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WO2019037049A1 - Arnsh du gène erbb2 humain et applications - Google Patents

Arnsh du gène erbb2 humain et applications Download PDF

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Publication number
WO2019037049A1
WO2019037049A1 PCT/CN2017/098906 CN2017098906W WO2019037049A1 WO 2019037049 A1 WO2019037049 A1 WO 2019037049A1 CN 2017098906 W CN2017098906 W CN 2017098906W WO 2019037049 A1 WO2019037049 A1 WO 2019037049A1
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WO
WIPO (PCT)
Prior art keywords
erbb2
shrna
erbb2 gene
gene
human
Prior art date
Application number
PCT/CN2017/098906
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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/CN2017/098906 priority Critical patent/WO2019037049A1/fr
Publication of WO2019037049A1 publication Critical patent/WO2019037049A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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 relates to a human ERBB2 gene, and more particularly to a shRNA (short hairpin RNA) of a human ERBB2 gene and use thereof.
  • shRNA short hairpin RNA
  • ERBB2 is a 185 kDa cell membrane receptor encoded by the proto-oncogene ERBB2 and is a member of the epidermal growth factor receptor (EGFR) family.
  • the family includes four members, ERBB1 (also known as EGFR, HERl), ERBB2 (also known as HER2), ERBB3 (HER3), and ERBB4 (HER4).
  • ERBB2 is involved in the regulation of growth and development of normal tissues, promotes cell division and secretion of proteolytic enzymes, and enhances cell motility, but under pathological conditions, ERBB2 expression increases, thereby promoting tumor invasion and metastasis.
  • ERBB2 has intracellular tyrosine kinase-like activity, plays a role in embryonic development and differentiation, is widely expressed in epithelial cells of human tissues, and is closely related to the survival and evolution of various tumors. . Therefore, the research on ERBB2 can greatly promote the development of tumor prevention and control, but the lack of vectors for specifically inhibiting the expression of ERBB2 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 the human ERBB2 gene.
  • the second technical problem to be solved by the present invention is to provide a shRNA of human ERBB2 gene.
  • the present invention provides a shRNA of a human ERBB2 gene, including:
  • sequence shown in SEQ ID NO: 1 and ERBB2-RNAi consisting of the sequence shown in SEQ ID NO: 2.
  • the shRNA provided by the invention has high transduction efficiency and can efficiently and specifically inhibit the ERBB2 group of Raji cells. Due to the advantages of expression, it can be used as a powerful tool for the preparation of drugs for the treatment of diseases related to abnormal expression of ERBB2 gene.
  • Figure 1 is a schematic diagram showing the results of Quantitative PCR detection of Raji cells after transduction of pLKO-ERBB2 vector.
  • 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 ERBB2-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 ERBB2-RNAi.
  • Age I and EcoR I double-cleavage pLKO.l-puro vector, Fermentas restriction enzymes Age I and EcoR I double digestion, 50 reaction system is as follows: 5 ⁇ L 10x FastDigest Buffer, 2 ⁇ L Age I , 2 EcoR I, 2 g pLK0.1-puro plasmid, ddH20 supplemented to 20ul, 37 ° C reaction for 30 min; [0019] 2) ligation, 2 L annealing of phosphorylated double-stranded DNA oligos, 1 ligase buffer, 1 double-digested pLKO. l-puro plasmid, 1 ligase, 5 L ddH20, reaction at 16 ° C for 5 h, Obtaining the ligation product pLKO-ERBB2 vector;
  • Plasmid Mini Kit for endotoxin-free pLKO-ERBB2 vector extraction Plasmid Mini Kit for endotoxin-free pLKO-ERBB2 vector extraction.
  • Raji cells were cultured, and 5,000,000 cells with good growth state were taken, and the cells were collected by centrifugation, then resuspended in 500 ⁇ L ⁇ PBS, mixed with 20 pL KO-ERBB2 vector, and then added to an electric shock cup, and electrotransformed using 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 ERBB2 gene.
  • Raji cells inoculated with Raji cells and transduced pLKO-ERBB2 vector were separately cultured into cell culture flasks, and after 24 h of culture, total RNA of each group was extracted with Trizol, and PrimeScrip RT reagent was used.
  • Kit reverse-transcribes mRNA into cDNA, and is diluted with 90 RNase-Free dH20 and stored at -20 °C.
  • ERBB2 For the template, GAPDH was used as the internal reference, and the relative expression of ERBB2 was detected by real-time quantitative PCR (QPCR). The reaction conditions were set: 95 ° C for 10 s, 1 cycle; 95 ° C for 5 s, 54 ° C for 30 s, for 40 cycles. The relative expression of ERBB2 gene in each group was detected by SYBR Primescript RT-PCR Kit. The results are shown in Figure 1. The results showed that the expression of ERBB2 gene was significantly inhibited in Raji cells transfected with pLKO-ERBB2 vector, and the inhibitory efficiency of the interference fragment on the target gene was 84.4% ⁇ 4.1%, which proved that the pLKO-ERBB2 vector used in this experiment can specifically inhibit shRNA. The expression of the ERBB2 gene is very significant.
  • the shRNA provided by the invention has the advantages of high transduction efficiency, high-efficiency and specific inhibition of ERBB2 gene expression in Raji cells, and can be used as a powerful tool for preparing a medicament for treating ERBB2 gene expression-related diseases.

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

Abstract

L'invention concerne un ARNsh d'un gène ERBB2 humain. Un brin sens positif d'une séquence codante de l'ARNsh est présenté dans SEQ ID NO : 1, et un brin antisens de la séquence codante de l'ARNsh est présenté dans SEQ ID NO : 2. L'ARNsh du gène ERBB2 humain peut être utilisé pour la préparation de médicaments pour le traitement de maladies liées à des anomalies d'expression d'un gène ERBB2.
PCT/CN2017/098906 2017-08-24 2017-08-24 Arnsh du gène erbb2 humain et applications WO2019037049A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/098906 WO2019037049A1 (fr) 2017-08-24 2017-08-24 Arnsh du gène erbb2 humain et applications

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/098906 WO2019037049A1 (fr) 2017-08-24 2017-08-24 Arnsh du gène erbb2 humain et applications

Publications (1)

Publication Number Publication Date
WO2019037049A1 true WO2019037049A1 (fr) 2019-02-28

Family

ID=65438318

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/098906 WO2019037049A1 (fr) 2017-08-24 2017-08-24 Arnsh du gène erbb2 humain et applications

Country Status (1)

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WO (1) WO2019037049A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101918844A (zh) * 2007-06-18 2010-12-15 米迪缪尼有限公司 表达epha2和erbb2的细胞的协同治疗

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101918844A (zh) * 2007-06-18 2010-12-15 米迪缪尼有限公司 表达epha2和erbb2的细胞的协同治疗

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 1 August 2016 (2016-08-01), TAL, M., XP055578017, Database accession no. AH002823 *
WEI BAO: "HER2-mediated upregulation of MMP-1 is involved in gastric cancer cell invasion", ARCHIEVES OF BIOCHEMISTRY AND BIOPHYSICS, 31 July 2010 (2010-07-31), XP027084046 *

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