A nucleic acid aptamer VACA01 of Helicobacter pylori VacA and its application

Abstract

The present invention relates to a nucleic acid aptamer VACA01 specific for Helicobacter pylori VacA and its applications, belonging to the field of biotechnology. Through in vitro SELEX screening technology, using carboxyl magnetic beads as the solid-phase medium and Helicobacter pylori VacA protein as the target, a nucleic acid aptamer that specifically binds to Helicobacter pylori VacA is screened from the ssDNA library. The nucleic acid aptamer VACA01 of the present invention can bind to Helicobacter pylori VacA protein with high affinity and high specificity, and this nucleic acid aptamer VACA01 provides a new option for the preparation of detection reagents, purification reagents and antagonists for Helicobacter pylori VacA protein.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a nucleic acid aptamer VACA01 of Helicobacter pylori VacA and its application. Background Art

[0002] Helicobacter pylori (Hp) is a Gram-negative bacterium that can cause persistent gastric mucosal infection, leading to inflammation, gastric and duodenal ulcers, MALT lymphoma, and gastric cancer. Helicobacter pylori produces many virulence factors involved in the pathogenesis of the disease. Among them, Vacuolating cytotoxin (VacA) is an exotoxin of about 90 kDa and has been shown to be associated with gastric inflammation and ulcers in animal disease models. VacA has two functional domains (p33-37 and p55-58). The p33-37 domain contributes to cytotoxicity, and the p55-58 domain can mediate the binding of Helicobacter pylori to target cell receptors, such as sphingomyelin, fibronectin, receptor protein-tyrosine phosphatase α (RPTPα), RPTPβ, low-density lipoprotein receptor-related protein 1 (LRP1). A large number of studies have shown that VacA disrupts cell functions (such as autophagy, ion channel formation, intracellular vesicle trafficking, and antigen presentation), ultimately leading to cell death. The latest research findings show that the toxic effects of VacA are not limited to the site of infection, but can also cause a variety of systemic symptoms and complications, and even mental symptoms, such as anxiety and anorexia.

[0003] Due to the important role of VacA in the pathogenic mechanism of Helicobacter pylori, precise treatment targeting VacA has received more attention at present. Some research reports suggest that antagonists of VacA toxin can be developed starting from the structural characteristics of the VacA protein or the signal pathways of its interaction with host cells for the treatment of VacA-related diseases. However, unfortunately, the current related research is in the preliminary exploration stage. Among them, antibody-based drugs have relatively high success hopes, but the inherent disadvantages of antibodies, such as high preparation costs, inconvenient storage and transportation, have become the main obstacles to their further research and development.

[0004] In the field of Helicobacter pylori etiology detection, there are two major categories of techniques, including non-invasive and invasive techniques. Non-invasive Helicobacter pylori detection techniques are more suitable for bedside use due to their convenient operation, and can even be carried out by families or individuals. Currently, the mainstream non-invasive Helicobacter pylori detection techniques include 13 C breath test and fecal Hp antigen detection. Among them, 13The C breath test has a certain degree of radioactivity, which more or less damages the health of infants and minors. Moreover, taking antibiotics recently and the degree of fasting will also affect the interpretation of test results. The fecal Hp antigen detection is convenient to operate and easy to promote. However, the mucus dissolving agent (N-acetylcysteine) in the reagent will inhibit the activity of protein molecules, which affects the activity of antibodies in the detection reagent and reduces the accuracy of the method. Therefore, establishing a VacA protein detection kit with strong anti-interference ability and high sensitivity is expected to be widely applied and promoted in the fields of Helicobacter pylori immediate infection diagnosis, disease condition and prognosis assessment, etc.

[0005] Aptamers are also known as "synthetic antibodies" and "chemical antibodies". Their chemical essence is a single-stranded oligonucleic acid molecule (ssDNA or RNA) folded into a specific three-dimensional structure to bind to the target substance with high affinity and high specificity. Aptamers are obtained through an in vitro screening process of the Systematic evolution of ligands by exponential enrichment (SELEX) technology. Nucleic acid aptamers have the characteristics of high affinity, high specificity, can be synthesized in vitro, can modify their functions and pharmacokinetic properties, have no immunogenicity, and are economical. Based on the above advantages, using nucleic acid aptamers as recognition elements, simple and accurate detection new technologies and efficient and economical affinity purification systems can also be developed. The developed nucleic acid aptamer drugs can specifically block the functions of targets. Therefore, screening nucleic acid aptamers with high specificity and high affinity for binding Helicobacter pylori VacA protein has important scientific research, clinical and market values. Summary of the Invention

[0006] The purpose of the present invention is to provide a nucleic acid aptamer VACA01 of Helicobacter pylori VacA with high specificity and high affinity; another purpose of the present invention is to provide the applications of the nucleic acid aptamer VACA01 in the preparation of Helicobacter pylori VacA protein detection reagents or kits, in the preparation of reagents for separating and purifying Helicobacter pylori VacA protein in samples, and in the preparation of VacA protein antagonists, etc.

[0007] The purpose of the present invention is achieved through the following technical solutions: A nucleic acid aptamer VACA01 of Helicobacter pylori VacA, and its sequence is shown as follows:

[0008] 5’-CATTCGTATGGACTGCGGCTATGACTGATCATGGGCTTCTTGTTCTTAGATATCCTGGCAGCAAGTTCAAAGTACG-3’(SEQ ID NO:1)

[0009] Preferably, the 5'-end or 3'-end of the nucleic acid aptamer VACA01 is modified with FITC, amino, biotin, or digoxin groups.

[0010] Preferably, the nucleic acid aptamer VACA01 can be prepared by PCR amplification or in vitro synthesis.

[0011] Preferably, the application of the nucleic acid aptamer VACA01 of Helicobacter pylori VacA is in the preparation of a detection reagent for Helicobacter pylori VacA protein.

[0012] Preferably, the application of the nucleic acid aptamer VACA01 of Helicobacter pylori VacA is in the isolation and purification of Helicobacter pylori VacA protein.

[0013] Preferably, the application of the nucleic acid aptamer VACA01 of Helicobacter pylori VacA is in an antagonist targeting Helicobacter pylori VacA protein.

[0014] The nucleic acid aptamer VACA01 of Helicobacter pylori VacA is based on the in vitro SELEX screening technology of nucleic acid aptamers. Using carboxyl magnetic beads as the solid-phase medium and Helicobacter pylori VacA as the target, a nucleic acid aptamer that specifically binds to Helicobacter pylori VacA is screened from the ssDNA library through Helicobacter pylori VacA magnetic beads.

[0015] Compared with the prior art, the advantages of the present invention are as follows:

[0016] 1. The nucleic acid aptamer VACA01 of the present invention is non-toxic, has a small molecular weight, good permeability, and is easy to synthesize and label.

[0017] 2. The synthesis cost of the nucleic acid aptamer VACA01 of the present invention is lower than that of antibody preparation, and it has a short cycle and good reproducibility.

[0018] 3. The nucleic acid aptamer VACA01 of the present invention can bind to Helicobacter pylori VacA with high affinity, reaching the pM level, and the dissociation constant is 23.91 pM.

[0019] 4. The nucleic acid aptamer VACA01 of the present invention can specifically bind to Helicobacter pylori VacA in an N-acetylcysteine environment and does not bind to other Helicobacter pylori toxins and structural proteins.

[0020] 5. The nucleic acid aptamer VACA01 of the present invention has broad application prospects and important scientific, social, and economic values in the fields of separation and purification of Helicobacter pylori VacA, diagnosis and treatment of Helicobacter pylori infection, etc. Brief Description of the Drawings

[0021] Figure 1 It is a bioinformatics simulation diagram of the secondary structure of the nucleic acid aptamer VACA01.

[0022] Figure 2 It is a specific diagram of the nucleic acid aptamer VACA01 analyzed by the fluorescence binding rate experiment. In Figure 2 , the abscissa is the analyzed protein, and the ordinate is the fluorescence binding rate.

[0023] Figure 3 It is a curve plotted for the dissociation constant of the nucleic acid aptamer VACA01 binding to Helicobacter pylori VacA analyzed by the fluorescence binding rate experiment. The dissociation constant (Kd) is 23.91 pM. In Figure 3 , the abscissa is the DNA concentration (pM), and the ordinate is the fluorescence binding rate.

[0024] Figure 4 It is the dose - effect result of the nucleic acid aptamer VACA01 inhibiting the vacuolating toxicity of Helicobacter pylori VacA determined by the neutral red method. Specific Embodiments

[0025] The content of the present invention will be described in detail below in conjunction with the accompanying drawings of the specification and examples:

[0026] A nucleic acid aptamer VACA01 against Helicobacter pylori VacA has the following sequence:

[0027] 5’-CATTCGTATGGACTGCGGCTATGACTGATCATGGGCTTCTTGTTCTTAGATATCCTGGCAGCAAGTTCAAAGTACG-3’ (SEQ ID NO:1).

[0028] The nucleic acid aptamer VACA01 against Helicobacter pylori VacA, under the conditions of 25 °C, 100 mM Na + , 1 mM Mg 2+ , its spatial structure is as shown in Figure 1 . The nucleic acid aptamer VACA01 against Helicobacter pylori VacA is chemically modified with FITC, amino group, biotin, or digoxin at the 5’-end or 3’-end of the nucleic acid aptamer VACA01.

[0029] The nucleic acid aptamer VACA01 against Helicobacter pylori VacA is chemically modified with FITC, amino group, biotin, or digoxin for the product obtained by structural modification such as truncation, extension, or partial base substitution of the nucleic acid aptamer VACA01.

[0030] The nucleic acid aptamer VACA01 of Helicobacter pylori VacA is obtained by screening from a random ssDNA library with carboxyl magnetic beads as the solid-phase medium and Helicobacter pylori VacA protein as the target based on the in vitro SELEX screening technology of nucleic acid aptamers.

[0031] The screening method of the nucleic acid aptamer VACA01 of Helicobacter pylori VacA comprises the following steps:

[0032] (1) Preparation of the screening library: Prepare a random ssDNA library shown in the following sequence: 5’-CATTCGTATGGACTGCGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNCAGCAAGTTCAAAGTACG-3’;

[0033] (2) Couple Helicobacter pylori VacA protein with carboxyl magnetic beads to prepare Helicobacter pylori VacA magnetic beads;

[0034] (3) Perform heat activation treatment on the random ssDNA library;

[0035] (4) Incubate the ssDNA library after step (3) with the Helicobacter pylori VacA magnetic beads obtained in step (2);

[0036] (5) Magnetically separate the Helicobacter pylori VacA magnetic beads after step (4), wash away the ssDNA that is not bound, weakly bound and non-specifically bound on the surface of the Helicobacter pylori VacA magnetic beads; heat the Helicobacter pylori VacA magnetic beads, collect the specifically bound ssDNA, namely the ssDNA enriched library;

[0037] (6) PCR amplification: Perform PCR amplification on the ssDNA enriched library obtained in step (5), wherein the primers used for PCR amplification are:

[0038] Primer P1: 5’-FAM-CATTCGTATGGACTGCGG-3’ (SEQ ID NO:2)

[0039] Primer P2: 5’-Biotin-CGTACTTTGAACTTGCTG-3’ (SEQ ID NO:3);

[0040] (7) Purification of the PCR product: Purify the PCR product using a small fragment purification kit; incubate the purified dsDNA with streptavidin magnetic beads, after the streptavidin magnetic beads bound with dsDNA are washed and the dsDNA is denatured, separate with a magnetic stand and collect the supernatant; the supernatant is obtained as the secondary ssDNA library for the next round of screening after ethanol precipitation;

[0041] (8) Cyclic screening: The FAM-labeled secondary ssDNA library obtained in step (7) is used as the secondary library for the next round of screening, and the screening process of steps (3) to (7) is repeated.

[0042] Example 1: Screening of nucleic acid aptamer VACA01

[0043] The screening method of nucleic acid aptamer VACA01 of Helicobacter pylori VacA includes the following steps:

[0044] (1) Preparation of the initial ssDNA library for screening: Design an ssDNA library with a primer-binding region (18 fixed nucleotide sequences) at both ends and a random region (40 random nucleotide sequences) in the middle, and commission Sangon Biotech Co., Ltd. to synthesize it. Its sequence is as follows

[0045] 5’-CATTCGTATGGACTGCGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCAGCAAGTTCAAAGTACG-3’.

[0046] (2) Coupling of Helicobacter pylori VacA protein with carboxyl magnetic beads: The Helicobacter pylori VacA protein (His Tag) is purchased from ABCAM Company in the UK, prepared using an Escherichia coli expression system, with a purity > 90%. The carboxyl magnetic beads and coupling reagents are purchased from Bangs Laboratories Company in the US. The operation refers to the instructions provided by the manufacturer; the change in the protein concentration in the Helicobacter pylori VacA solution before and after coupling is measured by the BCA method. After calculation, the coupling efficiency of the magnetic beads is 81.6%; the Helicobacter pylori VacA magnetic beads are dispersed in 1×PBS buffer and stored at 4°C.

[0047] (3) Dissolve 2 nM of the initial ssDNA library in 500 μL of selection buffer (60 mM Tris-HCl, 100 mM NaCl, 1.5 mM MgCl2, 5 mM KCl, pH 7.4), and then perform heat activation treatment (95°C for 5 min, ice-water bath for 10 min, room temperature for 10 min).

[0048] (4) Mix the ssDNA library after step (3) with the Helicobacter pylori VacA magnetic beads obtained in step (2) (the Helicobacter pylori VacA loading is 80 ng) and yeast tRNA (the molar amount is 5 times that of the ssDNA library), and incubate at room temperature for 1 h.

[0049] (5) Magnetically separate the Helicobacter pylori VacA magnetic beads after step (4), and wash away the ssDNA that is unbound, weakly bound, and non-specifically bound to the surface of the Helicobacter pylori VacA magnetic beads with a selection buffer containing 0.2% BSA; then resuspend the Helicobacter pylori VacA magnetic beads in 200 μL of ddH2O, incubate in a hot water bath at 100 °C for 10 min, place on a magnetic rack for 1 - 2 min, collect the supernatant, and obtain the ssDNA specifically bound to the Helicobacter pylori VacA magnetic beads, namely the ssDNA enrichment library.

[0050] (6) PCR amplification: Add the ssDNA enrichment library obtained in step (5) to 1 mL of PCRmix reagent; after vortexing and mixing evenly, dispense into tubes at 50 μL per tube for PCR amplification. The amplification conditions are: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 S, annealing at 62 °C for 30 S, extension at 72 °C for 30 S, for 13 - 25 cycles.

[0051] Among them, 1 mL of PCRmix contains: 100 μL of 10×PCR buffer; 1 μL each of pfu enzyme and Taq enzyme; 20 μL of dNTP; 3 μL each of primer P1: 5’-FAM-CATTCGTATGGACTGCGG-3’ and primer P2: 5’-Biotin-CGTACTTTGAACTTGCTG-3’; both primer P1 and primer P2 were synthesized by Sangon Biotech Co., Ltd.

[0052] (7) Purification of PCR products: The PCR products labeled with biotin and the fluorescent group FAM at both ends are purified using a small fragment purification kit (the small fragment purification kit was purchased from Sangon Biotech Co., Ltd.). Incubate the purified dsDNA with streptavidin magnetic beads (purchased from Invitrogen-Dynal) at 37 °C for 20 min, wash the streptavidin magnetic beads bound to dsDNA 3 times with a washing buffer (5 mM Tris-HCl, pH 7.4, 1.2 M NaCl, 500 μM EDTA), then incubate with 50 μL of NaOH solution (0.2 M) at 37 °C for 30 min to denature the dsDNA; separate with a magnetic rack, collect the supernatant, and the supernatant is precipitated with ethanol to obtain the FAM-labeled secondary ssDNA library, which is dissolved in the selection buffer as the secondary library for the next round of screening.

[0053] (8) The screening process is carried out for 11 rounds. Starting from the second round, the dosage of the secondary library is 40 pM each time.

[0054] Example 2: Analysis of the sequence of nucleic acid aptamer VACA01

[0055] (1) After 11 rounds of screening, the enriched ssDNA library was collected, and the library sequence was analyzed by high-throughput sequencing technology. The analysis process was as follows: PCR amplification of the enriched library, adding sequencing adapters and Index parts; selecting and purifying the library by gel electrophoresis; quantitatively analyzing the purified library using Qbit; quality control of the library using Agilent 2100 Bioanalyzer with Agilent High Sensitivity DNA Kit; quantitatively analyzing the library using Quant-iT PicogGreen dsDNA Assay Kit; using Illumina Novaseq6000 platform, performing bridge PCR amplification, sequencing primer annealing, and sequencing by synthesis with the single-stranded library as the template; and performing alignment and enrichment analysis on the sequencing results.

[0056] (2) According to the enrichment degree of the aptamer in the library, ssDNA with a high enrichment degree was selected as the candidate aptamer. Among them, the aptamer VACA01 accounted for 18.2% in the enriched library, and its sequence was shown as SEQ ID NO:1.

[0057] (3) Using the UNAFold web platform to analyze the secondary structure of the aptamer VACA01 sequence under the conditions of 25 °C, 100 mM Na + , 1 mM Mg 2+ . The schematic diagram of the secondary structure of the aptamer VACA01 sequence was shown as Figure 1 . The stem-loop structure enables the aptamer VACA01 to have a lower free energy and a stable secondary structure, ensuring its binding ability to the target.

[0058] Example 3: Specificity analysis of aptamer VACA01

[0059] (1) Chemically synthesize FAM-labeled aptamer VACA01 in vitro and dissolve it in the selection buffer. The selection buffer dissolved N-acetylcysteine (NAC) with a final concentration of 0.1%. The pure product of N-acetylcysteine was purchased from Shanghai Yisheng Biotechnology Co., Ltd.

[0060] (2) Referring to step (2) in Example 1, BSA, Helicobacter pylori CagA, HpaA, BabA, and GroEL proteins were respectively coupled with carboxyl magnetic beads to prepare BSA magnetic beads, Helicobacter pylori VacA magnetic beads, HpaA magnetic beads, BabA magnetic beads, and GroEL magnetic beads. Among them, the BSA was purchased from Sigma Company, and the Helicobacter pylori CagA, HpaA, BabA, and GroEL proteins were all purchased from ABCAM Company, UK.

[0061] (3) Take 200 μL of the aptamer VACA01 solution obtained in step (1) and mix it with the BSA magnetic beads, Helicobacter pylori CagA magnetic beads, HpaA magnetic beads, BabA magnetic beads, GroEL magnetic beads, and Helicobacter pylori VacA magnetic beads prepared in step (2), and incubate at room temperature for 1 h in a dark box, with blank magnetic beads as the control.

[0062] (4) Wash the above magnetic beads from step (3) three times with 0.1% PBST. The aptamer bound to the magnetic beads is eluted with 200 μL of selection buffer by boiling at 100 °C for 5 min.

[0063] (5) Use a fluorescence quantitative analyzer to measure the fluorescence intensities of the initial solution and the eluate respectively, and calculate the fluorescence binding rate = (initial fluorescence intensity - eluted fluorescence intensity) / initial fluorescence intensity × 100%. The calculated value is used to preliminarily represent the binding rate of the aptamer VACA01 to the target molecule.

[0064] As Figure 2 shown, the binding rate of the aptamer VACA01 to Helicobacter pylori VacA is significantly higher than its binding rates to BSA, Helicobacter pylori CagA, HpaA, BabA, and GroEL proteins, indicating that the aptamer VACA01 has good specificity in binding to Helicobacter pylori VacA in a 0.1% N-acetylcysteine environment. Further illustration shows that the aptamer VACA01 has the potential in establishing a fecal VacA antigen detection kit.

[0065] Example 4: Affinity analysis of aptamer VACA01

[0066] (1) Take FAM-labeled aptamer solutions with different concentrations and mix them with Helicobacter pylori VacA magnetic beads, and incubate at room temperature for 1 h in a dark box.

[0067] (2) Refer to steps (4) and (5) in Example 3, and experimentally obtain and calculate the fluorescence binding rates of aptamer solutions with different concentrations to Helicobacter pylori VacA magnetic beads.

[0068] (3) Use the calculated values of the fluorescence binding rates to plot a saturation binding curve of the aptamer binding to Helicobacter pylori VacA, and calculate the dissociation constant of the aptamer binding to Helicobacter pylori VacA through nonlinear regression analysis.

[0069] As Figure 3 shown, we obtained a saturation binding curve of the aptamer VACA01. After calculation, the dissociation constant of the aptamer VACA01 is 23.91 pM, indicating that the aptamer VACA01 has a strong binding ability to Helicobacter pylori VacA, and the dissociation constant is at the picomolar level.

[0070] Example 5: Inhibitory effect of nucleic acid aptamer VACA01 on the virulence of VacA

[0071] (1) The human gastric cancer cell line AZ-521 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. and cultured in MEME medium (purchased from Sigma) containing 10% fetal bovine serum (FBS, purchased from Sigma).

[0072] (2) The toxicity of VacA was evaluated using AZ-521 cells. AZ-521 cells were seeded in 48-well plates at a concentration of 1×10 5 cells / well, 250 μL per well. They were cultured as a monolayer in 48-well culture plates under the conditions of 5% CO2 and 37 °C for 24 h.

[0073] (3) The concentrations of nucleic acid aptamer VACA01 added to each group were 0 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM in sequence. VacA protein with a final concentration of 120 nM was added to each well, and a blank control group without VacA protein and nucleic acid aptamer VACA01 was set. Incubation continued for 5 h.

[0074] (4) The cells were incubated with 50 μL of freshly prepared 0.05% neutral red (purchased from Beyotime) in 1×PBS containing 0.3% BSA, and then washed 3 times with 0.1 mL of 1×PBS containing 0.3% BSA. After adding 0.1 mL of 70% ethanol to 0.4% hydrochloric acid solution, the absorbance (OD) at 540 nm was measured using a spectrophotometer.

[0075] As Figure 4 shown, the abscissa is the concentration of nucleic acid aptamer VACA01, and the ordinate is the OD value at 540 nm. As the concentration of nucleic acid aptamer VACA01 increased, the effect of VacA protein causing vacuoles decreased significantly (P < 0.05). The above results indicate that nucleic acid aptamer VACA01 can significantly inhibit the toxicity of VacA protein in a dose-dependent manner in in vitro experiments and is a potential VacA protein inhibitor.

[0076] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several changes, improvements, and modifications can still be made, and these changes, improvements, and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A nucleic acid aptamer VACA01 of Helicobacter pylori VacA, characterized in that: Its sequence is shown as follows: 5’-CATTCGTATGGACTGCGGCTATGACTGATCATGGGCTTCTTGTTCTTAGATATCCTGGCAGCAAGTTCAAAGTACG-3’.

2. The nucleic acid aptamer VACA01 of Helicobacter pylori VacA according to claim 1, characterized in that: Modify the 5'-end or 3'-end of the nucleic acid aptamer VACA01 with FITC, amino, biotin, or digoxin groups.

3. The nucleic acid aptamer VACA01 of Helicobacter pylori VacA according to claim 1, characterized in that: The nucleic acid aptamer VACA01 can be prepared by PCR amplification or in vitro synthesis.

4. Use of the nucleic acid aptamer VACA01 of Helicobacter pylori VacA according to claim 1 or 2 in the preparation of a detection reagent for Helicobacter pylori VacA protein.

5. Use of the nucleic acid aptamer VACA01 of Helicobacter pylori VacA according to claim 1 or 2 in the isolation and purification of Helicobacter pylori VacA protein.

6. Use of the nucleic acid aptamer VACA01 of Helicobacter pylori VacA according to claim 1 or 2 in the preparation of a Helicobacter pylori VacA antagonist.

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