Method for closing target gene and removing HBV e antigen based on base editing technology

A base editing and target gene technology, applied in genetic engineering, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as HBV cannot be completely cured, viral DNA double-strand breaks, etc., and achieve high editing efficiency and precision, The effect of less dependence and strong security

Pending Publication Date: 2022-05-31
SHAOXING WOMEN & CHILDRENS HOSPITAL
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Problems solved by technology

The increase of alternative drugs can help the problem of drug resistance, but these drugs can only inhibit the replication of HBV, and are ineffective against covalently closed circular DNA (cccDNA), and cannot completely cure HBV
Many research teams have tried to use CRISPR / Cas9 to edit the HBV genome to achieve antiviral effects, but it caused double-strand breaks in the viral DNA and uncontrollable unknown mutations

Method used

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  • Method for closing target gene and removing HBV e antigen based on base editing technology
  • Method for closing target gene and removing HBV e antigen based on base editing technology
  • Method for closing target gene and removing HBV e antigen based on base editing technology

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Embodiment Construction

[0028] The invention will be further described below in combination with specific embodiments.

[0029] Such as Figure 1-3 As shown, a method based on base editing technology to close the target gene to clear HBV e antigen, analyze the positive strand of the HBV genome contained in HepG2.215 and HepAD38, determine the target of base editing, design and synthesize ABE8e and BE4 -max paired universal sgRNA primers targeting the edited inactive PreC ATG codon, connected to the pGL3-U6-sgRNA-PGK-EGFP or pGL3-U6sgRNA-accg-puro vector recovered after digestion with BsaI, transformed into Escherichia coli DH5α, Pick positive colonies and clones, and extract the plasmids in a small way. After the enzyme digestion and sequencing are identified correctly, the plasmids are extracted in large quantities for later use.

[0030] The method for constructing a carrier comprises the steps:

[0031] a: Prepare sgRNA plasmid

[0032] (1) Using the default parameters of DNAstar, analyze the p...

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Abstract

The invention belongs to the technical field of pathogen biological treatment, and particularly relates to a method for closing a target gene and removing an HBV e antigen based on a base editing technology, which comprises the following steps: analyzing a positive chain of HBV genomes contained in HepG2.215 and HepAD38, determining a base editing target spot, designing and synthesizing a universal sgRNA primer of an ATG codon of targeted editing inactivated PreC paired with ABE8e and BE4-max, and detecting the HBV e antigen. Connecting to a carrier recovered by BsaI enzyme digestion, transforming Escherichia coli DH5alpha, selecting positive bacterial colonies for cloning, extracting small plasmids, and extracting large plasmids for later use after enzyme digestion and sequencing identification are correct. By changing HBV genetic information, preventing virus protein synthesis and blocking virus replication, a method and a strategy for thoroughly curing and removing the HBV e antigen are explored, and the method and the strategy have great theoretical significance and clinical practice value.

Description

technical field [0001] The invention belongs to the technical field of pathogen biological treatment, and in particular relates to a method for shutting down target genes and clearing HBV e antigen based on base editing technology. Background technique [0002] About half of the world's population is in a high-incidence area of ​​hepatitis B virus (HBV), 2 billion people have been infected, about 350 million are chronically infected, and about 1 million people die from HBV infection every year, ranking seventh among the top ten deadly diseases in the world . HBV occult infection is very common, as high as 7.5-16% in the Asian population. The coverage rate of HBV vaccine in my country is more than 90%, the successful blocking rate of mothers and infants is as high as 95%, and the number of infected people has dropped significantly. In 2016, WHO first proposed the ambitious goal of eliminating viral hepatitis globally by 2030. In my country, the HBV infection rate is high, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/66C12N9/22
CPCC12N15/1131C12N15/85C12N15/66C12N9/22C12N2310/20C12N2800/107
Inventor 钦博丁金龙吴云路曹杰
Owner SHAOXING WOMEN & CHILDRENS HOSPITAL
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