A group of nucleic acid aptamers specifically binding to Mycobacterium tuberculosis and their applications

Abstract

The present invention discloses a group of nucleic acid aptamers specifically binding to Mycobacterium tuberculosis and their applications. The ssDNA aptamer of the Mycobacterium tuberculosis standard strain H37Rv of the present invention has strong affinity and high specificity, and the aptamer can detect Mycobacterium tuberculosis with high specificity, which can provide a favorable basis for the preparation of sensors for detecting Mycobacterium tuberculosis and laboratory diagnosis of tuberculosis.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and medical microorganisms, and relates to two nucleic acid aptamers specifically binding to mycobacterium tuberculosis cells and uses thereof. Background Art

[0002] Tuberculosis is a chronic infectious disease that seriously endangers human health and is a public health and social issue of global concern. In recent years, due to the lack of effective and inexpensive diagnostic equipment, the emergence of drug-resistant tuberculosis strains, population mobility and other factors, the incidence of tuberculosis has increased significantly worldwide, and the situation of tuberculosis has become more severe. Rapid and accurate early diagnosis is an effective measure to prevent and treat tuberculosis and one of the key steps to control the spread of tuberculosis.

[0003] At present, chest X-ray, smear microscopy and culture are the most commonly used methods for diagnosing tuberculosis. Chest X-ray can only detect a large number of Mycobacterium tuberculosis after it is produced, and it is difficult to diagnose HIV patients. Smear microscopy is simple and fast, but the detection rate is only 50%. The culture method has a high detection rate, but it is time-consuming and labor-intensive, and it cannot specifically identify Mycobacterium tuberculosis. It takes 4 to 6 weeks to detect Mycobacterium tuberculosis and requires a high level of technical ability. The BACTEC MGIT 960 fully automatic detection system shortens the detection time to 10 days, but it still requires the cultivation of Mycobacterium tuberculosis, and it cannot specifically identify Mycobacterium tuberculosis, and it cannot distinguish between BCG and Mycobacterium tuberculosis and Mycobacterium smegmatis. The detection time cannot meet clinical requirements, and the equipment and reagents are very expensive. Multi-channel serial piezoelectric quartz crystal (MSPQC) sensors have made some progress in the field of rapid bacterial detection due to their high sensitivity, strong stability, low cost, and easy operation. They are mainly based on the changes in the electrical parameters of the culture medium caused by bacterial growth metabolism, but the metabolic method can only shorten the detection time of Mycobacterium tuberculosis to 7 days. After combining with the phage lysis method, the detection time is shortened to 30 hours. However, the detection time of these two methods is still limited by the culture time of bacteria, and they are also unable to specifically identify Mycobacterium tuberculosis and cannot distinguish between BCG and Mycobacterium tuberculosis. Immunological methods are based on the sensitive reaction of antigens and antibodies, which greatly improves the detection limit, but the instability of antibodies at room temperature limits the promotion and use of these methods. Molecular biology methods shorten the detection time and improve the sensitivity, but they cannot be popularized due to the need for harsh experimental conditions and high costs.

[0004] The study of aptamers is a new hot area. Aptamers are a type of single-stranded nucleic acid (DNA or RNA) that can bind to many target molecules (proteins, drugs, inorganic or organic molecules) with high affinity and specificity. So far, the method for discovering high-affinity and specific aptamers is to use the ligand index enrichment system. Due to the high specificity and high affinity of aptamers in binding to targets (small molecules, proteins, whole cells, viruses, bacteria), they are widely used in medical research in detection and drug research. Aptamers targeting Mycobacterium tuberculosis phosphokinase 2 (PPK2), aptamers targeting Mycobacterium tuberculosis early secretory proteins CFP-10 and ESAT-6, and Mycobacterium tuberculosis bacterial aptamers used in the prevention and treatment of Mycobacterium tuberculosis in mice. However, Mycobacterium tuberculosis bacterial aptamers used for clinical detection have not been reported. Therefore, it is necessary to develop an aptamer that can specifically recognize Mycobacterium tuberculosis and be used for clinical detection. Summary of the invention

[0005] The first object of the present invention is to provide a nucleic acid aptamer that can specifically bind to Mycobacterium tuberculosis.

[0006] The core sequences of the nucleic acid aptamer specifically binding to Mycobacterium tuberculosis of the present invention are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0007] The nucleic acid aptamer sequence is as follows:

[0008] 5′-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3′, wherein N35 is the core sequence, and the core sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0009] A certain position on the nucleotide sequence of the nucleic acid aptamer specifically binding to Mycobacterium tuberculosis of the present invention can be phosphorylated, oxygen-methylated, methylated, amino-methylated, sulfhydryl-methylated or isotope-methylated.

[0010] The nucleotide sequence of the nucleic acid aptamer specifically binding to Mycobacterium tuberculosis of the present invention can be combined with biotin, digoxin, fluorescent substances, nano-luminescent materials, polyethylene glycol, peptides, proteins, enzymes or folic acid markers.

[0011] The nucleotide sequence of the nucleic acid aptamer of the present invention may also include any one of the following three sequences:

[0012] (1) having a nucleotide sequence homology of more than 60% with the aforementioned nucleic acid aptamer;

[0013] (2) a sequence that hybridizes with the nucleotide sequence of the aforementioned nucleic acid aptamer;

[0014] (3) An RNA sequence transcribed from the aforementioned nucleotide sequence of the nucleic acid aptamer.

[0015] The second object of the present invention is to provide the application of the nucleic acid aptamer of the present invention, specifically including the application in preparing tuberculosis diagnostic reagents, the application in preparing drugs for preventing or treating tuberculosis, and the application in preparing probes for sensors for detecting Mycobacterium tuberculosis.

[0016] The present invention adopts the following method to obtain a DNA aptamer that specifically recognizes Mycobacterium tuberculosis H37Rv: using an in vitro SELEX screening technology of nucleic acid aptamers, taking Mycobacterium tuberculosis as a target, in order to obtain an aptamer that binds to mycobacteria with high specificity, taking a mixture of Mycobacterium smegmatis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella enteritidis as a counter-screening target, and screening out a nucleic acid aptamer that specifically binds to Mycobacterium tuberculosis from an in vitro synthesized random oligomeric DNA library (5′-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3′, N35 as a core sequence), the core sequence of which is as follows:

[0017]

[0018] The selected sequences were used

[0019] Primer P1 (5′-GGGAGCTCAGAATAAACGCTCAA-3′) and

[0020] Primer P2 (5′-GATCC GGGCCTCATGTCGAA-3′) was used for amplification. Symmetric PCR amplification and asymmetric PCR amplification were performed with the following procedures: 95°C for 5 min, 95°C for 30 s, 65°C for 30 s, 72°C for 30 s, 72°C for 5 min, and 18-40 cycles; the number of cycles was adjusted according to the specific amplification effect. The PCR product was detected by 3% agarose gel electrophoresis and recovered by gel purification kit (operated according to the kit product manual). After purification of the PCR amplification product, 1 μL was taken and connected to the vector PGM-T under the action of T4 DNA ligase, and then transformed into competent cells. An appropriate volume was evenly coated on an LA plate containing IPTG, x-gal, and antibiotics (Amp). After 12 to 16 hours, a single white colony on the screening plate was picked with an inoculation loop and placed in LB liquid medium containing Amp, and cultured overnight at 37°C. 1 mL of the bacterial solution was placed in a centrifuge tube, sealed with a film, and sent to Shanghai Shenggong Biotechnology Co., Ltd. (hereinafter referred to as Shanghai Shenggong) for sequencing.

[0021] The present invention uses the SELEX technology to screen with a reverse screening target different from the prior art, and screens out nucleic acid aptamers that specifically bind to Mycobacterium tuberculosis from a new in vitro synthesized random oligomeric DNA library, thereby obtaining a new nucleic acid aptamer sequence with extremely strong specificity. The nucleic acid aptamer sequence of the present invention is selected from a naturally occurring or artificially synthesized sequence, or the same sequence from any other source.

[0022] The aptamers screened by the present invention can specifically bind to Mycobacterium tuberculosis, and the nucleic acid aptamer sequence provided by the present invention has a very strong affinity with Mycobacterium tuberculosis as a target, and has the advantage of strong specificity; therefore, Mycobacterium tuberculosis can be detected with high specificity, which can provide a favorable basis for the preparation of Mycobacterium tuberculosis preparations, laboratory diagnosis of tuberculosis, and preparation of drugs for the prevention or treatment of tuberculosis. In addition, the sequence can be synthesized in large quantities and quickly in vitro, and the preparation method is simple and relatively easy to obtain. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The secondary structure of the core sequence of SEQ ID NO: 1 and SEQ ID NO: 2 of the aptamer of the present invention has high affinity and can specifically recognize Mycobacterium tuberculosis;

[0024] Figure 2 , the left figure shows the binding rate of Mycobacterium tuberculosis and aptamers, and the right figure shows the enrichment of aptamers in the library analyzed by flow cytometry;

[0025] Figure 3 The mechanism diagram of the SWCNTs / Apt / IDE-MSPQC sensor prepared by the present invention for detecting Mycobacterium tuberculosis;

[0026] Figure 4 The impedance value change curve of the sensor of the present invention, (a) bare gold interdigitated electrode, (b) modified with aptamer, (c) combined with carbon nanotubes, (d) aptamer captures Mycobacterium tuberculosis and releases carbon nanotubes;

[0027] Figure 5 The frequency shift curve of the SWCNTs / Apt / IDE-MSPQC sensor of the present invention for detecting different bacteria, with the bacterial concentration being 1×10 6 cfu / mL: (a) blank control, (b) Escherichia coli, (c) Pseudomonas aeruginosa, (d) Mycobacterium smegmatis, (e) Staphylococcus aureus, (f) BCG, (g) Mycobacterium tuberculosis. DETAILED DESCRIPTION

[0028] Example 1: Construction of random single-stranded DNA library and primers

[0029] A DNA library containing 78 nt base sequence (5′-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3′) was constructed. The upstream primer was 5′-GGGAGCTCAGAATAAACGCTCAA-3′, the downstream primer was 5′-GAT CCGGGCCTCATGTCG AA-3′, N35 was a random core sequence, and the library capacity was 4 35 The above libraries and primers were synthesized by Shanghai Bioengineering.

[0030] Example 2: SELEX screening

[0031] In the first four rounds, the ssDNA library was first placed in 400 μL selection buffer (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.1% NaN3), heated at 95°C for 5 min, and then cooled in ice for 10 min for use; Mycobacterium tuberculosis was taken, washed with physiological saline, centrifuged, and the supernatant was discarded, and repeated three times; 100 μL of the prepared ssDNA library solution was added to the centrifuge tube containing the precipitate, incubated at 37°C for 45 min; centrifuged at 6000 rpm for 10 min, and the supernatant was discarded. The supernatant was discarded, 100 μL of double distilled water was added, and the supernatant was centrifuged at 10,000 rpm for 15 min at 95°C water bath for 5 min. The supernatant was the ssDNA sequence that specifically binds to M. tuberculosis. In the last ten rounds of screening, the ssDNA library was first incubated with a mixture of Mycobacterium smegmatis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella enteritidis, centrifuged, and the supernatant was collected and then incubated with Mycobacterium tuberculosis to prepare the ssDNA sequence that specifically binds to Mycobacterium tuberculosis. Symmetrical and asymmetric PCR amplifications were performed using this sequence as a template, with the program being 95°C for 5 min, 95°C for 30 s, 65°C for 30 s, 72°C for 30 s, 72°C for 5 min, and 18-40 cycles; the number of cycles was adjusted according to the specific amplification effect. The PCR product was detected by 3% agarose gel electrophoresis and recovered by gel purification kit. After 14 rounds of screening, the target ssDNA single strand was obtained, and the screening was terminated.

[0032] Example 3: Binding rate of aptamer library to Mycobacterium tuberculosis

[0033] The ssDNA library obtained in the 2nd, 4th, 6th, 8th, 10th, 12th, 13th, and 14th rounds was amplified by asymmetric PCR using upstream and downstream primers (Biotin-RP) modified with fluorescein at the 5' end. The obtained ssDNA was incubated with Mycobacterium tuberculosis and centrifuged to collect the supernatant and the bacterial precipitate bound to the ssDNA. The fluorescence value of the supernatant was measured by a fluorescence spectrophotometer as F1. The bacterial precipitate bound to the ssDNA was resuspended in 100 μL of double distilled water, heated in a 100°C water bath for 10 minutes, and then placed in a high-speed centrifuge for centrifugation at 10,000 rpm and 4°C for 15 minutes. The supernatant was collected and transferred to a new centrifuge tube, and the fluorescence value F2 was measured. The binding rate R = F2 / (F l +F2)x100%, the binding rate was basically stable after the 13th round, indicating that the binding of ssDNA library and Mycobacterium tuberculosis had reached equilibrium, and the screening was ended.

[0034] Example 4: Demonstration of enrichment of target sequence using flow cytometry

[0035] To demonstrate the enrichment of the target sequence during the screening process, the capture of Mycobacterium tuberculosis by the target sequence was monitored by flow cytometry. 6 cfu / mL) in 200 μL selection buffer and incubate on a shaker at 100 rpm and 37°C for 40 min. The above sample was placed in a flow cytometer and 10,000 cells were collected for analysis. The test showed that with the increase in screening rounds, high-affinity and high-specificity aptamers were continuously enriched.

[0036] Example 5: Aptamer Cloning

[0037] Amplification and purification of the 14th round of screening products: The ssDNA sequences obtained in the 14th round of screening were amplified by PCR. The PCR products were recovered and purified using the UNIQ.10 DNA gel recovery kit. The purified PCR products were cloned: 1 μl of the amplified product was ligated to the vector PGM-T under the action of T4 DNA ligase, and then transformed into competent cells. An appropriate volume was evenly coated on an LA plate containing IPTG, x-gal, and antibiotics (Amp) (which were purchased from Shanghai Bioengineering). The culture dish was inverted and cultured at 37°C for 12-16 hours for "blue-white screening". 40 clones were selected for culture and 1 mL of the culture solution from each clone was sent to Shanghai Bioengineering for sequencing. The homology of the primary structure of the measured nucleic acid aptamer sequence was analyzed using DNAMAN software, and its secondary structure was simulated online using Mfold sever analysis software.

[0038] Example 6: Determination of aptamer affinity

[0039] Different concentration gradients of carboxyfluorescein (FAM) labeled aptamer solutions (concentrations ranged from 0 to 120 nM) were mixed with a fixed amount of Mycobacterium tuberculosis (10 7 cfu / mL) in a centrifuge tube, add selection buffer to 500μL, and incubate at 37℃, 100r / min shaking for 45min. Centrifuge at 8000rpm for 10min, discard the supernatant, resuspend the bacteria with 500μL washing buffer (50mMTris-HCl (pH 7.4), 100mM NaCl, 5mM KCl, 1mM MgCl2, 0.1% NaN3, 0.2% bovine serum albumin), discard the supernatant, and repeat washing 3 times. The bacterial precipitate bound to the aptamer was resuspended in 100μL sterile deionized water, and the centrifuge tube was placed in a water bath at 100℃ for 5min, immediately cooled to room temperature in an ice bath, and centrifuged at 10000rpm for 15min. The supernatant was transferred to a micro-cuvette, and its fluorescence value was measured by a fluorescence spectrophotometer. The obtained fluorescence values ​​were analyzed by nonlinear regression using software Origin8.0 according to the formula: Y = X × Bmax / (K d +X) to calculate the K of the aptamer d The results show that the affinity of SEQ ID NO: 1 is the best, and the affinity of SEQ ID NO: 2 is the second best. The results are shown in Table 2.

[0040] Example 7: Preparation and application of sensor for detecting Mycobacterium tuberculosis

[0041] The aptamer selected in Example 6 was fixed on the interdigitated gold electrode after thiol modification, and connected to the carbon nanotubes through π-π bonds as a probe. The interdigitated gold electrode was connected to the multi-channel piezoelectric sensor to design the SWCNTs / Apt / IDE-MSPQC sensor. When Mycobacterium tuberculosis is present, the aptamer specifically binds to Mycobacterium tuberculosis and the carbon nanotubes are detached, thereby causing an increase in the surface impedance value of the interdigitated electrode and an increase in the frequency shift value of the sensor. The sensor can detect Mycobacterium tuberculosis within 1 hour, and the detection limit is 100 cfu / mL. The results are shown in Figure 4 and 5 .

[0042] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

[0043] Table 1: Amounts of H37Rv and ssDNA libraries added for 14 rounds of screening

[0044]

[0045] Table 2: Dissociation equilibrium constants / nM for 12 aptamers containing the following core sequences

[0046]

Claims

1. Use of a group of nucleic acid aptamers that specifically bind to Mycobacterium tuberculosis in the preparation of tuberculosis diagnostic reagents, characterized in that: The nucleic acid aptamer sequence is as follows: 5'-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3', wherein N35 is the core sequence, and the core sequence is shown in SEQ ID NO: 1 and SEQ ID NO:

2.

2. Use of a group of nucleic acid aptamers that specifically bind to Mycobacterium tuberculosis in the preparation of probes for sensors for detecting Mycobacterium tuberculosis, characterized in that: The aptamer sequences are as follows: 5'-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3', wherein N35 is the core sequence, and the core sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

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