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Recombinant HBV (Hepatitis B Virus) vector for expressing specific HBVshRNA (Hepatitis B Virus Short Hairpin Ribonucleic Acid) as well as construction method and application of vector

A specific and carrier technology, applied in the field of molecular biology, can solve the problems of incomplete inhibition, inability to synthesize and replicate, etc.

Inactive Publication Date: 2014-09-17
ARMY MEDICAL UNIV
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Problems solved by technology

Studies have found that the RCDNA synthesis efficiency of mutant rHBV missing certain sequences is higher. When rHBV and HBV coexist in cells, rHBV has the following characteristics: ① High synthesis efficiency, rHBV will be dominant in number; ② rHBV itself cannot synthesize and replicate The necessary Core and Pol proteins need to use the Core and Pol proteins of wild HBV to replicate, thereby competitively inhibiting the replication of wild HBV, but cannot completely inhibit

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  • Recombinant HBV (Hepatitis B Virus) vector for expressing specific HBVshRNA (Hepatitis B Virus Short Hairpin Ribonucleic Acid) as well as construction method and application of vector
  • Recombinant HBV (Hepatitis B Virus) vector for expressing specific HBVshRNA (Hepatitis B Virus Short Hairpin Ribonucleic Acid) as well as construction method and application of vector
  • Recombinant HBV (Hepatitis B Virus) vector for expressing specific HBVshRNA (Hepatitis B Virus Short Hairpin Ribonucleic Acid) as well as construction method and application of vector

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[0026] In order to make the object, technical solution and advantages of the present invention clearer, preferred embodiments of the present invention are described in detail below. The experimental methods not specified in the preferred embodiment are usually carried out according to conventional conditions, or according to the conditions suggested by the manufacturer. This patent project is funded by the National Natural Science Foundation of China, project number: No.30800981.

[0027] The LJ196 and LJ96 plasmids used in the present invention were donated by Professor Daniel D. Loeb, and are described in published literature (Liu N, Ji L, Maguire ML, Loeb DD. cis-Acting sequences that contribute to the synthesis of relaxed- circular DNA of human hepatitis B virus. J Virol. Jan 2004; 78(2): 642-649.).

[0028] 1. Deletion mutant rHBV construction process

[0029] On the basis of the plasmid LJ196 (SEQ ID NO.1) that does not express any viral protein, the DNA fragment of the...

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Abstract

The invention relates to a recombinant HBV vector expressing specific HBV shRNA and its construction method and application. After the recombinant HBV vector deletes 2111-2643, 187-680 or 1060-1500 bases from the LJ196 vector, it replaces and recognizes at the deleted sequence The shRNA transcriptional structural unit of the deletion sequence is obtained, and the preparation method is simple. The recombinant HBV vector can utilize the Core protein and Pol protein of the wild-type hepatitis B virus genome to efficiently synthesize RCDNA, and the synthesis efficiency is higher than that of the wild-type hepatitis B virus. Genomic DNA competes for the replication of wild-type hepatitis B virus, and at the same time utilizes the characteristics of high synthesis efficiency of recombinant HBV vectors to express shRNA efficiently, and then interferes with the replication of hepatitis B virus DNA and the synthesis of HBsAg, which can be used to prepare the genome of interference hepatitis B virus Drugs for DNA replication provide new ideas for the treatment of chronic hepatitis B virus infection.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a recombinant HBV vector expressing specific HBV shRNA, and also to a construction method and application of the vector. Background technique [0002] Hepatitis B virus (hepatitis B virus) is a DNA virus that causes acute and chronic hepatitis in humans, also known as Dan's particles, or HBV for short. HBV belongs to the hepadnaviridae family. Its genome is about 3.2kb long and has four ORFs, which encode the following proteins: Core protein and pre-core protein, Pol protein, X protein and S protein (L, M, S). Core is the nucleocapsid protein; X protein is important for virus replication and is related to the occurrence of liver cancer; S protein is the envelope protein of the virus and is related to the virus entering cells. [0003] The persistence of HBV supercoiled covalent, closed, and circular DNA molecules (covalently closed circular DNA, cccDNA) is the root cause of HBV ...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/113A61K48/00A61P31/20
Inventor 李俊刚毛青王宇明
Owner ARMY MEDICAL UNIV
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