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Establishment method and application of hepatitis B virus recombinant cccDNA mouse model

A technology of hepatitis B virus and mouse model, applied in the field of hepatitis B virus recombinant cccDNA mouse model and its establishment, and the field of hepatitis B virus recombinant cccDNA mouse model, which can solve the problem of inappropriate cccDNA targeting research and limited application And other issues

Pending Publication Date: 2021-11-12
FUDAN UNIV +1
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Problems solved by technology

[0005] In recent years, the recombinant cccDNA (rcccDNA) mouse model has developed rapidly. rcccDNA can effectively support virus replication and protein expression, and establish chronic infection. However, in the reported rcccDNA mouse model, the rcccDNA library in mouse liver cells The level and duration of rcccDNA are still not suitable for cccDNA targeting studies, although rcccDNA bands can be clearly detected by Southern blot within a week of modeling, but the rcccDNA levels drop rapidly after that, limiting their usefulness in the study of cccDNA-related biological mechanisms and Applications in the development of new cccDNA-targeted drugs

Method used

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  • Establishment method and application of hepatitis B virus recombinant cccDNA mouse model
  • Establishment method and application of hepatitis B virus recombinant cccDNA mouse model
  • Establishment method and application of hepatitis B virus recombinant cccDNA mouse model

Examples

Experimental program
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Effect test

Embodiment 1A

[0022] The establishment of embodiment 1AAV-rcccDNA mouse model

[0023] ①Construction of pAAV8-rcccDNA plasmid: AAV8 vector was obtained from pAAV8-CAG-RFP plasmid by XhoI / BamHI double enzyme digestion, and the HBV single-copy genome sequence with loxP sites at both ends was amplified from prcccDNA plasmid and cloned into pAAV8 vector;

[0024] ② Virus packaging: pAAV8-rcccDNA, pRC and pHelper plasmids were co-transfected into HEK293T cells, and the transfection reagent was PEI. After 72 hours of transfection, collect the cells together with the medium into a 50ml centrifuge tube;

[0025] ③ Virus purification: Purify by chloroform treatment-PEG / NaCl precipitation-chloroform extraction method, collect cells and culture medium, add 1 / 10 volume of chloroform, shake vigorously in a shaker at 37°C for 1 hour, add solid chloride Sodium to a final concentration of 1mol / L, shake to dissolve, centrifuge at 12000r / min at 4°C for 15min, take out the upper aqueous phase, add PEG8000 t...

Embodiment 2

[0027] Example 2 Detection of rcccDNA in liver of AAV8-rcccDNA mouse model

[0028] ① Model establishment: 8-week-old male Alb-Cre mice received 2×10 11 After injection of vg AAV8-rcccDNA virus, the livers were sacrificed at corresponding time points;

[0029] ② DNA extraction: Weigh 100 mg of mouse liver tissue, place it in ice-precooled 100 μl PBS, cut it into small pieces as much as possible with scissors, grind it with a tissue grinder, and pass it through a 100 μm pore size cell sieve to obtain liver tissue. cell suspension. Extraction by Hirt extraction;

[0030]③Southern blot detection of rcccDNA: Southern Blot detection (DIG High Prime DNA Labeling and Detection Starter Kit II manual) was performed using Roche’s digoxin probe labeling and detection system; within 30 weeks, obvious signals of rcccDNA could be detected; rcccDNA strips The band can be linearized by EcoRI endonuclease, but insensitive to T5 exonuclease, confirming its supercoiled structure (e.g. figur...

Embodiment 3

[0032] Example 3 Detection of virological indicators in serum and liver of AAV8-rcccDNA mouse model

[0033] ①Model establishment: male Alb-Cre mice of specified age were injected with specified dose of AAV8-rcccDNA virus to establish the model;

[0034] ② Serum virus index detection: blood was collected from the infraorbital canthus venous plexus once every two weeks after injection. The blood collected was 200 μl of non-anticoagulated whole blood. In the detection of HBsAg, HBeAg, alanine aminotransferase ALT, and HBV DNA, the results showed that the serum HBsAg and HBeAg of the AAV-rcccDNA mouse model were at 10 4 IU / ml and 10 3 COI levels (eg image 3 A, shown in B), and keep positive in the whole experimental period (51 weeks); Serum HBV DNA starting amount is at 10 4 IU / ml, and then gradually decreased (such as image 3 Shown in C), AAV8-rcccDNA mouse model serum virus antigen and DNA expression levels are related to the dose of recombinant virus that is injected, an...

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Abstract

The invention belongs to the technical field of microbial animal cell lines, and particularly relates to a hepatitis B virus recombinant cccDNA (rcccDNA) mouse model established through mediation of a type 8 adeno-associated virus vector as well as an establishment method and application thereof. According to the invention, the type 8 adeno-associated virus vector is utilized, an HBV single-copy genome with loxP sites at two ends is brought into an Alb-Cre transgenic mouse, and the AAV-rcccDNA mouse model is established. The established AAV-rcccDNA mouse model has the advantages that (1) the modeling success rate is high; 2) the expression level of rcccDNA in the liver of the model mouse is high, the maintenance time is long, and a Southern blot method is easy to detect; 3) the expression level and the maintenance time of virus antigens in serum and liver of the model mouse are long; 4) the HBV specific T cell immunoreaction in the liver of the model mouse is weak, and the like. The mouse model established by the invention is suitable for screening and testing HBV cccDNA targeted drugs and immunomodulators and suitable models for researching chronic hepatitis B healing.

Description

technical field [0001] The invention belongs to the technical field of microbial animal cell lines, and relates to a hepatitis B virus recombinant cccDNA (rcccDNA) mouse model, in particular to a hepatitis B virus recombinant cccDNA (rcccDNA) mouse model established through the mediation of a type 8 adeno-associated virus vector and its method of establishment and use. Background technique [0002] The prior art discloses that hepatitis B caused by hepatitis B virus (Hepatitis B virus, HBV) infection is a major infectious disease that seriously endangers human health. According to statistics, the risk of liver cirrhosis, liver failure and liver cancer in patients with chronic hepatitis B (CHB) infection is significantly higher (>20%) than normal people. At present, there are more than 90 million chronic hepatitis B patients in my country, 28 million of whom need treatment. Although the treatment of chronic hepatitis B has made some progress in recent years, a series of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/51A01K67/027
CPCC07K14/005C12N15/86A01K67/0276C12N2730/10122C12N2750/14143A01K2227/105A01K2267/0337A01K2267/0393
Inventor 袁正宏邬敏张小楠陈捷亮
Owner FUDAN UNIV
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