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Hepatitis B virus vector and application thereof

A hepatitis B virus, hepatitis B virus technology, applied in the direction of virus/phage, vector, virus, etc., can solve the problem of low infectivity

Pending Publication Date: 2022-03-01
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no clinical strains of HBV genotype B or C that can infect highly efficiently, which may be related to the low infectivity of genotype B and C viruses in vitro

Method used

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  • Hepatitis B virus vector and application thereof
  • Hepatitis B virus vector and application thereof
  • Hepatitis B virus vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1.cHBV preparation

[0048] The present invention screened the serum HBV infectivity of 53 chronic HBV infected persons with high HBV DNA loads of B or C genotypes, and found that most of the specimens had low infectivity, and only one case of serum was in HepG2 / NTCP and differentiated HepaRG cells showed high infectivity. Subsequently, wild-type HBV and 5' preS1 (preS1) region deletion mutant HBV were simultaneously isolated from the serum.

[0049] In order to construct the HBV expression plasmid, we extracted the patient's serum HBV DNA, selected the whole gene clone, and connected it into the vector pcDNA3.1 zeo(-) to construct the HBV 1.1 ploid expression plasmid. Among them, clone 42.5 is a wild-type HBV strain, and clone 42.5-18 is a deletion-type HBV strain (ie ++++++ highly infectious clinical isolates in the present invention).

[0050] The specific preparation method is as follows:

[0051] 1) HepG2 cells were transfected with 2 μg of 1.1-ploidy...

Embodiment 2

[0056] Embodiment 2. Infection experiment

[0057] After cHBV was fully dissolved in FBS(-) / PEG(-) / RM, an appropriate amount of 40% PEG was added to adjust the final concentration of PEG in inoculum to 4%, and the same copy number of cHBV was used to infect cells under the condition of 4% PEG. Viruses were incubated for 16-20 hours.

[0058] 3. Detectable indicators after infection: secreted HBeAg and HBsAg levels in the supernatant, intracellular HBV DNA replication and cccDNA levels.

[0059] Secretible hepatitis B virus e antigen (HBeAg) and hepatitis B virus surface antigen (HBsAg) were detected by ELISA method. HepG2 / NTCP and differentiated HepaRG cells were infected with wild-type virus (clone 42.5) and deletion virus (clone 42.5-18) with the same copy number, and HBeAg secreted in the supernatant of clone 42.5-18 on day 15 after infection ( figure 1 ) and HBsAg ( figure 2 ) levels were significantly stronger than the wild-type clone 42.5.

[0060] For HBV in vitro ...

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Abstract

The invention belongs to the field of biological medicine, and relates to a hepatitis B virus vector and application thereof. Specifically, the invention comprises a mutant hepatitis B virus expression vector and an in-vitro high-infection susceptible C genotype hepatitis B virus strain prepared by using the hepatitis B virus expression vector. The hepatitis B virus expression vector disclosed by the invention contains a coding sequence of C genotype hepatitis B virus without a coding sequence of an S1 region before a 5'end. Tests show that the expression vector can be used for preparing high-infectivity C genotype hepatitis B virus isolates, and can be used for in-vitro infection experiment-based interaction between C genotype HBV and hosts, formation and degradation of cccDNA, screening of anti-HBV drugs and other related researches.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a carrier of hepatitis B virus (hepatitis B virus). Specifically, the present invention relates to a hepatitis B virus expression vector, using the hepatitis B virus expression vector to prepare in vitro highly infectious and sensitive C genotype hepatitis B virus strains, and C genotype hepatitis B virus (Hepatitis B virus, HBV ) clinical isolates, which can be used for related studies such as the interaction between genotype C HBV and the host, the formation and degradation of cccDNA, and the screening of anti-HBV drugs based on in vitro infection experiments. Background technique [0002] The prior art discloses that hepatitis B virus infection is a major global public health problem. Currently, about 250 million people are chronically infected with HBV, accounting for 3.5% of the world's total population. Persistent chronic HBV infection can lead to malignant outcomes such as liver ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N7/00C12Q1/70C12Q1/02C12R1/93
CPCC12N15/85C12N7/00C12Q1/706C12Q1/025C12N2800/107C12N2730/10121C12N2730/10152G01N2333/02
Inventor 张继明童舒平李菁
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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