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WO2019036869A1 - Arnsh du gène tl6 humain et ses applications - Google Patents

Arnsh du gène tl6 humain et ses applications Download PDF

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Publication number
WO2019036869A1
WO2019036869A1 PCT/CN2017/098367 CN2017098367W WO2019036869A1 WO 2019036869 A1 WO2019036869 A1 WO 2019036869A1 CN 2017098367 W CN2017098367 W CN 2017098367W WO 2019036869 A1 WO2019036869 A1 WO 2019036869A1
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WO
WIPO (PCT)
Prior art keywords
shrna
gene
expression
human
sequence
Prior art date
Application number
PCT/CN2017/098367
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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/CN2017/098367 priority Critical patent/WO2019036869A1/fr
Publication of WO2019036869A1 publication Critical patent/WO2019036869A1/fr

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Classifications

    • 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
    • A61P35/00—Antineoplastic agents
    • 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 genetic engineering technology, and relates to the construction and application of a shRNA expression sequence. Specifically, the present invention contemplates the synthesis of shRNA against the nucleotide sequence of the TL6 gene on the surface of human T cells, which can inhibit the expression of the human TL6 gene after transfer.
  • TL6 is a ligand for the surface molecule AITR on thymus-derived CD4+ CD25+ Treg cells. research shows
  • AITR/TL6 has many important biological activities, including cell proliferation, differentiation and survival. technical problem
  • the AITR/TL6 system participates in the role of Treg cells in the regulation of immune regulation, plays an important role in the immunotherapy of tumors, and has a good clinical transformation prospect.
  • the lack of vectors for specifically inhibiting the expression of TL6 gene in the prior art makes related research. Can't develop well.
  • shRNA a small hairpin RNA
  • RISC RNA-induced silencing complex
  • the present invention constructs a shRNA, and the sense strand template sequence of the shRNA is as shown in the sequence listing SEQ NO.
  • the antisense strand template sequence is shown in SEQ NO. 2 of the Sequence Listing.
  • the present invention designed a pair of TL6-shRNA targeting TL6 gene according to the mRNA sequence of human TL6 gene in GenBank database and the primer design principle of shRNA, and commissioned Shanghai Biotech to synthesize the TL6-shR.
  • the oligonucleotide of the TL6-shRNA designed above is routinely annealed, and then double-stranded, double-digested and ligated to the pSilencer 3.1-H1 hygro RNAi expression vector, and the ligation product is transformed into Escherichia coli. Single colonies were picked for PCR and sequencing, and positive clones and plasmids were obtained.
  • the present invention transduced the pSilencer3.1-H1 hygro RNAi expression vector containing TL6-shRNA into the Jurkat cell line, and the silencing efficiency of TL6 mRNA was 73.9%.
  • the TL6-shRNA provided by the invention has the advantages of high transduction efficiency, high-efficiency and specific inhibition of TL6 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 TL6 gene.
  • FIG. 1 is a schematic diagram showing the results of fluorescent quantitative PCR detection of TL6 gene expression by Jurkat cells transduced with TL6-shRNA expression 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 endotoxin-free plasmid extraction kit was purchased from Tiangen Biochemical (Beijing).
  • the complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
  • the "AA or NA" sequence was searched for and the 19-base sequence at the 3' end was recorded as a potential interference target site. Ensure that the target sequence has a GC content of 30% to 60% and is not on the 5' and 3' non-coding regions. NCBI BLAST confirmed that the selected sequences have no homology to other genes.
  • the target sequence obtained in this example is 5'- GCTCCCAATGCAAACTACA
  • the sense strand ⁇ ij of TL6-shRNA is shown in SEQ ID No: 1
  • the antisense strand sequence is shown in SEQ ID No: 2.
  • Jurkat cells were cultured, and 5,000,000 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.
  • the TL6-shRNA expression vector was mixed and added to the electric shock cup, and electroporated using the Invitrogen Neon electroporation system.
  • the electroporation procedure was: 2.1 KV, 25 ⁇ , pulse shock once; the cells were transferred to a 6 cm dish containing 5 mL DMEM complete medium. In the middle, the cells were gently shaken to mix the cells, and the expression of TL6 gene was detected 48 h later.
  • Example 4 Fluorescence quantitative PCR was used to detect the expression level of TL6 gene.
  • Jurkat cells were transfected with normal Jurkat cells and TL6-shRNA expression vector, total RNA was extracted from each group with RNeasy Mini Kit, mRNA was reverse transcribed into cDNA using PrimeScrip RT reagent Kit, and then 9 (L RNase) was added. -Free dH20 diluted cDNA, stored at -20 °C for later detection
  • ⁇ is the template, GAPDH is used as the internal reference, and the relative expression of TL6 is detected by real-time quantitative PCR (QPCR).
  • the reaction conditions are set: 95 ° C for 10 s, 1 cycle; 95 ° C 5 s, 60 ° C
  • the TL6-shRNA provided by the invention has the advantages of high transduction efficiency, high-efficiency and specific inhibition of TL6 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 TL6 gene.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un ARNsh du gène TL6 humain et ses applications. La séquence du brin sens de l'ARNsh est représentée par la SEQ ID NO : 1, et la séquence du brin antisens de l'ARNsh est représentée par la SEQ ID NO : 2. Au moyen de l'ARNsh de la présente invention, un vecteur d'expression d'ARNsh a été conçu, synthétisé et construit selon une séquence nucléotidique du gène TL6 humain.
PCT/CN2017/098367 2017-08-21 2017-08-21 Arnsh du gène tl6 humain et ses applications WO2019036869A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/098367 WO2019036869A1 (fr) 2017-08-21 2017-08-21 Arnsh du gène tl6 humain et ses applications

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/098367 WO2019036869A1 (fr) 2017-08-21 2017-08-21 Arnsh du gène tl6 humain et ses applications

Publications (1)

Publication Number Publication Date
WO2019036869A1 true WO2019036869A1 (fr) 2019-02-28

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PCT/CN2017/098367 WO2019036869A1 (fr) 2017-08-21 2017-08-21 Arnsh du gène tl6 humain et ses applications

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104080909A (zh) * 2011-11-30 2014-10-01 中外制药株式会社 包含进入细胞内以形成免疫复合体的搬运体(载体)的药物

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104080909A (zh) * 2011-11-30 2014-10-01 中外制药株式会社 包含进入细胞内以形成免疫复合体的搬运体(载体)的药物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Identification of a novel activation-inducible protein of the tumor necrosis factor receptor superfamily and its ligand", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 10, 5 March 1999 (1999-03-05), pages 6056 - 6061, XP002147323, ISSN: 1083-351X *

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