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B-type hepatitis virus micro ribonucleic-acid interference moleculars and its designing method

A microRNA, hepatitis B virus technology, applied in antiviral agents, sugar derivatives, pharmaceutical formulations, etc., can solve problems such as infection, human cancer virus, etc.

Inactive Publication Date: 2007-08-29
GUILIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

Lack or inappropriate expression of these miRNAs can lead to cancer and viral infections in humans

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0016] The design of embodiment 1 anti-HBV miR RNAi

[0017] 1. Use BioEdit and its auxiliary software Clustal W to compare 20 HBV sequences, including 10 HBV virus sequences isolated from Chinese, and get two conserved sequences from the X and S regions of HBV. Among them, the conserved sequence of the X region of HBV is 465bp in length and has the DNA sequence shown in sequence-5 in the sequence listing; the conserved sequence in the S region is 1203bp in length and has the DNA sequence shown in sequence-6 in the sequence listing.

[0018] 2. Go to Invitrogen's RNAi design software web page (https: / / rnaidesigner. invitrogen.com / rnaiexpress / ), paste the screened conserved sequence into the sequence box, select the "miR RNAi" type in the Target Design Options column (Target Design Options), and then follow the software guide to complete the anti-HBV miR RNAi design. The given Top Strand, Bottom Strand and ds Oligo represent template strand, coding strand and miR RNAi, res...

Embodiment 2

[0019] Example 2 Synthesis of anti-HBV miR RNAi

[0020] Anti-HBV miR RNAi template strand and coding strand oligonucleotides were synthesized, respectively. The two oligonucleotides are annealed to form a complementary double-stranded anti-HBV miR RNAi, and the protruding single-strand composed of four nucleotides at the 5' end is used for cloning anti-HBV miR RNAi.

Embodiment 3

[0021] Example 3 Cloning and packaging of anti-HBV miR RNAi

[0022] Using the kit from Invitrogen (BLOCK-iT TM Lentiviral Pol II miR RNAiExpression System) will act on the HBV X protein and coat protein 2 anti-HBV miR RNAi respectively or tandem cloned into the expression vector (pcDNA TM 6.2-GW / miR or pcDNA TM 6.2-GW / EmGFP-miR), expression clones were generated. Then, apply the adapter vector pDONR TM 221 The anti-HBV miR RNAi was transferred to the pLenti6 / V5-DEST vector to generate recombinant Lenti virus expression clones, which were then packaged into recombinant Lenti virus particles.

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Abstract

The present invention discloses an anti-hepatitis B virus minute ribonucleic acid interference molecule. It is characterized by that two double-stranded DNA oligonucleotides with gene target inactivation action can respectively act on X protein gene and coat protein gene of HBV, in which anti-HBV mi RNAi acting on X protein and coat protein of HBV respectively has DNA sequence structures shown by sequence-1 and sequence-2 in sequence table, and their action target points respectively have DNA sequence structures shown by sequence-3 and sequence-4 in the sequence table. Besides, said invention also provides the designing method of said anti-hepatitis B virus minute ribonucleic acid interference molecule and its concrete steps.

Description

technical field [0001] The invention relates to antiviral gene therapy, in particular to an anti-HBV microribonucleic acid interference molecule (miR RNAi) and a design method thereof. Background technique [0002] Hepatitis B is a worldwide infectious disease caused by HBV. According to World Health Organization estimates, more than 2 billion people in the world are infected with HBV, of which 350 million are chronic HBV carriers, and China accounts for about 1 / 3. At least 1.2 million patients with chronic HBV infection die from HBV-related diseases every year. Although vaccines play an important role in controlling HBV infection, there are still a large number of patients in need of effective treatment. Currently internationally recognized anti-HBV drugs such as α-interferon (IFN-α), lamivudine (lamivudine, 3TC) and so on are not satisfactory. Therefore, the research and development of effective drugs and methods for preventing and treating hepatitis B is still an urgen...

Claims

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Application Information

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IPC IPC(8): A61K48/00C12Q1/68C07H21/00C12N15/11A61P1/16A61P31/20C12N15/10C12N15/113
Inventor 刘永明
Owner GUILIN MEDICAL UNIVERSITY
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