DNA aptamer binding specifically to esat6 and use thereof

A DNA aptamer and specific technology, applied in the field of DNA aptamers, can solve problems such as lack of stability and reduced sensitivity, and achieve excellent stability, high exclusivity, and excellent binding ability

Pending Publication Date: 2020-05-29
MD APTUS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with TST, the probability of false positive results is low due to the use of M. tuberculosis-specific antigens, but the lack of stability is a disadvantage considering that tuberculosis endemic areas are mostly hot or humid
In addition, the secretion of interferon-γ is indirectly measured by stimulation in the blood, so there are disadvantages such as decreased sensitivity
[0004] Meanwhile, ESAT6 is known to be overexpressed in Mycobacterium tuberculosis H37Rv, which causes tuberculosis fatal to humans
The mechanism by which host disease is induced during infection is not known exactly, but it is speculated by IGRA that ESAT6 forms a complex with CFP10 or TB7.7, which is used as a M. tuberculosis-specific antigen against interferon-γ secretion, To mediate Th1 cell immune response

Method used

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  • DNA aptamer binding specifically to esat6 and use thereof
  • DNA aptamer binding specifically to esat6 and use thereof
  • DNA aptamer binding specifically to esat6 and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1. ESAT6 gene cloning

[0050] To amplify the gene for the 6 kDa early secretory antigen target (ESAT6), a forward primer including the BamH1 restriction enzyme recognition sequence [5'-CCC GGATCC ATG ACA GAG CAG CAG TGG AAT TT3'( SEQ ID NO:1)], the reverse primer includes the Hind3 restriction enzyme recognition sequence [5'CCC CCA AGC TTC TAT GCGAAC ATC CCA GTG A3'(SEQ ID NO:2)].

[0051] Genomic DNA of M. tuberculosis H37Rv was used as a template for gene amplification and was amplified by polymerase chain reaction (PCR) using i-pfu polymerase. Each process of PCR is as follows: 1) Incubate at 98°C for 20 seconds, as a process of denaturing double-stranded DNA into a template; 2) Incubate at 57°C for 20 seconds, as a process of annealing the template with primers; 3) Incubate at 72°C 30 seconds, as a process of extending new strands, and a cycle of repeating these processes 30 times.

[0052] The amplified ESAT6 gene was cloned into a (His)6-tag-contai...

Embodiment 2

[0053] Example 2. Expression of ESAT6 protein

[0054] BL21 (DE3) cells transformed with ESAT6 gene were cultured in Luria Bertani (LB) medium and incubated at 37° C. until the optical density (OD) at UV 600 nm reached 0.563. Thereafter, isopropyl-thio-b-D-galactopyranoside (IPTG) was added to a final concentration of 20 mM to induce the expression of the protein, followed by incubation at 37°C for 4 hours. The expression of the protein was confirmed by SDS PAGE, and the cells were separated from the medium using a centrifuge and washed once with PBS (10 mM sodium phosphate, 150 mM NaCl, pH 8.0) buffer.

Embodiment 3

[0055] Example 3. Purification of ESAT6 protein

[0056] In order to purify ESAT6 protein expressed in bacterial cells BL21(DE3) with high purity, cells were lysed in cell lysis buffer (20 mM Tris, 500 mM NaCl, 0.5 mM β-mercaptoethanol, 5% glycerol, pH 8.0) and then washed with Ultrasound for 15 minutes to rupture. A centrifuge was used at 18,000 rpm for 40 minutes to separate the protein in an aqueous solution from the cells.

[0057] After centrifugation, the pellet was added to and allowed to dissolve in refolding buffer containing 8M urea (8M urea, 20mM Tris-HCl, 500mM NaCl, 15mM imidazole, 0.5mM β-mercaptoethanol, 5% glycerol, pH 8.0) . Separation was performed at 18,000 rpm for 50 minutes using a centrifuge to separate proteins dissolved in an aqueous solution state from the cells.

[0058] Furthermore, in order to obtain high-purity proteins, the binding properties between nickel-nitrilotriacetic acid (Ni-NTA) and (His)6-tagged amino acids were utilized. Specifica...

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Abstract

The present invention relates to a DNA aptamer binding specifically to early secretory antigenic target 6 kDa (ESAT6), a biosensor for diagnosis of tuberculosis, comprising the same, and a method forproviding information for diagnosis of tuberculosis. The present inventors found that not only does a DNA aptamer according to the present invention have specific binding potential to ESAT6 protein, but also the binding affinity is excellent. When used, the DNA aptamer of the present invention can be thus expected to exhibit greater stability than a conventional ELISA method using an antibody. Hence, the aptamer is expected to find useful applications in the development of compositions for tuberculosis diagnosis, biosensors for tuberculosis diagnosis, and information providing methods for tuberculosis diagnosis.

Description

technical field [0001] The present invention relates to aptamers for protein detection, and more particularly, to DNA aptamers that specifically bind to a 6 kDa early secretory antigen target (ESAT6) known to be expressed in Mycobacterium tuberculosis H37Rv. Background technique [0002] Tuberculosis is a common and fatal disease caused by infection with the bacterium Mycobacterium tuberculosis. Symptoms of tuberculosis include sudden weight loss due to anorexia, high fever, and cough with bloody sputum. Diagnostic methods include X-ray examination, sputum smear examination, and polymerase chain reaction (PCR), and finally a combination of them is used to diagnose tuberculosis. Tuberculosis is known to occur frequently in developing countries, including in the African region, and was recently considered an eradicated disease. However, it is a dreadful disease that ranks among the top 10 causes of death worldwide in 2015 according to the statistics of the World Health Organ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/553G01N33/569
CPCC12N15/115C12N2310/16G01N33/5695G01N33/5308G01N33/553G01N2800/26
Inventor 金润根潘沧一张哲熏
Owner MD APTUS INC
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