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A kind of siRNA molecule targeting hepatitis B virus gene and application thereof

A hepatitis B virus, molecular technology, applied in DNA/RNA fragments, gene therapy, antiviral agents, etc., can solve problems such as high recurrence rate and virus mutation

Active Publication Date: 2015-09-02
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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  • A kind of siRNA molecule targeting hepatitis B virus gene and application thereof
  • A kind of siRNA molecule targeting hepatitis B virus gene and application thereof
  • A kind of siRNA molecule targeting hepatitis B virus gene and application thereof

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preparation example Construction

[0031] Various methods can be used to prepare siRNA, such as: chemical synthesis, in vitro transcription, enzyme-cut long-chain dsRNA, vector expression of siRNA, PCR synthesis of siRNA expression elements, etc. The emergence of these methods provides researchers with options. Better access to gene silencing efficiency.

[0032] The siRNA molecule of the present invention can be used as an active ingredient of an anti-HBV drug.

[0033] As another expression form of the siRNA molecule, it can be prepared in the form of a DNA expression cassette, such as: U6 promoter-siRNA transcription template-H1 promoter.

[0034] In short, "U6 promoter-siRNA transcription template-H1 promoter" is abbreviated as "U6-siRNA transcription template-H1" or "U6-siRNA-H1" hereinafter, and they represent the same meaning and scope.

[0035] For the purpose of application, the siRNA molecule, the DNA expression cassette expressing the siRNA molecule, or the plasmid containing the siRNA molecule expr...

Embodiment 1

[0045] Preparation of siRNA all-site molecular library

[0046] 1. Main instruments, reagents and materials

[0047] 1.1 Instruments: PCR instrument (ABI Company); electrotransfer instrument (Bio-Rad Company); centrifuge (Eppendorf Company), long-wavelength ultraviolet lamp, etc.

[0048] 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 (Biometics ); Agarose (BBI company); dNTP (Shanghai Sangong); Phenol chloroform extraction reagent (Shanghai Sangong); 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 Extraction Kit...

Embodiment 2

[0104] Preparation of siRNA expression cassette and screening of target sites

[0105] 1. Main instruments, reagents and materials

[0106] 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.

[0107] 1.2 Materials and reagents: 1kb plus DNA Ladder (Invitrogen); Pfu DNA polymerase (Biometics); agarose (BBI); dNTP (Shanghai Sangong); Maike Corporation), Lipofectamin TM 2000 (invitrogen company), DMEM medium (Gibco company), TurboCapture mRNA kit (QIAGEN company), SensiMix TM One-Step Kit (Quantace Company), etc. Other biochemical reagents were purchased from Shanghai Sangong.

[0108] 1.3PCR primers (synthesized by Biometics):

[0109] 5'U6 promoter primer: 5'-AAGGTCGGGCAGGAAGAGGGC-3'

[0110] 3'H1 promoter primer: 5'-TATTTGCATGTCGCTATGTGTTCT-3'

[0111] HBV polymerase forward primer: 5'-TGTGGTTATCCTGC...

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Abstract

The invention relates to a double-stranded small interfering RNA (siRNA) molecule of a targeted hepatitis B virus (HBV) polymerase gene and application thereof. The siRNA molecule has the sense strand of SEQ ID NO:3 and the antisense strand of SEQ ID NO:4, wherein the antisense strand can be specifically bonded with the mRNA of the HBV polymerase gene to degrade the mRNA, thereby interfering with a translation process after transcription to fulfill the aim of resisting HBV.

Description

technical field [0001] The invention relates to a siRNA molecule targeting hepatitis B virus gene and application thereof. Background technique [0002] Hepatitis, that is, inflammation of the liver, has a variety of pathogenic factors - such as viruses, bacteria, parasites, chemical poisons, drugs and poisons, alcohol, etc., which attack the liver, destroying the cells of the liver and impairing the function of the liver. It can cause A series of symptoms of discomfort in the body, as well as abnormalities in liver function indicators. In most cases, hepatitis refers to viral hepatitis caused by hepatitis A, B, C, D, E and other hepatitis viruses. [0003] Hepatitis B caused by hepatitis B virus (Hepatitis B virus, HBV) infection is an infectious disease that seriously threatens global human health, and is also the most serious type of viral hepatitis. It can cause chronic liver disease, and patients die of The risk of cirrhosis and liver cancer is extremely high. [000...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K48/00A61P1/16A61P31/20
Inventor 李铁军陆毅祥孙云成唐小军王晋康朱远源付博峻M·格拉汉姆
Owner BIOMICS BIOTECH