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Reagent and method for preparation of HBV persistently infected animal model

A technology for constructing and replicating sequences, which is applied in the fields of biotechnology and virology, and can solve problems such as complex systems, PCR quantitative methods not widely accepted, and difficulty in forming cccDNA

Active Publication Date: 2015-01-14
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, there are technical difficulties in the detection of low-abundance cccDNA. Due to the complexity of the system and lack of accuracy, a series of PCR quantitative methods developed in recent years are not widely accepted.
[0006] In the prior art, it is generally believed that cccDNA is difficult to form in mouse hepatocytes, and the expression based on HBV transgenes cannot reflect the true intracellular viral replication cycle

Method used

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  • Reagent and method for preparation of HBV persistently infected animal model
  • Reagent and method for preparation of HBV persistently infected animal model
  • Reagent and method for preparation of HBV persistently infected animal model

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0088] Preparation of animal models or cell models

[0089] After the transgenic construct is obtained, cells, such as hepatocytes, can be transfected with the construct or an expression vector containing the construct by conventional methods. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art, such as microinjection, electroporation, and the like.

[0090] The constructs or expression vectors containing the constructs can also be used to transfect animals, such as mice. As a preferred mode of the present invention, the construct or the expression vector containing the construct is transfected into non-human mammals by means of tail vein high pressure injection

[0091] The non-human mammals of the present invention can be mice, sheep, cows, pigs, rabbits, and the like. Preferred are mice, including mice, rats or other types of mice.

Embodiment 1

[0124] Example 1. Molecular biological model of Cre-induced rcccDNA

[0125] The HBV genome is about 3.2Kb, which is extremely compact, and even overlaps multiple reading frames in some regions. It is worth noting that there are some cryptic RNA splicing sites in the HBV genome transcription process, and the possible biological significance is not clear. The inventors inserted a hybrid intron sequence (chimeric Intron) between the HBsAg coding frame nt202-203 of the HBV wild virus genome (ayw3 subtype): including 5'Intron, LoxP-1, the basic sequence of plasmid replication pUC Ori, antibiotic selection gene (Amp(R)), eukaryotic gene tailing signal (polyA processing site), LoxP-2, and 3'Intron.

[0126] In the case of non-induction by recombinase Cre, due to the introduction of the eukaryotic gene tailing signal, the transcription initiated by the HBV upstream promoter is terminated within the hybrid intron sequence, and the HBV genome is blocked and cannot be produced. Virus ...

Embodiment 2

[0132] Example 2. Expression and viral replication of rcccDNA based on recombinase Cre

[0133] Based on the strategies discussed in the Examples, the inventors constructed prcccDNA ( figure 1 B). HepG2 cells were transfected in vitro with prcccDNA and recombinant Cre expression plasmid (pCMV-Cre). The same cells transfected with payw1.3 and prcccDNA alone were used as controls. The presence of HBV secreted antigens was detected by conventional enzyme-linked immunosorbent assay (ELISA).

[0134] As a result, the HBV secreted antigens HBeAg, HBsAg and LHBsAg were detected in the culture supernatant of HepG2 cells after transfection ( figure 2 A). However, in the culture medium of cells transfected with prcccDNA alone, both HBsAg and LHBsAg were negative. This experiment confirmed that the built-in LoxP hybrid intron can be efficiently transcribed and spliced ​​in the HBV genome.

[0135] Further, the present inventors applied the above in vitro transfection experiments t...

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Abstract

The invention relates to a reagent and method for preparation of an HBV (hepatitis B virus) persistently infected animal model. According to the invention, a construct able to conditionally induce HBV covalently closed circular DNA (cccDNA) molecules in the liver of mice is constructed, and the HBV persistently infected animal model is successfully established.

Description

technical field [0001] The present invention belongs to the fields of biotechnology and virology; more particularly, the present invention relates to reagents and methods for preparing animal models of persistent HBV infection. Background technique [0002] Chronic hepatitis B virus (HBV) infection is still a worldwide public health problem, and about 1 million people die each year from liver failure, cirrhosis and liver cancer caused by hepatitis B infection. China is a high-incidence area of ​​hepatitis B. It is of great social significance to study chronic HBV infection and develop effective antiviral therapy. [0003] HBV susceptible hosts are limited to primates such as humans and chimpanzees, and the lack of suitable experimental animal models has always been a bottleneck in HBV virology and immunology research. Mice are ideal experimental model animals, however HBV does not infect mice. Through transgenic technology, viral antigens can be used as "self" proteins to ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N15/51C12N15/10A01K67/027
Inventor 邓强蓝柯谢幼华汪垣齐治华李改云
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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