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
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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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