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Construction and application of IFNAR2 gene-targeting RNA interference expression vector

A technology for expressing vectors and genes, applied in the field of molecular biology, can solve the problem that there is no guidance for IFNAR2 gene interference research.

Active Publication Date: 2014-07-30
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

At present, the use of RNA interference to study gene function has become one of the most important methods in the study of gene function, but there is no guidance for the study of IFNAR2 gene interference

Method used

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  • Construction and application of IFNAR2 gene-targeting RNA interference expression vector
  • Construction and application of IFNAR2 gene-targeting RNA interference expression vector
  • Construction and application of IFNAR2 gene-targeting RNA interference expression vector

Examples

Experimental program
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Embodiment 1

[0032] Embodiment 1 is aimed at the construction of the RNA interference plasmid vector of IFNAR2 gene

[0033] 1. Design and synthesis of oligonucleotide sequences expressing siRNA of IFNAR2 gene

[0034] According to the nucleotide sequence of the IFNAR2 gene (GenBank No: HQ665551), the DNA sequences of several siRNAs targeting the IFNAR2 gene mRNA sequence were designed and screened, and they were separated to form an auxiliary sequence (loop region) of a "hairpin" structure. The positive-sense strand of the "hairpin" siRNA was added at both ends for pSilencer4.1-CMV neo (purchased from Ambion, schematic diagram as shown in figure 1 shown), the complementary sequence of the sense strand is used as the antisense strand, and the linker sequences for pSilencer4.1-CMV neo are added to the two ends of the antisense strand respectively, and the two anneal to form a linker sequence for pSilencer4.1- shRNA for the linker of CMV neo.

[0035] Further screening obtained three pairs...

Embodiment 2

[0050] Example 2 Detection of interference efficiency against IFNAR2 gene mRNA interference plasmid

[0051] 1. ST cell (porcine testis cell) culture, plasmid transfection and extraction of total RNA and total protein

[0052] 1. The ST cell line comes from the China Center for Type Culture Collection (CCTCC, Wuhan), and is subcultured and preserved by our laboratory according to conventional methods.

[0053] 2. Using Promega's TransFast TM Transfection Reagent (Promega Product No. E2431) is used to transfer three kinds of interfering plasmid vectors that interfere with IFNAR2 gene expression into ST cells. The specific transfer steps refer to the actual instructions, which are briefly described as follows:

[0054] (1) Cell seeding: In order to obtain the best transfection efficiency, the cell density should be 50-80%. In a 24-well cell plate, the ideal condition is to inoculate 8×10 cells per well 18-24 hours before transfection. 4 ~2×10 5 cells;

[0055] (2)TransFast...

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Abstract

The invention belongs to the field of molecular biology, and discloses construction and application of an IFNAR2 gene-targeting RNA interference expression vector. The method disclosed by the invention comprises the step of inserting IFNAR2 gene-targeting siRNA oligonucleotide fragments obtained by screening in a vector system to construct an IFNAR2 gene-targeting RNA interference plasmid vector. Experiments indicate that the expression vector constructed by the method disclosed by the invention is capable of efficiently expressing a specific siRNA sequence in ST cells, and the sequence is capable of being combined with mRNA of the IFNAR2 gene, so that the expression quantity of the IFNAR2 gene is remarkably reduced, and the purpose of inhibiting the expression of the IFNAR2 gene is achieved; a more reliable and convenient research means is provided for further research on the biological functions of the IFNAR2 gene, and the foundation is laid for research on the function of an IFNAR2 receptor in virus infection.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to the construction and application of an RNA interference expression vector targeting the IFNAR2 gene. Background technique [0002] The antiviral effect of interferon is that after the interferon molecule binds to the interferon receptor, it transmits the signal to the nucleus, 2,5-adenylate synthetase and protein kinase are activated, and finally blocks the translation of viral protein and the synthesis of viral RNA . Interferon receptors are the initial proteins of the interferon chain reaction. Human interferon receptors are divided into type I interferon receptors (which bind to interferon α and β) and type II interferon receptors (which bind to interferon γ to produce specific sensitivity). In addition, it has been confirmed that type II interferon receptors are also sensitive to interferon α and β, and both the α and β subtypes of interferon are sensitive to in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/64
Inventor 张建峰沈海燕张春红郭鹏举陈琴苓卢宇
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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