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RNA interference vector capable of simultaneously silencing multiple ligands of NKG2D, and construction method and application thereof

A technology of RNA interference and RNA structure, applied in the field of molecular biology, can solve the problems of difficult stability, short action time, and high cost, and achieve obvious effects, inhibit liver damage, and highly effective infection

Active Publication Date: 2012-06-20
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, antibody blocking is limited by the proteinaceous nature of the antibody and has a short duration of action, requiring multiple administrations and is expensive
As for the RNA interference method, since NKG2D is expressed on migratory lymphocytes, the RNA interference targeting NKG2D molecules loses its effect with the migration of lymphocytes, and the effect is difficult to stabilize

Method used

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  • RNA interference vector capable of simultaneously silencing multiple ligands of NKG2D, and construction method and application thereof
  • RNA interference vector capable of simultaneously silencing multiple ligands of NKG2D, and construction method and application thereof
  • RNA interference vector capable of simultaneously silencing multiple ligands of NKG2D, and construction method and application thereof

Examples

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Effect test

Embodiment 1

[0060] Example 1: Construction of an RNA interference vector that can simultaneously silence multiple ligands of NKG2D

[0061] The construction method of the present invention for constructing NKG2D multi-ligand RNA interference single carrier is further described in conjunction with the accompanying drawings as follows

[0062] Experimental Materials:

[0063]

[0064] experimental method:

[0065] 1. Construction of a single shRNA expression vector

[0066] (1) RNA interference sequence design:

[0067]According to experimental experience, Clontech online siRNA design tool was used to design 3 RNA interference sequences according to the gene sequences of Rae1, Mult1 and H60 published by GENEBANK (see figure 1 )

[0068] Rae1:

[0069] 1: GACCAAUGGUUACCCACAU (SEQ ID NO: 1)

[0070] 2: AGCCAAGAUCAACCUCAAG (SEQ ID NO: 2)

[0071] 3: CAGCAAGUGCUCUUUGCUA (SEQ ID NO: 3)

[0072] Mult1-

[0073] 1: GGUUAACAGGUCAGAGUCU (SEQ ID NO: 4)

[0074] 2: GACUAACACAACCGGAAAG (S...

Embodiment 2

[0143] Example 2: Application of RNA interference adenovirus shuttle vector that can simultaneously silence multiple ligands of NKG2D

[0144] The application of the present invention to construct a single carrier of NKG2D multi-ligand RNA interference is further described in conjunction with the accompanying drawings as follows

[0145] Experimental Materials:

[0146]

[0147]

[0148] experimental method:

[0149] 1. NKG2D multi-ligand (pRNAT-RMH), dual ligand (pRNAT-RMN, pRNAT-RNH, pRNAT-NMH), single ligand (pRNAT-RNN, pRNAT-NMN) constructed in the expanded culture Example 1, pRNAT-NNH) shRNA expression vector and nonsense control vector (pRNAT-NNN), the middle extraction plasmid;

[0150] 2. Take more than 10 μg of the plasmid obtained from the middle extraction and inject the mice with high pressure in the tail vein in vivo, and 3 days later, co-inject polyI:C / D-GalN (Xin Hou, Zhigang Tian, ​​et al. NKG2D-retinoic acid early inducible-1 recognition between natur...

Embodiment 3

[0155] Example 3: Application of RNA interference adenoviruses that can simultaneously silence multiple ligands of NKG2D

[0156] The application of the present invention to construct NKG2D multi-ligand RNA interference recombinant adenovirus is further described in conjunction with the accompanying drawings as follows

[0157] experimental method:

[0158] 1. Harvest NKG2D multi-ligand shRNA expression recombinant adenovirus Ad-shR(ael)M(Mult1)H(60) in Example 1 and control nonsense adenovirus Ad-shN(eg)N(eg)N( eg);

[0159] 2. Take 1*10 at different time points (3, 7, 10 days) in advance 10 The mice were injected with ifu virus into the tail vein, and then polyI:C / D-GalN was co-injected. The serum of the mice was collected 18 hours later, and the serum aminotransferase levels of the mice were detected. Experiments have shown that tail vein injection of NKG2D polyligand RNA interference recombinant adenovirus into mice 3-10 days in advance can effectively alleviate the ful...

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Abstract

The invention discloses a method for constructing a RNA interference vector capable of simultaneously silencing multiple ligands of NKG2D, and application of the RNA interference vector. The method constructs a single vector capable of encoding shRNAs of multiple ligand molecules (Rae1, Mult1 and H60) of NKG2D. The vector can be further constructed to produce an adenovirus vector encoding shRNAs of multiple ligands of NKG2D and having an infectious ability. The vector can silence different target genes at the same time, to achieve the purpose of disease treatment. The vector can be applied to lower liver local microenvironment, including lowering the expression levels of Rae1, Mult1 and H60 on the surfaces of liver parenchymal cells, and is significant in prevention and treatment of NKG2D-dependent fulminant hepatitis. The invention develops the multi-target RNA interference technique and has important practical significance in multi-target gene therapy.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a method for constructing an RNA interference vector capable of simultaneously silencing multiple ligands of NKG2D, and using the vector for treating fulminant hepatitis in mice induced by polyI:C combined with D-GalN, Significant curative effect can be achieved. Background technique [0002] RNA interference (RNAi) refers to the specific post-transcriptional gene silencing phenomenon induced by double-stranded RNA homologous to the target gene. a mechanism. Since its discovery in 1998, RNA interference technology has been used by researchers to specifically knock out or turn off the expression of specific genes. It has been widely used and has achieved rapid development. It was rated as one of the top ten in 2001 by "Science" magazine. It is one of the scientific progresses, and ranked first among the top ten scientific progresses in 2002. There are five main methods for the pr...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/113C12N15/861C12N15/66C12N7/01A61K48/00A61P1/16
Inventor 田志刚黄玫孙汭魏海明
Owner UNIV OF SCI & TECH OF CHINA
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