WO2019037049A1

WO2019037049A1 - Shrna of human erbb2 gene and applications

Info

Publication number: WO2019037049A1
Application number: PCT/CN2017/098906
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2017-08-24
Filing date: 2017-08-24
Publication date: 2019-02-28

Abstract

An shRNA of a human ERBB2 gene. A positive-sense strand of a coding sequence of the shRNA is shown in SEQ ID NO:1, and an antisense strand of the coding sequence of the shRNA is shown in SEQ ID NO:2. The shRNA of the human ERBB2 gene can be used for the preparation of drugs for treating diseases related to expression anomalies of an ERBB2 gene.

Description

shRNA and its application of human ERBB2 gene

Technical field

[0001] 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.

Background technique

[0002] 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).

[0003] 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.

technical problem

[0004] 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.

Problem solution

Technical solution

One of the technical problems to be solved by the present invention is to provide a shRNA of the human ERBB2 gene.

[0006] The second technical problem to be solved by the present invention is to provide a shRNA of human ERBB2 gene.

In the preparation of a medicament for treating diseases associated with abnormal expression of the ERBB2 gene.

[0007] In order to solve the above technical problem, the present invention provides a shRNA of a human ERBB2 gene, including:

The sequence shown in SEQ ID NO: 1 and ERBB2-RNAi consisting of the sequence shown in SEQ ID NO: 2.

Advantageous effects of the invention

Beneficial effect

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.

Brief description of the drawing

DRAWINGS

Figure 1 is a schematic diagram showing the results of Quantitative PCR detection of Raji cells after transduction of pLKO-ERBB2 vector.

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0011] Raji 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.

[0012] Example 1 shRNA sequence design

[0013] (1) Designing a silent sequence according to the characteristics of human ERBB2 gene, the principle of design: 19 bp specific binding E RBB2 sequence GC content is 45<3⁄4-55<3⁄4, annealing temperature 45 °C-65 °C, the same The first base that requires the start of the sequence is G. The selected fragments were BLAST aligned with the human genome to ensure specificity.

[0014] (2) Design a 59 bp sequence ERBB2-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 ERBB2-RNAi oligonucleotide chain was synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

Example 2 Construction of pLKO-ERBB2 Vector

[0016] The synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded ERBB2-RNAi.

[0017] 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:

[0018] 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 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;

[0020] 3) conversion;

[0021] 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-ERBB2 vector extraction.

Example 3 pLKO-ERBB2 vector transduced Raji cells.

[0023] 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 μL·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 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.

[0024] Example 4 Fluorescence quantitative PCR was used to detect the expression level of ERBB2 gene.

[0025] 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.

[0026] Take cDNA 1 of each group of cells

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.

[0027] The above is a further detailed description of the present invention in conjunction 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 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.

Claims

Claim

[Claim 1] A shRNA of the human ERBB2 gene, comprising: encoding by SEQ ID NO:

The sequence shown in 1 and the ERBB2-RNAi consisting of the sequence shown in SEQ ID NO: 2.

[Claim 2] The use of the shRNA of the human ERBB2 gene according to claim 1 for the preparation of a medicament for treating an abnormality-related disease of the ERBB2 gene.

[Claim 3] The use according to claim 2, wherein: the shRN of the human ERBB2 gene

A is ERBB2-RNAi.