio-tek.
- the complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
- the synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded BTLA-RNAi.
- Plasmid Mini Kit for endotoxin-free pLKO-BTLA vector extraction Plasmid Mini Kit for endotoxin-free pLKO-BTLA vector extraction.
- Example 3 The pLKO-BTLA vector transduced Jurkat cells.
- Jurkat cells cultured the growth state of the cell to take good 5,000,000, the cells were collected by centrifugation, then resuspended in 500 ⁇ L PBS is added and after mixing the cuvette 20 ⁇ ⁇ pLKO-BTLA vector, Invitrogen Neon electroporation application system Electrotransfer, electroporation procedure: 2.1 KV, 25 ⁇ , pulse shock once; Transfer the cells to a 6 cm dish containing 5 mL DMEM complete medium, gently shake the dish to mix the cells, and detect the expression of TL6 gene after 48 h.
- Example 4 Fluorescence quantitative PCR was used to detect the expression level of BTLA gene.
- Jurkat cells inoculated with Jurkat cells and transduced pLKO-BTLA vectors were separately cultured into cell culture flasks, and after 24 hours of culture, total RNA of each group was extracted with Trizol, and mRNA was reverse-transcribed into cDNA using PrimeScrip RT reagent Kit.
- the cDNA was diluted with 90 RNase-Free dH20 and stored at -20 °C.
- the shRNA provided by the present invention has the advantages of high transduction efficiency, and can efficiently and specifically inhibit the expression of BTLA gene in Jurkat cells, and can be used as a powerful tool for preparing a medicament for treating diseases related to abnormal expression of BTLA gene.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
L'invention concerne un shARN du gène BTLA humain et son utilisation. Le shARN du gène BTLA humain comprend une interférence ARN-BTLA consistant en une séquence codant pour SEQ ID NO : 1 et une séquence codant pour SEQ ID NO : 2. Le shARN du gène BTLA humain peut être utilisé dans la préparation d'un médicament destiné au traitement d'une maladie associée à une expression anormale du gène BTLA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/097432 WO2019033249A1 (fr) | 2017-08-14 | 2017-08-14 | Sharn du gène btla humain et utilisation correspondante |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/097432 WO2019033249A1 (fr) | 2017-08-14 | 2017-08-14 | Sharn du gène btla humain et utilisation correspondante |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2019033249A1 true WO2019033249A1 (fr) | 2019-02-21 |
Family
ID=65361641
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2017/097432 WO2019033249A1 (fr) | 2017-08-14 | 2017-08-14 | Sharn du gène btla humain et utilisation correspondante |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2019033249A1 (fr) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2803525A1 (fr) * | 2009-06-23 | 2011-01-13 | University Of Miami | Siarn cible contre un aptamere pour inhiber une degradation a mediation par un non-sens |
| WO2012012518A2 (fr) * | 2010-07-20 | 2012-01-26 | University Of Miami | Inhibition des voies de dégradation des arnm non-sens |
| CN107034193A (zh) * | 2016-02-03 | 2017-08-11 | 北京马力喏生物科技有限公司 | 治疗b细胞白血病及b细胞淋巴瘤的治疗组合物 |
-
2017
- 2017-08-14 WO PCT/CN2017/097432 patent/WO2019033249A1/fr active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2803525A1 (fr) * | 2009-06-23 | 2011-01-13 | University Of Miami | Siarn cible contre un aptamere pour inhiber une degradation a mediation par un non-sens |
| WO2012012518A2 (fr) * | 2010-07-20 | 2012-01-26 | University Of Miami | Inhibition des voies de dégradation des arnm non-sens |
| CN107034193A (zh) * | 2016-02-03 | 2017-08-11 | 北京马力喏生物科技有限公司 | 治疗b细胞白血病及b细胞淋巴瘤的治疗组合物 |
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