A double-stranded DNA molecule and its use in preparing medicine for inhibiting hepatitis B virus replication

A molecular and purpose technology, applied in the field of preparation of new drugs for the treatment of hepatitis B, can solve the problems of high price, limited siRNA methods, and unsuitability for large-scale preparation, and achieve low cost, simple preparation method, and the effect of inhibiting infection replication

Inactive Publication Date: 2006-09-20
FUDAN UNIV +1
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Problems solved by technology

For example, the methods currently used to obtain siRNA that can inhibit the replication of viral infection are limited and expensive, and are not suitable for large-scale preparation

Method used

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  • A double-stranded DNA molecule and its use in preparing medicine for inhibiting hepatitis B virus replication
  • A double-stranded DNA molecule and its use in preparing medicine for inhibiting hepatitis B virus replication
  • A double-stranded DNA molecule and its use in preparing medicine for inhibiting hepatitis B virus replication

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0052] 1. The construction process of human hepatitis B virus (HBV virus) X protein gene band stem-loop structure double-stranded RNA expression vector is as follows:

[0053] Using oligonucleotides 5'-GGAATTC ATG GCT GCT AGG CTG TG-3' and 5'-GGGGTACC GGCAGA GGT GAA AAA GTTG-3' as primers, using HBV genomic DNA as template, the HBV virus was amplified by PCR X protein gene, and EcoRI and KpnI sites were introduced at the 5' end and 3' end respectively, and then this gene was cloned into pUC-118 (purchased from TaKaRa Company) and pST (such as eg. Figure 7 , the construction process is as follows: artificially synthesized oligonucleotides

[0054] 5'-ggccgcaacggtaccaccaagcttagtggatccgaattccggct-3', this fragment was ligated between the SfiI / NotI restriction sites of the pSectag2a (Invitrogen Company) plasmid to obtain the pST plasmid. ) vector to obtain pUC-HBX and pST-HBX, cut pST-HBX with SalI to obtain a 1.5Kb fragment, clone this fragment into the SalI site of pUC-HBX, an...

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Abstract

The present invention relates to a method for preparing double-stranded RNA molecule for RNA interference of small interference RNA molecule by utilizing colibacillus fermentation and the application of these RNA molecules in preparation of medicine for curing hepatitis B. The Ids RNA or siRNA obtained by said invention is mixed with hepatitis B virus expression vector in proper proportion, and injected into the mouse body by vane caudalis hydraulic pressure method, and its result shows that the non-homologous sequence IdsRNA and siRNA basically do not inhibit the replication of hepatitis B virus in mouse body, and these RNA molecules are mixed with hepatitis B virus gene.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and biomedicine. It specifically relates to a method for preparing double-stranded RNA (dsRNA) molecules or small interfering RNA (siRNA) molecules for RNA interference by fermentation with Escherichia coli, and using these RNA molecules to prepare new drugs for treating hepatitis B. Background technique [0002] RNA interference (RNA interference, RNAi) refers to the phenomenon that double-stranded RNA (dsRNA) specifically induces the degradation of mRNA molecules with its homologous sequence, resulting in the inhibition of corresponding gene expression. It is a special post-transcriptional gene expression silencing (post transcriptional gene silence, PTGS) phenomenon. RNAi technology refers to the molecular biology technology developed based on the RNAi phenomenon to inhibit the expression of specific genes. The earliest report on the phenomenon of RNAi appeared in 1990, when two di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12P19/34A61K31/7088A61P1/16A61P31/12C12R1/19
Inventor 钱志康宣宝琴徐剑锋李琳闵太善程小伟史顺成黄伟达
Owner FUDAN UNIV
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