CN115678902A - Aptamer VACA01 of helicobacter pylori VacA and application thereof - Google Patents

Abstract

The invention relates to an aptamer VACA01 of helicobacter pylori VacA and application thereof, belonging to the technical field of biology. The invention obtains the nucleic acid aptamer specifically combined with the helicobacter pylori VacA from ssDNA library by using carboxyl magnetic beads as solid phase medium and helicobacter pylori VacA protein as a target through an in vitro SELEX screening technology; the aptamer VACA01 can be combined with helicobacter pylori VacA protein with high affinity and high specificity, and the aptamer VACA01 provides a new choice for preparing a detection reagent, a purification reagent and an antagonist of the helicobacter pylori VacA protein.

Description

Aptamer VACA01 of helicobacter pylori VacA and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a nucleic acid aptamer VACA01 of helicobacter pylori VacA and application thereof.

Background

Helicobacter pylori (Hp) is a gram-negative bacterium that causes persistent infection of the gastric mucosa, which can lead to inflammation, gastric and duodenal ulcers, MALT lymphoma, and gastric cancer. Helicobacter pylori produces a number of virulence factors that are involved in the pathogenesis of the disease. Among them, vacuolar cytotoxin (VacA), an exotoxin of about 90kDa, has been demonstrated to be associated with gastritis and ulcers in animal disease models. VacA has two functional domains (p 33-37 and p 55-58). The p33-37 domain contributes to cytotoxicity and the p55-58 domain mediates binding of helicobacter pylori to target cell receptors, such as sphingomyelin, fibronectin, receptor protein-tyrosine phosphatase alpha (RPTP α), RPTP β, low density lipoprotein receptor-related protein 1 (LRP 1). Numerous studies have shown that VacA disrupts cellular functions (e.g., autophagy, ion channel formation, intracellular vesicle trafficking antigen presentation) and ultimately leads to cell death. Recent studies have found that the toxic effects of VacA are not limited to the site of infection, but can lead to a variety of systemic symptoms and complications, and even psychiatric symptoms such as anxiety and anorexia.

Due to the important role of VacA in the pathogenesis of helicobacter pylori, precise treatment targeting VacA is now receiving more attention. It has been reported that antagonists of the VacA toxin can be developed based on the structural features of the VacA protein or its signaling pathway with host cells, and can be used for treating diseases related to VacA. Unfortunately, the related studies are in the preliminary exploration phase at present. Among them, the antibody drugs are expected to be successful, but the inherent disadvantages of high preparation cost and inconvenient storage and transportation of the antibody have become major obstacles for further development.

The detection field of the etiology of the helicobacter pylori comprises two main technologies, namely non-invasive and invasive. The noninvasive helicobacter pylori detection technology is more suitable for being carried out by a bed or even by families or individuals due to convenient operation. The mainstream noninvasive helicobacter pylori detection technology at present comprises ¹³ C breath test and fecesAnd detecting the Hp antigen. Wherein, ¹³ c, the breath test has certain radioactivity, so that more or less damages are caused to the health of infants and immature adults, and the judgment of test results is influenced by taking antibiotics and fasting degree in the near term; the fecal Hp antigen detection is convenient to operate and popularize, but a mucolytic agent (N-acetylcysteine) in the reagent can inhibit the activity of protein molecules, so that the activity of an antibody in the detection reagent is influenced, and the accuracy of the method is reduced. Therefore, the establishment of the VacA protein detection kit with strong anti-interference capability and high sensitivity is expected to be widely applied and popularized in the fields of helicobacter pylori immediate infection diagnosis, disease condition and prognosis evaluation and the like.

Aptamers are also known as "synthetic antibodies", "chemical antibodies", and their chemical nature is that a single-stranded oligonucleotide molecule (ssDNA or RNA) folds into a specific three-dimensional structure that binds to a target substance with high affinity and specificity. The aptamer was obtained by in vitro screening process of Systematic evolution of ligands by exponentiation (SELEX). The aptamer has the characteristics of high affinity, high specificity, capability of being synthesized in vitro, capability of changing the function and the pharmacokinetic characteristic thereof through modification, no immunogenicity, economy and the like. Based on the advantages, the aptamer is used as an identification element, a new simple and accurate detection technology and an efficient and economic affinity purification system can be developed, and the developed aptamer drug can specifically block the target function. Therefore, screening the aptamer which has high specificity and high affinity and is combined with the helicobacter pylori VacA protein has important scientific research, clinical and market values.

Disclosure of Invention

The invention aims to provide a nucleic acid aptamer VACA01 of helicobacter pylori VacA with high specificity and high affinity; the invention also aims to provide the application of the aptamer VACA01 in various aspects such as preparation of a helicobacter pylori VacA protein detection reagent or kit, preparation of a helicobacter pylori VacA protein separation and purification reagent in a sample, preparation of a VacA protein antagonist and the like.

The purpose of the invention is realized by the following technical scheme: an aptamer VACA01 of a helicobacter pylori VacA, the sequence of which is shown as follows:

5’-CATTCGTATGGACTGCGGCTATGACTGATCATGGGCTTCTTGTTCTTA GATATCCTGGCAGCAAGTTCAAAGTACG-3’(SEQ ID NO:1)

preferably, the 5 'end or the 3' end of the aptamer VACA01 is modified by FITC, amino, biotin or digoxigenin.

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

Preferably, the application of the aptamer VACA01 of the helicobacter pylori VacA in preparing a helicobacter pylori VacA protein detection reagent.

Preferably, the application of the aptamer VACA01 of the helicobacter pylori VacA in the separation and purification of the helicobacter pylori VacA protein.

Preferably, the application of the aptamer VACA01 of the helicobacter pylori VacA in an antagonist taking helicobacter pylori VacA protein as a target.

The aptamer VACA01 of the helicobacter pylori VacA is an in-vitro SELEX screening technology based on the aptamer, carboxyl magnetic beads are used as a solid phase medium, the helicobacter pylori VacA is used as a target, and the aptamer specifically bound with the helicobacter pylori VacA is obtained by screening a ssDNA library through the helicobacter pylori VacA magnetic beads.

Compared with the prior art, the invention has the advantages that:

1. the aptamer VACA01 disclosed by the invention is non-toxic, small in molecular weight, good in permeability and easy to synthesize and label.

2. The synthesis cost of the aptamer VACA01 is lower than that of antibody preparation, and the aptamer VACA01 has short period and good reproducibility.

3. The aptamer VACA01 of the invention can be combined with helicobacter pylori VacA with high affinity, the affinity reaches pM level, and the dissociation constant is 23.91pM.

4. The nucleic acid aptamer VACA01 of the 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.

5. The aptamer VACA01 disclosed by the invention has wide 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 and the like.

Drawings

FIG. 1 is a bioinformatics mimetic diagram of the secondary structure of aptamer VACA 01.

FIG. 2 is a diagram showing the specificity of the aptamer VACA01 analyzed by a fluorescence binding rate experiment. In FIG. 2, the abscissa represents the analyzed protein, and the ordinate represents the fluorescence binding rate.

FIG. 3 is a graph showing dissociation constants (Kd) of the aptamer VACA01 binding to helicobacter pylori VacA in a fluorescence binding rate experiment, and the dissociation constant (Kd) is 23.91pM. In FIG. 3, the abscissa represents the DNA concentration (pM) and the ordinate represents the fluorescence binding rate.

FIG. 4 shows the results of dose-effect of the aptamer VACA01 for inhibiting the vacuolar toxicity of helicobacter pylori VacA, measured by a neutral red method.

Detailed Description

The invention is described in detail below with reference to the drawings and examples:

an aptamer VACA01 of a helicobacter pylori VacA, which has the following sequence:

5’-CATTCGTATGGACTGCGGCTATGACTGATCATGGGCTTCTTGTTCTTA GATATCCTGGCAGCAAGTTCAAAGTACG-3’(SEQ ID NO:1)。

the aptamer VACA01 of the helicobacter pylori VacA at 25 ℃ and 100mM Na ⁺ ，1mM Mg ²⁺ The spatial structure of which is shown in figure 1, the aptamer VACA01 of the helicobacter pylori VacA, and the 5 'end or the 3' end of the aptamer VACA01 is chemically modified by FITC, amino, biotin and digoxin.

The aptamer VACA01 of the helicobacter pylori VacA is subjected to FITC, amino, biotin and digoxin chemical modification on a product obtained by carrying out truncation or extension or partial base replacement structural modification on the aptamer VACA 01.

The aptamer VACA01 of the helicobacter pylori VacA is obtained by screening a random ssDNA library through the helicobacter pylori VacA magnetic beads by taking carboxyl magnetic beads as a solid phase medium and taking helicobacter pylori VacA protein as a target based on an in-vitro SELEX screening technology of the aptamer.

The screening method of the aptamer VACA01 of the helicobacter pylori VacA comprises the following steps:

(1) Preparation of the screening library: a random ssDNA library of the following sequence was prepared: 5 '-cattcggactgcggnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnngcaagttcaaagtacg-3';

(2) Coupling helicobacter pylori VacA protein with carboxyl magnetic beads to prepare helicobacter pylori VacA magnetic beads;

(3) Subjecting the random ssDNA library to a thermal activation treatment;

(4) Incubating the ssDNA library obtained in the step (3) with the magnetic beads of helicobacter pylori VacA obtained in the step (2);

(5) Magnetically separating the helicobacter pylori VacA magnetic beads after the step (4), and washing ssDNA which is not combined, weakly combined and non-specifically combined on the surfaces of the helicobacter pylori VacA magnetic beads; heating the magnetic beads of the VacA of the helicobacter pylori, and collecting ssDNA (single-stranded deoxyribonucleic acid) which is specifically combined, namely a ssDNA enrichment library;

(6) And (3) PCR amplification: and (4) carrying out PCR amplification on the ssDNA enriched library obtained in the step (5), wherein primers used for the PCR amplification are as follows:

primer P1:5'-FAM-CATTCGTATGGACTGCGG-3' (SEQ ID NO: 2)

And (3) primer P2:5 '-Biotin-CGTACTTTGAACTGCTG-3' (SEQ ID NO: 3);

(7) And (3) purification of PCR products: purifying the PCR product by using a small fragment purification kit; incubating the purified dsDNA with streptavidin magnetic beads, washing the streptavidin magnetic beads combined with the dsDNA, melting the dsDNA, separating by using a magnetic frame, and collecting supernatant; precipitating the supernatant by ethanol to obtain a secondary ssDNA library for the next round of screening;

(8) And (3) circulating screening: and (3) taking the FAM-labeled secondary ssDNA library obtained in the step (7) as a secondary library for the next round of screening, and repeating the screening processes of the steps (3) to (7).

The first embodiment is as follows: screening of aptamer VACA01

The screening method of the aptamer VACA01 of the helicobacter pylori VacA comprises the following steps:

(1) Preparation of screening initial ssDNA libraries: the ssDNA library with primer binding regions (18 fixed nucleotide sequences) at both ends and random regions (40 random nucleotide sequences) in the middle was designed and synthesized by Competition Biotechnology engineering, inc., having the following sequences

5’-CATTCGTATGGACTGCGGNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNCAGCAAGTTCAAAGTACG-3’。

(2) Coupling of the helicobacter pylori VacA protein with the carboxyl magnetic beads: the helicobacter pylori VacA protein (His Tag) is purchased from ABCAM company in UK, is prepared by an Escherichia coli expression system, and has the purity of more than 90 percent, and the carboxyl magnetic beads and the coupling reagent are purchased from Bangs Laboratories company in the United states, and the operation refers to the instruction provided by a manufacturer; measuring the change of protein concentration in the helicobacter pylori VacA solution before and after coupling by using a BCA method, and calculating the coupling efficiency of the magnetic beads to be 81.6 percent; the magnetic beads of VacA of H.pylori were dispersed in 1 XPBS buffer and stored at 4 ℃.

(3) 2nM of the initial ssDNA library was dissolved in 500. Mu.L of selection buffer (60 mM Tris-HCl,100mM NaCl,1.5mM MgCl) ₂ 5mM KCl, pH 7.4), and then heat activated (95 ℃ for 5min, ice water bath for 10min, room temperature for 10 min).

(4) And (3) mixing the ssDNA library obtained in the step (3) with the helicobacter pylori VacA magnetic beads obtained in the step (2) (the loading of the helicobacter pylori VacA is 80 ng) and yeast tRNA (the molar quantity is 5 times of that of the ssDNA library) and incubating for 1h at room temperature.

(5) Magnetically separating the helicobacter pylori VacA magnetic beads after the step (4), and washing ssDNA (single stranded deoxyribonucleic acid) which is not bound, weakly bound and non-specifically bound on the surfaces of the helicobacter pylori VacA magnetic beads by using a selection buffer containing 0.2% BSA (bovine serum albumin); then, 200. Mu.L ddH was applied to the magnetic VacA beads of helicobacter pylori ₂ O resuspending, placing in a magnetic frame for 1-2min after bathing in hot water at 100 deg.C for 10min, collecting supernatant to obtain the productMagnetic bead-specifically bound ssDNA, i.e., a ssDNA-enriched library.

(6) And (3) PCR amplification: adding the ssDNA enrichment library obtained in the step (5) into 1mL of PCRmix reagent; after vortex oscillation and uniform mixing, subpackaging each tube by 50 mu L for PCR amplification, wherein the amplification conditions are as follows: pre-denaturing at 95 ℃ for 5 min; denaturation at 95 ℃ 30S, annealing at 62 ℃ 30S, extension at 72 ℃ 30S, and 13-25 cycles.

Wherein 1mL of PCRmix contains: 10 XPCR buffer 100. Mu.L; pfu enzyme and Taq enzyme each 1 μ L; dNTP 20 u L; primer P1:5'-FAM-CATTCGTATGGACTGCGG-3' and primer P2: 3. Mu.L each of 5 '-Biotin-CGTACTTGAACTTGCTG-3'; the primer P1 and the primer P2 are synthesized by the corporation of Biotechnology engineering, inc.

(7) And (3) purification of PCR products: PCR products labeled with biotin and a fluorophore FAM at both ends, respectively, were purified using a small fragment purification kit (the small fragment purification kit was purchased from Biotechnology, inc.), the purified dsDNA was incubated with streptavidin magnetic beads (purchased from Invitrogen-Dynal) at 37 ℃ for 20min, the dsDNA-bound streptavidin magnetic beads were washed 3 times with a washing buffer (5 mM Tris-HCl, pH 7.4,1.2M NaCl, 500. Mu.M EDTA), and then incubated with 50. Mu.L NaOH solution (0.2M) at 37 ℃ for 30min to melt the dsDNA; the supernatant was collected by magnetic frame separation and ethanol precipitated to obtain the FAM-labeled secondary ssDNA library and dissolved in selection buffer as the secondary library for the next round of screening.

(8) The screening process was carried out for 11 rounds. Starting from the second round, the secondary libraries were all used at 40pM.

Example two: analysis of aptamer VACA01 sequences

(1) After 11 rounds of screening, the enriched ssDNA library was collected and the library sequence was analyzed by high throughput sequencing techniques, the analysis procedure was: amplifying the enrichment library by PCR, and adding a sequencing joint and an Index part; selecting a purified library by gel electrophoresis; carrying out quantitative analysis on the purified library by utilizing Qbit; the library was quality controlled by an Agilent High Sensitivity DNA Kit using an Agilent 2100 Bioanalyzer; quantifying the library by using a Quant-iT PicogGreen dsDNA Assay Kit; performing bridge PCR amplification, sequencing primer annealing, synthesis and sequencing by using a single-chain library as a template by using an Illumina Novaseq6000 platform; and comparing and enriching the sequencing result.

(2) According to the enrichment degree of the aptamer in the library, ssDNA with high enrichment degree is selected as a candidate aptamer, wherein the aptamer VACA01 accounts for 18.2% of the enrichment library, and the sequence of the aptamer is shown as SEQ ID NO. 1.

(3) Analysis at 25 ℃ 100mM Na using UNAFold network platform ⁺ ，1mM Mg ²⁺ Under the conditions of (1), the secondary structure of the aptamer VACA01 sequence. A schematic diagram of the secondary structure of the sequence of the aptamer VACA01 was analyzed and shown in FIG. 1. The neck loop structure gives the aptamer VACA01 a low free energy and stable secondary structure, ensuring the ability to bind to the target.

Example three: specific analysis of aptamer VACA01

(1) FAM-labeled aptamer VACA01 was chemically synthesized in vitro and dissolved in selection buffer with a final concentration of 0.1% N-acetylcysteine (NAC), a pure product of N-acetylcysteine purchased from san Ding Saint Biotech, inc. in Shanghai.

(2) Referring to step (2) of example one, BSA, H.pylori CagA, hpaA, babA, and GroEL proteins were coupled with carboxyl magnetic beads to prepare BSA magnetic beads, H.pylori VacA magnetic beads, hpaA magnetic beads, babA magnetic beads, and GroEL magnetic beads, respectively. Wherein, the BSA is purchased from Sigma, and the helicobacter pylori CagA, hpaA, babA and GroEL proteins are purchased from ABCAM of UK.

(3) And (3) mixing 200 mu L of the solution of the aptamer VACA01 obtained in the step (1) with the BSA magnetic beads, the helicobacter pylori CagA magnetic beads, the HpaA magnetic beads, the BabA magnetic beads, the GroEL magnetic beads and the helicobacter pylori VacA magnetic beads prepared in the step (2), incubating for 1h in a cassette at room temperature, and setting blank magnetic beads as a control.

(4) Washing the magnetic beads of step (3) with 0.1% PBST for 3 times, and boiling the aptamers bound to the magnetic beads with 200. Mu.L of selection buffer at 100 ℃ for 5min for elution.

(5) The fluorescence intensities of the initial solution and the eluate were measured by a fluorescence quantitative analyzer, and the fluorescence binding rate = (initial fluorescence intensity-eluted fluorescence intensity)/initial fluorescence intensity × 100% was calculated, and the binding rate of the aptamer VACA01 to the target molecule was preliminarily represented by the calculated value.

As shown in FIG. 2, the binding rates of the aptamer VACA01 and the helicobacter pylori VacA are all significantly higher than those of the aptamer VACA01 and the BSA, the helicobacter pylori CagA, the HpaA, the BabA and the GroEL proteins, which indicates that the aptamer VACA01 has better specificity in binding with the helicobacter pylori VacA in 0.1% of N-acetylcysteine environment. Further illustrates that the aptamer VACA01 has potential in establishing a fecal VacA antigen detection kit.

Example four: affinity assay for aptamer VACA01

(1) FAM labeled aptamer solutions with different concentrations are respectively mixed with the magnetic beads of the helicobacter pylori VacA and incubated for 1h in a cassette at room temperature.

(2) Referring to the step (4) and the step (5) in the third example, the fluorescence binding rates of the aptamer solutions with different concentrations to the magnetic beads of helicobacter pylori VacA were experimentally obtained and calculated.

(3) The calculated value of the fluorescence binding rate is used to draw a saturation binding curve of the aptamer binding to the helicobacter pylori VacA, and the dissociation constant of the aptamer binding to the helicobacter pylori VacA is calculated through nonlinear regression analysis.

As shown in FIG. 3, we obtained a saturated binding curve of the aptamer VACA01, and calculated that the dissociation constant of the aptamer VACA01 is 23.91pM, which indicates that the aptamer VACA01 has strong binding ability to the VacA of helicobacter pylori, and the dissociation constant is in picomolar scale.

Example five: toxicity of VACA inhibited by aptamer VACA01

(1) Human gastric cancer cell line AZ-521 cells were purchased from Nanjing Kebai Biotech Co., ltd and cultured in MEME medium (purchased from Sigma) containing 10% fetal bovine serum (FBS, purchased from Sigma).

(2) AZ-521 cells were used to assess the toxic effects of VacA. AZ-521 cells at a concentration of 1X 10 ⁵ Cells/well, 48-well plates, per well250 μ L. Culture as a monolayer in 48-well plates in 5% CO ₂ And cultured at 37 ℃ for 24 hours.

(3) The aptamer VACA01 was added to each group at a concentration of 0. Mu.M, 1.25. Mu.M, 2.5. Mu.M, 5. Mu.M and 10. Mu.M, respectively, and VacA protein was added to each well at a final concentration of 120nM, to set a blank control group without the addition of VacA protein and aptamer VACA 01. The cultivation was continued for 5h.

(4) Cells were incubated with 50 μ L of freshly prepared 0.05% neutral red (purchased from Biyunnan), in 1 × PBS containing 0.3% BSA, and then washed 3 times with 0.1mL of 1 × PBS containing 0.3% BSA. After adding 0.1mL of 70% ethanol to 0.4% hydrochloric acid solution, absorbance (OD) at 540nm was measured with a spectrophotometer.

As shown in FIG. 4, the abscissa is the concentration of the aptamer VACA01, and the ordinate is the OD value at 540 nm. The result shows that the aptamer VACA01 can obviously inhibit the toxicity of the VacA protein in a dose-dependent manner in-vitro experiment and is a potential VacA protein inhibitor.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that a person skilled in the art may make several changes, improvements and modifications without departing from the spirit of the present invention, and these changes, improvements and modifications should be construed as the protection scope of the present invention.

Claims (6)

1. A nucleic acid aptamer VACA01 of helicobacter pylori VacA, characterized in that: its sequence is shown below:

5’-CATTCGTATGGACTGCGGCTATGACTGATCATGGGCTTCTTGTTCTTA GATATCCTGGCAGCAAGTTCAAAGTACG-3’。

2. the aptamer VacA01 of helicobacter pylori VacA according to claim 1, characterized in that: and performing FITC, amino, biotin and digoxigenin modification on the 5 'end or the 3' end of the aptamer VACA 01.

3. The aptamer VacA01 of helicobacter pylori VacA according to claim 1, characterized in that: the aptamer VACA01 can be prepared by means of PCR amplification or in vitro synthesis.

4. The use of the aptamer VacA01 of helicobacter pylori VacA according to claims 1 and 2 for the preparation of a reagent for detecting the helicobacter pylori VacA protein.

5. The use of the aptamer VacA01 of helicobacter pylori VacA according to claims 1 and 2 for the isolation and purification of helicobacter pylori VacA protein.

6. Use of the aptamer VacA01 of helicobacter pylori VacA according to claims 1 and 2 for the preparation of antagonists of helicobacter pylori VacA.

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