WO2019037052A1 - Arnsh pour le silençage ciblé de wucam - Google Patents
Arnsh pour le silençage ciblé de wucam Download PDFInfo
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- WO2019037052A1 WO2019037052A1 PCT/CN2017/098909 CN2017098909W WO2019037052A1 WO 2019037052 A1 WO2019037052 A1 WO 2019037052A1 CN 2017098909 W CN2017098909 W CN 2017098909W WO 2019037052 A1 WO2019037052 A1 WO 2019037052A1
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- WIPO (PCT)
- Prior art keywords
- wucam
- shrna
- vector
- cells
- psuper
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 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 belongs to the field of DNA recombination technology in bioengineering, and specifically relates to shRNA which specifically silences the target protein W UCAM.
- WUCAM is a newly discovered membrane protein molecule composed of immunoglobulin variable region (IgV), transmembrane region and immunoreceptor tyrosine-based inhibitor motif (ITIM). Expressed on Treg cells, memory T cells, activating T cells, and NK cell surfaces. The study found that: 1) WUCAM is highly constitutively expressed on Treg, and its expression is further up-regulated after activation, which is lowly expressed on memory T cells, while naive CD4+ CD25-T
- IgV immunoglobulin variable region
- ITIM immunoreceptor tyrosine-based inhibitor motif
- WUCAM-Fc fusion protein can promote DC secretion of IL-10 and reduce pro-inflammatory factor IL-12; 3) The ability of WUCAM-Fc fusion protein-treated mature DC to stimulate T cell proliferation is weakened , related to DC secretion of IL-10; 4) in vivo use of WUCAM protein can significantly reduce delayed hypersensitivity, its effect and CTLA-4
- WUCAM is an important immunomodulator and functions similarly to sputum, which inhibits the cytotoxicity of NK cells and the proliferation of CD4+ T cells and the production of cytokines.
- WUCAM is a substance that inhibits the action of T cells, and plays a role in inhibiting the excessive activation of T cells under normal conditions; however, this property also makes it easy to be utilized by tumor cells to achieve immune escape. Therefore, WU CAM is a potential target for tumor therapy and requires in-depth research. However, the lack of lentiviral vectors that specifically inhibit the expression of WUCAM gene in the prior art makes the research difficult to develop.
- the object of the present invention is to synthesize a specific base sequence capable of targeting silent WUCAM, and provide an effective means for further research on the role of WUCAM.
- the main construct required in the present invention is a silencing vector of WUCAM.
- the construction of the core silencing vector is divided into two major steps. The first step is to design a 19 bp shRNA core fragment based on the human WUCAM sequence.
- the synthesized fragment has appropriate restriction sites at both ends, and the target fragment is ligated to the pSUPER vector to form
- the core silencing vector after transformation, pick positive clones for PCR identification. The correct vector was identified, DNA sequencing was performed, and silencing efficiency was subsequently detected by immunoblotting.
- a 19 bp shRNA core fragment capable of targeting human WUCAM was designed and synthesized, and its sequence 'J is 5,-GGACGTACACTGGGAGAAT-3, (SEQ ID No: l).
- Construction of an expression vector that specifically silences WUCAM includes the following steps:
- the sequence of WUCAM is specifically identified based on the human WUCAM gene sequence using the corresponding software design.
- the second step the artificial synthesis of double-stranded shRNA fragments
- the linearized vector is recovered by using a recovery kit, and the recovered fragment is ligated with the shRNA fragment obtained by annealing, transformed, and the positive clone is identified, and the successfully constructed silencing vector is obtained by sequencing.
- the plasmid was extracted, and the total RNA was extracted after transfecting the cells. After reverse transcription into cDNA, the silencing efficiency was detected by quantitative PCR.
- the shRNA fragment synthesized in the present invention can specifically silence WUCAM.
- the experimental results show that the successful construction of the vector can specifically silence the WUCAM and reduce the expression of intracellular WUCAM. Therefore, the application of WUCAM silencing vector can study the function of WUCAM more elaborately and provide a theoretical basis for explaining related mechanisms.
- DRAWINGS 1 is a schematic diagram showing the results of quantitative PCR detection of WUCAM gene expression of Jurkat cells after transfection of pSUPER-WUCAM vector.
- Jurkat cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences, and the RNeasy Mini Kit was purchased from Qiagen.
- the complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
- a 59 bp sequence was designed in the form of Bgl II-GN18-TT-loop-81NC-Hind III, 81NC is the reverse complement of NG18, the Bgl II cleavage site at the 5' end and the Hind III cleavage site at the 3' end. Point, the obtained sequences are respectively SEQ ID NO:
- the synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded shRNA. Take 2 g
- the pSUPER vector, Bgl II and Hind III were digested, electrophoresed, and the linear pSUPER large fragment was recovered. Dilute the linearized pSUPER concentration to 100
- Ng/ ⁇ , and the double-stranded shRNA obtained by annealing were ligated overnight at 4 °C with T4 DNA ligase (NEB).
- the ligation product was transformed into ToplO competent cells and plated on LB plates with Kan resistance. Monoclonal shakes were picked on the transformation plates and cultured overnight before being sent to Shanghai Biotech for sequencing. The correct bacteria were sequenced for the extraction of endotoxin-free plasmids, and the pSUPER-WUCAM vector was obtained.
- Example 3 The pSUPER-WUCAM vector transduced Jurkat cells.
- Jurkat cells were cultured, and cells grown in good condition were inoculated into a six-well plate at 10 6 cells per well, and the cell density was about 60% after 18 h, using Lipofectamin.
- the extracted pSUPER-WUCAM vector 3 g was transduced into Jurkat cells. After 6 h, the medium was changed to fresh complete medium and cultured for 48 h.
- Example 4 Detection of WUCAM gene expression level
- Jurkat cells and Jurkat cells transduced with pSUPER-WUCAM vector were separately inoculated into 6-well plates. Cell density reaches ⁇ , with RNeasy Mini
- Kit extracts total RNA from each group of cells, using PrimeScrip RT reagent
- the shRNA fragment synthesized in the present invention can specifically silence WUCAM.
- the experimental results show that the successful construction of the vector can specifically silence the WUCAM and reduce the expression of intracellular WUCAM. Therefore, the application of WUCAM silencing vector can study the function of WUCAM more elaborately and provide a theoretical basis for explaining related mechanisms.
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- Wood Science & Technology (AREA)
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Abstract
L'invention concerne un ARNsh pour le silençage ciblé d'un gène WUCAM humain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/098909 WO2019037052A1 (fr) | 2017-08-24 | 2017-08-24 | Arnsh pour le silençage ciblé de wucam |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/098909 WO2019037052A1 (fr) | 2017-08-24 | 2017-08-24 | Arnsh pour le silençage ciblé de wucam |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2019037052A1 true WO2019037052A1 (fr) | 2019-02-28 |
Family
ID=65438347
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2017/098909 Ceased WO2019037052A1 (fr) | 2017-08-24 | 2017-08-24 | Arnsh pour le silençage ciblé de wucam |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2019037052A1 (fr) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017048824A1 (fr) * | 2015-09-14 | 2017-03-23 | Compass Therapeutics Llc | Compositions et procédés de traitement du cancer par antagonisme de la voie cd155/tighit et de tgf-ss |
-
2017
- 2017-08-24 WO PCT/CN2017/098909 patent/WO2019037052A1/fr not_active Ceased
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017048824A1 (fr) * | 2015-09-14 | 2017-03-23 | Compass Therapeutics Llc | Compositions et procédés de traitement du cancer par antagonisme de la voie cd155/tighit et de tgf-ss |
Non-Patent Citations (2)
| Title |
|---|
| DATABASE Nucleotide NCBI; 6 June 2016 (2016-06-06), XP055578701, Database accession no. XM_011512538 * |
| ZHANG, TAO ET AL.: "Increased Expression of TIGIT on CD4+ T Cells Ameliorates Immune-Mediated Bone Marrow Failure of Aplastic Anemia", JOURNAL OF CELLULAR BIOCHEMISTRY, vol. 115, no. 11, 10 September 2014 (2014-09-10), pages 1918 - 1927, XP055215805, ISSN: 1097-4644 * |
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