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Treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon

A start codon and base editing technology, which is applied in the therapeutic field of suppressing HBV surface antigen based on the base editing ATG start codon, can solve the problems of viral DNA double-strand breaks and the inability to completely eliminate and cure HBV, and achieve the goal of inhibiting Replication, no DNA double-strand breaks, strong safety effects

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

The increase of optional 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 eliminate and 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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  • Treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon
  • Treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon
  • Treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon

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Embodiment Construction

[0031] The invention will be further described below in combination with specific embodiments.

[0032] like Figure 1-6 As shown, first determine the target that can be used as base editing to treat HBV, the specific steps are as follows: study pBSK-HBV1.3-P-null (P-ORF start codon ATG point mutation), -S5, -S6, - The biological impact of S5 / S6 (the fifth and sixth ATG start codons of S-ORF) mutation on the life cycle of HBV, the research provides the basis for the base editing treatment of HBV. On the positive strand of the HBV genome, there are 31 ORFs initiated by ATG. like figure 1As shown in A, there are 9 ATGs in the S-ORF, which are named S1-S9 in sequence, and S1, S3 and S4 initiate the translation of L, M and S proteins, respectively. Design primers, use fusion PCR method to construct pBSK-HBV1.3-P-null, -S5, -S6, -S5 / S6 and other mutant viruses, sequence analysis and identification; ELISA and western-blot to detect the expression of HBs / c / e , Realtime-PCR, Sout...

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Abstract

The invention belongs to the technical field of pathogen biological treatment, and particularly relates to a treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon, which comprises the following steps: analyzing positive chains of HBV genomes contained in HepG2.215 and HepAD38, determining base editing target spots, designing and synthesizing universal sgRNA primers of ATG codons of targeted editing inactive PreS1, PreS2 and S genes paired with ABE8e and BE4-max, and detecting whether the sgRNA primers are matched with ABE8e and BE4-max or not. 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, virus protein synthesis is prevented, virus replication is blocked, a method and a strategy for thoroughly curing and removing HBV surface antigens are explored, and great theoretical significance and clinical practice value are achieved.

Description

technical field [0001] The invention belongs to the technical field of pathogen biological treatment, and in particular relates to a treatment method for suppressing HBV surface antigen based on base editing ATG initiation codon. 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. my country has a high HBV infection rate and a la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N15/66
CPCC12N15/85C12N15/65C12N15/66C07K14/005C12N2730/10122C12N2800/107Y02A50/30
Inventor 钦博沈舒冯黎莉宋小华
Owner SHAOXING WOMEN & CHILDRENS HOSPITAL
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