ShRNA sequence for targeted silencing of BRAF gene and application of shRNA sequence

A technology of targeted silencing and gene sequence, applied in the field of biomedicine, can solve the problem of short time to play, achieve huge social and economic benefits, inhibit human fibroblast aging, and have strong specificity.

Pending Publication Date: 2021-01-12
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the expression of siRNA in cells is transient, and the time to play a role is relatively short.

Method used

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  • ShRNA sequence for targeted silencing of BRAF gene and application of shRNA sequence
  • ShRNA sequence for targeted silencing of BRAF gene and application of shRNA sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Construction of lentiviral vector

[0019] Obtain the mRNA sequence of the BRAF gene (NM_004333.6) according to the GenBank database (http: / / www.ncbi.nlm.nih.gov / genbank), and select the target sequence according to the shRNA design principle. These two oligonucleotides The sequences of the chains are respectively marked as BRAF-1 and BRAF-2; wherein, the target sequence of BRAF-1 is shown in SEQ ID NO.1, namely: 5´-Tcggctgcggaccctgccatt-3´ (the corresponding coding region is 335-355 nucleotide position); the target sequence of BRAF-2 is shown in SEQ ID NO.2, namely: 5´-AtctccaggacctcagCGAGA-3´ (the corresponding coding region is the 1479-1499 nucleotide position).

[0020] Use the primer software to design the shRNA sequence, and perform sequence alignment on the NCBI website to verify its specificity. According to the target sequence BRAF-1, design and synthesize the following oligonucleotide sequence: the sense strand of shBRAF-1 shBRAF-1- F is shown in S...

Embodiment 2

[0022] Example 2: Preparation of cell lentivirus and transfection of cells with lentivirus

[0023] The lentivirus was packaged according to the instructions of Lipofectamine 2000 reagent. The cells transfected with the two lentiviral vectors prepared in Example 1 were used as the experimental group, and the cells transfected with the pLKO.1-TRC lentiviral empty vector were used as the control group;

[0024] Add 0.75 μg pLP1, 0.35 μg pLP2, 0.49 μg pLPSVG, and 0.61 μg lentiviral vector pLKO.1-TRC-shBRAF-1 to 0.5 mL low-serum OPTI-MEM medium, mix gently, and incubate at room temperature for 5 min, to obtain liquid A, ready to use.

[0025]Take 9 μL of liposome Lipofectmine 2000 and add it to 0.5 mL of OPTI-MEM medium, mix gently, and place at room temperature for 5 minutes to obtain solution B, which is ready for use.

[0026] Mix solution A and solution B at a ratio of 1:1 to obtain a mixed solution, and incubate at room temperature for 20 min; gently and thoroughly mix the ...

Embodiment 3

[0029] Example 3: Detection of protein expression level of BRAF gene in IMR90 cells by Western blotting.

[0030] The virus-infected IMR90 cells were collected, the total protein was extracted, the protein concentration was determined, the protein was denatured, and the gel was transferred to the membrane. After the membrane was blocked with 5% skimmed milk for 1 h, the BRAF antibody and β-actin antibody were used to incubate overnight, and then pepper Incubate the goat anti-mouse antibody against root peroxidase for 1 h at room temperature. After antibody incubation, wash the membrane with TBS-T solution at room temperature for 3 times, 4 min each time, and finally place the membrane in a darkened cassette and take it to the darkroom. During exposure, the film is fixed on the film, and the protein bands will be printed on the film under the action of the luminescent solution and the developing solution. The surviving IMR90 cells in the experimental group and the control group...

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Abstract

The invention belongs to the field of biological medicines, and particularly relates to two shRNA sequences for targeted silencing of a BRAF gene and application of the two shRNA sequences. ShRNA molecules aiming at the BRAF gene are designed and synthesized, and an oligonucleotide sequence for constructing shRNA is designed. The molecules are combined with mRNA of the BRAF gene, transcription ofthe molecules is effectively interfered, expression of the BRAF gene in IMR90 cells is reduced, and therefore, EGF induced human fibroblast aging is inhibited. The shRNA sequences have very importantsignificance in aging prevention / treatment, the shRNA molecules are further developed into clinical treatment medicine, and the shRNA sequences have huge potential value in future anti-aging targetedgene medicine research and development and aging related disease treatment and are expected to be applied to preparing an anti-aging medicine which is efficient, high in specificity and small in sideeffect.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an shRNA sequence targeting silencing of the BRAF gene and an application thereof. Background technique [0002] B-Raf proto-oncogene serine / threonine kinase (BRAF) gene, encoding B-Raf protein, belongs to the serine / threonine protein kinase of raf / mil family, and participates in the regulation of Ras / Raf / MAP / ERKs signaling pathway, in Plays an important role in cell growth, division, and differentiation. Ras / Raf / MAP / ERKs signaling pathway is the key signaling pathway of cell senescence and SASP factor-induced cell senescence. When cells encounter external stimuli and overactivate Ras, this signaling pathway is activated, and then activates the downstream senescence signaling pathway to induce cell senescence. BRaf is a key protein in this signaling pathway, and inhibiting BRaf can effectively block this signaling pathway, thereby inhibiting cell senescence. [0003] RNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C07K14/82C12N15/113C12N15/867A61K31/7105A61K48/00A61P39/06
CPCC12N9/12C07K14/82C12N15/1135C12N15/1137C12N15/86C12Y207/11001A61K31/7105A61P39/06C12N2740/15043C12N2310/14C12N2310/531
Inventor 朱炫垒尚东胜刘晗青
Owner JIANGSU UNIV
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