CN112175961A - Aptamer H11 with targeted inhibition effect on vibrio anguillarum and application thereof - Google Patents
Aptamer H11 with targeted inhibition effect on vibrio anguillarum and application thereof Download PDFInfo
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Abstract
The invention relates to a nucleic acid aptamer, in particular to a nucleic acid aptamer H11 with targeting inhibition effect on vibrio anguillarum, and the nucleic acid sequence is shown in SEQ. NO. 1. And the application of the aptamer H11 in preparing Vibrio anguillarum inhibitors. The invention takes vibrio anguillarum as a target, and selects an aptamer (H11) screened by a Cell-SELEX technology to analyze the antibacterial function of the aptamer. Has the advantages of short screening period, simple and convenient chemical synthesis, capability of replacing antibiotics and the like, and has better application prospect. Provides a theoretical basis for further research on inhibitory aptamers.
Description
Technical Field
The invention relates to a nucleic acid aptamer, in particular to a nucleic acid aptamer H11 with targeting inhibition effect on vibrio anguillarum and application thereof.
Background
Introduction to the word
Vibrio anguillarum is one of the most common conditional pathogenic bacteria in aquaculture, and aquaculture animal diseases caused by the vibrio anguillarum are prevalent in the global range, and more than 50 kinds of sea fresh water organisms including rainbow trout, eel, sweet fish, weever, cod, turbot, paralichthys olivaceus, yellow croaker and the like can be infected. At present, the number of infected vibriosis of cultured animals is increased day by day along with the development of aquaculture to high density and intensification, and huge economic loss is brought to aquaculture industry. Because of the serious harm of the vibrio anguillarum to the aquaculture, a large amount of antibiotics are mainly used for preventing and treating the vibrio, which not only causes the generation of bacterial drug resistance, but also brings huge risks and hidden dangers to the quality safety of aquatic products.
The aptamer is a single-stranded oligonucleotide sequence obtained by in vitro artificial synthesis and screening through a Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The polypeptide has the characteristics of high affinity and specificity, easy modification, high stability, no immunogenicity, no toxic or side effect and the like, and has attracted extensive attention in a plurality of fields of life science research, target identification, biomedicine, environmental monitoring and the like. With the improvement of the scientific and technical level, the research of the aptamer as a therapeutic agent is more and more extensive. In 1992 Bock LC et al isolated a thrombin-grade protease thrombin single-stranded DNA aptamer that inhibited thrombin-catalyzed fibrin-thrombosis in vitro. Clinical trials of the aptamer Pegaptanib in Ng EW et al in 2006 demonstrated that it was effective in treating age-related macular degeneration choroidal neovascularization. This is the first aptamer therapeutic approved for use in humans. Shuhao Zhu et al discovered in 2020 that aptamer BT200 has good inhibitory effects on von Willebrand factor in humans in vitro and can prevent arterial occlusion in non-human primates. Therefore, the study of aptamers as inhibitors is a promising development with potentially broad therapeutic benefits.
Disclosure of Invention
The invention aims to solve the technical problem of providing an aptamer H11 for targeted inhibition of vibrio anguillarum and application thereof.
The invention is realized by the following steps:
the invention firstly provides a nucleic acid aptamer H11 for targeted inhibition of vibrio anguillarum, and the nucleic acid sequence of the nucleic acid aptamer is shown in SEQ.NO. 1.
The invention also provides application of the aptamer H11 in preparation of a vibrio anguillarum inhibitor.
Further, the concentration of the aptamer H11 in the inhibitor is 0.5-1.5. mu.M.
Preferably, the concentration of the aptamer H11 in the inhibitor is 1. mu.M.
Specifically, the aptamer H11 is denatured in water bath at 95 ℃ for 5min, and then placed on ice for 10min; mixing 0.5-1.5 μ M aptamer H11100 μ L/well with 200 μ L/well of vibrio anguillarum bacterial liquid with OD 0.1, and culturing.
Further, the liquid culture medium LB liquid was used to dilute the Vibrio anguillarum liquid to OD 0.1.
Further, the aptamer H11 was used diluted in a 1 XTE buffer.
Further, the culture was carried out in a light incubator at 28 ℃ for 72 hours.
The invention has the following advantages: the invention takes vibrio anguillarum as a target, and selects an aptamer (H11) screened by a Cell-SELEX technology to analyze the antibacterial function of the aptamer. Has the advantages of short screening period, simple and convenient chemical synthesis, capability of replacing antibiotics and the like, and has better application prospect. Provides a theoretical basis for further research on inhibitory aptamers.
Drawings
The invention will be further described with reference to the following examples with reference to the accompanying drawings.
FIG. 1 is a graph showing the relationship between a biofilm of Vibrio anguillarum and time.
FIG. 2 shows the bacteriostatic ratios of different aptamers.
FIG. 3 shows the bacteriostatic rate of other aptamers.
FIG. 4 shows the bacteriostatic ratio of aptamers at different concentrations.
Detailed Description
1 SELEX screening of aptamers
(1) And (3) treatment of bacteria: taking cultured vibrio anguillarum in a centrifuge tube, centrifuging for 5min at 6000r/min, discarding the supernatant, washing the bacteria for 3 times, then adding sterile culture solution, mixing uniformly, measuring the OD value of the bacteria solution, taking vibrio anguillarum bacteria solution with the bacteria content of about 4 multiplied by 108, centrifuging for 5min at 6000r/min, discarding the supernatant, washing the bacteria precipitate for 3 times by 0.9% physiological saline, washing the bacteria precipitate for 1 time by 1 multiplied by binding buffer solution, and finally adding 100 mu L of 2 multiplied by binding buffer solution for resuspension.
(2) Combining: taking a ssDNA random library, diluting to 3 mu mol/L100 mu L with 2 Xbinding buffer solution, carrying out denaturation in a constant-temperature metal bath at 95 ℃ for 5min, then carrying out ice bath for 10min, then adding the ssDNA random library into the previous bacterial suspension, mixing uniformly, and binding for 2h in a shaking table at 30 ℃ and 100 r/min.
(3) Washing: after binding was complete, the cells were centrifuged at 6000r/min for 5min, the supernatant was discarded, the pellet containing the cells and their ssDNA-bound cells was washed 1 time with 200. mu.L of 1 Xbinding buffer.
(4) Separation: resuspending the bacterial pellet with 100 μ L of 1 × binding buffer solution, heating at 95 deg.C for 5min to denature ssDNA, separating from the bacteria, cooling, centrifuging at 15000r/min for 10min, collecting supernatant, separating to obtain ssDNA bound to target bacteria, which is the obtained screening product, packaging the screening product into 20 μ L per tube by PCR tube, and storing at-20 deg.C for use.
(5) Amplification: asymmetric PCR amplification was used to obtain the next-stage ssDNA library.
(6) And (3) electrophoresis detection: the prepared agarose gel was placed in an electrophoresis tank, and a 1 XTAE electrophoresis buffer solution was added to completely cover the agarose gel. And taking 2 mu L of Marker reference, taking 5 mu L of asymmetric PCR product and 2 mu L of loading buffer solution, uniformly mixing, adding into the hole, and carrying out electrophoresis under the conditions that the voltage is 90V and the time is 45 min. And finally, observing and photographing.
(7) And (4) repeated screening: if the PCR product shows a band after electrophoresis detection, indicating that the PCR effect is acceptable, the PCR product of the round can be used as a library for the next round of SELEX screening, and the screening processes of (1) to (6) above can be repeated. And (5) verifying the antibacterial performance of the aptamer obtained by screening.
2 verification of antibacterial Properties
2.1 materials
2.1.1 nucleic acid aptamers
The aptamer sequences are shown in Table 1 (the fixed sequences to which the primers bind are underlined at both ends).
The above-mentioned aptamers are synthesized by Biotechnology, Inc. The synthesized freeze-dried powder product is prepared into a stock solution with the concentration of 10 mu mol/L by using 1 XTE buffer solution, and the stock solution is stored in a refrigerator at the temperature of minus 20 ℃ for later use.
TABLE 1 sequence of Vibrio anguillarum aptamers
2.1.2 bacteria for experiments
Vibrio anguillarum (Vibrio anguillarum) was identified and supplied by disease laboratories of university of Collection.
2.1.3 media and reagents
LB solid medium: taking 5g of tryptone, 2.5g of yeast extract, 5.25 g of NaCl and 7.5g of agar powder. Dissolving in ultrapure water, adjusting pH to 7.0 with HCl or NaOH to 500mL, sterilizing at 121 deg.C for 45 min.
20 × binding buffer: collecting NaCl5.844g, KCl3.725g, Tris-HCl6.06g and MgCl 2.6H2O 2.033.033 g. Adding ultrapure water for dissolving, adjusting the pH to 7.0 by using HCl or NaOH, and fixing the volume to 100 mL. Diluted to 2 Xand 1 Xbinding buffer, and sterilized at 121 ℃ for use.
0.1g of crystal violet is weighed and dissolved in 20mL of 95% ethanol to prepare a solution A; 0.8g of ammonium oxalate was dissolved in 80ml of ddH2O to prepare solution B. Mixing solution A and solution B, filtering with analysis filter paper, and storing at room temperature.
Fixative (4% acetaldehyde) paraformaldehyde 40g, NaH2P042.965g,Na2HPO429.00g, 500ml ddH mixed2Placing in a constant-temperature magnetic stirrer, dissolving paraformaldehyde thoroughly at 60 ℃ for 2h, and dissolving ddH2And O is added to 1000mL to be stored in a brown reagent bottle at normal temperature in a dark place.
Measuring 33mL of acetic acid solution, adding ddH2And O is added to 100mL to be stored at 4 ℃ for later use.
Phosphate buffer (pH7.4) NaCl 0.8g, KC10.02g, Na2HP04.12H20, 0.363g, KH2PO40.024g ddH2Sterilizing with O constant volume of 100ml, and storing at 4 deg.CThe application is as follows.
2.2 Experimental methods
2.2.1 Effect of time on bacteriostatic Effect
After diluting the culture broth to OD 0.1 with fresh LB liquid medium, 200. mu.L/well was added to a 96-well plate and cultured. Culturing for different time (6, 12, 24, 36, 48, 60, 72h), taking out the sample, removing the bacterial liquid in the pore plate, adding phosphate buffer to wash the pore plate for 3 times, and sucking the pore plate by a pipette as far as possible.
Fixing a bacterial membrane: adding 200 μ L of fixing agent into each well, treating for 15min, fixing bacterial membrane structure on the wall surface of the culture well plate, sucking off the fixing agent, and air drying at room temperature.
Dyeing: adding 200 μ L/well staining solution, incubating for 5min, removing the staining solution, washing the well plate with phosphate buffer solution for 3 times, and drying at room temperature.
And (3) detection: adding 200 μ L/well of 33% acetic acid solution, incubating at room temperature for 15min to elute and dissolve the bacterial pellicle structure on the wall surface of the culture well plate, and measuring the absorbance value at 595 nm.
2.2.2 bacteriostatic Effect of different aptamers
Modifying the aptamer with metal at 95 deg.C for 5min, and standing on ice for 10min; the experimental group takes 100 mu L/hole of 1 mu M aptamer and adds the aptamer into a 96-well plate, then takes fresh LB liquid culture medium to dilute the bacterial liquid to OD 0.1, 200 mu L/hole adds the bacterial liquid into the 96-well plate and mixes and cultures; taking a blank control, adding 100 mu L/hole 2 Xbuffer solution into 200 mu L/hole diluted bacterial solution, uniformly mixing and culturing; 1uM of random library is taken at 100 mu L/hole, then 200 mu L of bacterial liquid diluted at hole is added, mixed and cultured, and cultured for 72h in an illumination incubator at 28 ℃. The sample was taken out and the post-treatment method was the same as 2.2.1.
The inhibition ratio (%) of the bacterial membrane was calculated as (A)C-A)/AC(A is absorbance of experimental group, ACAbsorbance obtained for blank control).
2.2.3 bacteriostatic Effect of aptamers of different concentrations
Denaturing the aptamer in water bath at 95 ℃ for 5min, and placing on ice for 10min; taking aptamers with different concentrations (0.5 mu M, 1 mu M and 1.5 mu M)100 mu L/hole and 200 mu L/hole of bacterial liquid OD 0.1 for mixed culture, taking 100 mu L/hole 2 Xbuffer solution as a blank control, adding 200 mu L/hole of bacterial liquid for mixed culture, and culturing for 72h in an illumination incubator at 28 ℃. Measuring the absorbance value at 595nm and calculating the bacteriostasis rate by the same method as 2.2.1 and 2.2.2.
3 results and analysis:
3.1 relationship of Vibrio anguillarum mycoderm to time variation
As shown in FIG. 1, the formation of Vibrio anguillarum biofilm was closely related to the culture time. In the initial culture stage, the OD595 value of the biofilm formed by the vibrio anguillarum is increased along with the prolonging of the culture time, the peak value is reached in about 48 hours, the culture is continued, and the OD595 value is reduced along with the prolonging of the culture time; the reaction is relatively stable for 60-72h, and a new balance is achieved.
According to the results, the subsequent experiment selects and cultures the vibrio anguillarum mycoderm formation effect measured after 72h to be the best because of the instability of the early stage of the mycoderm formation.
3.2 bacteriostatic ratio of different kinds of aptamers
As shown in figure 2, the three selected aptamers can reduce the formation of vibrio anguillarum mycoderm, but the bacteriostatic effects are different. Wherein the bacteriostasis rate of H11 is 35%, and the antibacterial agent can replace antibiotics. The random library has no obvious inhibition effect on the mycoderm of the vibrio anguillarum, and the inhibition effect is only 5.21 percent and is lower than the inhibition rates of the three aptamers.
The principle of bacteriostasis is as follows: the bacterial motor flagella are closely related, and the aptamer can be specifically combined with a site on the bacterial flagella to limit the movement of the bacterial flagella and reduce the formation of a biological membrane, so that the bacteriostatic effect is achieved.
The different aptamers have different bacteriostatic effects, and probably because different aptamers have different capacities of combining with bacterial targets, the aptamers with high bacteriostatic rate have stronger capacities of combining with the bacterial targets, and vice versa, the aptamers have weaker bacteriostatic rates.
3.3 bacteriostatic rate of the remaining aptamers
Three other aptamers with good affinity to vibrio anguillarum are selected for carrying out bacteriostasis tests, wherein H1 and H5 are the aptamers with the highest affinity in early stage screening of vibrio anguillarum (CN110578010A), but the effect is poor or even no effect is achieved in the aspect of bacteriostasis, as shown in figure 3. It can be seen that the strength of affinity is independent of the bacteriostatic activity, and that the bacteriostatic activity is not strong.
3.4 bacteriostatic rate of aptamers with different concentrations
As shown in FIG. 4, the inhibition rate of the aptamer against Vibrio anguillarum mycoderm increased with the increase of concentration, reached a peak at 1. mu.M, and then gradually decreased with the increase of concentration. Instead, aptamer H7 promoted biofilm growth at 0.5. mu.M.
The promoting effect at low concentrations may be due to the fact that lower concentrations of aptamers do not achieve bacteriostatic effects but provide nutritional effects to the bacteria; the bacteriostatic effect at high concentration is probably because the combination of the quantity of bacteria and the aptamers in an experimental system reaches a saturated state, and redundant aptamers cannot be combined with the bacteria, so that the bacteria are provided with a nutritional effect. Therefore, the aptamer needs to be controlled to have a certain concentration to generate a bacteriostatic action, and on the contrary, the aptamer generates a growth promoting action.
Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.
Claims (7)
1. A nucleic acid aptamer H11 for targeted inhibition of Vibrio anguillarum, wherein: the nucleic acid sequence is shown in SEQ NO. 1.
2. The use of the aptamer H11 of claim 1 in the preparation of Vibrio anguillarum inhibitor.
3. Use according to claim 2, characterized in that: the concentration of the aptamer H11 in the inhibitor is 0.5-1.5. mu.M.
4. Use according to claim 2, characterized in that: denaturing the aptamer H11 in water bath at 95 deg.C for 5min, placing on ice for 10min, and mixing 0.5-1.5 μ M aptamer H11100 μ L/well with 200 μ L/well of vibrio anguillarum bacterial liquid OD 0.1 for culture.
5. Use according to claim 4, characterized in that: the culture is carried out for 72h at 28 ℃ in a light incubator.
6. Use according to claim 4, characterized in that: and diluting the vibrio anguillarum bacterial liquid to OD 0.1 by adopting an LB liquid culture medium.
7. Use according to claim 4, characterized in that: the aptamer H11 was used diluted in 1 XTE buffer.
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| WO2022088481A1 (en) * | 2020-10-26 | 2022-05-05 | 集美大学 | Nucleic acid aptamer h7 for targeted inhibition of vibrio anguillarum and use thereof |
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| WO2022088481A1 (en) * | 2020-10-26 | 2022-05-05 | 集美大学 | Nucleic acid aptamer h7 for targeted inhibition of vibrio anguillarum and use thereof |
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