A kind of establishment method and application of aav-hbv recombinant virus and hepatitis B virus mouse model based on s gene break

An AAV-HBV, AAV-HBV1.04 technology, applied in the direction of single-stranded DNA virus, retro DNA virus, virus, etc., can solve the problem of not being HBV cccDNA, unable to judge the role of cccDNA and so on

Active Publication Date: 2022-05-31
WUHAN UNIV
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Problems solved by technology

All of the above models have deficiencies, either it is impossible to judge whether the cccDNA is functioning, or the formed cccDNA is a recombinant cccDNA, not the real HBV cccDNA

Method used

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  • A kind of establishment method and application of aav-hbv recombinant virus and hepatitis B virus mouse model based on s gene break
  • A kind of establishment method and application of aav-hbv recombinant virus and hepatitis B virus mouse model based on s gene break
  • A kind of establishment method and application of aav-hbv recombinant virus and hepatitis B virus mouse model based on s gene break

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Embodiment 1

[0035] The pAAV-HBV1.2 and pAAV-HBV1.04 plasmids were constructed. The pAAV-HBV 1.04 plasmid was constructed by homologous recombination technology, see the schematic diagram figure 1 , the pAAV-HBV1.04 vector plasmid sequence is shown in SEQ ID NO.3. The small S region of the HBV genome rcDNA was selected as the starting point and extended back to the starting point along the HBV genome rcDNA, and then about 127 bp (homology arm) was added as the end point. The obtained HBV genome DNA length divided by 3182 was 1.04 copies. The selection of starting point and ending point can be changed before and after the small S area.

[0036] 1. Amplify AAV linearized vector backbone fragment and HBV target fragment by PCR technology

[0037]The template used to amplify the AAV vector backbone is pAAV-TBG>hSLC10A1[NM_003049.3]:WPRE (the AAV vector backbone sequence is a commonly used sequence). The template used to amplify the HBV target fragment is the HBV 1.3 or diploid plasmid store...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a method for establishing a mouse model of AAV‑HBV recombinant virus and hepatitis B virus based on S gene disruption and its application. The invention adopts the method of homologous recombination to construct the pAAV-HBV1.04 plasmid, and uses the method of co-transfecting 293T cells with three plasmids to package and produce the recombinant virus in vitro. C57BL / 6 mice with normal immune system were injected with the recombinant virus through the tail vein to form a mouse model of HBV chronic infection in which HBV cccDNA persisted. The mouse model in the present invention can produce real HBV cccDNA and can function as the only transcription template. The novel HBV chronic infection mouse model constructed by the invention can be used to study the mechanism of HBV cccDNA formation and maintenance under long-term chronic infection conditions, to study problems related to the immune system, and to find and evaluate new anti-HBV drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and application for establishing an AAV-HBV recombinant virus and a hepatitis B virus mouse model based on S gene fragmentation. Background technique [0002] Hepatitis B virus (HBV) is a hepatotropic DNA virus, the genome is partially double-stranded relaxed circular DNA (Relaxed circular DNA, rcDNA), the full length is about 3.2kb, is the smallest known DNA virus (Ganem, D. and A.M. Prince, Hepatitis B virus infection--natural history and clinical consequences. N Engl J Med, 2004.350(11):p.1118-29.). After HBV infection of host cells, its rcDNA will be repaired by DNA damage repair system into covalently closed circular DNA (cccDNA) in the nucleus of host cells, and cccDNA is the template for viral transcription. Although vaccines are effective in preventing HBV infection, they do not help the approximately 250 million chronic hepatitis B patients worldwide (W...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/36C12N15/864A01K67/027
CPCC12N7/00C12N15/86C07K14/005A01K67/0278C12N2730/10122C12N2750/14121C12N2750/14143A01K2207/15A01K2217/052A01K2227/105A01K2267/0337
Inventor 夏宇尘李聪钟有权
Owner WUHAN UNIV
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