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Small interfering RNA, short hairpin RNA, carrier and application for mammalian r-spondin3 gene target

A technology of short hairpins and carriers, applied in the direction of DNA/RNA fragments, carriers, nucleic acid carriers, etc., can solve the problems of short-term inhibition of target gene expression, low transfection efficiency, and non-permanent expression of siRNA, and achieve the goal of promoting the recovery process Effect

Active Publication Date: 2018-04-20
JIAXING NO 1 HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Initially, RNA interference sampling was the method of synthesizing siRNA in vitro, but its application was limited by the disadvantages of low transfection efficiency, persistent expression of siRNA transferred into cells, and short-term inhibition of target gene expression.

Method used

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  • Small interfering RNA, short hairpin RNA, carrier and application for mammalian r-spondin3 gene target
  • Small interfering RNA, short hairpin RNA, carrier and application for mammalian r-spondin3 gene target
  • Small interfering RNA, short hairpin RNA, carrier and application for mammalian r-spondin3 gene target

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 siRNA sequence design

[0031] Apply the principles of siRNA design (Petri S, siRNA design principles and off-target effects. Methods Mol Biol. 2013; 986:59-71) to screen siRNA sequences: (1) start from the AUG start codon of the transcript (mRNA), search for "AA" double-linked sequence, and write down the 19-23 nucleotide sequence at its 3' end as the candidate target site of siRNA; (2) the GC content of the siRNA sequence is between 45% and 55%; (3) the length 19-23 bp; (4) no inverted repeat sequence; (5) no more than 9 consecutive GC sequences; (6) the 5' end of the sense strand is preferably G / C; (7) antisense The 5' end of the strand is preferably A / U; (8) the 15-19 bases of the sense strand preferably contain more than 3 A / U; (9) the 5' end of the antisense strand is best among the 7 bases There are more than 5 A / U.

[0032]Use Blast (www.ncbi.nlm.nig.gov / Blast) to perform homology analysis on the candidate sequence and the genome database to ensure...

Embodiment 2

[0036] Example 2 shRNA design and synthesis

[0037] Apply the shRNA design principles (Sun G, Molecular Properties, Functional Mechanisms, and Applications of Sliced ​​siRNA. Mol Ther Nucleic Acids. 2015 Jan 20; 4:e221.), according to the above siRNA-R-Spondin3 sequence, design the shRNA sequence: (1) two Both ends of the complementary oligonucleotides must have restriction enzyme sites; (2) A C is immediately below the restriction site to ensure transcription; (3) the shRNA target sequence starts with a G base and ends with Ensure RNA polymerase transcription; (3) The loop adapter sequence inserted by shRNA should be close to the middle of the oligonucleotide, and 5`-TCAAGAG-3` is the most effective; (4) shRNA can only have a specific and unique loop structure; (5) 5-6 Ts are inserted at the end of the shRNA fragment to ensure that RNA polymerase III terminates transcription; (6) the G / C content is 40% to 50%; (7) the shRNA sequence should avoid more than three consecutive...

Embodiment 3

[0042] Example 3 Plasmid vector construction

[0043] The above shRNA-R-Spondin3 was synthesized into an oligo DNA single-stranded fragment and annealed to form a double-stranded DNA, which was connected to the pGCL-GFP vector (Shanghai Jikai) to construct the plasmid vector pGCL-GFP-shRNA-R-Spondin3 (see figure 1 ). The plasmid vector will transcribe shRNA in the cell, and produce siRNA-R-Spondin3 targeting the R-Spondin3 gene target after intracellular modification. The annealing and connection process is as follows:

[0044] 1. Annealing into double-stranded DNA

[0045] 1) Establish the following annealing reaction system (room temperature) in a 0.5ml sterile centrifuge tube:

[0046]

[0047] 2) Incubate at 95°C for 4 minutes and at 70°C for 10 minutes;

[0048] 3) Take out the centrifuge tube, place it at room temperature for 5-10 minutes, and cool to room temperature;

[0049] 4) Briefly centrifuge and mix well.

[0050] 2. Ligation of double-stranded DNA to ...

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Abstract

The invention discloses a small interfering RNA, a short hairpin RNA and a vector aiming at a target spot of a mammalian R Spondin3 gene and application thereof. The small interfering RNA comprises a positive-sense strand and an antisense chain. The short hairpin RNA can be synthesized by a chemical synthesis method based on the small interfering RNA. The short hairpin RNA can be further linked to a virus expression vector or a non-viral expression vector to form a vector targeting the R-Spondin3 gene target spot of the mammalian. The virus expression vector is a slow virus vector or adenovirus vector, and the non-viral expression vector is a plasmid vector. The vector of the short hairpin RNA can be used for preparing the gene medicine for treating liver fibrosis. The RNA interference fragment designed by the invention aiming at R-Spondin3 gene target spot can promote the activated hepatic stellate cells to go back into a stationary state or apoptosis, so as to effectively promote the recovery of liver fibrosis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a small interfering RNA, a short hairpin RNA, a carrier and an application for a mammalian R-Spondin3 gene target. Background technique [0002] Hepatic fibrosis is the wound healing response of the liver to chronic liver injury caused by various reasons, resulting in a large amount of fibrous tissue hyperplasia and precipitation in the hepatic lobule and portal area, and the pathological feature is the synthesis of various components of the extracellular matrix mainly composed of collagen Increased, relatively insufficient degradation, but did not form interlobular septa, if further developed into cirrhosis. Liver fibrosis is a reversible process, and the prevention and early intervention of liver fibrosis are the best measures to stabilize the disease and prevent the development of liver fibrosis to cirrhosis and liver cancer. [0003] Hepatic stellate cells are the main cells tha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/85A61K48/00A61K31/713A61P1/16
CPCC12N15/11C12N15/113C12N15/85C12N2310/141C12N2310/531C12N2320/30C12N2330/30C12N2800/107
Inventor 虞玲华
Owner JIAXING NO 1 HOSPITAL
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