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WO2019033249A1 - Sharn du gène btla humain et utilisation correspondante - Google Patents

Sharn du gène btla humain et utilisation correspondante Download PDF

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Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
btla
shrna
gene
btla gene
human
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.)
Ceased
Application number
PCT/CN2017/097432
Other languages
English (en)
Chinese (zh)
Inventor
毛吉炎
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Biocan Technologies Co Ltd
Original Assignee
Shenzhen Biocan Technologies Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Shenzhen Biocan Technologies Co Ltd filed Critical Shenzhen Biocan Technologies Co Ltd
Priority to PCT/CN2017/097432 priority Critical patent/WO2019033249A1/fr
Publication of WO2019033249A1 publication Critical patent/WO2019033249A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12N15/11—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.

Landscapes

  • 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)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (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.
PCT/CN2017/097432 2017-08-14 2017-08-14 Sharn du gène btla humain et utilisation correspondante Ceased WO2019033249A1 (fr)

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 Ceased 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)

* Cited by examiner, † Cited by third party
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
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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