Protein molecule for appearance methylated modification on DNA of HBV and application of protein molecule
A methylation and protein technology, applied in peptide/protein components, recombinant DNA technology, DNA/RNA fragments, etc., can solve problems such as rebound, death due to pathological consequences, inability to clear covalently closed circular DNA, etc., and achieve infection. strong effect
Active Publication Date: 2018-03-06
FUDAN UNIV
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Problems solved by technology
[0003] Nucleoside (acid) drugs (NAs) are the most commonly used anti-HBV infection drugs in clinic, including lamivudine (LAM), entecavir (entecavir, ETV), telbivudine (tebivudine, LdT) and albino Adefovir dipivoxil (ADV), these drugs directly act on the reverse transcriptase and DNA polymerase of HBV, can significantly inhibit the replication of the virus but cannot clear the covalently closed circular DNA (covalently closed circular DNA) in liver cells , cccDNA)
Moreover, the above-mentioned drugs will have a strong rebound phenomenon after stopping the drug, causing serious pathological consequences and even death.
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Embodiment Construction
[0028] 1. Screening and construction of HBV stably transfected cell lines
[0029] The present invention uses pcDNA3.1 to construct the 1.3 ploidy of the whole HBV genome. After transfecting HepG2 cells, the HBV virus can be replicated autonomously. After G418 screening, the cell strain HepG2-HBV 1.3 that stably secretes virus particles and marker antigens is obtained, and the cell strain The HBV virus relied on is the C genotype representative of the strains circulating in East Asia.
[0030] 2. Design and synthesis of HBV X gene promoter specific binding
[0031] Using ZF Tools Ver3.0 software, according to the CpG site of HBV XP region, select the best sequence and design the specific binding protein. The amino acid sequences of the selected DNA binding protein-mediated DNA methylase against HBV XP are SEQ.ID.No.3 and SEQ.ID.No.5. The corresponding DNA-binding domain nucleotide sequences are SEQ.ID.No.4, SEQ.ID.No.6. Use Reverse Translate software to convert to nucleotid...
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The invention belongs to the technical field of genetic biological medicines and in particular discloses a protein molecule for appearance methylated modification on DNA of an HBV and application of the protein molecule. According to principles of bioinformatics, epigenetic engineering and immunology, a completely novel protein molecule which has targeting specificity affinity interaction with hepatitis B virus genes and is capable of implementing appearance methylated modification on DNA of the HBV and furthermore silencing HBV genes is designed; the protein is cloned into various expressionvectors and are introduced into bodies or cells of human beings and animals, the protein molecule with HBV targeting specificity is efficiently expressed, and efficient methylated modification is achieved for the HBV genes; in-vivo and in-vitro cell experiments on animals show that the protein molecule has a function of efficiently blocking HBV gene expression for a long time, then continuous silencing of viruses in bodies of chronic affected patients is achieved, an intracellular immunotherapy effect of preventing hepatitis B viruses is achieved, and the protein molecule has wide applicationprospects.
Description
technical field [0001] The invention belongs to the technical field of genetic engineering biomedicine, and specifically relates to a protein molecule capable of performing apparent methylation modification on the DNA of the hepatitis B virus gene and its application. Background technique [0002] During the pathogenic process of hepatitis B virus (HBV), it usually presents a typical three-step pathological process of chronic hepatitis, liver cirrhosis and liver cancer. Mutations at the level of chromosome and gene sequence can occur in the body, including chromosome deletion and duplication. Chronic HBV infection has become a major global health problem due to changes in traditional molecular genetics such as row, aneuploidy, gene expansion and mutation. At present, nearly 350 million people in the world are chronically infected with hepatitis B virus. my country is also a high-incidence area of hepatitis B, with about 120 million hepatitis B carriers. Among them, 1 / 4 of...
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Login to View More IPC IPC(8): C07K14/00C12N15/11C12N15/861C12N15/867C12N15/85A61K38/16A61K48/00A61P1/16A61P31/20
Inventor 朱乃硕王桂良
Owner FUDAN UNIV