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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 problems such as brain nerve damage, high liver toxicity, and toxic reactions, and achieve improved targeting, strong affinity, and simple preparation Effect

Active Publication Date: 2012-03-14
武汉顺可达生物科技有限公司
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  • Abstract
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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

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Embodiment 1D

[0055] The screening of embodiment 1 DNA adapter

[0056] The present invention can specifically bind to the DNA aptamer of virulent Mycobacterium tuberculosis H37Rv, which is 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 with a length of 88 bases: 5'-GCG GAATTCTAATACGACTCACTATAGGGAACAGTCCGAGCC-N30-GGGTCAATGCGT CATA-3', where N stands for A, T, C, G four random bases. 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 enzyme cutting 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 preservation of dsDNA and ssDNA libraries

[0060] Before each ...

Embodiment 2

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

[0076] Use biotin-labeled downstream primers (5'-GCGGGATCCTATGACGCATTGACCC-3') to obtain a large number of biotin-labeled ssDNA adapters by asymmetric PCR amplification, according to 1X10 per well 5 Coat the ELISA plate with different bacteria, overnight at 4°C; wash 6 times with PBS (PBST) containing 0.05% Tween-20; add 100 μL of PBS containing salmon sperm DNA (100 μg / mL) to each well for blocking, 37°C, 1h , after washing 6 times with PBST, add 100 μL of biotin-labeled ssDNA (40 pmol) containing biotin-labeled each round to each well, 37°C, 1 h; wash 6 times with PBST; add 1:1000 diluted HR to each well Labeled streptavidin 37°C, 30min; TMB color development.

[0077] The binding ability of aptamer ZXL1 to virulent Mycobacterium tuberculosis H37Rv, BCG and Mycobacterium smegmatis was detected by flow cytometry. First, a large number of FAM-labeled aptamers ZXL1 were asymmetri...

Embodiment 3

[0080]The application of embodiment 3ZXL1 in the diagnosis of tuberculosis

[0081] Serum collection of tuberculosis patients (Wuhan Medical Treatment Center, including 17 cases of acute untreated tuberculosis patients, 18 cases of tuberculosis patients repeatedly hospitalized for chemotherapy, 24 cases of male patients, 11 cases of female patients, the youngest age of patients is 11 years old , the oldest age is 57 years old) and 14 cases of healthy volunteer serum (Zhongnan Hospital of Wuhan University).

[0082] Add serum samples to the wells of the microtiter plate to coat, add biotin-labeled aptamer ZXL1, add HRP-labeled streptavidin, and incubate at 37°C for 1 hour; after washing with PBS three times, add TMB for color development 5 Minutes; 2M sulfuric acid terminates the reaction and reads with a microplate reader; detects, and the results are shown in 5, compared with healthy volunteers, the OD450 of the ManLAM antibody in the serum of tuberculosis patients all signif...

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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, in particular to a single-stranded DNA aptamer that can be used for the treatment and diagnosis of tuberculosis caused by human Mycobacterium Tuberculosis (H37Rv) [ATCC 93009 (4)], The present invention also relates to the application of the aptamer in diagnosis or treatment of tuberculosis infection. Background technique [0002] At present, tuberculosis is still one of the major 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 in the world 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 great challenges, and there is ...

Claims

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Application Information

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Patent Type & Authority Applications(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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