CN115820652B - Group of nucleic acid aptamer for specifically recognizing human chorionic gonadotrophin and application thereof - Google Patents
Group of nucleic acid aptamer for specifically recognizing human chorionic gonadotrophin and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine, and in particular relates to a group of nucleic acid aptamers for specifically recognizing human chorionic gonadotrophin and application thereof. Specifically, the invention provides a nucleic acid aptamer, and the sequence of the nucleic acid aptamer is shown in any one of SEQ ID NO. 1-6. The nucleic acid aptamer is a DNA sequence, can be directly used for diagnosis, and can also be used as a molecular probe to construct a biological detection sensor and the like.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a group of nucleic acid aptamers for specifically recognizing human chorionic gonadotrophin and application thereof.
Background
Human chorionic gonadotrophin (human chorionic gonadotropin, hcg) is a glycoprotein hormone secreted by trophoblast cells of placental chorionic vesicles and consists of two distinct subunits, the alpha and beta subunits, and 244 amino acids, with a molecular weight of about 36.7kda. Structurally, the α subunit is similar to many hormones such as thyroid stimulating hormone, follicle stimulating hormone, etc., whereas the β subunit is hcg-specific, so the clinical hcg assay mainly uses the specificity of the β subunit. The placenta can produce hcg, trophoblastoma, germ cell tumor containing trophoblast tissue, and some non-trophoblastoma can also produce hcg. The serum of pregnant women contains mainly intact molecules hcg, whose concentration increases exponentially in early pregnancy and is time dependent, which plays an important role in maintaining pregnancy.
If the hcg value changes irregularly, the specific time is too high or too low, which indicates pregnancy abnormality. Abnormal high detection results indicate the possibility of choriocarcinoma, grape embryo or multiple pregnancy, and low detection results indicate threatened/early abortion, ectopic pregnancy, gestational toxicosis or intrauterine death of the fetus. By combining alpha fetoprotein detection with accurate gestational age, pregnant woman weight and other parameters, detection of hcg +beta is helpful for evaluating the risk of the trisomy 21 syndrome in the middle gestation period, the serum alpha fetoprotein concentration of the trisomy 21 pregnant woman is reduced, and the maternal serum hcg +beta concentration can reach twice the normal median value. The monitoring of human chorionic gonadotrophin level can predict the occurrence of pregnancy-induced hypertension and has important guiding significance for diagnosing the disease course of gestational hypertension. hcg is also an important serum and urine tumor marker, and pregnancy-independent elevated hcg concentrations are also seen in germ cell, ovarian, bladder, pancreatic, gastric, lung and liver tumor patients. The detection methods commonly used at present are as follows: latex set inhibition and hemagglutination inhibition assays, radioimmunoassay (RIA), adsorption assay (ELISA), monoclonal antibody colloidal gold assay.
The exponential enrichment ligand system evolution technology, abbreviated as SELEX (Systematic volution of Ligands by EXponential Enrichment) technology, is a high-throughput biological library screening technology which has emerged and developed rapidly in more than ten years. By using a large-capacity random oligonucleotide library (ssDNA library and RNA library), combining with a PCR in-vitro amplification technology, exponentially enriching oligonucleotides specifically combined with target molecules, and repeatedly screening and amplifying in vitro, the finally obtained aptamer is combined with the target molecules with high specificity and high affinity based on a space structure.
The aptamer has the advantages of accurate identification, no immunogenicity, easy in vitro synthesis and modification, and the like, is also called as an artificial substitute antibody, and has wide application prospect in the aspects of basic medicine, clinical diagnosis, new medicine research and development and the like.
Disclosure of Invention
The invention takes human chorionic gonadotrophin as a target through a SELEX technology, and a group of nucleic acid aptamers HCG-1, HCG-3, HCG-4, HCG-5, HCG-7 and HCG-15 for specifically identifying the human chorionic gonadotrophin (human chorionic gonadotropin, HCG) and application thereof in identifying the human chorionic gonadotrophin are obtained through screening. The nucleic acid aptamer is a DNA sequence, can be directly used for diagnosis, and can also be used as a molecular probe to construct a biological detection sensor and the like. The nucleic acid aptamer and the truncated sequence thereof are identified to be capable of specifically recognizing human chorionic gonadotrophin without binding to other unrelated proteins (BSA proteins), and the control nucleic acid sequence is not bound to human chorionic gonadotrophin.
The specific technical scheme is as follows:
in one aspect, the invention provides a nucleic acid aptamer, wherein the sequence of the nucleic acid aptamer is shown in any one of SEQ ID NO. 1-6.
Preferably, the nucleic acid aptamer may also be a nucleic acid molecule that specifically binds hcg by adding or deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides relative to any one of the sequences set forth in SEQ ID nos. 1-6;
preferably, the nucleic acid aptamer may also be a nucleic acid molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to any of the sequences shown in SEQ ID nos. 1-6;
preferably, the sequence of the nucleic acid aptamer is also reverse complementary to any of the sequences shown in SEQ ID NO. 1-6.
Preferably, the nucleic acid comprises DNA, RNA, or a hybrid of both.
Preferably, the nucleic acid aptamer is DNA.
By "homology" is meant sequence similarity, which can be assessed visually or by computer software. Using computer software, homology between two or more sequences can be expressed in percent (%), which can be used to evaluate homology between related sequences.
The term "aptamer", as used herein, refers to a single stranded oligonucleotide that specifically binds to a target molecule. Principle of recognition of target molecules by nucleic acid aptamers: the single-stranded oligonucleotide forms a specific three-dimensional structure after self-adaptive folding through nucleotide base complementary pairing, hydrogen bond, pi-pi accumulation, electrostatic acting force and other interaction forces, and the three-dimensional structure is specifically combined with a target molecule through intermolecular acting force. Its binding dissociation constant can reach nanomolar and picomolar levels, comparable to monoclonal antibodies. The target molecule of interest for the present invention is hcg.
In the present invention, the terms "HCG", "HCG", "human chorionic gonadotropin", "human chorionic gonadotrophin" are used interchangeably with the same meaning.
As used herein, the term "specific binding" refers to a non-random binding reaction between a nucleic acid aptamer provided by the invention and hcg. The nucleic acid aptamer provided by the invention is not combined with other proteins.
In another aspect, the present invention provides a derivative of a nucleic acid aptamer comprising a nucleic acid aptamer and a detectable label labeling the nucleic acid aptamer.
Preferably, the detectable label comprises an enzyme (e.g., peroxidase, alkaline phosphatase, luciferase, etc.), a radionuclide (e.g. 3 H、 125 I、 35 S、 14 C、 32 P, etc.), fluorescent dyes (e.g., FITC, TRITC, PE, texas Red, cy7, alexa 750, VIC, JOE, TET, CY, etc.), acridine esters, magnetic beads, colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and avidin (e.g., streptavidin), biotin, for binding the label modifications described above.
As used herein, the term "detectable label" refers to any substance that is detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means.
The detectable label as used in the specific embodiments of the present invention is biotin, the method of which labels the nucleic acid aptamer is conventional in the art.
Preferably, the aptamer or derivative thereof may be immobilized on a suitable solid support in daily storage to facilitate more convenient, visual detection, identification hcg; alternatively, the nucleic acid aptamer, the detectable label-labeled nucleic acid aptamer, may be stored in a suitable liquid to maintain its stability, e.g., water, buffer.
In another aspect, the invention provides a method of preparing a probe targeting hcg, the method comprising obtaining a nucleic acid aptamer of the invention and labelling the nucleic acid aptamer with a detectable label.
Preferably, the nucleic acid aptamer obtained according to the invention can be obtained by chemical synthesis and biological amplification methods.
Preferably, the biological amplification method comprises any one of the conventional nucleic acid amplification techniques in the art, in particular, the biological amplification method comprises: and (3) variable temperature amplification and constant temperature amplification. The temperature-variable amplification mainly comprises classical polymerase chain reaction (Polymerase Chain Reaction, abbreviated as PCR) and ligase chain reaction (Ligase Chain Reaction, abbreviated as LCR), while the isothermal amplification comprises strand displacement amplification (Strand displacement amplification, abbreviated as SDA), rolling circle amplification (Rolling Circle amplification, abbreviated as RCA), loop-mediated amplification (Loop Mediated Amplification, abbreviated as LAMP), helicase-dependent isothermal amplification (Helicase-dependent Isothermal DNA Amplification, abbreviated as HDA), nucleic acid sequence-dependent amplification (Nucleic acid sequence based amplification, abbreviated as NASBA), transcription-dependent amplification system (transition-based Amplification System, abbreviated as TAS) and the like.
Preferably, the step of labelling the nucleic acid aptamer with a detectable label may use any alternative method in the art.
In another aspect, the invention provides a method of detecting hcg, comprising contacting an analyte with a nucleic acid aptamer of the invention or a derivative thereof.
Preferably, the test object comprises a sample collected from a human body.
Preferably, the sample comprises peripheral blood, tissue, blood, serum, plasma, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid, and bronchoalveolar lavage fluid.
Preferably, the sample comprises urine and blood.
Preferably, the method is of non-diagnostic interest.
Preferably, the aptamer or derivative thereof is denatured.
More preferably, the specific steps of the denaturation treatment are: dissolving the aptamer in a buffer solution, and cooling after denaturation at 100 ℃;
preferably, the composition of the buffer is 50mM HEPES,100mM NaCl,2mM MgCl 2 ，5mM KCl，1mM CaCl 2 。
Preferably, the contacting is maintained for at least 5, 10, 15, 20, 30, 40 minutes or more.
Preferably, the method further comprises the step of visualizing the detection result using a reporting group. Specifically, for example, when biotin is used as a detectable label, a reporter group HRP enzyme is used.
In another aspect, the invention also provides the use of the nucleic acid aptamer of the invention and derivatives thereof for specific binding hcg.
Preferably, the specific binding occurs in vitro for non-diagnostic purposes.
Preferably, the detection comprises a quantitative or qualitative detection, the result of which comprises the "presence" or "absence" hcg of the analyte.
In another aspect, the invention also provides the use of the aptamer of the invention and derivatives thereof in the preparation of a product for diagnosing hcg-related symptoms.
Preferably, the hcg-associated symptoms include conception, choriocarcinoma, grape embryo, multiple gestations, threatened/early abortion, ectopic pregnancy, gestational toxicosis, fetal intrauterine death, trisomy 21 syndrome, hypertensive disorder of pregnancy, germ cell tumor, ovarian tumor, bladder tumor, pancreatic tumor, gastric tumor, lung tumor, and liver tumor.
Preferably, the product comprises a kit, a test strip or a biosensor.
Drawings
FIG. 1 is a graph showing the results of an EMSA experiment in which HCG-1 specifically binds human chorionic gonadotrophin hCG.
FIG. 2 is a graph showing the results of an EMSA experiment in which HCG-3 specifically binds human chorionic gonadotropin hCG.
FIG. 3 is a graph showing the results of an EMSA experiment in which HCG-4 specifically binds human chorionic gonadotropin hCG.
FIG. 4 is a graph showing the results of an EMSA experiment in which HCG-5 specifically binds human chorionic gonadotrophin hCG.
FIG. 5 is a graph showing the results of an EMSA experiment in which HCG-7 specifically binds human chorionic gonadotrophin hCG.
FIG. 6 is a graph showing the results of an EMSA experiment in which HCG-15 specifically binds human chorionic gonadotrophin hCG.
FIG. 7 is a statistical plot of results of ELISA assays.
Detailed Description
The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
Example 1 EMSA experiment to verify the specificity of the aptamer
1. Experimental materials
(1) Human chorionic gonadotrophin: hua Yangzheng Dragon number: 201215
(2) Bovine Serum Albumin (BSA): beijing Zhongsheng Aubang Bio Inc. number: 01.10001D
(3) Biotin-labeled nucleic acid aptamer: the specific sequences of the biochemical synthesis are shown in Table 1
(4) HRP enzyme: solarbio cat No. 898800
TABLE 1 sequence of nucleic acid aptamers
2. Experimental method
2.1 labeling biotin with a certain concentration respectivelyThe noted nucleic acid aptamers HCG-1, HCG-3, HCG-4, HCG-5, HCG-7, HCG-15 and the biotin-labeled irrelevant control sequence AAAA were dissolved in a suitable volume of buffer (50mM HEPES,100mM NaCl,2mM MgCl) 2 ，5mM KCl，1mM CaCl 2 ) Denaturation is carried out for 5min at 100 ℃, and then the mixture is immediately placed on ice for full cooling;
2.2 incubating the denatured biotin-labeled aptamer with human chorionic gonadotrophin (hCG) protein (or unrelated protein BSA) at 37 ℃ for 40min;
2.3 adding 10 XDNA loading buffer solution into the co-incubation system of aptamer and human chorionic gonadotrophin hCG, and separating by 6% natural PAGE gel electrophoresis;
2.4, rotating the die after discharging the adhesive, and sealing the liquid for 30min after ultraviolet crosslinking for 2 min;
2.5 placing the membrane into HRP enzyme diluted according to a ratio of 1:1000, incubating for 40min at room temperature, taking out the membrane and washing the membrane for 2 times, each time for 10min;
2.6 color development with TMB substrate and leaving a plot.
3. Experimental results
The results of EMSA experiments for HCG-1, HCG-3, HCG-4, HCG-5, HCG-7, HCG-15 specifically binding human chorionic gonadotrophin hCG are shown in FIGS. 1-6 in sequence.
Example 2 enzyme-Linked method for verifying the specificity of aptamer
1. Experimental materials
As in example 1.
2. Experimental method
2.1 a quantity of human chorionic gonadotrophin hCG was dissolved in carbonate buffer pH 9.7 and added to the ELISA strip at 100 μl/well and coated overnight at 4deg.C;
2.2, removing the coating liquid, adding 100 mu l of blocking liquid containing 2% BSA into each hole, and blocking for 60min at room temperature;
2.3 dissolving a concentration of the biotin-labeled aptamer HCG-1, HCG-3, HCG-4, HCG-5, HCG-7, HCG-15 and the biotin-labeled irrelevant control sequence AAAA, respectively, in a suitable volume of buffer (50 mM HEPES,100mM NaCl，2mM MgCl 2 ，5mM KCl，1mM CaCl 2 ) Denaturation is carried out for 5min at 100 ℃, and then the mixture is immediately placed on ice for full cooling;
2.4 adding the biotin-labeled HCG aptamer subjected to denaturation treatment and an irrelevant control sequence Bio-AAAA into an enzyme-linked strip, and incubating the aptamer and the coated human chorionic gonadotrophin hCG protein for 30min at 37 ℃;
2.5, discarding the liquid in the holes, washing each hole with 200 μl of washing liquid, repeating the washing for 3 times, and completely spin-drying the liquid in the holes after the last washing;
2.6 adding 100 μl of HRP enzyme diluted 1:100 into each well, incubating at room temperature for 40min, discarding the liquid in the well, and washing the plate for 5 times, the method is the same as above;
2.7 adding 100 μl TMB chromogenic substrate per well, developing at 37deg.C in dark, adding 10 μl stop solution when there is a significant color change, and reading by ELISA.
3. Experimental results
The results of the ELISA are shown in FIG. 7, which demonstrates that the HCG-1, HCG-3, HCG-4, HCG-5, HCG-7, and HCG-15 aptamers can specifically bind to human chorionic gonadotrophin as compared to the unrelated and control sequences.
Claims (13)
1. A nucleic acid aptamer, the sequence of which is shown in SEQ ID NO. 3.
2. A derivative of a nucleic acid aptamer comprising the nucleic acid aptamer of claim 1 and a detectable label that labels the nucleic acid aptamer.
3. The derivative of a nucleic acid aptamer of claim 2, wherein the detectable label comprises an enzyme, a radionuclide, a fluorescent dye, an acridine ester compound, a magnetic bead, a colloidal gold, a colored glass, a plastic bead, an avidin, or biotin.
4. A method of making a probe targeted to hcg, the method comprising obtaining the nucleic acid aptamer of claim 1 and labeling the nucleic acid aptamer with a detectable label.
5. The method of claim 4, wherein the method of obtaining the aptamer of claim 1 comprises a chemical synthesis method or a biological amplification method.
6. The method of claim 5, wherein the biological amplification method comprises temperature swing amplification and isothermal amplification; the temperature swing amplification includes polymerase chain reaction and ligase chain reaction, and the isothermal amplification includes strand displacement amplification, rolling circle amplification, loop-mediated amplification, helicase-dependent isothermal amplification, nucleic acid sequence-dependent amplification, or transcription-dependent amplification systems.
7. A method of detecting hcg for non-diagnostic purposes, the method comprising contacting an analyte with the nucleic acid aptamer of claim 1 or contacting an analyte with a derivative of the nucleic acid aptamer of claim 2.
8. The method of claim 7, wherein the test object comprises a sample collected from a human body.
9. The method of claim 8, wherein the sample comprises blood, serum, plasma, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid, and bronchoalveolar lavage fluid.
10. The method of claim 9, wherein the sample comprises urine and blood.
11. Use of a nucleic acid aptamer of claim 1 or a derivative of a nucleic acid aptamer of claim 2 in the preparation of a product that specifically binds hcg or detects hcg.
12. Use of the aptamer of claim 1 or the derivative of the aptamer of claim 2 for the preparation of a product for diagnosing hcg-related symptoms.
13. The use of claim 12, wherein the hcg-associated symptoms comprise conception, choriocarcinoma, grape embryo, multiple gestation, threatened/early abortion, ectopic pregnancy, gestational toxicosis, fetal intrauterine death, trisomy 21 syndrome, hypertensive gestational disorder, germ cell tumor, ovarian tumor, bladder tumor, pancreatic tumor, gastric tumor, lung tumor, and liver tumor.
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| US5837456A (en) * | 1990-06-11 | 1998-11-17 | Nexstar Pharmaceuticals, Inc. | High affinity oligonucleotide ligands to chorionic gonadotropin hormone and related glycoprotein hormones |
| CN101144814A (en) * | 2007-10-22 | 2008-03-19 | 中国人民解放军第三军医大学第一附属医院 | Method for detecting, identifying and/ or quantifying compound using adapter type reagent |
| CN102417920A (en) * | 2011-12-07 | 2012-04-18 | 浙江工业大学 | Method for preparing anti-human chorionic gonadotrophin (hCG) monoclonal antibody fragment |
| CN113264985A (en) * | 2021-02-26 | 2021-08-17 | 长沙市信励致和科技有限责任公司 | Human chorionic gonadotropin peptide aptamer and preparation method and application thereof |
| CN114720527A (en) * | 2022-04-07 | 2022-07-08 | 广州大学 | Electrochemical aptamer detection method of glycoprotein |
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