Construction and application of model for researching interaction between HBV cccDNA and host

A MC-HBV and model technology, applied in the field of bioengineering, can solve the problems of low copy number of cccDNA, no direct labeling and detection of HBVcccDNA, low transfection rate, etc., and achieve the effect of a reliable detection method

Active Publication Date: 2019-12-20
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently, HBV infectious cloning models mainly include >1.0 copy expression plasmids (such as HBV1.2, HBV1.3 expression plasmids) and newly developed cccDNA minicircle vector plasmids (including prcccDNA / pCMV-Cre system, HBVcircle system), prcccDNA / The recombinant rcccDNA (recombinant cccDNA) produced by the pCMV-Cre system transcribes pgRNA and undergoes alternative splicing to produce wild-type cccDNA. However, prcccDNA and pCMV-Cre plasmids are required to co-transfect liver cell lines to obtain rcccDNA, and the transfection rate is low. The shortcoming of producing cccDNA copy number is small, which limits its further application
Although the HBVcircle system highly simulates cccDNA in vitro, the residual attR sequence in the genome cannot be removed, and it has always existed in the progeny virus, with potential unknown effects
In addition, there is currently no method for direct labeling and detection of HBV cccDNA, which limits the exploration of the interaction between HBV cccDNA and liver cells

Method used

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  • Construction and application of model for researching interaction between HBV cccDNA and host
  • Construction and application of model for researching interaction between HBV cccDNA and host
  • Construction and application of model for researching interaction between HBV cccDNA and host

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] The construction of embodiment 1MC-HBV eukaryotic expression plasmid

[0084] 1. Using high-fidelity DNA polymerase to amplify the backbone fragments of HBV-Intron and MN0501 vectors;

[0085] 1) HBV Intron-attR sequence:

[0086] 5'-cgtgtacag (S gene)-

[0087]

[0088] 2) High-fidelity DNA polymerase amplification system:

[0089]

[0090] PCR amplification conditions: pre-denaturation at 98°C for 3 min, followed by denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 30 s and repeated 30 cycles, and finally extension at 72°C for 8 min.

[0091] 2. DNA gel electrophoresis, gel cutting recovery and quantification;

[0092]3. Infusion cloning technology connection, construction of eukaryotic expression plasmid pmini-MC-HBV-Intron

[0093]

[0094] Incubate at 50°C for 15 minutes;

[0095] 4. Transform DH5a competent, select and culture in Kan+ resistant LB culture plate;

[0096] 5. Pick a single clone, screen positive clones b...

Embodiment 2

[0102] Embodiment 2 MC-HBV alternative splicing ability and supporting HBV replication ability analysis

[0103] HuH7 cells were digested to make a concentration of 2×10 5 cells / ml of cell suspension, seeded into cell culture plates or dishes as needed. After culturing overnight for 16h-24h, the MC-HBV, PP and pCMV-Cre / prcccDNA plasmids were transfected into HuH7 cells for 48h, respectively. ELISA was used to detect HBsAg and HBeAg in the culture supernatant, and the pgRNA was detected by RT-PCR and PCR amplification using P1P2 primers. Whether alternative splicing occurs, whether the offspring cccDNA can be produced.

[0104] 1. ELISA detection of HBsAg in the supernatant

[0105] 1) Add 20 μl sample diluent to each well;

[0106] 2) Add 100 μl of negative and positive control serum or supernatant to be tested in corresponding wells respectively, and react at 37°C for 1 hour;

[0107] 3) Add 50ul of HRP enzyme-labeled antibody, and continue to react at 37°C for 30min;

...

Embodiment 3I

[0144] Example 3 Intron-attR sequence optimization restores pgRNA alternative splicing ability

[0145] The RNA secondary structure has an important influence on the efficiency of pre-mRNA alternative splicing. In Example 2, the 5'Intron-attR-3'Intron structure has more stem-loop structures, and the secondary structure changes from a single rod structure to two hairpins structure, and may be the main reason for the inhibition of RNA alternative splicing. According to the principle of complementary base pairing, add gtcgcgcccgggga sequence, changing its structure to a single rod-like structure similar to the intron of the 5'Intron-loxp-3'Intron and pCI-Neo vectors. Subsequent verification steps are the same as in Example 2.

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to construction and application of a model for researching interaction between HBV cccDNA and a host. The invention provides the construction and the application of the minicircle DNA model highly simulating hepatitis B virus genome cccDNA. By constructing a pmini-MC-HBV-Intron plasmid, a high-purity MC-HBV minicircle plasmid is obtained in vitro; and by optimizing a chimeric intron sequence and a secondary structure of RNA, efficient and variable splicing of the chimeric intron on the RNA level is achieved, and the MC-HBV capable of being copied at a high level in vitro is obtained. The MC-HBV can highly simulate the physiological activity between an HBV virus and host cells. The invention further discloses an in-vitro marker MC-HBV model and a detection technology, and provides the excellent model for revealing the interaction relationship between the HBV cccDNA and the host, and a function regulation mechanism, and exploring a new HBV treatment target.

Description

technical field [0001] The disclosure belongs to the technical field of bioengineering, and specifically relates to a method for constructing an HBV cccDNA microcircle model (MC-HBV) that can support high-level and continuous replication of HBV and the application of various labeling methods to explore the interaction between cccDNA and liver cells. Background technique [0002] The information disclosed in this Background section is only intended to increase the understanding of the general background of the disclosure, and is not necessarily to be taken as an acknowledgment or any form of suggestion that the information constitutes prior art that is already known to those skilled in the art. [0003] Hepatitis B virus (Hepatitis B virus, HBV) is a DNA virus and belongs to the hepadnaviridae family. Its chronic infection is closely related to liver cirrhosis and liver cancer. HCC) is the most important predisposing factor. The HBV genome is a loose circular DNA (RC-DNA), w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/85G01N33/569C12Q1/02A61K45/00A61P31/20A61P1/16
CPCA61K45/00A61P1/16A61P31/20C12N7/00C12N15/85C12N2730/10121C12N2730/10131C12N2730/10151G01N33/5067G01N33/56983
Inventor 武专昌马春红王鑫高立芬梁晓红王丽媛孙杨
Owner SHANDONG UNIV
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