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siRNA molecule interfering HBV gene and application thereof

A molecular and genetic technology, applied in the field of siRNA molecules that interfere with HBV genes, can solve problems such as high recurrence rate and virus mutation

Active Publication Date: 2011-02-23
BIOMICS BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, oral nucleoside antiviral drug - lamivudine, the main function is to inhibit the replication of HBV, but the relapse rate is high after stopping the drug, and long-term use can lead to virus mutation, and the virus polymerase gene occurs after 6 months of medication. Drug resistance caused by mutations poses great challenges to clinical antiviral treatment

Method used

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  • siRNA molecule interfering HBV gene and application thereof
  • siRNA molecule interfering HBV gene and application thereof
  • siRNA molecule interfering HBV gene and application thereof

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preparation example Construction

[0030] The preparation of siRNA can use a variety of methods, such as: chemical synthesis, in vitro transcription, digestion of long-chain dsRNA, vector expression of siRNA, PCR synthesis of siRNA expression elements, etc. The emergence of these methods provides researchers with a choice. Better obtain gene silencing efficiency.

[0031] The siRNA molecules of the present invention can be used as effective ingredients of anti-HBV drugs.

[0032] As another expression form of the siRNA molecule, it can be prepared into a DNA expression box form, such as: U6 promoter-siRNA transcription template-H1 promoter.

[0033] In short, “U6 promoter-siRNA transcription template-H1 promoter” is abbreviated as “U6-siRNA transcription template-H1” or “U6-siRNA-H1” hereinafter, and they have the same meaning and scope.

[0034] For application purposes, siRNA molecules, DNA expression cassettes expressing siRNA molecules, or plasmids containing siRNA molecular expression cassettes can be used as drug...

Embodiment 1

[0044] Preparation of siRNA full-site molecular library

[0045] 1. Main instruments, reagents and materials

[0046] 1.1 Instrument: PCR instrument (ABI company); electroporation instrument (Bio-Rad company); centrifuge (Eppendorf company), long-wavelength ultraviolet lamp, etc.

[0047] 1.2 Materials and reagents: HepG22.2.15 cells (preserved by Biomec); 1kb plus DNA Ladder (invitrogen); DNase I (Roche); MnCl 2 (BBI company); phosphate linker (Sigma-aldrich company); ATP (BBI company); BSA (NEB company); BmsbI (NEB company); T4DNA ligase (NEB company); Tag DNA polymerase (Biomec company) ); Agarose (BBI Company); dNTP (Shanghai Shenggong); Phenol Chloroform Extraction Reagent (Shanghai Shenggong); Low Molecular Weight DNA Ladder (NEB Company); EcoP15I (NEB Company); T4DNA Polymerase (NEB Company); FokI enzyme (NEB company); SfiI enzyme (NEB company); competent cells (invitrogen company); pU6H1-GFP expression vector (NT Oimcs company). Gel extraction kit: QIAEX II Gel Extration Ki...

Embodiment 2

[0103] Preparation of siRNA expression cassette and screening of target sites

[0104] 1. Main instruments, reagents and materials

[0105] 1.1 Instruments: PCR instrument (ABI company); real-time quantitative PCR instrument (Bio-Rad company); gel electrophoresis equipment (Beijing Liuyi); long-wavelength ultraviolet lamp; cell incubator (Thermo company), etc.

[0106] 1.2 Materials and reagents: 1kb plus DNA Ladder (invitrogen company); Pfu DNA polymerase (Biomec company); agarose (BBI company); dNTP (Shanghai Shenggong); agarose gel purification kit (BIO Maike), Lipofectamin TM 2000 (invitrogen company), DMEM medium (Gibco company), TurboCapturemRNA kit (QIAGEN company), SensiMix TM One-Step Kit (Quantace Company), etc. Other biochemical reagents were purchased from Shanghai Shenggong.

[0107] 1.3 PCR primers (synthesized by BioMec):

[0108] 5’U6 promoter primer: 5’-AAGGTCGGGCAGGAAGAGGGC-3’

[0109] 3’H1 promoter primer: 5’-TATTTGCATGTCGCTATGTGTTCT-3’

[0110] HBV polymerase forw...

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Abstract

The invention relates to an siRNA molecule interfering an HBV gene and application thereof. In the siRNA molecule, a positive-sense strand is shown in SEQ ID NO:3, and an antisense strand is shown in SEQ ID NO:4, wherein the antisense strand can be specifically combined with mRNA of an HBV polymerase gene to degrade the mRNA, so that the translation process after transcription is interfered to fulfill the aim of resisting hepatitis B virus.

Description

Technical field [0001] The invention relates to a siRNA molecule that interferes with HBV gene and its application. Background technique [0002] Hepatitis, that is liver inflammation, has a variety of pathogenic factors-such as viruses, bacteria, parasites, chemical poisons, drugs and poisons, alcohol, etc., which invade the liver, cause liver cells to be destroyed, and liver functions are damaged. It can cause A series of physical discomforts and abnormal liver function indicators. In most cases, hepatitis refers to viral hepatitis caused by hepatitis viruses such as type A, type B, type C, type D, and type E. [0003] Hepatitis B virus (Hepatitis B virus, HBV) infection caused by hepatitis B is an infectious disease that seriously threatens human health worldwide, and it is also the most serious type of viral hepatitis. It can cause chronic liver disease and patients die The risk of liver cirrhosis and liver cancer is extremely high. [0004] HBV belongs to the Hepadnaviridae f...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61P1/16A61K48/00A61P31/20
Inventor 唐小军陆毅祥李铁军孙云成王晋康朱远源付博峻M·格拉汉姆
Owner BIOMICS BIOTECH
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