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Aptamer sequence of hepatitis B virus (HBV) core antigen and application of nucleic aptamer sequence

A nucleic acid aptamer and core antigen technology, which can be used in antiviral agents, pharmaceutical formulations, microbial measurement/inspection, etc., and can solve the problems of nucleic acid aptamers, reports or publications of hepatitis B virus.

Active Publication Date: 2012-08-29
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As far as hepatitis B virus is concerned, studies have reported peptide aptamers targeting the core antigen of hepatitis B virus, but so far there has been no report or publication of nucleic acid aptamers targeting hepatitis B virus

Method used

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  • Aptamer sequence of hepatitis B virus (HBV) core antigen and application of nucleic aptamer sequence
  • Aptamer sequence of hepatitis B virus (HBV) core antigen and application of nucleic aptamer sequence

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1: HBc gene cloning

[0026] Using known molecular cloning techniques, based on the recombinant plasmid pBS_HBV3.6II containing double copies of the HBV genome sequence, primers were designed according to the HBc gene sequence:

[0027] HBc_L: 5'-GCCCATATGGACATTGACCCGTA-3'

[0028] HBc_R: 5'-GCCCTCGAGTCAAAACAACAGTAGTTT-3'

[0029] Configure PCR system (total volume 50ul): H 2 O 38ul, 10×PCR buffer 5ul, 25mM MgCl 2 3ul, dNTP(10mM) 1ul, primer mix 1.5ul (H 2 O: 100uM HBc_L: 100uM HBc_R=4:0.5:0.5), DNA template 1ul, PrimeStar enzyme 0.5ul. Amplify on a PCR machine under the following conditions: heat pre-denaturation at 94°C for 2 min, denature at 94°C for 30 s, anneal at 55°C for 30 s, extend at 72°C for 20 s, 30 cycles, extend at 72°C for 8 min, and hold at 4°C for <1 h.

[0030] After the reaction, 5 ul of the product was electrophoresed in 1% agarose gel at 100V constant voltage, and after identifying and amplifying a DNA fragment consistent with the...

Embodiment 2

[0033] Embodiment 2: HBc expression and purification

[0034] A single colony of Escherichia coli containing the recombinant expression plasmid pET28a-HBc was inoculated into 5 mL of LB medium containing Kan antibiotics, and cultured overnight at 37°C. Transfer the overnight culture to 200 mL of fresh medium according to the volume ratio of 1:100, culture for 2-4 hours, and wait until OD 600 When the value reaches 0.6, add IPTG (final concentration is 0.1mM), induce culture at 30°C for 8h. The cells were collected by centrifugation at 4000rpm for 30min.

[0035] Resuspend the bacteria with 5mL lysate (50mM Tris-HCl (pH8.5~9.0), 2mM EDTA, 100mM NaCl, 0.5% Triton X-100, 1mg / ml lysozyme), and divide into three 2ml tubes, -80 degrees for 30 minutes. 100w ultrasonic crushing in ice bath (2s~5s, 60~80 times). Mix 1ml supernatant with 500ul Ni-NTA Agarose beads, add to the purification column, wash with 2ml PBS+Mg (1mM MgCl 2 ) after washing twice, resuspend with 2ml PBS+Mg an...

Embodiment 3

[0038] Example 3: Nucleic acid aptamer screening (one round)

[0039] First round oligo DNA library sequence: 5'-acgctcggatgccactacagNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNctcatggacgtgctggtgac-3';

[0040] Primers used for enrichment:

[0041] Aptamer_L: 5'-FAM-acgctcggatgccactacag-3';

[0042] Aptamer_R: 5'-biotin-gtcaccagcacgtccatgag-3';

[0043] Determination of OD of oligomeric DNA library 260 Afterwards, it was centrifuged and dried. Dissolve the library with 300ul PBS+Mg, take 200pmol (2.5nmol in the first round), put it on ice at 95°C for 8min, and then centrifuge quickly.

[0044] Reverse screening: Aspirate 200pmol of the library into the reverse screening target equivalent to 40pmol of the positive screening target, and make up to 800ul with PBS+Mg. Shake at 37°C for 30 minutes. Inhale the Milipore ultrafiltration tube for quick centrifugation (<1000rpm), and take the filtrate.

[0045] Positive sieve: Take 40pmol positive sieve target, centrifuge at 800rpm...

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Abstract

The invention belongs to the field of molecular immunity and relates to an aptamer sequence for a hepatitis B virus (HBV) core antigen and an application of the nucleic aptamer sequence. According to the invention, a gene-recombination plasmid is adopted to express the core antigen, and an aptamer specially bound with the core antigen is screened, so as to prepare a characteristic sequence CATTT with the special binding HBV core antigen. The characteristic sequence is the base of the special binding of the aptamer and the HBc (hepatitis B core) antigen, can be used for drug design and the preparation of drugs and other products; and the aptamer with the characteristic sequence can be used as a probe or therapeutic target resisting to HBV to design and prepare drugs or agents resisting to HBV.

Description

Technical field [0001] The invention is a molecular immune field, involving a sequence and use of a nucleic acid with a core antigen of hepatitis B virus. Background technique [0002] Hepatitis B (hepatitis B) is the main infectious disease that seriously endangers the health of our people. According to statistics from the World Health Organization (WHO), hepatitis B virus (hepatitis B virus, HBV) carriers in my country reached 130 million, as many as 30 million patients with liver disease. Due to 300,000 people with various types of liver disease.Liver disease has become one of the main diseases that threaten China's population health today. The existence of huge number of patients with chronic liver disease, and the emergence of new cases of hepatitis every year, has caused great consumption to social medical resources.Studies have shown that hepatitis B virus is the main pathogenic primer of hepatitis B, and the pathogen is clear.At present, the treatment effect of hepatitis ...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/11C12Q1/70C12Q1/68A61K48/00A61P31/20
Inventor 刘杰张骏张作伟
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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