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RNA interference target for treating hepatitis B virus infection

A technology of RNA interference and target sequence, applied in DNA/RNA fragments, gene therapy, antiviral agents, etc., can solve the problems of differences in inhibition efficiency

Active Publication Date: 2009-12-16
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, not all RNAi targets that meet the conventional design requirements can effectively inhibit the expression of target genes, and the inhibition efficiency varies among different targets

Method used

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  • RNA interference target for treating hepatitis B virus infection
  • RNA interference target for treating hepatitis B virus infection
  • RNA interference target for treating hepatitis B virus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] Example 1. Design and Construction of siRNA Expression Vector Plasmids Targeting HBV

[0138] The design of the RNA interference target sequence targeting HBV: take the HBV reference sequence as the target sequence, select a well-conserved region to walk and design the siRNA sequence; perform a BLAST search on the siRNA sequence obtained from the primary selection in GenBank, select and non- The target sequence has 3 or more bases different sequences as candidate sequences.

[0139] Construction of siRNA expression plasmid: In this embodiment, the pSUPER vector (Cat.No VEC-PBS-0001 / 0002 of oligoengine company) was used as an example to construct the expression vector of siRNA. For the specific construction process, please refer to the pSUPER vector experiment guide of the company ( www.oligoengine.com), the construction brief process can be seen schematically figure 1 . The primers with RNA interference sequences were synthesized, and the complementary primers were an...

Embodiment 2

[0141] Example 2. Co-transfection experiment screening to obtain RNA interference targets that can inhibit HBV

[0142] pN31-N10 plasmid (this plasmid contains about 1.4 times the HBV genome (GenBank accession number: AY707087). The construction method is briefly described as follows: mammalian cell expression vector plasmid pCDNA3.1 (+) (Invitrogen Company Cat.No V790-20) and After SpeI digestion, the recovered vector was self-ligated to obtain plasmid pN31. Respectively, N1f (ACT AGT GGA TCC TTC GCGGGA CGT CC) / N1r (GAA TTC CAC TGC ATG GCC TGA G), N2f (GAA TTCCAC TGC CTT CCA CC) / N2r (GAT ATC CAC ATT GTG TAA ATG G), N3f (GAT ATC CTG CCT TAA TGC CTT TG) / N3r (GGG CCC ACA AAT TGTTGA CAC C) these 3 pairs of primers were used for PCR amplification of HBV genome, and the products were cloned into T The vectors were pTN1, pTN2, and pTN3. The fragment obtained after pTN3 was digested with EcoRV and ApaI was ligated with the vector recovered after pN31 was digested with EcoRV and ApaI ...

Embodiment 3

[0189] Example 3. Construction of recombinant viral expression vector expressing siRNA

[0190] We take the construction of a recombinant lentiviral expression vector that can express siRNA targeting siHBV7 and siHBV12 as an example.

[0191] Expression vector: In this example, the expression vector pDEST-MR of the lentiviral system we used (patent application number: 200510112917.1; publication number: CN1948475) contains an expression cassette that can be used to express siRNA controlled by the H1 promoter.

[0192] Construction method of expression vector pDEST-siHBV7, pDEST-siHBV12:

[0193] Synthesize siHBV7 and siHBV12 gene fragments respectively (the RNA interference target sequence siHBV7 (SEQ ID NO: 7) and siHBV12 (SEQ ID NO: 12) shown in Example 2 are respectively included here as examples, but other present inventions can also be included RNA interference target sequence provided), an Age I site was added to the 5' end of the fragment, and a Sma I site was added to...

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Abstract

The invention relates to an RNA interference target for treating hepatitis B virus infection with 42 different targeting hepatitis B viruses (HBV), which can be used for preparing a medicament for treating the hepatitis B virus infection. The invention provides a recombinant expression vector of siRNA and / or miRNA and / or ribozyme and / or antisense oligonucleotide, which can be used for expressing targeting HBV. The invention relates to a cell which has the function of inhibiting the expression of HBV virus gene and can express and / or is induced with the siRNA and / or the miRNA and / or the ribozyme and / or the antisense oligonucleotide and / or the medication obtained according to the RNA interference target.

Description

technical field [0001] The present invention relates generally to the fields of molecular biology, cell biology and gene therapy. More specifically, the present invention relates to 42 targets of RNA interference (RNAi) that can be used for the treatment of hepatitis B virus infection and the recombinant expression vectors using these targets and the recombinant expression vectors that can be used in hepatitis B obtained in various ways using these targets. Drugs and methods for the treatment of hepatitis virus infection. Background technique [0002] Hepatitis B virus infection is one of the most important public health problems worldwide. Related diseases caused by HBV infection include acute or chronic hepatitis B, and liver cirrhosis (HC) and hepatocellular carcinoma (HCC) caused by the further development of chronic hepatitis B. The range of HBV infection is very wide. At present, there are about 400 million HBV infected people in the world, and about 2 billion people...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85C12N15/867C12N9/22C12N5/10A61K31/713A61K38/46A61K48/00A61P31/20C12N15/113
Inventor 程通张雅丽蔡毅君苗季张军夏宁邵
Owner XIAMEN UNIV
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