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Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof

A technology of hepatitis B virus and surface antigen, which is applied in the fields of genetic engineering and biomedicine, can solve the problems of chronic HBV infection and the lack of long-term effective antiviral drugs, and achieves the effect of simple method and low cost.

Inactive Publication Date: 2012-03-28
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the planned immunization of hepatitis B vaccine in China has greatly reduced the incidence of hepatitis B, there is still a lack of long-term effective antiviral drugs for the existing chronic HBV infection, and most of the drugs do not specifically act on the liver

Method used

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  • Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof
  • Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof
  • Nucleic acid aptamer combining hepatitis B virus surface antigen and sequence thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1H1

[0017] Example 1H1 protein specifically binds to the SELEX screening of RNA aptamers

[0018] SELEX screening process such as figure 1 As shown, chemically synthesized initial oligonucleotide random library and primers, the sequence is as follows: 5'-TTAATACGACTCACTATAGTTGATTGCGTGTCAATCATGG-25N-GGTCATGTGTATGTTGGGGATTAGGACCTGATTGAGTTCAGCCCACATAC-3' (25N represents 25 random nucleotides);

[0019] Primer 1: 5′-TTAATACGACTCACTATAGTTGATTGCGTGTCAATC-3′,

[0020] Primer 2: 5'-GTATGTGGGCTGAACTCAAT-3'. The single-stranded DNA library is amplified into double-stranded DNA, and the product is subjected to 2% agarose gel electrophoresis and then gel-cut to recover and purify; the recovered double-stranded DNA is used as a template to transcribe a single-stranded RNA random library in vitro, and the transcript is purified by PAGE . 68 μg RNA library was reverse-screened with nitrocellulose membrane to remove membrane-bound RNA molecules, then incubated with 2 μg HBsAg at 37°C for 40 mi...

Embodiment 2

[0021] Secondary structure analysis of embodiment 2 RNA aptamers

[0022] The library obtained in the 12th round of screening was cloned into the pMD19-T vector, transformed into Escherichia coli DH5α, and 48 clones were randomly selected and sequenced. Obtain the sequence information of the screened aptamers, predict the RNA secondary structure of all sequencing clones through the structure prediction software RNA Structure Program, and obtain an aptamer that can form a special stem-loop in the random sequence region (23-47nt) structure, such as figure 2 shown.

Embodiment 3

[0023] Example 3 Obtaining of HBsAg binding suitable gametes with high specificity and high affinity

[0024] Take 1 μg of the RNA aptamers with the above secondary structures, digest them with calf intestinal alkaline phosphatase (CIP) at 37°C for 1 h, purify and recover the dephosphorylated RNA; label [γ- 32 P]ATP at the end of dephosphorylated RNA molecules. 10nmol of radioactively labeled RNA aptamers were incubated with different concentrations (10-200nM) of HBsAg at 37°C for 40min, and the reaction solution of each group was filtered through a nitrocellulose membrane, the filter membrane was washed, dried, and the filter membrane was measured by a liquid scintillation counter. The residual radioactive dose on the same sample was measured twice in parallel. The dissociation constants of each aptamer and HBsAg were calculated, and the aptamer with the highest affinity was obtained, which was named S-A22. 32 P-marked fifth-round library R5, sixth-round library R6, tenth-r...

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Abstract

The invention discloses a nucleic acid aptamer of a targeted human infectious hepatitis B virus cell and a sequence thereof and belongs to the fields of gene engineering and biological medicine. According to the invention, a novel combining chemical technology SELEX is utilized to screen out an RNA aptamer capable of specifically binding to a hepatitis B virus surface antigen from single chain RNA random library by using the hepatitis B virus surface antigen as a target protein. The RNA aptamer has a sequence of 5'-GUUGAUUGCGUGUCAAUCAUGGCCGUCUAUAAUGAUCGUAAACGACGGGUCAUGUGUAUGUUGGGGAUUGGGACCUGAUUGAGUUCAGCCCACAUAC-3', can form a special loop structure in a random sequence area thereof and combine with the hepatitis B virus surface antigen singularly and with high affinity or combine to hepatocyte infected by the hepatitis B virus by target bonding. The RNA aptamer provides a specific efficient mark molecule for hepatitis B diagnosis and treatment and also provides a new option for exploiting diagnostic reagent and treatment medicament for chronic hepatitis B.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and biomedicine. The invention relates to a nucleic acid aptamer targeted to infect hepatitis B virus hepatocytes obtained by screening with SELEX technology, and the nucleotide sequence of the aptamer. Background technique [0002] Hepatitis B Virus (HBV) infection is one of the important causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. About 75% of the 350 million chronic HBV infected people in the world are distributed in Asia, and my country is the global HBV Countries with the highest number of infections. Therefore, research on the prevention and treatment of hepatitis B has always been an important field of medical and health research in my country. Although the planned immunization of hepatitis B vaccine in China has greatly reduced the incidence of hepatitis B, there is still a lack of long-term effective antiviral drugs for the existing chronic HBV infection...

Claims

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Application Information

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IPC IPC(8): C12N15/115
Inventor 杨燕杨东亮刘嘉
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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