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, carrier technology, applied in the field of molecular biology, which can solve the problems of inability to synthesize replication and complete inhibition.

Inactive Publication Date: 2013-04-03
ARMY MEDICAL UNIV
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Problems solved by technology

Studies have found that the RC DNA 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 quantity; ② rHBV itself cannot synthesize The Core and Pol proteins necessary for replication 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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[0027] 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 for which specific conditions are not indicated in the preferred embodiments are usually carried out according to conventional conditions, or according to the conditions suggested by the manufacturer.

[0028] The LJ196 and LJ96 plasmids used in the present invention were donated by Professor Daniel D. Loeb, and are described in the 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.).

[0029] 1. Deletion mutant rHBV construction process

[0030] On the basis of the plasmid LJ196 (SEQ ID NO.1) that does not express any viral protein, the DNA fragment of the LJ196 plasmid is deleted by reverse PCR technology, so that the RC DNA...

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Abstract

The invention relates to a recombinant HBV (Hepatitis B Virus) vector for expressing specific HBVshRNA (Hepatitis B Virus Short Hairpin Ribonucleic Acid) as well as a construction method and application of the vector. The recombinant HBV vector is formed by deleting 2111st-2643rd bases, 187th-680th bases or 1060th-1500th bases from an LJ196 vector, and replacing shRNA transcription structure units of the deleted sequences at the deleted sequence deleting positions. The preparation method is simple; RCDNA (Relaxed Circular Deoxyribonucleic Acid) can be efficiently synthesized using Core protein and Pol protein of wild-type hepatitis B virus genome by adopting the recombinant HBV vector, and the synthesis efficiency is higher than that of the wild-type hepatitis B virus genome DNA, the RCDNA synthesis can compete with the duplication of the wild-type hepatitis B virus, simultaneously, the shRNA can be efficiently expressed by utilizing the characteristics of high synthesis efficiency of the recombinant HBV vector, and then the DNA duplication and HBsAg synthesis of the hepatitis B virus can be disturbed. The vector can be applied to preparation a medicine for disturbing the DNA duplication of the hepatitis B virus genome, and a new idea for treating the chronic hepatitis B virus infection is provided.

Description

[0001] Technical field [0002] The present invention involves the field of molecular biology, which specializes in the restructuring HBV carrier that expresses the specific HBV ShRNA, and also involves the construction method and application of the carrier. Background technique [0003] Hepatitis B Virus is a DNA virus that causes human acute and chronic hepatitis, also known as Dan's particles, referred to as HBV.HBV is a hepatic DNA virus (Hepadnaviridae), with a genome leader of about 3.2kb, four ORF, encoding the following protein: Core protein and Pre-Core protein, POL protein, X protein and S protein (L, M, S).Core is a nucleus shell protein; X protein is important to copy virus, and it is related to the occurrence of liver cancer; S protein is a virus envelope protein, which is related to the virus entering cells. [0004] HBV ultra -spiral covalent, closed, and ring -shaped DNA molecules (CCCDNA) are the root causes of HBV chronic infection. The relaxation ring DNA (RC D...

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Application Information

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Patent Type & Authority Applications(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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