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Construction method of bismall molecule interference RNA carrier and its application

A technology of small molecule interference and construction method, which is applied in the field of double siRNA vector construction to achieve the effects of no toxic side effects, strong specificity and inhibition of replication

Inactive Publication Date: 2006-11-29
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using the same vector to express two kinds of double small molecule interfering RNA vectors to inhibit the antiviral treatment of the mixed infection of the two viruses has not been reported so far.

Method used

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  • Construction method of bismall molecule interference RNA carrier and its application

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Embodiment 1

[0029] Example 1. Construction of dual siRNA production vectors

[0030] 1) Construction of dual siRNA production vector Primer synthesis: a pair of primers were designed, upstream primers:

[0031] 5'-GCTGATGACGTCAGTGGAAAGACGCG-3' downstream primer:

[0032] 5'-TCAGCGAATTCACGCCAAGCTTTTCC-3'. The upstream primer contains a sequence of U6 promoter and introduces a restriction endonuclease site of AatII, and the downstream primer is a sequence between 788 and 807 on HBssiRNA, and introduces a restriction endonuclease site of EcoRI. Then send it to Invitrogen Company for synthesis.

[0033] 2) siRNA-generated template PCR amplification: HBssiRNA was used as a template, and the above-mentioned synthesized primers were used to amplify to obtain a PCR product. Electrophoresis was performed on the PCR product with 1% agarose gel, and the DNA purification and recovery kit from Qiagen Company was used to purify.

[0034] 3) Cloning and sequencing: The recovered DNA fragments AatII ...

Embodiment 2

[0035] Example 2. Inhibition of the replication of HBV by double siRNA production vectors

[0036] 1) Preparation of transfection plasmid solution: 1 mg-10 mg of HBV-HIV siRNA plasmid was dissolved in 1 mL of sterilized water.

[0037] 2) Transfection: Take 1uL-10uL of the above solution plus 5uL-10uL 5mg / mL cationic liposome (Xiamen Sun Horse Biotechnology Co., Ltd.) to transfect HepG2.2.15 cells grown on a 6-well tissue culture dish (with A vector expressing an irrelevant siRNA served as a negative control).

[0038] 3) Extraction of HBV replication intermediates: HepG2.2.15 cells cultured in a 6-well plate were washed once with cold PBS (pH7.4) after being cultured for 48 hours, and then 300uL / well of cell lysate was added (the main component of the lysate was 50mMT -Cl, pH7.4, 1mM EDTA, 1%Nonidet P-40) placed at room temperature (20-25°C) for 15min to 30min to fully lyse; transfer the cell lysate to a 1.5mLEP tube and centrifuge at 14,000rpm for 1min; Add 10mM MgCl and 1...

Embodiment 3

[0040] Example 3. Inhibition of HIV replication by dual siRNA production vectors

[0041] 1) Preparation of transfection plasmid solution: 1 mg-10 mg of HBV-HIV siRNA plasmid was dissolved in 1 mL of sterilized water. pNL4-3 (purchased from NIH in the United States) pNL4-3 is an infectious plasmid of HIV-1, which can replicate and produce infectious virus particles in HEK293T cells. Also use sterilized water to make a solution of 1mg-10mg / mL.

[0042] 2) Transfection: Take 1uL-5uL HBV-HIV siRNA plasmid solution and 1uL-5uL pNL4-3 plasmid solution plus 5uL-10uL 5mg / mL cationic liposome (Xiamen Taiyangma Biotechnology Co., Ltd.) to co-transfect and grow in 6 wells HEK293T cells on tissue culture dishes (and a vector expressing an irrelevant siRNA as a negative control).

[0043] 3) Determination of HIV inhibition efficiency: After the transfected cells were cultured for 72 hours, the concentration of HIV-1p24 protein in the cell culture supernatant was measured with a p24 prot...

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Abstract

The invention discloses a construction method and application for a double micro-molecule disturbance RNA carrier. It includes the following steps: compounding construction primer of double siRNA producing carrier, PCR expanding of siRNA producing module; cloning and sequencing. The invention has the advantages of simple method to construct siRNA carrier, convenient to operate, and high RNA disturbance efficiency. It could make the gene expression that has the same source sequence of siRNA dumbness and would not influence to other functions of the gene. It has strong specificity, and no poison function. It is a good method to cure virus mixed infection by the double siRNA.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a method for constructing a dual siRNA carrier, and the use of the carrier in the preparation or prevention of antiviral drugs. Background technique [0002] 70% of human infectious diseases are caused by viruses. Viruses are highly infectious and spread widely. Influenza, atypical pneumonia, measles, Japanese encephalitis, hepatitis B, polio, rabies, AIDS and other viral diseases Diseases seriously threaten human health and affect the development of the world economy. Take hepatitis B virus and human immunodeficiency virus as examples: Hepatitis B virus (HBV) infection can cause acute and chronic liver lesions. Every year, more than 1 million people die from liver cirrhosis or liver cancer caused by hepatitis B virus infection all over the world. The virus infects as many as 50,000 people each year. Worldwide, about 2...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/11A61K48/00A61P1/16A61P31/14C12N15/113C12N15/66
Inventor 吴建国邬开朗朱应
Owner WUHAN UNIV
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