WO2018170764A1 - Vecteur pour arni et son application - Google Patents
Vecteur pour arni et son application Download PDFInfo
- Publication number
- WO2018170764A1 WO2018170764A1 PCT/CN2017/077606 CN2017077606W WO2018170764A1 WO 2018170764 A1 WO2018170764 A1 WO 2018170764A1 CN 2017077606 W CN2017077606 W CN 2017077606W WO 2018170764 A1 WO2018170764 A1 WO 2018170764A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sequence
- vector
- gene
- rnai
- rna interference
- Prior art date
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- 239000013598 vector Substances 0.000 title claims abstract description 43
- 108091030071 RNAI Proteins 0.000 title claims description 9
- 230000009368 gene silencing by RNA Effects 0.000 claims abstract description 31
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 30
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 claims abstract description 23
- 239000002773 nucleotide Substances 0.000 claims abstract description 13
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 13
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims abstract description 7
- 230000000295 complement effect Effects 0.000 claims abstract description 4
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract 2
- 108091027967 Small hairpin RNA Proteins 0.000 claims abstract 2
- 239000004055 small Interfering RNA Substances 0.000 claims abstract 2
- 230000014509 gene expression Effects 0.000 claims description 15
- 108091033319 polynucleotide Proteins 0.000 claims description 7
- 239000002157 polynucleotide Substances 0.000 claims description 7
- 102000040430 polynucleotide Human genes 0.000 claims description 7
- 238000013461 design Methods 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- 108020004999 messenger RNA Proteins 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 201000010099 disease Diseases 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 238000012163 sequencing technique Methods 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 4
- 102000012410 DNA Ligases Human genes 0.000 claims description 3
- 108010061982 DNA Ligases Proteins 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 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 3
- 229960000723 ampicillin Drugs 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 230000010261 cell growth Effects 0.000 claims description 2
- 238000003776 cleavage reaction Methods 0.000 claims description 2
- 238000001962 electrophoresis Methods 0.000 claims description 2
- 230000007017 scission Effects 0.000 claims description 2
- 230000002159 abnormal effect Effects 0.000 claims 1
- 238000005215 recombination Methods 0.000 claims 1
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- 238000011084 recovery Methods 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 abstract description 3
- 102000004190 Enzymes Human genes 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract 2
- 210000004027 cell Anatomy 0.000 description 16
- 230000002401 inhibitory effect Effects 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 2
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 2
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
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- 238000001890 transfection Methods 0.000 description 2
- 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 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
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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
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
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- SPTYHKZRPFATHJ-HYZXJONISA-N dT6 Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)CO)[C@@H](O)C1 SPTYHKZRPFATHJ-HYZXJONISA-N 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000001280 germinal center Anatomy 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
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- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention belongs to the field of genetic engineering, and relates to a vector for RNAi and application thereof, and particularly to an RNA interference vector of GP34 gene and 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. OX40/GP34 is an important co-stimulatory molecule that plays an important role in the body's immune response and various diseases, and its interaction can promote CD+4.
- Activation, proliferation, and migration of T cells prolong their life span and promote the formation of germinal centers and the differentiation and maturation of DCs.
- GP34 can synergistically stimulate the activation of T cells, promote the production of high-valent antibodies and class switching of B cells, mediate the infiltration of OX40+ T cells into the inflammatory reaction site, and play an important role in the immunotherapy of tumors.
- the research can achieve clinical transformation, but the lack of vectors specifically inhibiting the expression of GP34 gene in the prior art makes the related research not well developed.
- the object of the present invention is an RNA interference vector of GP34 gene and a construction method and application thereof.
- an isolated polynucleotide is provided, the nucleotide sequence of which is set forth in SEQ ID NO: 1.
- a recombinant vector comprising the nucleotide sequence set forth in SEQ ID NO: 2, wherein the recombinant vector is a pLVX-shRNA1 vector as a backbone vector, in a polyclonal enzyme GP34- consisting of Bam HI restriction site + target nucleotide sequence + stem loop structure sequence + target nucleotide sequence complementary sequence + termination site sequence + EcoR I restriction site at the cleavage site RNAi sequence.
- RNA interference vector of the aforementioned GP34 gene including the following steps:
- RNA interference vector of GP34 gene The synthesized GP34-RNAi sequence and the extracted plasmid pLVX-shRNA1 were digested with restriction endonucleases Bam HI and EcoR I, electrophoresis and gelatinization. The vector was recovered, and the GP34-RNAi sequence was ligated into the SJpLVX-shRNA1 expression vector by T4 DNA Ligase to obtain a ligation product; the ligation product was transformed into competent E. coli Stbl3 and uniformly coated onto an ampicillin-containing LB medium plate. The positive monoclonal colonies were picked and cultured and identified by PCR. The preliminary identification results indicated that the GP34-RNAi sequence was inserted into the successful bacterial solution for sequencing and identification;
- RNA interference vector of GP34 gene Extraction of RNA interference vector of GP34 gene: The GP34-RNAi sequence was confirmed to be inserted into a successful bacterial cell expansion culture, and a large amount of recombinant plasmid was extracted to obtain an RNA interference vector of GP34 gene.
- the present invention utilizes RNAi technology to construct an RNA interference vector targeting GP34 gene expression, and after successful identification, electrotransformation of DC2.4 cells, using the real-time fluorescent quantitative PCR technique to verify the inhibitory effect of GP34 gene expression from the mRNA level, the experiment As a result, it was confirmed that the GP34-RNAi sequence provided by the present invention was successfully inserted into the pLVX-shRNA1 expression vector, and the inhibitory effect on the expression of the GP34 gene was remarkable.
- polynucleotide of the polynucleotide represented by 1 or the RNA interference vector of the aforementioned GP34 gene for the drug of the GP34 gene expression abnormality-related disease.
- RNA interference vector of the GP34 gene provided by the invention has the advantages of high transfection efficiency, low dosage, high and specific inhibition of GP34 gene expression in DC2.4 cells, and can be used as a powerful tool for preparing and treating the G P34 gene.
- a drug that expresses an abnormally related disease A drug that expresses an abnormally related disease.
- FIG. 1 is a schematic diagram showing the results of real-time quantitative PCR detection of RNA interference vector cells transfected with GP34 gene.
- DC2.4 cells were purchased from Wuxi Yingnuui Biomedical Technology Co., Ltd., RNAi vector pLVX-shRNAl was purchased from Clontech, RNeasy MiniKit was purchased from Qiagen, and endotoxin free plasmid extraction kit was purchased from Omega bio-tek.
- the complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
- GP34 mRNA The complete sequence of GP34 mRNA was found in GenBank. According to the full sequence of GP34 mRNA, the interference fragment was designed according to the design principle of RNAi fragment, NCBI BLSAT was used for homology comparison, and the target nucleotide sequence was obtained after confirming specificity.
- the GP34-RNAi sequence was designed as follows: Bam
- the designed GP34-RNAi sequence chain was synthesized by Shanghai Biotech Engineering Services Co., Ltd.
- the synthesized GP34-RNAi sequence and the extracted plasmid pLVX-shRNA1 were digested with restriction endonucleases Bam HI and EcoR I, electrophoresed, and the gel was recovered, and the GP34-RNAi sequence was further amplified by T4 DNA Ligase.
- An RNA interference vector that forms the GP34 gene is ligated into the vector pLVX-shRNA1.
- the ligation product was transformed into competent E. coli Stbl3, plated on a plate containing ampicillin LB medium, and cultured at 37 ° C for 14 h. Pick up the grown colonies and send them to Shanghai Biotech for sequencing. The sequencing results are fully consistent with the designed sequence and can be used in subsequent experiments.
- DC2.4 cells were cultured, and 35,000 cells in good growth state were taken, and the cells were collected by centrifugation, and then resuspended in 500 L of PBS with 20 g.
- RNA interference vector of GP34 gene was mixed and added to the electric shock cup, and the electric rotation was performed by BTX ECM830 electro-rotation instrument.
- the electroporation procedure was: 2.1 KV, 25 ⁇ , pulse electric shock once; the cells were transferred to 5 mL DMEM. In a 6 cm dish of complete medium, the cells were gently shaken to mix the cells, and the expression of GP34 gene was detected 48 h later.
- DC2.4 cells of normal DC2.4 cells and RNA interference vector of GP34 gene were cultured for 48 h, respectively, and PrimeScrip RT reagent was used for He 1 J
- Kit reverse transcribes mRNA into cDNA, reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, ⁇ . After the reverse transcription was completed, the cDNA was diluted with 90 ⁇ l of RNase-Free dH20 and stored at -20 ° C for later detection. Take the cDNA of each group of cells
- ⁇ is the template, GAPDH is used as the internal reference, and the relative expression of GP34 is detected by real-time quantitative PCR (QPCR).
- the reaction conditions are set: 95 ° C for 10 s, 1 cycle; 95 ° C 5 s, 54 ° C
- the NP34 gene RNA interference vector provided by the invention has the advantages of high transfection efficiency, low dosage, high and specific inhibition of GP34 gene expression in DC2.4 cells, and can be used as a powerful tool for preparing and treating G P34 gene.
- a drug that expresses an abnormally related disease is a drug that expresses an abnormally related disease.
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
L'invention concerne : une séquence GP34-ARNi et un vecteur recombinant contenant la séquence, la séquence GP34-ARNi étant telle que représentée dans la SEQ ID NO:1 ; un vecteur recombinant contenant une séquence telle que représentée dans la SEQ ID NO:2 ; et un vecteur d'ARN interférent du gène GP34 et sa méthode de préparation. La méthode de préparation comprend : l'utilisation de pLVX-shRNA1 en tant que vecteur squelette, et l'insertion dans un site recombinant d'une séquence d'oligonucléotides d'ARNsh consistant en un site de coupe de l'enzyme Bam HI + une séquence nucléotidique cible + une séquence de structure tige-boucle + une séquence complémentaire de séquence nucléotidique cible + une séquence de site de terminaison + un site de coupe de l'enzyme EcoR I en connexion séquentielle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/077606 WO2018170764A1 (fr) | 2017-03-22 | 2017-03-22 | Vecteur pour arni et son application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/077606 WO2018170764A1 (fr) | 2017-03-22 | 2017-03-22 | Vecteur pour arni et son application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2018170764A1 true WO2018170764A1 (fr) | 2018-09-27 |
Family
ID=63586177
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2017/077606 WO2018170764A1 (fr) | 2017-03-22 | 2017-03-22 | Vecteur pour arni et son application |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2018170764A1 (fr) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948859A (zh) * | 2010-08-17 | 2011-01-19 | 深圳市疾病预防控制中心 | 特异性抑制SET蛋白表达的shRNA表达载体及其构建方法和应用 |
| WO2016022468A1 (fr) * | 2014-08-04 | 2016-02-11 | Baylor Research Institute | Anticorps anti-ox40l antagonistes et leurs procédés d'utilisation |
-
2017
- 2017-03-22 WO PCT/CN2017/077606 patent/WO2018170764A1/fr active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948859A (zh) * | 2010-08-17 | 2011-01-19 | 深圳市疾病预防控制中心 | 特异性抑制SET蛋白表达的shRNA表达载体及其构建方法和应用 |
| WO2016022468A1 (fr) * | 2014-08-04 | 2016-02-11 | Baylor Research Institute | Anticorps anti-ox40l antagonistes et leurs procédés d'utilisation |
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