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Small interference RNA molecule SiRNA capable of attacking human hepatitis B virus and application thereof

A hepatitis B virus, small interference technology, applied in the field of nucleic acid, can solve the problems of uncertain effect of lamivudine, low efficiency of hepatitis B, poor specificity of hepatitis B virus, etc.

Inactive Publication Date: 2007-06-27
丽水瑞德佳生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Their common shortcoming is poor specificity against hepatitis B virus, and the effective rate (30%-40%) of both treatment hepatitis B is lower, and the negative conversion rate to the surface antigen of hepatitis B virus is lower, al Interferon is 6%, and the effect of lamivudine is uncertain

Method used

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  • Small interference RNA molecule SiRNA capable of attacking human hepatitis B virus and application thereof
  • Small interference RNA molecule SiRNA capable of attacking human hepatitis B virus and application thereof
  • Small interference RNA molecule SiRNA capable of attacking human hepatitis B virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1 Synthesis of siRNA

[0104] The synthesis of siRNA can be entrusted to commercial companies that openly carry out synthesis business, such as Dharmacon in the United States, specifically through www.dharmacon.com It was known that all siRNA molecules were deprotected at the 2-position, desalted, purified, and annealed to form double strands, and then dissolved in DEPC-treated distilled water. The synthetic methods used are general methods, such as the methods disclosed in the following documents: Scaringe SA, Wincott FE, and Caruthers MH. Novel RNA Synthesis Method Using 5′-Silyl-2′-Orthoester Protecting Groups.J Am Chem Soc 1998 ;120:11820-11821.

[0105]The preparation method of the small interfering RNA molecule (SiRNA) provided by the present invention can also adopt the existing solid-phase chemical synthesis method. This method is available in: Wincott F, DiRenzo A, Shaffer C, Grimm S, Tracz D, Workman C, Sweedler D, Gonzalez C, Scaringe S and Usman N....

Embodiment 2

[0115] Example 2 Transfection of SiRNA

[0116] 1. The cell model of hepatitis B virus: the cell model of hepatitis B virus is HepG2.2.15 cell line. This cell line was formed in 1987 when Dr. Acs' laboratory transformed the full length of AYW subtype hepatitis B virus into HepG2. It has been the standard cell used to screen anti-hepatitis virus drugs in vitro. The cells were cultured with RPMI 1640 medium containing 10% fetal bovine serum in vitro and were regularly screened with G418 to maintain high expression of hepatitis virus. (Sells MA, Chen ML, Acs G. Production of hepatitis B particles in HepG2 cells transfected with cloned hepatitis B virus DNA. PNAS1987; 84: 1005-1009.)

[0117] 2. SiRNA transfection: SiRNA was transferred into HepG2.2.15 cells by means of Oligofectamine from Invitrogen (www.invitrogen.com), and the specific steps were carried out according to the instructions. Briefly described as follows, 5x10 4 The HepG2.2.15 cells were seeded on 24-well cultur...

Embodiment 3

[0120] Example 3 Inhibitory effect of siRNA on virus replication and pathogenicity-related protein synthesis in hepatitis B virus cell model

[0121] 1. Selection of the hepatitis B virus cell model: In this example, the hepatitis B virus cell model was used as the standard HepG.2.2.15 cell line, which was formed by screening after liver cancer cells were transfected with the whole genome of the AYW subtype of hepatitis B virus. Hepatitis virus can stably replicate and multiply in this cell line, and can produce various antigenic proteins of the virus.

[0122] 2. Selection of SiRNA: As mentioned above, the hepatitis B virus genome expresses five kinds of mRNA molecules, each mRNA molecule is responsible for encoding different proteins, and the positions of transcription initiation and termination on the genomic DNA molecule are also different. Therefore, the positions of the coding regions of different proteins corresponding to the genomic DNA are different, among which the c...

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Abstract

The invention provides a small interference RNA molecule SiRNA capable of attacking human hepatitis B virus, which is double chain RNA molecule whose sequence has at least 70% of consanguinity degree with the sequence (I), wherein the antisense chain of the sequence (I) and one nucleic acid mutant has no consanguinity with the known human gene and gene expression segment. The SiRNA can be used for preparing medicament or preparation for prevention or treating hepatitis B and any diseases relating to hepatitis B viral infection.

Description

technical field [0001] The invention relates to the technical field of nucleic acid, in particular to a small interfering RNA molecule (SiRNA) for attacking human hepatitis B virus and an application thereof. Background technique [0002] Hepatitis B is a disease that seriously endangers the health of all human beings. Currently, there are two types of drugs clinically used to treat hepatitis B. One is alpha interferon, which has no direct effect on hepatitis B virus, and its antiviral The effect is carried out by inducing liver cells to form an antiviral state; the second type of lamivudine is a non-specific reverse transcriptase inhibitor, and its main function is to inhibit the reverse transcription process of RNA viruses to achieve Inhibits viral replication. Their common shortcoming is poor specificity against hepatitis B virus, and the effective rate (30%-40%) of both treatment hepatitis B is lower, and the negative conversion rate to the surface antigen of hepatitis ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00C07H21/02A61K31/7088A61P31/12A61P1/16
Inventor 丁佳逸郑树
Owner 丽水瑞德佳生物医药有限公司
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