WO2019000152A1 - Cellule cho exprimant le gène rhbdd1 et son utilisation - Google Patents
Cellule cho exprimant le gène rhbdd1 et son utilisation Download PDFInfo
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- WO2019000152A1 WO2019000152A1 PCT/CN2017/089932 CN2017089932W WO2019000152A1 WO 2019000152 A1 WO2019000152 A1 WO 2019000152A1 CN 2017089932 W CN2017089932 W CN 2017089932W WO 2019000152 A1 WO2019000152 A1 WO 2019000152A1
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- WIPO (PCT)
- Prior art keywords
- rhbdd1
- gene
- cells
- cho
- pcag
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims description 11
- 210000004027 cell Anatomy 0.000 claims abstract description 42
- 101000709000 Homo sapiens Rhomboid-related protein 4 Proteins 0.000 claims abstract description 41
- 102100032690 Rhomboid-related protein 4 Human genes 0.000 claims abstract description 37
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims abstract description 28
- 230000014509 gene expression Effects 0.000 claims abstract description 26
- 101150100996 RHBDD1 gene Proteins 0.000 claims abstract description 24
- 239000013604 expression vector Substances 0.000 claims abstract description 21
- 229930189065 blasticidin Natural products 0.000 claims abstract description 4
- 239000000047 product Substances 0.000 claims description 11
- 239000002299 complementary DNA Substances 0.000 claims description 9
- 238000003753 real-time PCR Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 102000012410 DNA Ligases Human genes 0.000 claims description 6
- 108010061982 DNA Ligases Proteins 0.000 claims description 6
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 238000012163 sequencing technique Methods 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 5
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 4
- 229960000723 ampicillin Drugs 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 4
- 238000010367 cloning Methods 0.000 claims description 4
- 230000029087 digestion Effects 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 claims description 2
- 238000011530 RNeasy Mini Kit Methods 0.000 claims description 2
- 230000009471 action Effects 0.000 claims description 2
- 238000000137 annealing Methods 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000004925 denaturation Methods 0.000 claims description 2
- 230000036425 denaturation Effects 0.000 claims description 2
- 238000001976 enzyme digestion Methods 0.000 claims description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 claims description 2
- 108020004999 messenger RNA Proteins 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 238000003259 recombinant expression Methods 0.000 claims description 2
- 230000035939 shock Effects 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 230000026683 transduction Effects 0.000 claims description 2
- 238000010361 transduction Methods 0.000 claims description 2
- 238000011144 upstream manufacturing Methods 0.000 claims description 2
- 101150084750 1 gene Proteins 0.000 claims 3
- 230000001351 cycling effect Effects 0.000 claims 1
- 239000012264 purified product Substances 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 102000049820 human RHBDD1 Human genes 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 210000003289 regulatory T cell Anatomy 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 2
- 210000000712 G cell Anatomy 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 1
- 241001330975 Magnaporthe oryzae Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
Definitions
- the present invention belongs to the field of recombinant cell technology, and relates to a CHO cell expressing an RHBDD1 gene and an application thereof.
- RHBDD1 is the 18th member of the tumor necrosis factor receptor (TNFR) superfamily and is a thymus-derived CD4+
- RHBDD1/RHBDD1L A surface molecule on CD25+ Treg cells with a ligand of RHBDD1L. Studies have shown that RHBDD1/RHBDD1L has many important biological activities, including cell proliferation, differentiation and survival. Since RHBDD 1 is mainly expressed on resting Treg cells, anti-RHBDD 1 antibody (DTA-1) can eliminate the immunosuppressive effects of Tre g cells.
- DTA-1 anti-RHBDD 1 antibody
- the HBDD1/RHBDD1L system is involved in the regulation of Treg cells and has a good clinical transformation prospect.
- the lack of cells expressing the RHBDD1 gene in the prior art has hindered the progress of related research.
- the object of the present invention is to provide a CHO cell expressing the RHBDD1 gene, which is achieved by the following technical solutions:
- a CHO cell expressing the RHBDD1 gene, the recombinant CHO cell with high expression of RHBDD1 is CH
- the 0 cell is a host cell
- the exogenous expression vector for transfecting the host cell is an expression vector comprising the full-length gene of RHBDD1.
- the nucleotide sequence of the full-length RHBDD1 gene is shown in SEQ ID No: 1.
- the expression vector comprising the full-length gene of RHBDD1 is pCAG(m)-IRESblast, and the expression vector is through EcoR
- the present invention constructs the eukaryotic expression vector pCAG(m)-IRESblast of human RHBDD1, which is confirmed by clone sequencing to be identical to the human RHBDD1 sequence in the NCBI database; transfected CHO cells by liposome method, blasticidal A cell line capable of stably growing and surviving was obtained by restriction enzyme screening, and a recombinant CHO/RHBDD1 cell line stably and highly expressing RHBDD1 was selected by quantitative PCR detection; a recombinant CHO/RHBDD1 cell line containing the full length of RHBDD1 gene constructed by the present invention was constructed. It can stably express RHBDD1 and has a complete molecular structure.
- CHO cells as host cells have many advantages, including relatively low expression of the cells themselves, and exogenous recombinant genes have higher amplification and expression ability, and RHBDD1 expressed by recombinant CHO/RHBDD1 cells.
- the relative expression level was significantly higher than that of CHO, and R HBDD1 occupied the dominant expression level compared with other expression products.
- FIG. 1 is a schematic representation of the results of detection of RHBDD1 expression levels by fluorescent quantitative PCR after screening of CHO cells by blasticidin.
- the present invention is based on the construction of the eukaryotic expression vector pCAG(m)-IRESblast-RHBDD1 of the full-length RHBDD1 gene, and transfects CHO cells to obtain a stable CHO/RHBDD1 cell line stably expressing RHBDD1.
- the explanation of the embodiments of the invention is not to be construed as limiting.
- the pCAG(m)-IRESblast-based vector was used, and the EcoR I and Spel cleavage sites were specifically selected as the multiple cloning site for the foreign gene.
- Amplification was carried out by using the PrimeSTAR enzyme of Dalian Bao Biotech Co., Ltd., and the PCR reaction system was: upstream primer 4 ⁇ , downstream primer 4 L, 2 ⁇ Premix PrimeSTAR 25 L, cDNA: 1 ⁇ , plus ddH20 to 50 L; 98 ° C for 2 min, After 30 cycles (denaturation at 98 °C for 10 s, annealing at 60 °C for 10 s, extension at 72 °C for 1 min), and finally extension at 72 °C for 5 min, a large number of target fragments were obtained, and the product was expanded using a commercial PCR purification kit. The product is purified.
- DNA/plasmid 2 g, lOxFastDigest Buffer 2 L, supplement ddH20 to 2 ( ⁇ L; reaction at 37 ° C for 30 min, and the product was purified by commercial PCR product purification kit.
- the DNA ligase Buffer ⁇ obtained the recombinant pCAG(m)-IRESblast-RHBDD1 expression vector.
- CHO cells were seeded in 6-well plates at 10 6 cells per well, 18
- CHO cells and CHO/RHBDD1 cells were seeded separately into 6-well plates. Cell density reached 80 ⁇ 3 ⁇ 4-90 ⁇ 3 ⁇ 4 ⁇ , total RNA was extracted from each group using RNeasy Mini Kit, using PrimeScrip RT reagent
- Kit reverse transcribes mRNA into cDNA and stores at -20 °C.
- RHBDD1 As a template, GAPDH was used as an internal reference, and the relative expression of RHBDD1 was detected by real-time fluorescent quantitative PCR.
- the reaction conditions were set: 95. C 30s, 1 cycle, 58. C 30s 40 cycles, 95. C 5s, 60 ° C lmin, 95. C 15s , the results are shown in Figure 1. It can be seen that the expression level of RHBDD1 gene of CHO/RHBDD1 cells is 140 times higher than that of C HO cells, indicating that the cDNA sequence of RHBDD1 gene provided by the present invention is successfully inserted into the pCAG(m)-IRESblast vector, and can specifically and efficiently promote the RHBDD1 gene. High expression.
- the present invention constructs the eukaryotic expression vector pCAG(m)-IRESblast of human RHBDD1, which is confirmed by cloning and sequencing to be identical to the human RHBDD1 sequence in the NCBI database; transfected CHO cells by liposome method, and killed by rice blast fungus A cell line capable of stably growing and surviving was obtained by restriction enzyme screening, and a recombinant CHO/RHBDD1 cell line stably and highly expressing RHBDD1 was selected by quantitative PCR detection; a recombinant CHO/RHBDD1 cell line containing the full length of RHBDD1 gene constructed by the present invention was constructed. It can stably express RHBDD1 and has a complete molecular structure.
- CHO cells as host cells have many advantages, including a relatively low level of protein expression in the cell itself, and a higher amplification and expression ability of the foreign recombinant gene, and RHBDD1 expressed by the recombinant CHO/RHBDD1 cells.
- the relative expression level was significantly higher than that of CHO, and R HBDD1 occupied the dominant expression level compared with other expression products.
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
L'invention concerne une cellule CHO exprimant le gène RHBDD1. Le vecteur d'expression eucaryote pCAG(m)-IRESblast-RHBDD1 du gène RHBDD1 pleine longueur est construit à l'aide du vecteur d'expression eucaryote pCAG(m)-IRESblast, et est en outre utilisé pour transfecter des cellules CHO. La blasticidine est utilisée pour cribler la lignée cellulaire modifiée CHO/RHBDD1 exprimant fortement le gène RHBDD1 pleine longueur, et il a été confirmé que la lignée cellulaire peut augmenter significativement l'expression du RHBDD1 pleine longueur.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/089932 WO2019000152A1 (fr) | 2017-06-26 | 2017-06-26 | Cellule cho exprimant le gène rhbdd1 et son utilisation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/089932 WO2019000152A1 (fr) | 2017-06-26 | 2017-06-26 | Cellule cho exprimant le gène rhbdd1 et son utilisation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2019000152A1 true WO2019000152A1 (fr) | 2019-01-03 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2017/089932 WO2019000152A1 (fr) | 2017-06-26 | 2017-06-26 | Cellule cho exprimant le gène rhbdd1 et son utilisation |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2019000152A1 (fr) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003093466A1 (fr) * | 2002-05-02 | 2003-11-13 | Bayer Healthcare Ag | Proteine humaine associee au rhomboide |
-
2017
- 2017-06-26 WO PCT/CN2017/089932 patent/WO2019000152A1/fr active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003093466A1 (fr) * | 2002-05-02 | 2003-11-13 | Bayer Healthcare Ag | Proteine humaine associee au rhomboide |
Non-Patent Citations (3)
| Title |
|---|
| DATABASE Genbank (online) 6 June 2016 (2016-06-06), "Predicted: Homos sapiens rhomboid domain cobtaining 1 (RHBDD1), transcript variant X20, mRNA", Database accession no. XM-017005091.1 * |
| SONG, WEI ET AL.: "Study of epitope tagging of intramembrane serine protease RHBDD1 by improved somatic cell knock in approach", JOURNAL OF MEDICAL RESEARCH, vol. 40, no. 11, 30 November 2011 (2011-11-30), pages 21 - 24, ISSN: 1673-548X * |
| YANG, LIN ET AL: "Construction, identification and expression of recombinant eukaryotic vector pCAG-IRES-SHIP-GFP on porliferation of leukemia cell line K562", CHINA BIOTECHNOLOGY, vol. 29, no. 6, 31 December 2009 (2009-12-31), pages 14 - 19, ISSN: 1671-8135 * |
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