DNA aptamer binding specifically to tb7.7 and use thereof

A DNA aptamer and specific technology, applied in the field of DNA aptamers, to achieve the effect of eliminating false positive reactions, excellent binding ability and high exclusivity

Active 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

The mechanism by which host disease is induced during infection is not known exactly, but it is speculated by IGRA that TB7.7 forms a complex with ESAT6, which is used as a Mycobacterium tuberculosis-specific antigen against interferon-γ secretion to mediate induce Th1 cell immune response

Method used

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  • DNA aptamer binding specifically to tb7.7 and use thereof
  • DNA aptamer binding specifically to tb7.7 and use thereof
  • DNA aptamer binding specifically to tb7.7 and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1.TB7.7 gene cloning

[0050] To amplify the gene for the 7.7kDa tuberculosis-specific antigen (TB7.7), a forward primer including the BamH1 restriction enzyme recognition sequence [5'-CCC GGA TCC ATG AGC GGC CAC GCG TTG3' was used (SEQ ID NO:1)], the reverse primer includes the Xho1 restriction enzyme recognition sequence [5'CCC CCC TCG AGT CAC GGC GGA TCA CCCC3'(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 to denature double-stranded DNA into a template; 2) Incubate at 55°C for 20 seconds, as a process for 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 TB7.7 gene was cloned into a (His)6-tag-containing...

Embodiment 2

[0053] Expression of embodiment 2.TB7.7 protein

[0054] BL21(DE3) cells transformed with TB7.7 gene were cultured in Luria Bertani(LB) medium and incubated at 37°C until the optical density (OD) at UV 600nm 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. results, such as Figure 1b As shown, it was confirmed that the recombinant TB7.7 protein was overexpressed.

Embodiment 3

[0055] Purification of embodiment 3.TB7.7 protein

[0056] To purify TB7.7 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), It was then ruptured by ultrasonication for 15 minutes. A centrifuge was used at 18,000 rpm for 40 minutes to separate the protein in an aqueous solution from the cells.

[0057] 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. Specifically, as shown in FIG. 1 , a Ni-NTA column was connected to a fast protein liquid chromatography (FPLC) system, and TB7.7 in an aqueous solution state was flowed into the column, thereby allowing TB7.7 to bind thereto. Since the (His)6-tag of the protein bound to the Ni-NTA column competes with the imidazole compound to separate the target protein from the column, the elution b...

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Abstract

The present invention relates to a DNA aptamer binding specifically to tuberculosis specific antigen 7.7 kDa (TB7.7), a biosensor for diagnosis of tuberculosis, comprising the same, and a method for providing 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 TB7.7 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 an aptamer for protein detection, more specifically, a DNA aptamer specifically binding to a 7.7 kDa tuberculosis-specific antigen (TB7.7) 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 Organizati...

Claims

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Application Information

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