WO2019037130A1 - Arnsh du gène ampar humain et application associée - Google Patents

Arnsh du gène ampar humain et application associée Download PDF

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Publication number
WO2019037130A1
WO2019037130A1 PCT/CN2017/099174 CN2017099174W WO2019037130A1 WO 2019037130 A1 WO2019037130 A1 WO 2019037130A1 CN 2017099174 W CN2017099174 W CN 2017099174W WO 2019037130 A1 WO2019037130 A1 WO 2019037130A1
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WO
WIPO (PCT)
Prior art keywords
ampar
shrna
gene
expression
human
Prior art date
Application number
PCT/CN2017/099174
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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/CN2017/099174 priority Critical patent/WO2019037130A1/fr
Publication of WO2019037130A1 publication Critical patent/WO2019037130A1/fr

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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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • AD Alzheimer's disease
  • This disease is common in the elderly. It is a central nervous system degenerative degenerative disease with progressive cognitive impairment and memory impairment. The clinical manifestations are the deterioration of cognitive and memory functions, the progressive decline of daily living ability, and Various neuropsychiatric symptoms and behavioral disorders. More onset in the old age, latent onset, slow and irreversible, clinically based on intelligent damage.
  • AMPAR glutamate a-amino-3-carbo-5-methyl-4-iso-propionate receptor
  • a-amino-3-hy droxy-5-methyl-4 -isoxazole propionic acid receptor AMPAR is a key mechanism regulating central synaptic plasticity.
  • AMPAR is one of the ionotropic glutamate receptors and mediates the central component of central excitatory postsynaptic currents and is essential for synaptic transmission.
  • 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 SEQ NO.
  • the antisense strand template sequence is shown in SEQ NO. 2 of the Sequence Listing.
  • the present invention designed a pair of AMPAR-shRNA targeting AMPAR gene according to the mRNA sequence of human AMPAR gene in GenBank database and the primer design principle of shRNA, and commissioned Shanghai Biotech to synthesize the AMPAR-shRNA.
  • the oligonucleotide of the AMPAR-shRNA designed above is routinely annealed and then double-stranded, 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 will contain AM-shRNA-containing pSilencer3.1-Hl hygro
  • RNAi expression vector was transduced into the Jurkat cell line, and the silencing efficiency of AMPAR mRNA was 77.2 ⁇ 3 ⁇ 4.
  • the AMPAR-shRNA provided by the present invention has the advantages of high transduction efficiency, high-efficiency and specific inhibition of the expression of the AMPAR gene of the Jurkat cell, and can be used as a powerful tool for preparing a medicament for treating an abnormal expression of AMPAR gene.
  • FIG. 1 is a schematic diagram showing the results of fluorescent quantitative PCR detection of AMPAR gene expression by Jurkat cells transduced with AMPAR-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.
  • Example 1 Design of shRNA Oligonucleotide Sequences Targeting AMPAR Gene
  • 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. It is ensured that the GC content of the target sequence should be around 30% to 60% and 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'- GTGTCTGCAGGTCTGAGTA
  • the sense strand sequence ⁇ ij of AMPAR-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 AMPAR-shRNA expression vector was mixed and added to the electric shock cup.
  • the Invitrogen Neon electroporation system was used for electroporation.
  • 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. Medium, gently shake the dish to mix the cells, and detect AMPAR gene expression 48 h later.
  • Example 4 Fluorescence quantitative PCR was used to detect the AMPAR gene expression level.
  • Jurkat cells were transfected with normal Jurkat cells and AMPAR-shRNA expression vector, and total RNA was extracted from each group using RNeasy Mini Kit, using PrimeScrip RT reagent.
  • Kit reverse-transcribes mRNA into cDNA, and then cDNA is diluted with 90 ⁇ M of RNase-Free dH20 and stored at -20 °C for later detection.
  • ⁇ is the template, GAPDH is used as the internal reference, and the relative expression of AMPAR is detected by real-time quantitative PCR (QPCR).
  • the reaction conditions are set: 95°C for 10s, 1 cycle; 95°C for 5s, 60°C for 30s, for 40 cycles.
  • the relative expression of AMPAR 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 AMPAR gene was significantly inhibited in Jurkat cells transduced with AMPAR-shRNA expression vector, and the inhibitory efficiency of the interference fragment on the target gene was 77.2% ⁇ 3.4%, which proved that the AMPAR-shRNA expression vector used in this experiment carries shRNA energy. It specifically inhibits the expression of the AMPAR gene, and the inhibitory effect is very remarkable.
  • the AMPAR-shRNA provided by the present invention has the advantages of high transduction efficiency, high-efficiency and specific inhibition of the expression of the AMPAR gene of Jurkat cells, and can be used as a powerful tool for preparing a medicament for treating an abnormality in AMPAR gene expression.

Abstract

La présente invention concerne un ARNsh d'un gène AMPAR humain et une application associée. L'ARNsh inhibe l'expression d'un gène AMPAR, la séquence de brin sens de l'ARNsh est représentée par SEQ ID NO : 1, et la séquence de brin antisens est représentée par SEQ ID NO : 2.
PCT/CN2017/099174 2017-08-25 2017-08-25 Arnsh du gène ampar humain et application associée WO2019037130A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/099174 WO2019037130A1 (fr) 2017-08-25 2017-08-25 Arnsh du gène ampar humain et application associée

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/099174 WO2019037130A1 (fr) 2017-08-25 2017-08-25 Arnsh du gène ampar humain et application associée

Publications (1)

Publication Number Publication Date
WO2019037130A1 true WO2019037130A1 (fr) 2019-02-28

Family

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WO (1) WO2019037130A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2338492A1 (fr) * 2009-12-24 2011-06-29 Universidad del Pais Vasco Procédés et compositions pour le traitement de la maladie d'Alzheimer
WO2012047355A2 (fr) * 2010-07-16 2012-04-12 The Research Foundation Of State University Of New York Inhibiteurs d'acide nucléique sélectifs de sous-unité de récepteurs de glutamate

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2338492A1 (fr) * 2009-12-24 2011-06-29 Universidad del Pais Vasco Procédés et compositions pour le traitement de la maladie d'Alzheimer
WO2012047355A2 (fr) * 2010-07-16 2012-04-12 The Research Foundation Of State University Of New York Inhibiteurs d'acide nucléique sélectifs de sous-unité de récepteurs de glutamate

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE Database Nucleotide 6 June 2016 (2016-06-06), ANONYMOUS: "PREDICTED: Homo sapiens calcium voltage-gated channel auxiliary subunit gamma 2 (CACNG2), transcript variant X1, mRNA.", XP055576961, retrieved from NCBI Genbank Database accession no. XM_017028531. 1 *
TRACY: "Acute knockdown of AMPA receptors reveals a trans-synaptic sig- nal for presynaptic maturation", EMBO J., vol. 30, no. 8, 4 March 2011 (2011-03-04) - 20 April 2011 (2011-04-20), pages 1577 - 1592, XP055576789, ISSN: 0261-4189, DOI: DOI10.1038/emboj.2011.59 *
XU: "The regulation of PTEN on expression and positioning of Ampa-glur2 in Rat primary Hippocainpal Neuron after stretch damage", CFDT- MEDICAL AND HEALTH SCIENCES, no. 2, 15 February 2011 (2011-02-15), ISSN: 1674-022X *
ZHENG: "Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice va- riant", J NEUROPHYSIOL., vol. 108, no. 1, 4 April 2012 (2012-04-04) - 1 July 2012 (2012-07-01), pages 101 - 111, XP055576799, ISSN: 0022-3077, DOI: doi.org/10.1152/jn.01097.2011 *

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