A nucleic acid aptamer CAGA03 for Helicobacter pylori CagA and its application

Abstract

The present invention relates to a nucleic acid aptamer CAGA03 of Helicobacter pylori CagA and an application thereof, and belongs to the field of biotechnology. The present invention uses an in vitro SELEX screening technology, carboxyl magnetic beads as a solid phase medium, takes Helicobacter pylori CagA protein as a target, and obtains a nucleic acid aptamer specifically binding to Helicobacter pylori CagA from a ssDNA library through Helicobacter pylori CagA magnetic beads; the nucleic acid aptamer CAGA03 of the present invention can bind to Helicobacter pylori CagA protein with high affinity and high specificity, and the nucleic acid aptamer CAGA03 provides a new choice for preparing a detection reagent, a purification reagent, a targeting vector and an antagonist of Helicobacter pylori CagA protein.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a nucleic acid aptamer CAGA03 of Helicobacter pylori CagA and an application thereof. Background Art

[0002] Helicobacter pylori (Hp) is a Gram-negative helicobacter. The global infection rate of Hp is about 50%, and it is a gastric pathogen unique to humans. Most Hp-infected people are asymptomatic, and some show chronic gastritis, peptic ulcers, and gastric mucosal atrophy, and may develop into mucosa-associated lymphoid tissue lymphoma, gastric adenocarcinoma, and primary non-Hodgkin's lymphoma. The inflammation and carcinogenesis of Hp infection are mainly mediated by various toxin molecules secreted by Hp. These toxin molecules can induce pathological processes such as hyperplasia, apoptosis, and excessive inflammatory response of gastric mucosal epithelial cells.

[0003] There are large differences in the pathogenicity and carcinogenicity of Hp, which is related to the types of virulence factors secreted by it. Cytotoxin-associated gene A (CagA) is an important virulence factor secreted by Hp. Depending on whether CagA protein is secreted, Hp strains can be classified into type I strains (CagA positive) and type II strains (CagA negative). Compared with type II strains, type I strains can cause more severe gastric mucosal damage, gastritis, and a higher incidence of gastric cancer. Epidemiological surveys show that the incidence of gastric cancer in Eastern countries is significantly higher than that in Western countries, which is related to the higher infection rate of CagA-positive Hp strains.

[0004] The CagA protein is secreted by the type IV secretion system encoded by the ACG pathogenicity island gene (cagPAI) and transferred into gastric epithelial cells. After entering the gastric epithelial cells, the CagA protein interacts with a variety of proteins in the cells and disrupts the normal signal transduction pathways of the cells, causing a series of functional disorders in the gastric epithelial cells, leading to cell lesions and even transformation. In Hp-positive gastric epithelial cells, the CagA protein will translocate to the cell surface. Therefore, CagA is considered to be a precise targeted recognition marker for Hp-positive gastric cancer cells. Therefore, specific CagA recognition molecules have the potential to become drug carriers for Hp-positive gastric cancer and an important tool for studying the function of CagA protein.

[0005] In the field of pathogen detection, clinical detection of Hp infection is mainly through C14 breath test and detection of Hp-specific antibodies. Among them, C14 breath test has certain radioactivity, which may cause more or less damage to the health of infants and minors; and recent use of antibiotics and fasting degree will also affect the interpretation of breath test results. In terms of Hp-specific antibody detection, due to the strong immunogenicity of CagA protein, CagA-specific antibodies have become an important serological marker for Hp infection. However, even if Hp bacteria have been eradicated in the body and CagA protein has disappeared in the body, CagA antibodies will continue to be positive for several years, which makes CagA antibody positivity unable to be used as a sign of active Hp infection. Therefore, the establishment of a highly sensitive CagA protein detection kit will hopefully replace CagA antibodies and become an effective auxiliary diagnostic method for Hp immediate infection diagnosis, disease condition and prognosis assessment.

[0006] 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 that binds to the target substance with high affinity and high specificity. Aptamers are obtained through the in vitro screening process of systematic evolution of ligands by exponential enrichment (SELEX). Nucleic acid aptamers have the characteristics of high affinity, high specificity, in vitro synthesis, modification to change their functions and pharmacokinetic properties, non-immunogenicity, and economy. Based on the above advantages, nucleic acid aptamer drugs developed can specifically block the target function; using nucleic acid aptamers as recognition elements, simple and accurate detection technologies and efficient and economical affinity purification systems can also be developed. Therefore, screening nucleic acid aptamers that bind to Helicobacter pylori CagA protein with high specificity and high affinity has important scientific research, clinical and market value. Summary of the invention

[0007] The object of the present invention is to provide a nucleic acid aptamer CAGA03 for Helicobacter pylori CagA with high specificity and high affinity; another object of the present invention is to provide multiple applications of the nucleic acid aptamer CAGA03 in preparing Helicobacter pylori CagA protein separation and purification reagents in samples, in preparing Helicobacter pylori CagA protein detection reagents or kits, and in preparing CagA protein targeting vectors or antagonists.

[0008] The purpose of the present invention is achieved through the following technical solution: A nucleic acid aptamer CAGA03 of Helicobacter pylori CagA, whose sequence is as follows:

[0009] 5'-CATCCACATACAGCATTTAGCGAATCAGCCCGGGCAGTGAGTGCCGCGTAGTCCCGTACATACAATGAGCCTAGTT-3'(SEQIDNO:1)

[0010] The Helicobacter pylori CagA aptamer CAGA03 is based on the in vitro SELEX screening technology of nucleic acid aptamers, using carboxyl magnetic beads as solid phase medium and Helicobacter pylori CagA as target, and obtaining nucleic acid aptamers that specifically bind to Helicobacter pylori CagA from the ssDNA library through Helicobacter pylori CagA magnetic beads.

[0011] The application of the nucleic acid aptamer CAGA03 of Helicobacter pylori CagA is used in the separation and purification of Helicobacter pylori CagA protein.

[0012] The application of the nucleic acid aptamer CAGA03 of Helicobacter pylori CagA is used in the preparation of a Helicobacter pylori CagA protein detection reagent.

[0013] The application of the nucleic acid aptamer CAGA03 of Helicobacter pylori CagA is used in a gastric cancer treatment vector targeting the Helicobacter pylori CagA protein.

[0014] The application of the nucleic acid aptamer CAGA03 of Helicobacter pylori CagA is used in antagonists targeting Helicobacter pylori CagA protein.

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

[0016] 1. The nucleic acid aptamer CAGA03 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 CAGA03 of the present invention is lower than that of antibody preparation, and has a short cycle and good reproducibility.

[0018] 3. The nucleic acid aptamer CAGA03 of the present invention can bind to Helicobacter pylori CagA with high affinity and high specificity, with a dissociation constant of 81.59 pM, and it has no recognition function for other toxins and structural proteins of Hp.

[0019] 4. The nucleic acid aptamer CAGA03 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 CagA, diagnosis and treatment of Helicobacter pylori infection, and Helicobacter pylori CagA-mediated targeted therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a bioinformatics simulation diagram of the secondary structure of the nucleic acid aptamer CAGA03.

[0021] Figure 2 Specificity diagram of nucleic acid aptamer CAGA03 for fluorescence binding rate analysis. Figure 2 In the figure, the horizontal axis is the analyzed protein, and the vertical axis is the fluorescence binding rate.

[0022] Figure 3 The dissociation constant curve of nucleic acid aptamer CAGA03 binding to Helicobacter pylori CagA was drawn for fluorescence binding rate experiment. The dissociation constant (Kd) was 81.59 pM. Figure 3 In the figure, the horizontal axis is the DNA concentration (pM) and the vertical axis is the fluorescence binding rate.

[0023] Figure 4 The SDS-PAGE gel electrophoresis diagram of nucleic acid aptamer CAGA03 affinity-purified Helicobacter pylori CagA. Figure 4 In the figure, “M” is the molecular weight standard; “A” is the protein elution solution after affinity purification. DETAILED DESCRIPTION

[0024] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0025] A nucleic acid aptamer CAGA03 for Helicobacter pylori CagA, whose sequence is as follows:

[0026] 5'-CATCCACATACAGCATTTAGCGAATCAGCCCGGGCAGTGAGTGCCGCGTAGTCCCGTACATACAATGAGCCTAGTT-3' (SEQ ID NO: 1).

[0027] The aptamer CAGA03 of Helicobacter pylori CagA has a spatial structure under the conditions of 25°C, 100mM Na+, 1mM Mg2+. Figure 1 The nucleic acid aptamer CAGA03 of Helicobacter pylori CagA is chemically modified with FITC, amino, biotin or digoxigenin at the 5' end or 3' end of the nucleic acid aptamer CAGA03.

[0028] The nucleic acid aptamer CAGA03 of Helicobacter pylori CagA is subjected to FITC, amino, biotin, and digoxigenin chemical modification to obtain a product obtained by truncating or extending the nucleic acid aptamer CAGA03 or structurally modifying the nucleic acid aptamer CAGA03 by partial base substitution.

[0029] The Helicobacter pylori CagA aptamer CAGA03 is based on the in vitro SELEX screening technology of nucleic acid aptamers, using carboxyl magnetic beads as solid phase medium, taking Helicobacter pylori CagA protein as target, and is obtained by screening from a random ssDNA library through Helicobacter pylori CagA magnetic beads.

[0030] The method for screening the nucleic acid aptamer CAGA03 of Helicobacter pylori CagA comprises the following steps:

[0031] (1) Preparation of screening library: prepare a random ssDNA library with the following sequence: 5′-CATCCACATACAGCATTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCATACAATGAGCCTAGTT-3′;

[0032] (2) coupling Helicobacter pylori CagA protein with carboxyl magnetic beads to prepare Helicobacter pylori CagA magnetic beads;

[0033] (3) heat-activating the random ssDNA library;

[0034] (4) incubating the ssDNA library after step (3) with the Helicobacter pylori CagA magnetic beads obtained in step (2);

[0035] (5) magnetically separating the Helicobacter pylori CagA magnetic beads after step (4), washing away the unbound, weakly bound and non-specifically bound ssDNA on the surface of the Helicobacter pylori CagA magnetic beads; heating the Helicobacter pylori CagA magnetic beads to collect the specifically bound ssDNA, i.e., the ssDNA enriched library;

[0036] (6) PCR amplification: The ssDNA enriched library obtained in step (5) is subjected to PCR amplification, wherein the primers used for PCR amplification are:

[0037] Primer P1: 5'-FAM-CATCCACATACAGCATTT-3' (SEQ ID NO: 2)

[0038] Primer P2: 5'-Biotin-AACTAGGCTCATTGTATG-3' (SEQ ID NO: 3);

[0039] (7) Purification of PCR products: Purify the PCR products using a small fragment purification kit; incubate the purified dsDNA with streptavidin magnetic beads, wash the streptavidin magnetic beads bound to the dsDNA, and after the dsDNA is melted, separate them using a magnetic rack and collect the supernatant; precipitate the supernatant with ethanol to obtain a secondary ssDNA library for the next round of screening;

[0040] (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.

[0041] Example 1: Screening of nucleic acid aptamer CAGA03

[0042] The method for screening the nucleic acid aptamer CAGA03 of Helicobacter pylori CagA comprises the following steps:

[0043] (1) Preparation of the initial ssDNA library for screening: A ssDNA library with primer binding regions (18 fixed nucleotide sequences) at both ends and a random region (40 random nucleotide sequences) in the middle was designed and commissioned to be synthesized by Sangon Biotech Co., Ltd. The sequence is as follows:

[0044] 5'-CATCCACATACAGCATTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCATACAATGAGCCTAGTT-3'.

[0045] (2) Coupling of Helicobacter pylori CagA protein with carboxyl magnetic beads: The Helicobacter pylori CagA protein was purchased from ABCAM, UK, and was prepared using an Escherichia coli expression system with a purity of >90%. The carboxyl magnetic beads and their coupling reagents were purchased from Bangs Laboratories, USA, and the operation was performed according to the instructions provided by the manufacturer. The protein concentration in the Helicobacter pylori CagA solution before and after coupling was determined by the BCA method, and the coupling efficiency of the magnetic beads was calculated to be 85%. The Helicobacter pylori CagA magnetic beads were dispersed in PBS buffer and stored at 4°C.

[0046] (3) Take 2 nM of the initial ssDNA library and dissolve it in 500 μL of selection buffer (40 mM Tris-HCl, 100 mM NaCl, 1.5 mM MgCl2, 4 mM KCl, pH 7.4), and then treat it with heat activation (95°C for 5 min, ice water bath for 10 min, room temperature for 10 min).

[0047] (4) The ssDNA library after step (3) was mixed with the Helicobacter pylori CagA magnetic beads (Helicobacter pylori CagA loading amount was 100 ng) and yeast tRNA (molar amount was 5 times that of the ssDNA library) obtained in step (2) and incubated at room temperature for 1 h.

[0048] (5) Magnetic separation of the Helicobacter pylori CagA magnetic beads after step (4), washing the unbound, weakly bound and non-specifically bound ssDNA on the surface of the Helicobacter pylori CagA magnetic beads with a selection buffer containing 0.2% BSA; then resuspending the Helicobacter pylori CagA magnetic beads with 200 μL ddH2O, hot water bath at 100°C for 10 minutes, placing on a magnetic stand for 1-2 minutes, collecting the supernatant, and obtaining the ssDNA specifically bound to the Helicobacter pylori CagA magnetic beads, i.e., the ssDNA enriched library.

[0049] (6) PCR amplification: Add the ssDNA enriched library obtained in step (5) to 1 mL of PCR mix; vortex to mix, and then dispense 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 56°C for 30 s, and extension at 72°C for 30 s, for 15-25 cycles.

[0050] 1 mL PCR mix contains: 100 μL 10×PCR buffer; 1 μL pfu enzyme and 1 μL Taq enzyme; 20 μL dNTP; 3 μL each of primer P1: 5'-FAM-CATCCACATACAGCATTT-3' and primer P2: 5'-Biotin-AACTAGGCTCATTGTATG-3'; the primer P1 and primer P2 were commissioned to be synthesized by Sangon Biotech Co., Ltd.

[0051] (7) Purification of PCR products: The PCR products labeled with biotin and the fluorescent group FAM at both ends were purified using a small fragment purification kit (the small fragment purification kit was purchased from Sangon Biotechnology Co., Ltd.). The purified dsDNA was incubated with streptavidin magnetic beads (purchased from Invitrogen-Dynal) at 37°C for 20 min. The streptavidin magnetic beads bound to the dsDNA were washed three times with a washing buffer (5 mM Tris-HCl, pH 7.4, 1.2 M NaCl, 500 μM EDTA), and then incubated with 50 μL NaOH solution (0.2 M) at 37°C for 30 min to denature the dsDNA. The supernatant was separated using a magnetic rack and collected. The supernatant was precipitated with ethanol to obtain a FAM-labeled secondary ssDNA library, which was dissolved in a selection buffer as the secondary library for the next round of screening.

[0052] (8) The screening process was carried out for 9 rounds in total. Starting from the second round, the dosage of the secondary library was 40 pM.

[0053] Example 2: Analysis of nucleic acid aptamer CAGA03 sequence

[0054] (1) After 9 rounds of screening, the enriched ssDNA library was collected and the library sequence was analyzed using high-throughput sequencing technology. The analysis process was as follows: PCR amplification of the enriched library, and addition of sequencing adapters and index parts; selection and purification of the library by gel electrophoresis; quantitative analysis of the purified library using Qbit; quality control of the library using Agilent 2100 Bioanalyzer through Agilent High Sensitivity DNA Kit; quantification of the library using Quant-iT Picog Greends DNA Assay Kit; using the Illumina Novasek 6000 platform, bridge PCR amplification, sequencing primer annealing, and sequencing by synthesis were performed using the single-stranded library as a template; and the sequencing results were compared and enrichment analyzed.

[0055] (2) According to the enrichment degree of nucleic acid aptamers in the library, ssDNA with high enrichment degree was selected as candidate nucleic acid aptamers, among which nucleic acid aptamer CAGA03 accounted for 11.7% in the enriched library, and its sequence is shown in SEQ ID NO: 1.

[0056] (3) The secondary structure of the nucleic acid aptamer CAGA03 sequence was analyzed using the UNAFold network platform under the conditions of 25°C, 100 mM Na+, and 1 mM Mg2+. The secondary structure diagram of the nucleic acid aptamer CAGA03 sequence is as follows: Figure 1 shown.

[0057] Example 3: Specificity analysis of nucleic acid aptamer CAGA03

[0058] (1) The FAM-labeled nucleic acid aptamer CAGA03 was chemically synthesized in vitro and dissolved in a selection buffer.

[0059] (2) Referring to step (2) in Example 1, BSA, Helicobacter pylori VacA, HpaA, BabA, and GroEL proteins were coupled to carboxyl magnetic beads to prepare BSA magnetic beads, Helicobacter pylori VacA magnetic beads, HpaA magnetic beads, BabA magnetic beads, and GroEL magnetic beads. Wherein, the BSA was purchased from Sigma, and the Helicobacter pylori VacA, HpaA, BabA, and GroEL proteins were purchased from ABCAM, UK.

[0060] (3) Take 200 μL of the nucleic acid aptamer CAGA03 solution obtained in step (1) and mix it with the BSA magnetic beads, Helicobacter pylori VacA magnetic beads, HpaA magnetic beads, BabA magnetic beads, GroEL magnetic beads and Helicobacter pylori CagA magnetic beads prepared in step (2), respectively, and incubate at room temperature in a dark box for 1 h, with blank magnetic beads as a control.

[0061] (4) The magnetic beads prepared in step (3) were washed three times with 0.1% PBST, and the nucleic acid aptamers bound to the magnetic beads were eluted with 200 μL of selection buffer by boiling at 100° C. for 5 min.

[0062] (5) The fluorescence intensity of the initial solution and the elution solution was measured using a fluorescence quantifier, and the fluorescence binding rate was calculated as (initial fluorescence intensity - elution fluorescence intensity) / initial fluorescence intensity × 100%. The calculated value was used to preliminarily represent the binding rate of the nucleic acid aptamer CAGA03 with the target molecule.

[0063] like Figure 2 As shown in the figure, the binding rate of nucleic acid aptamer CAGA03 to Helicobacter pylori CagA was significantly higher than that to BSA Helicobacter pylori VacA, HpaA, BabA, and GroEL proteins, indicating that the binding of nucleic acid aptamer CAGA03 to Helicobacter pylori CagA has good specificity.

[0064] Example 4: Affinity analysis of nucleic acid aptamer CAGA03

[0065] (1) Different concentrations of FAM-labeled aptamer solutions were mixed with Helicobacter pylori CagA magnetic beads and incubated in a dark box at room temperature for 1 h.

[0066] (2) Referring to step (4) and step (5) in Example 3, the fluorescence binding rates of nucleic acid aptamer solutions with different concentrations and Helicobacter pylori CagA magnetic beads were experimentally obtained and calculated.

[0067] (3) Using the calculated value of the fluorescence binding rate, the saturation binding curve of the aptamer binding to Helicobacter pylori CagA was drawn, and the dissociation constant of the aptamer binding to Helicobacter pylori CagA was calculated by nonlinear regression analysis.

[0068] like Figure 3 As shown, we obtained the saturation binding curve of the nucleic acid aptamer CAGA03. The dissociation constant of the nucleic acid aptamer CAGA03 was calculated to be 81.59 pM, indicating that the nucleic acid aptamer CAGA03 has a strong binding ability with Helicobacter pylori CagA, and the dissociation constant is at the picomolar level.

[0069] Example 5: Purification of recombinant Helicobacter pylori CagA protein using CAGA03-based affinity chromatography

[0070] (1) Preparation of CAGA03 affinity column: Dissolve the biotinylated nucleic acid aptamer CAGA03 in buffer A (20 mM NaH2PO4, 150 mM NaCl, pH 7.4) to a final concentration of 20 nM; then inject it onto a pre-packed column of streptavidin agarose gel and circulate it at a flow rate of 2 mL / min using a peristaltic pump; wash the nonspecific CAGA03 on the matrix surface with buffer (10 mM CaCl2, 5 mM MgCl2, 2 M NaCl).

[0071] (2) The Helicobacter pylori CagA protein was recombinantly expressed using an E. coli system. After the cells were broken by ultrasonication, the supernatant was centrifuged at 12,000 rpm for 30 min and the supernatant containing the Helicobacter pylori CagA protein was collected.

[0072] (3) Using G25 molecular sieve chromatography, the recombinant Helicobacter pylori CagA protein solution was replaced with a selection buffer to prepare a loading Helicobacter pylori CagA solution.

[0073] (4) The Helicobacter pylori CagA solution was added to the CAGA03 affinity column via a peristaltic pump, and the flow rate was calculated based on a contact time (I / O) of 8 min.

[0074] (5) Equilibrate with 20 column volumes of equilibration solution (0.05 M Tris-HCl, 0.01 M CaCl2, pH 7.4); elute with elution solution (0.02 M Tris-HCl, 0.01 M EDTA, pH 7.4), and monitor the elution of Helicobacter pylori CagA protein by measuring the absorbance at a wavelength of 280 nM.

[0075] (6) Analyze the protein purification status by SDS-PAGE gel electrophoresis.

[0076] like Figure 4 The image of the SDS-PAGE electrophoresis gel of the protein purification product. The lanes from left to right represent the migration results of the following starting products: "A" is the protein elution solution after affinity purification; "M" is the molecular weight standard. Image scanning analysis shows that after affinity purification by nucleic acid aptamer CAGA03, the purity of Helicobacter pylori CagA reaches more than 95%.

[0077] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A nucleic acid aptamer CAGA03 for Helicobacter pylori CagA, characterized in that: Its sequence is as follows: 5'-CATCCACATACAGCATTTAGCGAATCAGCCCGGGCAGTGAGTGCCGCGTAGTCCCGTACATACAATGAGCCTAGTT-3'.

2. The nucleic acid aptamer CAGA03 of Helicobacter pylori CagA according to claim 1, characterized in that: The 5' end or 3' end of the nucleic acid aptamer CAGA03 is modified with FITC, amino, biotin or digoxigenin groups.

3. The nucleic acid aptamer CAGA03 of Helicobacter pylori CagA according to claim 1, characterized in that: The nucleic acid aptamer CAGA03 can be prepared by PCR amplification or in vitro synthesis.

4. Use of the nucleic acid aptamer CAGA03 of Helicobacter pylori CagA according to claim 1 or 2 in separating and purifying Helicobacter pylori CagA protein.

5. Use of the nucleic acid aptamer CAGA03 of Helicobacter pylori CagA according to claim 1 or 2 in the preparation of a Helicobacter pylori CagA protein detection reagent.

6. Use of the nucleic acid aptamer CAGA03 of Helicobacter pylori CagA according to claim 1 or 2 in the preparation of Helicobacter pylori CagA targeting vector.

7. Use of the nucleic acid aptamer CAGA03 of Helicobacter pylori CagA according to claim 1 or 2 in the preparation of a Helicobacter pylori CagA antagonist.

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