WO2019033245A1 - Arnsh du gène clock humain et utilisation correspondante - Google Patents

Arnsh du gène clock humain et utilisation correspondante Download PDF

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Publication number
WO2019033245A1
WO2019033245A1 PCT/CN2017/097428 CN2017097428W WO2019033245A1 WO 2019033245 A1 WO2019033245 A1 WO 2019033245A1 CN 2017097428 W CN2017097428 W CN 2017097428W WO 2019033245 A1 WO2019033245 A1 WO 2019033245A1
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WO
WIPO (PCT)
Prior art keywords
clock
shrna
clock gene
gene
human
Prior art date
Application number
PCT/CN2017/097428
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English (en)
Chinese (zh)
Inventor
毛吉炎
Original Assignee
深圳市博奥康生物科技有限公司
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 深圳市博奥康生物科技有限公司 filed Critical 深圳市博奥康生物科技有限公司
Priority to PCT/CN2017/097428 priority Critical patent/WO2019033245A1/fr
Publication of WO2019033245A1 publication Critical patent/WO2019033245A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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 CLOCK gene, and more particularly to a shRNA (short hairpin RNA) of a human CLOCK gene and use thereof.
  • Biological rhythm refers to the cyclical changes in the process of long-term biological evolution in order to adapt to the natural cycle.
  • the living organisms from single cells to higher animals and plants and all human life activities are formed according to certain rules.
  • the circadian rhythm is a kind of biological rhythm, which is a rhythm exhibited by the combination of exogenous factors and endogenous factors. Its endogenous factor is a closed-loop oscillating system consisting of a set of biorhythm genes and their gene products interacting.
  • the biological rhythm gene is an important internal fixed material basis of circadian rhythm, which plays a key role in maintaining the body's body temperature, breathing, sleep, eating, blood pressure, blood sugar, endocrine and other homeostasis.
  • These biorhythm genes include
  • the CLOCK gene plays a central role in this system. Therefore, the study of the CLOCK gene is very important, but the lack of a vector that specifically inhibits the expression of the CLOCK gene in the prior art makes the related research not well developed.
  • One of the technical problems to be solved by the present invention is to provide a shRNA of a human CLOCK gene.
  • the second technical problem to be solved by the present invention is to provide a shRNA of human CLOCK gene.
  • the present invention provides a shRNA of a human CLOCK gene, comprising: a CLOCK-RNAi consisting of the sequence of SEQ ID NO: 1 and the sequence encoding SEQ ID NO: 2.
  • the shRNA provided by the invention has the advantages of high transduction efficiency, high and specific inhibition of CLOCK gene expression in C6 cells, and can be used as a powerful tool for preparing a medicament for treating diseases related to abnormal CLOCK gene expression.
  • Figure 1 is a schematic diagram showing the results of CLOCK gene expression of C6 cells after transduction of pLKO-CLOCK vector.
  • C6 cells were purchased from ATCC, Trizol was purchased from Invitrogen, Endo-free Plasmid Mini
  • Kit was purchased from Omega bio-tek.
  • the complete medium described below is a cell culture medium to which 10% fetal calf serum is added.
  • the CLOCK-RNAi oligonucleotide chain was synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • the synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded CLOCK-RNAi.
  • Plasmid Mini Kit for endotoxin-free pLKO-CLOCK vector extraction Plasmid Mini Kit for endotoxin-free pLKO-CLOCK vector extraction.
  • Example 3 pLKO-CLOCK vector transduced C6 cells.
  • C6 cells were cultured, and 5,000,000 cells with good growth state were taken, and the cells were collected by centrifugation, and then resuspended in 500 ⁇ L of PBS, mixed with 20 pL KO-CLOCK carrier, and then added to an electric shock cup, and electroporated by Invitrogen Neon electrotransfer system.
  • 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 hours.
  • Example 4 Fluorescence quantitative PCR was used to detect the expression level of CLOCK gene.
  • C6 cells inoculated with C6 cells and transduced pLKO-CLOCK vector into cell culture flasks were cultured for 24 h, and 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 L of RNase-Free dH20 and stored at -20 °C.
  • the shRNA provided by the invention has the advantages of high transduction efficiency, high-efficiency and specific inhibition of CLOCK gene expression of C6 cells, and can be used as a powerful tool for preparing a medicament for treating diseases related to abnormal expression of CLOCK gene.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un ARNsh du gène CLOCK humain et son utilisation. L'ARNsh du gène CLOCK humain comprend un ARNi de CLOCK constitué d'une séquence codant pour SEQ ID NO : 1 et d'une séquence codant pour SEQ ID NO : 2. L'ARNsh du gène CLOCK humain peut être utilisé dans la préparation d'un médicament destiné au traitement d'une maladie associée à une expression anormale du gène CLOCK.
PCT/CN2017/097428 2017-08-14 2017-08-14 Arnsh du gène clock humain et utilisation correspondante WO2019033245A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/097428 WO2019033245A1 (fr) 2017-08-14 2017-08-14 Arnsh du gène clock humain et utilisation correspondante

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/097428 WO2019033245A1 (fr) 2017-08-14 2017-08-14 Arnsh du gène clock humain et utilisation correspondante

Publications (1)

Publication Number Publication Date
WO2019033245A1 true WO2019033245A1 (fr) 2019-02-21

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Family Applications (1)

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WO (1) WO2019033245A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020183250A1 (en) * 1997-07-08 2002-12-05 Duckworth David Malcolm Novel use
EP2315600A2 (fr) * 2008-07-24 2011-05-04 The Regents of the University of California Compositions et procédés se rapportant à la fonction sirt1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020183250A1 (en) * 1997-07-08 2002-12-05 Duckworth David Malcolm Novel use
EP2315600A2 (fr) * 2008-07-24 2011-05-04 The Regents of the University of California Compositions et procédés se rapportant à la fonction sirt1

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MUKHERJEE, S. ET AL.: "Knockdown of Clock in the Ventral Tegmental Area Through RNA Interference Results in a Mixed State of Mania and Depression-Like Behavior", BIOL PSYCHIATRY, vol. 68, 31 December 2010 (2010-12-31), pages 503 - 511, XP028147467, ISSN: 0006-3223 *
STEEVES, T.D.L. ET AL.: "Molecular Cloning and Characterization of the Human CLOCK Gene : Expression in the Suprachiasmatic Nuclei", GENOMICS, vol. 57, 31 December 1999 (1999-12-31), pages 189 - 200, XP004444923, ISSN: 0888-7543 *

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