CN111849995B - Aptamer TLH01 of thermolabile hemolysin TLH and application thereof - Google Patents

Aptamer TLH01 of thermolabile hemolysin TLH and application thereof Download PDF

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CN111849995B
CN111849995B CN202010774476.6A CN202010774476A CN111849995B CN 111849995 B CN111849995 B CN 111849995B CN 202010774476 A CN202010774476 A CN 202010774476A CN 111849995 B CN111849995 B CN 111849995B
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杨湘越
王娅
黄来荣
曹鹏
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Abstract

The invention relates to an aptamer TLH01 of thermolabile hemolysin TLH and application thereof, wherein the sequence of the aptamer TLH01 comprises: 5'-GCGCCACCCCGACAGCGGTAGACAAACTC-3' are provided. The aptamer TLH01 disclosed by the invention can be combined with thermolabile hemolysin TLH with high affinity and high specificity, and has wide application value and wide market prospect in the fields of environmental monitoring, clinical medicine, research on the biological function of Vibrio parahaemolyticus TLH and the like.

Description

Aptamer TLH01 of thermolabile hemolysin TLH and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to an aptamer TLH01 of thermolabile hemolysin TLH and application thereof.
Background
Vibrio parahaemolyticus, also known as halophilic bacteria, belongs to the genus Vibrio of the family Vibrionaceae, is a gram-negative bacterium that infects livestock and humans, and is widely distributed in coastal seawater, submarine sediments, seafood and pickled foods. The main clinical symptoms of microbial food poisoning are acute enteritis, which is manifested by stomachache, diarrhea and vomiting, the incubation period is usually 8-20h, the average period is 12h, the course of disease is generally 2-3 days, the microbial food poisoning can be completely recovered, the prognosis is good, and a few serious patients die due to untimely treatment. Vibrio parahaemolyticus poisoning is associated with food containing the bacteria, and eating or eating uncooked seafood contaminated or pickled food is the main transmission path. Known important infectious sources are crabs, shrimps, scallops, oysters, clams, jellyfishes, cuttlefish slices, pickles, broilers and the like which are polluted by germs, and the optimal growth temperature of the vibrio parahaemolyticus is 37 ℃, so the high incidence period of the vibrio parahaemolyticus poisoning is in summer and autumn, and the peak period is 8 months. In recent years, vibrio parahemolyticus infection ranks the first in food poisoning in China, so that the establishment of a rapid and convenient detection method is an effective way for preventing vibrio parahemolyticus poisoning.
The currently established detection method for vibrio parahaemolyticus includes: pathogen detection methods such as enrichment culture method and maximum possible counting method, antigen and antibody detection methods such as enzyme-linked immunosorbent assay, chip method, radioactive colloidal gold method, immunosensor method and immunomagnetic bead method, and pathogen gene detection methods such as PCR method, multiplex PCR, fluorescent quantitative PCR, nano PCR, EMA-PCR method, nucleic acid probe method and LAMP method. These methods are still required to be further improved in terms of rapidity, simplicity, sensitivity, and the like.
The thermolabile hemolysin (TLH) of Vibrio parahaemolyticus is an atypical phospholipase and has hemolytic activity. The TLH protein is used for immunizing a mouse to prepare an anti-TLH polyclonal antibody, and an enzyme-linked immunosorbent assay kit can be established. TLH has species specificity, and can be used for detecting vibrio parahaemolyticus and providing basis for infection condition by detecting TLH. However, the antibody as a recognition element for TLH detection has the defects of complex preparation, high price, reagent cold chain storage requirement and the like.
Disclosure of Invention
Based on this, it was an object of the present invention to provide a nucleic acid aptamer TLH01, which is capable of specifically recognizing the thermolabile hemolysin TLH of Vibrio parahaemolyticus.
The specific technical scheme is as follows:
a nucleic acid aptamer TLH01 of thermolysin TLH, wherein the sequence of the nucleic acid aptamer TLH01 comprises a sequence shown as SEQ ID NO. 1. Wherein, the SEQ ID NO.1 is shown as follows:
5'-GCGCCACCCCGACAGCGGTAGACAAACTC-3'(SEQ ID NO.1)。
in some of these embodiments, 100mM Na is at 25 deg.C+，1mM Mg2+Under the conditions of (a), the spatial structure of the aptamer TLH01 is as follows:
Figure BDA0002617876480000021
in some of these embodiments, the 5 'end or the 3' end of the aptamer TLH01 is chemically modified with FITC, amino, biotin, or digoxigenin.
Another objective of the invention is to provide an aptamer, which is a sequence obtained by deleting or replacing 1-3 bases of the sequence part of SEQ ID NO.1 of the aptamer TLH 01. Further, as shown in FIG. 4, the bases of the sequence shown by SEQ ID NO.1 are numbered in the order of 1 → 29 from the 5 'end → the 3' end, and the aptamer is a sequence obtained by deleting or substituting 1 to 3 bases (i.e., 1, 2 or 3 bases) from the 5 th to 14 th bases and/or the 22 th to 26 th bases of the sequence part of SEQ ID NO.1 of the aptamer TLH 01.
The invention also aims to provide an application of the aptamer TLH01 or the aptamer in preparation of a detection kit for detecting thermolysin TLH.
The invention also aims to provide an application of the aptamer TLH01 or the aptamer in preparation of a molecular probe for detecting thermolysin TLH.
The invention also aims to provide an application of the aptamer TLH01 or the aptamer in preparation of a medicine for detecting thermolysin TLH.
The invention also aims to provide application of the aptamer TLH01 or the aptamer in preparation of a detection kit, a molecular probe or a medicament for detecting vibrio parahaemolyticus.
Another object of the present invention is to provide an application of the aptamer TLH01 or the aptamer TLH for detecting Vibrio parahaemolyticus or thermolysin TLH for non-disease diagnosis and treatment purposes.
It is a further object of the present invention to provide a test kit comprising the aptamer TLH01 as described above or the aptamer as described above.
Compared with the prior art, the invention has the following beneficial effects:
the aptamer TLH01 disclosed by the invention can identify thermolabile hemolysin TLH of vibrio parahemolyticus with high affinity and high specificity, does not have an identification function on other vibrio parahemolyticus hemolysins, and has wide application value and wide market prospect in the fields of environmental monitoring, clinical medicine, research on biological functions of vibrio parahemolyticus TLH and the like.
Compared with protein antibodies, the aptamer TLH01 is more stable, can be directly synthesized and labeled in vitro, does not need secondary antibodies, and is simple and rapid to operate.
Compared with an antibody, the aptamer TLH01 has the advantages of low preparation cost, short period and good reproducibility.
Drawings
FIG. 1 shows the results of TLH detection of Vibrio parahaemolyticus in gel migration retardation assay;
FIG. 2 shows the result of TLH detection of Vibrio parahaemolyticus by ELISA;
FIG. 3 is a saturation binding curve for aptamer TLH 01;
FIG. 4 shows the spatial structure of aptamer TLH 01.
Detailed Description
Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
The present invention will be described in further detail with reference to specific examples.
The aptamer TLH01 of the invention is obtained by taking TLH expressed by pichia pastoris as a target and screening by the inventor of the invention. The TLH01 is 29 bases in length and has the following sequence:
5'-GCGCCACCCCGACAGCGGTAGACAAACTC-3'(SEQ ID NO:1)。
the aptamer TLH01 of Vibrio parahaemolyticus TLH was analyzed at 25 ℃ and 100mM Na according to mfold platform analysis+，1mM Mg2+Under the conditions of (a), the spatial structure thereof is as follows:
Figure BDA0002617876480000051
example one detection of Vibrio parahaemolyticus TLH by gel migration retardation assay with aptamer TLH01 as recognition element
(1) The TLH01 sequence was synthesized, labeled at its 5' end with the fluorophore FITC (synthesized by Shanghai Biotech Co., Ltd.) and had the sequence: 5'-GCGCCACCCCGACAGCGGTAGACAAACTC-3' are provided.
(2) The FITC-labeled aptamer TLH01 was dissolved in an appropriate volume of buffer (50mM Tris-HCl, 100mM NaCl, 1mM MgCl) 25mM KCl, pH 7.4), the concentration of aptamer TLH01 was 1 mM; and then subjected to a thermal activation treatment. The method of the thermal activation treatment is as follows: after denaturation at 95 ℃ for 5min, the mixture was immediately placed in an ice-water bath for 10min and then at room temperature for 10 min.
(3) The FITC-labeled TLH01 after the heat activation treatment was incubated with two hemolysin mixtures of TLH-containing and non-TLH-containing vibrio parahaemolyticus, respectively, at room temperature in a cassette for 1h, respectively, "TLH + Thermostable Direct Hemolysin (TDH) + thermostable direct hemolysin-related hemolysin (TRH)" and "TDH + TRH", prepared in equimolar ratios. The TLH, TDH and TRH are expressed, purified and stored by a pichia pastoris system by Fuzhou Jinju medical inspection institute Limited, and the purity is more than 95 percent.
(4) The co-incubation system of aptamer TLH01 with the toxin mixture was separated by electrophoresis on a 15% PAGE gel with 10 Xloading buffer.
(5) Observed in a fluorescent gel imaging system and photographed.
As shown in fig. 1, the gel migration retardation experiment was: the FITC labeled aptamer TLH01 can be combined with TLH-containing vibrio parahemolyticus hemolysin mixture, namely, the TLH01 nucleic acid electrophoresis band generates migration retardation; but not with the TLH-free Vibrio parahaemolyticus hemolysin mixture, i.e.the TLH01 nucleic acid electrophoresis band did not undergo migration retardation. This indicates that the aptamer TLH01 is used as a recognition element, TLH can be specifically detected by a gel migration inhibition assay, and TLH01 has high binding affinity and good specificity with TLH.
Example two: detecting the vibrio parahaemolyticus TLH by adopting an enzyme-linked immunosorbent assay method by taking a biotin-labeled aptamer TLH01 as an identification element
(1) The TLH01 sequence was synthesized, and biotin (synthesized by Shanghai Biotech) was labeled at its 5' end, and the sequence was: 5'-GCGCCACCCCGACAGCGGTAGACAAACTC-3', respectively;
(2) dissolving yeast-expressed Vibrio parahaemolyticus TLH, TDH, TRH in carbonate buffer solution with pH of 9.7, adding into enzyme-linked strip at a molar concentration of 100mM per well, and packaging in wet box at 4 deg.C overnight;
(3) discarding the coating solution, adding 100 μ l of maleic acid blocking solution containing 1% casein into each well, and blocking at room temperature for 1 h;
(4) biotin-labeled TLH01 was dissolved in an appropriate volume of buffer (50mM Tris-HCl, 100mM NaCl, 1mM MgCl)25mM KCl, pH 7.4), the concentration of aptamer TLH01 was 1mM, followed by heat activation treatment. The method of the thermal activation treatment is as follows: after denaturation at 95 ℃ for 5min, immediately placing in an ice water bath for 10min, and then placing at room temperature for 10 min;
(5) adding the biotin label TLH01 subjected to heat activation treatment into the enzyme-linked strip according to the amount of 100 mu l/hole, and incubating the nucleic acid sequence and the coated protein for 2h at 37 ℃;
(6) discarding liquid in the holes, washing each hole with 300 mul of phosphate buffer solution, repeating the washing for 3 times, and completely drying the liquid in the holes after the last washing;
(7) Adding 100 μ l of HRP enzyme diluted with phosphate buffer solution at a ratio of 1: 100 into each well, incubating at room temperature for 40min, discarding the liquid in the well, and washing the plate for 5 times in the same manner as above;
(8) mu.l of TMB color developing solution (purchased from Beijing Tiangen biotechnology Co., Ltd.) was added to each well, color development was performed at 37 ℃ in the dark, and when there was a significant color change, 10. mu.l of stop solution was added, and reading was performed by an enzyme-linked immunosorbent assay.
As shown in FIG. 2, enzyme-linked immunosorbent assay found that aptamer TLH01 can specifically bind to Vibrio parahaemolyticus TLH, but not to Vibrio parahaemolyticus TDH and TRH, indicating that aptamer TLH01 has high affinity and good specificity to TLH.
Example three: determination of the dissociation constant (KD value) for the binding of the aptamer TLH01 to Vibrio parahaemolyticus TLH
(1) Coupling of vibrio parahaemolyticus TLH with carboxyl magnetic beads: the carboxyl magnetic beads and the coupling reagent thereof are purchased from Bangs Laboratories, Inc. in the United states. The coupling of TLH to magnetic beads was performed according to the manufacturer's instructions. Measuring the change of protein concentration in TLH solution before and after coupling by BCA method, and calculating the coupling efficiency of magnetic beads to be 81%; the TLH magnetic beads were dispersed in PBS buffer and stored at 4 ℃.
(2) Solutions of FITC-labeled aptamer TLH01 at different concentrations were mixed with the TLH magnetic beads and incubated in a cassette at room temperature for 1 hour.
(3) The magnetic beads obtained in step (2) were washed 3 times with 0.1% PBST, and the aptamer TLH01 bound to the magnetic beads was eluted by boiling 200. mu.L of phosphate buffer at 100 ℃ for 5 min.
(4) The fluorescence binding rate of the aptamer TLH01 solution with different concentrations and TLH magnetic beads is obtained and calculated, the fluorescence binding rate is calculated as (initial fluorescence intensity-elution fluorescence intensity)/initial fluorescence intensity multiplied by 100%, and the calculated value is used for preliminarily representing the binding rate of the aptamer TLH01 and the target molecule.
(5) The calculated value of the fluorescence binding rate was used to plot a saturation binding curve for binding of aptamer TLH01 to TLH, and the dissociation constant for binding of aptamer TLH01 to TLH was calculated by nonlinear regression analysis.
As shown in FIG. 3, the saturated binding curve of aptamer TLH01, which was calculated to have a dissociation constant of 17.21nM for aptamer TLH01, shows that aptamer TLH01 binds to Vibrio parahaemolyticus TLH strongly with a dissociation constant on the nanomolar scale.
In a word, the aptamer TLH01 can specifically recognize the Vibrio parahemolyticus TLH, and the aptamer TLH01 is used as a recognition element, so that a gel migration blocking experiment and an enzyme-linked immunosorbent assay can be used for detecting the Vibrio parahemolyticus TLH. Therefore, the aptamer TLH01 has wide application potential and value in the aspect of developing various Vibrio parahaemolyticus TLH detection methods.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Fuzhou national institute of medicine, Inc
<120> thermolysin TLH-labile aptamer TLH01 and application thereof
<130> 2020-08-04
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 1
gcgccacccc gacagcggta gacaaactc 29

Claims (9)

1. An aptamer TLH01 of thermolysin TLH, having the sequence shown in SEQ ID No. 1.
2. According to claimThe aptamer TLH01 according to claim 1, wherein the aptamer TLH01 is 100mM Na at 25 ℃ +，1mM Mg2+Under the conditions (a), the spatial structure of the aptamer TLH01 is:
Figure 289196DEST_PATH_IMAGE001
。
3. the aptamer TLH01 of claim 1 or 2, wherein the aptamer TLH01 is chemically modified at the 5 'end or 3' end with FITC, amino, biotin or digoxigenin.
4. Use of the aptamer TLH01 of any one of claims 1 to 3 in the preparation of a test kit for the detection of thermolysin TLH.
5. Use of the aptamer TLH01 of any one of claims 1 to 3 in the preparation of a molecular probe for detecting thermolysin TLH.
6. Use of the aptamer TLH01 of any one of claims 1 to 3 in the preparation of a medicament for the detection of thermolysin TLH.
7. Use of the aptamer TLH01 of any one of claims 1 to 3 in the preparation of a detection kit, a molecular probe or a medicament for detecting Vibrio parahaemolyticus.
8. Use of the aptamer TLH01 of any of claims 1 to 3 for the detection of Vibrio parahaemolyticus or thermostable direct hemolysin TLH for non-disease diagnostic and therapeutic purposes.
9. A detection kit comprising the aptamer TLH01 according to any one of claims 1 to 3.
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