Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for modifying hepatitis B virus with N6-methyladenine in vitro

A technology of hepatitis B virus and methyl adenine, which is applied in the field of molecular biology, can solve the problems of low degree of methylation modification, inability to obtain a large amount of methylated HBV DNA, and has not yet been involved, and achieve the effect of simple operation

Inactive Publication Date: 2020-04-24
CHONGQING ACADEMY OF SCI & TECH
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on HBV epigenetic marks only focuses on the methylation of the carbon atom at the 5th position of cytosine, that is, the 5mC modification, and has not yet involved the 6mA modification.
Moreover, the only research on 5mC modification of HBV is based on the extraction of HBV DNA with a very small amount and a very low degree of methylation modification from the body, and it is impossible to obtain a large amount of methylated HBV DNA. The study of viruses poses great limitations

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for modifying hepatitis B virus with N6-methyladenine in vitro
  • Method for modifying hepatitis B virus with N6-methyladenine in vitro
  • Method for modifying hepatitis B virus with N6-methyladenine in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The method for in vitro N6-methyladenine modification of hepatitis B virus provided by the invention comprises the following steps:

[0021] 1. HBV viral genome cloning

[0022] The patient's serum DNA sample was provided by the Second Affiliated Hospital of Chongqing Medical University, China, and the patient number is 11061012. Using PrimeSTAR Max DNA polymerase, serum DNA template, front primer HBVF: 5'-CCGGAA AGCTTA TGCTCTTCTTTT TCACCT CTGCCT ARTCAT C-3', back primer HBVR: 5'-CCGGAG AGCTCA TGCTCT TCAAAAAGTTGC ATGGTG CTGGT-3' obtained by PCR amplification The full-length sequence of HBV with a length of 3.2kb. Then HBV was cloned into pEASY-Blunt vector to obtain pEASY-HBV positive plasmid. Through Sanger sequencing analysis and inspection, the present invention successfully obtains the full-length sequence of HBV DNA.

[0023] 2. In vitro circularization of HBV cccDNA

[0024] Using the successfully constructed pEASY-HBV plasmid of the present invention as a tem...

Embodiment 2

[0031] The method for in vitro N6-methyladenine modification of hepatitis B virus provided by the invention comprises the following steps:

[0032] 1. HBV viral genome cloning

[0033]The patient's serum DNA sample was provided by the Second Affiliated Hospital of Chongqing Medical University, China, and the patient number is 11061012. Using PrimeSTAR Max DNA polymerase, serum DNA template, front primer HBVF: 5'-CCGGAA AGCTTA TGCTCTTCTTTT TCACCT CTGCCT ARTCAT C-3', back primer HBVR: 5'-CCGGAG AGCTCA TGCTCT TCAAAAAGTTGC ATGGTG CTGGT-3' obtained by PCR amplification The full-length sequence of HBV with a length of 3.2kb. Then HBV was cloned into pEASY-Blunt vector to obtain pEASY-HBV positive plasmid. Through Sanger sequencing analysis and inspection, the present invention successfully obtains the full-length sequence of HBV DNA.

[0034] 2. In vitro circularization of HBV cccDNA

[0035] Using the successfully constructed pEASY-HBV plasmid of the present invention as a temp...

Embodiment 3

[0042] The method for in vitro N6-methyladenine modification of hepatitis B virus provided by the invention comprises the following steps:

[0043] 1. HBV viral genome cloning

[0044] The patient's serum DNA sample was provided by the Second Affiliated Hospital of Chongqing Medical University, China, and the patient number is 11061012. Using PrimeSTAR Max DNA polymerase, serum DNA template, front primer HBVF: 5'-CCGGAA AGCTTA TGCTCTTCTTTT TCACCT CTGCCT ARTCAT C-3', back primer HBVR: 5'-CCGGAG AGCTCA TGCTCT TCAAAAAGTTGC ATGGTG CTGGT-3' obtained by PCR amplification The full-length sequence of HBV with a length of 3.2kb. Then HBV was cloned into pEASY-Blunt vector to obtain pEASY-HBV positive plasmid. Through Sanger sequencing analysis and inspection, the present invention successfully obtains the full-length sequence of HBV DNA.

[0045] 2. In vitro circularization of HBV cccDNA

[0046] Using the pEASY-HBV plasmid successfully constructed in the present invention as a tem...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for modifying HBV with 6mA in vitro, comprising the following steps: (1) constructing a pEASY-HBV positive plasmid: cloning HBV into a pEASY-Blunt vector to obtain thepEASY-HBV positive plasmid; (2) preparing HBV cccDNA: taking the pEASY-HBV plasmid as a template, and adopting a PCR amplification method to obtain an HBV DNA amplification product; carrying out a digestion reaction on the amplification product and then carrying out a cyclization reaction to obtain HBV cccDNA; (3) extracting HepG2 nucleoprotein from human hepatocellular carcinoma cells: isolatingcytoplasmic protein and nucleoprotein of HepG2 cells and collecting HepG2 nucleoprotein; and (4) performing 6mA methylated modification of HBV cccDNA in vitro: taking HepG2 nucleoprotein as an enzyme, SAM as a methyl donor and HBV cccDNA as a substrate, incubating at 35-39 DEG C for 10-15 hours, and carrying out in-vitro 6mA methylated modification. The method provided by the invention is simpleto operate, successfully carries out 6mA methylated modification of HBV cccDNA in vitro for the first time, can be adopted to obtain a large number of 6mA modified HBV cccDNA products, and lays a foundation for the research of hepatitis B virus.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for modifying hepatitis B virus with N6-methyladenine in vitro. Background technique [0002] DNA methylation is a common and important epigenetic modification that can alter genetic expression without changing the DNA sequence. DNA methylation in a broad sense refers to the specific bases on the DNA sequence under the catalysis of DNA methyltransferase (DNA methyltransferase, DNMT), with S-adenosylmethionine (S-adenosylmethionine, SAM) as the Methyl donor, the process of chemical modification to obtain a methyl group through covalent bonding. A large number of studies have shown that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the interaction between DNA and proteins, thereby controlling gene expression. DNA 6mA modification (DNA N6-methyladenine, 6mA) is a methylation (N6-adenine DNA methylation) modificati...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/10C12N7/00C12R1/93
CPCC12N7/00C12N15/10C12N15/1096C12N2310/3521C12N2730/10151
Inventor 张华堂唐雨微赵中华魏青绿
Owner CHONGQING ACADEMY OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com