Mycobacterium tuberculosis surface lipolysaccharide-antistatic nucleic acid aptamer and application thereof

A technology of mycobacterium tuberculosis and nucleic acid aptamer, which is applied in the field of microbial immunity and clinical treatment, can solve the problems of high liver toxicity, brain nerve damage, toxic reaction, etc., and achieve improved targeting, simple preparation, and strong affinity Effect

Active Publication Date: 2013-07-24
武汉顺可达生物科技有限公司
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0003] In terms of tuberculosis treatment, rifampicin, isoniazid, streptomycin, pyrazinamide and ethambutol are currently the most commonly used drugs for tuberculosis treatment. Pyrazinamide has liver toxicity to patients, and the combination of the two has greater liver toxicity (Capelle P, Dhumeaux D, Mora M, Feldmann G, Berthelot P.Effect of rifampicin on liver function in man.Gut.1972, 13(5 ): 366-371); although streptomycin can effectively kill Mycobacterium tuberculosis by inhibiting protein synthesis, the drug can cause irreversible damage to the eighth cranial nerve, including ataxia, vertigo, tinnitus, deafness, etc. At the same time, similar to other aminoglycosides, it can cause renal toxicity (Fürst G, Maurer J, Schlegel J. Monitoring ototoxic side effects in streptomycin therapy of tuberculosis patients with transitory evoked otoacoustic emissions TEOAE.Pneumologie.1995, 49(11): 590-595); while ethambutol is mainly used in combination with isoniazid, rifampicin, and pyrazinamide in the intensive phase of treatment, but its toxic side effects are proportional to its dose, and can cause severe optic neuritis (Lim SA. "Ethambutol-associated optic neuropathy". Ann. Acad. Med. Singap. 2006, 35(4): 274-278)
At the same time, due to various factors such as irregular drug use, the drug resistance rate of the above tuberculosis drugs is getting higher and higher.
Although people continue to develop new drugs such as moxifloxacin and R207910, the current development trend of new anti-tuberculosis drugs is generally slow, and there is an urgent need to develop new anti-tuberculosis drugs

Method used

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  • Mycobacterium tuberculosis surface lipolysaccharide-antistatic nucleic acid aptamer and application thereof
  • Mycobacterium tuberculosis surface lipolysaccharide-antistatic nucleic acid aptamer and application thereof
  • Mycobacterium tuberculosis surface lipolysaccharide-antistatic nucleic acid aptamer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0055] Example 1 Screening of DNA aptamers

[0056] The present invention can specifically bind to the DNA aptamer of virulent Mycobacterium tuberculosis H37Rv, which can be obtained by screening according to the following steps:

[0057] 1. Construction of random single-stranded DNA library and primers

[0058] Construction of a single-stranded DNA library of 88 bases: 5’-GCG GAATTC TAATACGACTCACTATAGGGAACAGTCCGAGCC-N30-GGGTCAATGCGT CATA-3’, where N represents four random bases, A, T, C, and G. The upstream primer is: 5’-GCG GAATTC TAATACGACTCACTATAGGGAACAGTCCGAGCC-3’, the underlined part is the DNA restriction site of EcoRI; the downstream primer is: 5’-GCG GGATCC TATGACGCATTGACCC-3', the underlined part is the DNA restriction site of BamHI. The random single-stranded DNA library and primers can be synthesized by Shanghai Boya Biological Co., Ltd.

[0059] 2. PCR amplification and storage of dsDNA and ssDNA libraries

[0060] Before each round of screening, the ssDNA library is amp...

Embodiment 2

[0075] Example 2 Detection of the binding force between aptamer ZXL1 and different bacteria

[0076] Using biotin-labeled downstream primers (5’-GCGGGATCCTATGACGCATTGACCC-3’) through asymmetric PCR amplification to obtain a large number of biotin-labeled ssDNA aptamers, according to 1X10 per well 5 ELISA plates were coated with different bacteria at 4°C overnight; washed 6 times with PBS (PBST) containing 0.05% Tween-20; 100μL of PBS containing salmon sperm DNA (100μg / mL) was added to each well for blocking, 37°C, 1h After washing 6 times with PBST, add 100μL of biotin-labeled ssDNA (40pmol) for each round of biotin-labeled ssDNA (40pmol), 37℃, 1h; wash 6 times with PBST; add 1:1000 diluted HR to each well The labeled streptavidin is 37°C, 30min; TMB develops color.

[0077] Flow cytometry was used to detect the binding ability of aptamer ZXL1 with virulent Mycobacterium tuberculosis H37Rv, BCG and Mycobacterium smegmatis. First, a large number of FAM-labeled aptamers ZXL1 were am...

Embodiment 3

[0080] Example 3 Application of ZXL1 in the diagnosis of tuberculosis

[0081] Collect sera of tuberculosis patients (Wuhan Medical Treatment Center, including 17 acute untreated tuberculosis patients, 18 tuberculosis patients repeatedly hospitalized for chemotherapy, 24 male patients, 11 female patients, the minimum age of patients is 11 years old , The maximum age is 57 years old) and 14 healthy volunteers (Department of Laboratory Medicine, Zhongnan Hospital, Wuhan University).

[0082] Add the serum sample coating to the wells of the microtiter plate, add the biotin-labeled aptamer ZXL1, add HRP-labeled streptavidin, and incubate at 37°C for 1 hour; wash with PBS three times and add TMB for color development 5 Minutes; after the reaction was terminated by 2M sulfuric acid, the microplate reader reads; the results are shown in 5, compared with healthy volunteers, the OD450 of ManLAM antibody in the serum of tuberculosis patients was significantly increased (p=0.0023). The resul...

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Abstract

The invention discloses a mycobacterium tuberculosis surface lipolysaccharide-antistatic nucleic acid aptamer and application thereof. The aptamer is a small molecular single-stranded deoxyribonucleic acid (ssDNA) which is specific to toxic mycobacterium tuberculosis surface lipolysaccharide ManLAM (Mannosylated Lipoarabinomannan) and has tuberculosis infection resisting and cellular immunity enhancing functions, and the nucleotide sequence of the ssDNA is shown as SEQIDNo.1. The small molecular ssDNA has a novel target spot and a novel structure which are different from those of the conventional anti-tuberculosis medicament, and has the immunologic suppression function of directly enclosing the ManLAM. The aptamer is easy to prepare and has low price; the obtained ssDNA aptamer is specifically combined on the surface of toxic mycobacterium tuberculosis; and the treatment targeting is further enhanced. The problems of increasing medicament tolerance and large side effect existing in the conventional treatment of tuberculosis are solved, and the aptamer can be taken as an effective novel anti-tuberculosis medicament or a tuberculosis diagnosis reagent.

Description

Technical field [0001] The invention belongs to the field of microbial immunity and clinical treatment, and specifically relates to a single-stranded DNA aptamer that can be used to treat and diagnose tuberculosis caused by human Mycobacterium Tuberculosis (H37Rv) [ATCC 93009(4)], The invention also relates to the application of the aptamer in the diagnosis or treatment of tuberculosis infection. Background technique [0002] At present, tuberculosis is still one of the main infectious diseases threatening human life and health, and it is also one of the main factors leading to human death. According to WHO statistics, about 2 billion people worldwide are currently infected with tuberculosis. In 2008, about 11.1 million people worldwide suffered from active tuberculosis, and about 1.8 million people died of tuberculosis. With the continuous emergence of drug-resistant strains of tuberculosis, the treatment of tuberculosis is facing huge challenges, and there is an urgent need to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12Q1/68C12Q1/04A61K31/7088A61P31/06C12N15/10C12R1/32
Inventor 章晓联潘勤王其龙
Owner 武汉顺可达生物科技有限公司
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