WO2019037130A1

WO2019037130A1 - Shrna of human ampar gene and application thereof

Info

Publication number: WO2019037130A1
Application number: PCT/CN2017/099174
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2017-08-25
Filing date: 2017-08-25
Publication date: 2019-02-28

Abstract

Provided are shRNA of a human AMPAR gene and an application thereof. The shRNA inhibits the expression of an AMPAR gene, the sense strand sequence of the shRNA is represented by SEQ ID NO: 1, and the antisense strand sequence is represented by SEQ ID NO: 2.

Description

Human AMPAR gene shRNA and its application

[0001] 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 AMPAR gene on the surface of human T cells, which can inhibit the expression of the human AMPAR gene after transfection.

Background technique

[0002] Alzheimer's disease (AD) is the most common cause of senile dementia. 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.

[0003] There is increasing evidence that 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.

technical problem

[0004] The physiological function of AMPAR depends on the number of receptors, subunit composition and protein phosphorylation to regulate AMPAR dysfunction, leading to decreased synaptic plasticity of neurons, causing AD cognitive impairment, which plays an important role in the pathogenesis of AD. Role, therefore, is essential for the role of AMPAR in Alzheimer's disease and its use as a therapeutic target. The lack of vectors specifically inhibiting the expression of AMPAR gene in the prior art makes the relevant studies incomprehensible. exhibition.

[0005] shRNA, a small hairpin RNA, is an exogenous RNA sequence with a stem-loop structure that can be processed into siRNA in a cell, and the siRNA is then combined with an enzyme to form an RNA-induced silencing complex (RISC). It is a good way to reduce gene expression by activating homologous mRNA and inducing its degradation. Problem solution

Technical solution The present invention constructs a shRNA, and the sense strand template sequence of the shRNA is SEQ NO.

As shown in Figure 1, the antisense strand template sequence is shown in SEQ NO. 2 of the Sequence Listing. The shRNA

It can reduce the protein expression level of human AMPAR gene.

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.

In the present invention, 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

The RNAi expression vector was transduced into the Jurkat cell line, and the silencing efficiency of AMPAR mRNA was 77.2 < 3⁄4. Advantageous effects of the invention

Beneficial effect

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.

Brief description of the drawing

DRAWINGS

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.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0013] 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.

[0014] Example 1 Design of shRNA Oligonucleotide Sequences Targeting AMPAR Gene [0015] Starting from the AUG initiation codon of mRNA, 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

-3', the sense strand sequence 歹 ij of AMPAR-shRNA is shown in SEQ ID No: 1, and the antisense strand sequence is shown in SEQ ID No: 2.

Example 2 Construction of AMPAR-shRNA Expression Vector

[0017] An equal amount of 10 mmol/L DNA oligonucleotide single-stranded fragment was mixed, heated in a TE buffer at 95 ° C for 5 min, and slowly lowered to room temperature. The pSilencer 3.1-H1 hygro expression vector was digested with BamHI and Hindlll, and the fragment was ligated with the vector by T4 ligase. The ligation product was then transformed into E. coli ToplO and single clones were picked for sequencing.

[0018] The clones with the correct sequencing were selected and inoculated into 5 mL of medium, and cultured overnight, without endotoxin extraction plasmid, which is an AMPAR-shRNA expression vector.

Example 3 Jurkat Cell Transduction

[0020] 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 μ¥Ό, 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.

[0021] Example 4 Fluorescence quantitative PCR was used to detect the AMPAR gene expression level.

[0022] 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 μM of RNase-Free dH20 and stored at -20 °C for later detection.

[0023] taking cDNA of each group of cells

Ιμί 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.

[0024] The above is a further detailed description of the present invention in conjunction with the specific preferred embodiments, and it is not intended that the specific embodiments of the invention are limited to the description. It will be apparent to those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the invention.

Industrial applicability

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.

Claims

Claim

[Claim 1] A shRNA which inhibits expression of an AMPAR gene, wherein the shRNA has a sense strand sequence as shown in SEQ ID NO: 1, and the shRNA antisense strand sequence is as SEQ ID NO:

2 is shown.

[Claim 2] The use of shRNA according to claim 1 for the preparation of a reagent for reducing cellular AMPAR mRNA.

[Claim 3] The use of shRNA according to claim 1 for the preparation of an inhibitory cell AMPAR protein expression reagent.

[Claim 4] The use of shRNA according to claim 1 for the preparation of a medicament for a disease associated with abnormal expression of AMPAR gene.