WO2019033245A1

WO2019033245A1 - Shrna of human clock gene and use thereof

Info

Publication number: WO2019033245A1
Application number: PCT/CN2017/097428
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2017-08-14
Filing date: 2017-08-14
Publication date: 2019-02-21

Abstract

Disclosed are an shRNA of the human CLOCK gene and the use thereof. The shRNA of the human CLOCK gene comprises a CLOCK-RNAi consisting of a sequence encoding SEQ ID NO: 1 and a sequence encoding SEQ ID NO: 2. The shRNA of the human CLOCK gene can be used in the preparation of a drug for treating a disease associated with abnormal expression of the CLOCK gene.

Description

Title: Inventive Name: shRNA and application of human CLOCK gene

[0001] 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.

Background technique

[0002] 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. Among them, 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

CLOCK Per p _er 2, Per3, Cryl, Cry2, BMAL1, Timless, etc.

technical problem

[0003] 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.

Problem solution

Technical solution

One of the technical problems to be solved by the present invention is to provide a shRNA of a human CLOCK gene.

[0005] The second technical problem to be solved by the present invention is to provide a shRNA of human CLOCK gene.

In the preparation of a medicament for treating a disease associated with abnormal expression of the CLOCK gene.

In order to solve the above technical problems, 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. Advantageous effects of the invention

Beneficial effect 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.

Brief description of the drawing

DRAWINGS

[0008] Figure 1 is a schematic diagram showing the results of CLOCK gene expression of C6 cells after transduction of pLKO-CLOCK vector.

BEST MODE FOR CARRYING OUT THE INVENTION

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

[0010] 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.

[0011] Example 1 shRNA sequence design of CLOCK gene

[0012] (1) According to the characteristics of human CLOCK gene design silence sequence, the design principle: 19bp specific binding CLOCK sequence GC content is 45<3⁄4-55<3⁄4, annealing temperature 45 °C-65 °C, the same The first base of the start of the sequence is required to be G. The selected fragments were BLAST aligned with the human genome to ensure specificity.

[0013] (2) Design a 59 bp sequence CLOCK-RNAi in the form of Age I-GN18-TT-loop-81NC-EcoR I, 81NC is the reverse sequence of NG18, and the 5' end is the Age I restriction site, 3' The end is an EcoR I restriction site, the sense strand sequence is shown in SEQ ID No: 1, and the antisense strand sequence is as SEQ ID No:

2 is shown. The CLOCK-RNAi oligonucleotide chain was synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

Example 2 Construction of pLKO-CLOCK Vector

[0015] The synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded CLOCK-RNAi.

[0016] pLKO.l-puro vector (Sigma Aldrich) full length 7086 bp, restriction endonuclease site selection Age

I and EcoR I are used as insertion sites for DNA fragments, and the specific steps for constructing each vector using different shRNA corresponding DNA fragments are as follows:

[0017] 1) Age l 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 μL 10x FastDigest Buffer, 2 μL Age I , 2 L EcoR I, 2 g pLK0.1-puro plasmid, ddH20 supplemented to 20 ul, 37 ° C reaction for 30 min;

[0018] 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-CLOCK vector;

[0019] 3) conversion;

[0020] 4) Pick positive clone culture and perform sequencing to verify that the insertion sequence is completely correct, then use Endo-free

Plasmid Mini Kit for endotoxin-free pLKO-CLOCK vector extraction.

Example 3 pLKO-CLOCK vector transduced C6 cells.

[0022] 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 μL 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 μΐΌ, 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.

[0023] Example 4 Fluorescence quantitative PCR was used to detect the expression level of CLOCK gene.

[0024] 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.

[0025] taking cDNA 1 of each group of cells

For the template, GAPDH was used as the internal reference, and the relative expression of CLOCK 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 CLOCK 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 CLOCK gene was significantly inhibited in C6 cells transduced with pLKO-CLOCK vector, and the inhibitory efficiency of the interference fragment on the target gene was 78.5%±3.9<3⁄4, which proved that the pLKO-CLOCK vector used in this experiment can be specific to shRNA. The expression of the CLOCK gene was inhibited, and the inhibitory effect was very remarkable.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. For those skilled in the art to which the present invention pertains, a number of simple deductions or substitutions may be made without departing from the inventive concept. All should be considered as belonging to the scope of protection of the present invention.

Industrial applicability

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.

Claims

Claim

[Claim 1] A shRNA of a human CLOCK gene, comprising: encoded by SEQ ID NO:

The sequence shown in 1 and the CLOCK-RNAi consisting of the sequence shown in SEQ ID NO: 2.

[Claim 2] The use of the shRNA of the human CLOCK gene according to claim 1 for the preparation of a medicament for treating a disease associated with abnormal expression of the CLOCK gene.

[Claim 3] The use according to claim 2, wherein: the shR of the human CLOCK gene

NA is CLOCK-RNAi.