Reagent and method for preparing hbv persistent infection 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
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0088] Preparation of animal models or cell models
[0089] After the transgenic construct is obtained, the construct or the expression vector containing the construct can be transfected into cells, such as liver cells, by conventional methods. Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art, such as microinjection, electroporation and the like.
[0090] The construct or the expression vector containing the construct 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 a non-human mammal by tail vein high pressure injection
[0091] The non-human mammals described in the present invention can be mice, sheep, cattle, pigs, rabbits and the like. Preferred are murines, including mice, rats or other types of murines.
Embodiment 1
[0124] Embodiment 1, the molecular biology model of Cre-induced rcccDNA
[0125] The HBV genome is about 3.2Kb, extremely compact, and even overlaps multiple reading frames in some regions. It is worth noting that there are some hidden RNA splicing sites in the HBV genome transcription process, and the possible biological significance is not clear. The inventor 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, and the basic sequence pUC for plasmid replication 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 of recombinase Cre, due to the introduction of the eukaryotic gene tailing signal, the transcription initiated by the upstream promoter of HBV is terminated in the hybrid intron sequence, and the HBV genome is therefore blocked and unable to produce V...
Embodiment 2
[0132] Embodiment 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 with prcccDNA and recombinase Cre expression plasmid (pCMV-Cre) in vitro. The same kind of cells transfected with payw1.3 and prcccDNA alone were used as controls. The conventional enzyme-linked immunosorbent assay (ELISA) method was used to detect the presence of HBV secreted antigens.
[0134] As a result, HBV secreted antigens HBeAg, HBsAg and LHBsAg were detected in the culture supernatant of HepG2 cells after transfection ( figure 2 A). However, both HBsAg and LHBsAg were negative in the culture medium of cells transfected with prcccDNA alone. This experiment confirmed that the built-in LoxP heterozygous intron can be effectively transcribed and spliced in the HBV genome.
[0135] Furthermore, the inventors used the above-mentioned in vit...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More