WO2019000145A1 - Procédé de construction de cellule cho recombinée exprimant fortement l'ampar - Google Patents
Procédé de construction de cellule cho recombinée exprimant fortement l'ampar Download PDFInfo
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- WO2019000145A1 WO2019000145A1 PCT/CN2017/089925 CN2017089925W WO2019000145A1 WO 2019000145 A1 WO2019000145 A1 WO 2019000145A1 CN 2017089925 W CN2017089925 W CN 2017089925W WO 2019000145 A1 WO2019000145 A1 WO 2019000145A1
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- ampar
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- 238000000034 method Methods 0.000 title claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims abstract description 44
- 230000014509 gene expression Effects 0.000 claims abstract description 35
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims abstract description 24
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 24
- 239000013604 expression vector Substances 0.000 claims abstract description 19
- 229930189065 blasticidin Natural products 0.000 claims abstract description 4
- 239000000047 product Substances 0.000 claims description 10
- 239000002299 complementary DNA Substances 0.000 claims description 9
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 8
- 239000013598 vector Substances 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 238000003753 real-time PCR 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
- 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
- 238000001514 detection method Methods 0.000 claims description 5
- 230000029087 digestion Effects 0.000 claims description 5
- 239000007788 liquid Substances 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
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 2
- 108020004414 DNA 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
- 238000003776 cleavage reaction Methods 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
- 239000012264 purified product Substances 0.000 claims description 2
- 238000003259 recombinant expression Methods 0.000 claims description 2
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- 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
- 239000000126 substance Substances 0.000 claims 1
- 208000024827 Alzheimer disease Diseases 0.000 description 6
- 102000008298 human alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptor Human genes 0.000 description 4
- 108010035933 human alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptor Proteins 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 101150029129 AR gene Proteins 0.000 description 2
- 208000028698 Cognitive impairment Diseases 0.000 description 2
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000003956 synaptic plasticity Effects 0.000 description 2
- 102000006541 Ionotropic Glutamate Receptors Human genes 0.000 description 1
- 108010008812 Ionotropic Glutamate Receptors Proteins 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
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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
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
Definitions
- the present invention belongs to the field of recombinant cell technology, and relates to a method for constructing recombinant CHO cells with high AMPAR expression.
- AD Alzheimer's disease
- This disease is common in the elderly. It is a central nervous system degenerative degenerative disease with progressive cognitive impairment and memory impairment. The clinical manifestations are the deterioration of cognitive and memory functions, the progressive decline of daily living ability, and Various neuropsychiatric symptoms and behavioral disorders. More onset in the old age, latent onset, slow and irreversible, clinically based on intelligent damage.
- AMPAR glutamate a-amino-3-carbo-5-methyl-4-iso-propionate receptor
- a-amino-3-hy droxy-5-methyl-4 -isoxazole propionic acid receptor AMPAR is a key mechanism regulating central synaptic plasticity.
- AMPAR is one of the ionotropic glutamate receptors and mediates the central component of central excitatory postsynaptic currents and is essential for synaptic transmission.
- AMPAR The physiological function of AMPAR depends on the number of receptors, subunit composition and protein phosphorylation to regulate AMPAR dysfunction, leading to decreased synaptic plasticity of neurons, causing AD cognitive impairment, which plays an important role in the pathogenesis of AD.
- the role therefore, is essential for the role of AMPAR in Alzheimer's disease and its use as a therapeutic target, and the cells that express the AMPAR gene required for these related studies are still lacking in the prior art.
- the object of the present invention is to provide a method for constructing recombinant CH0 cells with high AMPAR expression, which is achieved by the following technical schemes:
- a method for constructing recombinant CH0 cells with high AMPAR expression the recombinant CH0 cells highly expressed by AMPAR are CH0 cells as host cells, and the exogenous expression vector for transfecting host cells comprises AMP.
- An expression vector for the AR full-length gene is an expression vector for the AR full-length gene.
- the expression vector comprising the full-length gene of AMPAR is pCAG(m)-IRESblast, and the expression vector is through EcoR
- the full-length AMPAR gene was cloned into the eukaryotic expression vector pCAG(m)-IRESblast by I and Spel restriction sites.
- the present invention constructs the eukaryotic expression vector pCAG(m)-IRESblast of human AMPAR, which is confirmed by cloning and sequencing to be identical to the human AMPAR sequence in the NCBI database; transfected CHO cells by liposome method, blasticidal Resistant CHO/AMPAR cell line stably and highly expressed AMPAR was screened by restriction endonuclease screening, and recombinant CHO/AMPAR cells containing AMP AR gene full length were constructed according to the present invention.
- the strain is capable of stably expressing AMPAR and has a complete molecular structure.
- CHO cells as host cells have many advantages, including relatively low levels of protein expression in cells themselves, and higher amplification and expression ability of exogenous recombinant genes, AMPAR expressed by recombinant CHO/AMPAR cells.
- the relative expression level was significantly higher than that of CHO, and AMP AR occupied the dominant expression level compared with other expression products.
- FIG. 1 is a schematic diagram showing the results of fluorescent quantitative PCR detection of AMPAR expression levels after screening of CHO cells by blasticidin.
- the present invention is based on the construction of the eukaryotic expression vector pCAG(m)-IRESblast-AMPAR of the AMPAR full-length gene, transfecting CHO cells to obtain a stable, high expression AMPAR-recombinant CHO/AMPAR cell line.
- the following is an explanation of the embodiments of the present invention and is not 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.
- AMPAR-F 5'-GCGAATTCATGGGGCTCAACTGGAAAAATC - 3'
- AMPAR-R 5'- AACTAGTTTATACGGGGGTGGTCCGG -3,.
- Amplification was carried out by using the PrimeSTAR enzyme of Dalian Bao Biotechnology Co., Ltd., and the PCR reaction system was: upstream primer 4 ⁇ , downstream primer 4 L, 2 ⁇ Premix PrimeSTAR 25 L, cDNA: 1 ⁇ , supplement ddH20 to 50 L; 98 ° C 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 purified by commercial PCR.
- the kit purifies the amplified product.
- DNA ligase Buffer ⁇ obtained recombinant pCAG(m)-IRESblast-AMPAR expression vector.
- CHO cells were seeded in 6-well plates at 10 6 cells per well, 18
- Lipofectamine 3000 was transduced into CHO cells, and after continuing to culture for 24 h, blasticidin was added to a final concentration of 5 g/mL, and the cells were selected.
- CHO cells and CHO/AMPAR cells were seeded separately into 6-well plates. The cell density reached ⁇ , and the total RNA of each group was extracted with RNeasy Mini Kit, using PrimeScrip RT reagent.
- Kit reverse transcribes mRNA into cDNA and stores at -20 °C.
- the present invention constructs the eukaryotic expression vector pCAG(m)-IRESblast of human AMPAR, which is confirmed by clone sequencing to be identical to the human AMPAR sequence in the NCBI database; transfected CHO cells by liposome method, blasticidal Resistant CHO/AMPAR cell line stably and highly expressed AMPAR was screened by restriction endonuclease screening, and recombinant CHO/AMPAR cells containing AMP AR gene full length were constructed according to the present invention.
- the strain is capable of stably expressing AMPAR and has a complete molecular structure.
- CHO cells as host cells have many advantages, including a relatively low amount of protein expression in the cell itself, and a higher amplification and expression ability of the foreign recombinant gene, and AMPAR expressed by the recombinant CHO/AMPAR cells.
- the relative expression level was significantly higher than that of CHO, and AMP AR occupied the dominant expression level compared with other expression products.
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
L'invention concerne un procédé de construction d'une cellule CHO recombinée exprimant fortement l'AMPAR. Le vecteur d'expression eucaryote du gène AMPAR pleine longueur pCAG(m)-IRESblast-AMPAR est construit à l'aide du vecteur d'expression eucaryote pCAG(m)-IRESblast et est en outre utilisé pour transfecter les cellules CHO, et la blasticidine est utilisée pour cribler celles-ci pour obtenir la lignée cellulaire modifiée CHO/AMPAR exprimant fortement le gène AMPAR pleine longueur, et il a été confirmé que la lignée cellulaire peut considérablement augmenter l'expression de l'AMPAR pleine longueur.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/089925 WO2019000145A1 (fr) | 2017-06-26 | 2017-06-26 | Procédé de construction de cellule cho recombinée exprimant fortement l'ampar |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/089925 WO2019000145A1 (fr) | 2017-06-26 | 2017-06-26 | Procédé de construction de cellule cho recombinée exprimant fortement l'ampar |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2019000145A1 true WO2019000145A1 (fr) | 2019-01-03 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2017/089925 WO2019000145A1 (fr) | 2017-06-26 | 2017-06-26 | Procédé de construction de cellule cho recombinée exprimant fortement l'ampar |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2019000145A1 (fr) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110606885A (zh) * | 2019-08-12 | 2019-12-24 | 陕西脉元生物科技有限公司 | 人体液中抗ampa1自身抗体的检测材料、制备方法及应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1065215A1 (fr) * | 1999-06-30 | 2001-01-03 | Boehringer Ingelheim International GmbH | Récepteurs AMPA homomères et hétéromères |
| EP2338492A1 (fr) * | 2009-12-24 | 2011-06-29 | Universidad del Pais Vasco | Procédés et compositions pour le traitement de la maladie d'Alzheimer |
| CN104338135A (zh) * | 2013-08-09 | 2015-02-11 | 中国科学院上海生命科学研究院 | 抑郁症的调节因子及其应用 |
| US20170176459A1 (en) * | 2015-12-22 | 2017-06-22 | Grace Laboratories, Llc | Neurotoxicity biomarkers for diagnosis and treatment of acute traumatic brain and spinal cord injury |
-
2017
- 2017-06-26 WO PCT/CN2017/089925 patent/WO2019000145A1/fr active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1065215A1 (fr) * | 1999-06-30 | 2001-01-03 | Boehringer Ingelheim International GmbH | Récepteurs AMPA homomères et hétéromères |
| EP2338492A1 (fr) * | 2009-12-24 | 2011-06-29 | Universidad del Pais Vasco | Procédés et compositions pour le traitement de la maladie d'Alzheimer |
| CN104338135A (zh) * | 2013-08-09 | 2015-02-11 | 中国科学院上海生命科学研究院 | 抑郁症的调节因子及其应用 |
| US20170176459A1 (en) * | 2015-12-22 | 2017-06-22 | Grace Laboratories, Llc | Neurotoxicity biomarkers for diagnosis and treatment of acute traumatic brain and spinal cord injury |
Non-Patent Citations (1)
| Title |
|---|
| DATABASE Genbank (online) MENG, H. ET AL.: "Homo sapiens calcium voltage-gated channel auxilary subunit gamma 2 (CACNG2), mRNA", Database accession no. NM_006078.4 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110606885A (zh) * | 2019-08-12 | 2019-12-24 | 陕西脉元生物科技有限公司 | 人体液中抗ampa1自身抗体的检测材料、制备方法及应用 |
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