Nucleic acid aptamer for targeted specific recognition of probiotic bacillus licheniformis and application of nucleic acid aptamer
High-affinity nucleic acid aptamers Apt-1, Apt-2, and Apt-3 were obtained through screening and sequencing, which solved the uncertainty of Bacillus licheniformis detection and application in aquaculture, achieved efficient and low-cost targeted identification, and improved the healthy development of aquaculture.
Patent Information
- Application Number
- CN202510356131.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-25
- Publication Date
- 2025-07-25
AI Technical Summary
In the prior art, the detection and application of Bacillus licheniformis in aquaculture have problems such as poor resuscitation effect, undetermined viable bacterial amount, uncertain survival time of strains, and lack of highly specific targeted aptamers.
By constructing a random library, nucleic acid aptamers Apt-1, Apt-2, and Apt-3 targeting the specifically identifying aquaculture probiotic Bacillus licheniformis were screened out. Sub-library was prepared using specific primer pairs, and multiple rounds of screening and sequencing were performed to obtain nucleic acid aptamers with high affinity and low free energy, which were used to target the recognition of Bacillus licheniformis.
It provides a high affinity and low cost targeted and specifically identifying Bacillus licheniformis, solves the uncertainty of Bacillus licheniformis application in aquaculture, reduces the cost of synthesis and use, and provides convenience for aquaculture.
Smart Images
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Figure SMS_3
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and relates to a nucleic acid aptamer that specifically recognizes the probiotic Bacillus licheniformis and its application. Background Art
[0002] With the rapid economic development, the living standards of people have been greatly improved, and the consumption of aquatic products as a high-quality protein source has been increasing year by year. Due to the deterioration of the aquaculture water environment caused by long-term high-density aquaculture, it is often accompanied by the breeding of harmful microorganisms and serious aquaculture diseases, which brings great harm to the aquaculture industry. Therefore, using beneficial microbial agents to regulate the aquaculture water quality and bottom sediment, improve the living environment of the aquaculture organisms, inhibit the breeding of harmful microorganisms, reduce the incidence rate, and reduce the use of drugs such as antibiotics and disinfectants has become one of the effective ways to solve such problems and ensure the healthy development of the aquaculture industry. It has been found that Bacillus licheniformis can produce a variety of antibacterial substances, has good protease, lipase and amylase activities, and has the effect of controlling bacteria with bacteria. Therefore, it is widely used in medicine, feed additives, pesticides, veterinary drugs and other aspects. At present, the development of Bacillus licheniformis as medicine and pesticide has been quite mature, and Bacillus licheniformis has achieved effects such as promoting animal growth, reducing diseases and purifying water quality in aquaculture applications. There are many varieties of microecological agents on the market, and they are all provided to farmers in a dormant state (spores), and directly splashed into the pond, which has problems such as poor resuscitation effect, uncertain viable bacteria count, and uncertain survival time of the strains. Therefore, it is of great significance to find and determine a target aptamer suitable for detecting Bacillus licheniformis in aquaculture water.
[0003] An aptamer is a short single-stranded DNA or RNA oligonucleotide that can specifically select and recognize a variety of target substances. At present, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method is used to select aptamers from a random library. A random library refers to a gene mixture containing random nucleotides by randomly introducing nucleotides into the gene sequence to meet the need to independently design specific sequences for research. Each gene molecule in this mixture contains a certain number of random mutation points in the region where random mutation is required. Usually, each gene contains less than 20 mutation points on average. However, due to the immaturity of the SELEX technology, differences exist between bacterial cells due to different batch fermentations, it is easily degraded by nucleases widely present in the bacterial cells after binding to the target bacterial cells, and the high technical requirements for operators, etc., there is a wide market demand and important application value for determining a target aptamer with high specificity to the probiotic Bacillus licheniformis in aquaculture. At present, there is no high-specificity aptamer for the probiotic Bacillus licheniformis in aquaculture. Summary of the Invention
[0004] In order to overcome the deficiencies of the prior art, the present invention provides nucleic acid aptamers that specifically recognize the probiotic Bacillus licheniformis. The present invention provides nucleic acid aptamers that specifically recognize Bacillus licheniformis with high affinity and low free energy, which provides great convenience for specific applications. The provided nucleic acid aptamers that specifically recognize Bacillus licheniformis have specific recognition of Bacillus licheniformis.
[0005] The above object of the present invention is achieved by the following technical solutions:
[0006] A nucleic acid aptamer Apt-1 that specifically recognizes the probiotic Bacillus licheniformis in aquaculture, and its nucleotide sequence is shown in SEQ ID NO.1.
[0007] A nucleic acid aptamer Apt-2 that specifically recognizes the probiotic Bacillus licheniformis in aquaculture, and its nucleotide sequence is shown in SEQ ID NO.2.
[0008] A nucleic acid aptamer Apt-3 that specifically recognizes the probiotic Bacillus licheniformis in aquaculture, and its nucleotide sequence is shown in SEQ ID NO.3.
[0009] The present invention also requests protection for the application of the nucleic acid aptamers Apt-1, Apt-2, and Apt-3 that specifically recognize the probiotic Bacillus licheniformis in aquaculture in the targeted recognition of Bacillus licheniformis in aquaculture.
[0010] The present invention also provides a screening method for obtaining the above-mentioned nucleic acid aptamers that specifically recognize the probiotic Bacillus licheniformis in aquaculture, including constructing a random library; preparing a biotin-labeled secondary library based on specific primer pairs; and performing screening: using Bacillus licheniformis as the positive screening strain, and Bacillus subtilis, Lactobacillus plantarum, Lactobacillus paracasei, Alcaligenes faecalis, Vibrio alginolyticus, and Vibrio splendidus as the negative screening strains. The 1st - 9th rounds of screening are positive screening, the 9th - 16th rounds are negative screening, and the 17th - 19th rounds are positive screening.
[0011] The screening method for nucleic acid aptamers that specifically recognize the probiotic Bacillus licheniformis in aquaculture is specifically as follows: preparing a secondary library with primer pairs; using Bacillus subtilis, Lactobacillus plantarum, Lactobacillus paracasei, Alcaligenes faecalis, Vibrio alginolyticus, and Vibrio splendidus to obtain an ssDNA library that specifically binds to Bacillus licheniformis; performing sequencing, and after sequencing, selecting sequences with low free energy to obtain high-affinity specific sequences that target Bacillus licheniformis, and determining them as nucleic acid aptamers that specifically recognize Bacillus licheniformis.
[0012] Among the primer pairs, the forward primer sequence is as shown in SEQ ID NO.4 (5’FAM-AGATAGTTACAGTCCACAGGAGC), and the reverse primer sequence is as shown in SEQ ID NO.5 (5’Biotin-CTGAAGTCTCCAGAT GAACGTG).
[0013] The random library is 5’-AGATAGTTACAGTCCACAGGAGC-N40-CTGAAGTCTC CAGATGAACGTG-3’. There is a 40-nucleotide random region in the middle, and the 5’-end and 3’-ends are fixed regions of 23 and 22 nucleotides respectively, with the total sequence length being 85 nucleotides.
[0014] The ssDNA library that specifically binds to Bacillus licheniformis is the ssDNA library obtained in the 19th round of screening.
[0015] The sequencing is specifically as follows: After amplifying the ssDNA enriched in the 19th round, it is ligated to a T-vector, transferred into Escherichia coli, and then sequenced. 23 nucleic acid aptamers that specifically bind to Bacillus licheniformis are obtained (the nucleotide sequences are as shown in SEQ ID NO.1 - SEQ ID NO.23).
[0016] Obtaining the high-affinity specific sequences targeting Bacillus licheniformis specifically includes: 23 nucleic acid aptamers that specifically bind to Bacillus licheniformis prepared with primer pairs respectively. Six different counter-screening bacteria are used, and the fluorescence intensity is used to determine the specific nucleic acid aptamers with high affinity targeting Bacillus licheniformis, which are Apt-1, Apt-2, and Apt-3. Among the primer pairs, the forward primer sequence is as shown in SEQ ID NO.4 (5’FAM-AGATAGTTACAGTCCACAGGAGC), and the reverse primer sequence is as shown in SEQ ID NO.5 (5’Biotin-CTGAAGTCTCCAGAT GAACGTG).
[0017] Among them, the nucleic acid aptamer Apt-3 targeting and specifically recognizing Bacillus licheniformis as an aquaculture probiotic with the sequence shown in SEQ ID N0.3 has the optimal affinity for targeting and recognizing Bacillus licheniformis.
[0018] The present invention also claims the application of the nucleic acid aptamers Apt-1, Apt-2, and Apt-3 in targeting and recognizing Bacillus licheniformis.
[0019] The beneficial effects of the present invention compared with the prior art are:
[0020] The present invention provides three nucleic acid aptamers that have target-specific recognition of Bacillus licheniformis and are specifically targeted at probiotic Bacillus licheniformis in aquaculture. Compared with antibody substances, the nucleic acid aptamers specifically targeted at probiotic Bacillus licheniformis in aquaculture screened in the present invention greatly reduce the synthesis and use costs of the sequences in subsequent applications, providing great convenience for specific applications. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 It is a graph showing the change in fluorescence intensity after the nucleic acid aptamers screened in rounds 1-19 in Example 1 of the present invention are fluorescently labeled and incubated with Bacillus licheniformis.
[0022] Figure 2 It is the secondary structure diagrams of three nucleic acid aptamers Apt-1, Apt-2, and Apt-3 that are specifically targeted at probiotic Bacillus licheniformis provided by the present invention and have good affinity and low free energy. Figure a is the secondary structure diagram of Apt-1, Figure b is the secondary structure diagram of Apt-2, and Figure c is the secondary structure diagram of Apt-3. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0023] The present invention will be described in detail below through specific examples, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels. The present invention provides the initial library sequence of nucleic acid aptamers, the screening method, and the nucleic acid aptamers targeted at Bacillus licheniformis.
[0024] Example 1
[0025] S1. Preparation of Bacillus licheniformis
[0026] 2 mL of Bacillus licheniformis in the logarithmic growth phase (China Center for Agricultural Culture Collection, ATCC11946) was inoculated into LB medium and cultured at 37 °C and 180 rpm for 12 h, and then the cell precipitate was collected by centrifugation.
[0027] S2. Design a random ssDNA library sequence with a total sequence length of 85 nucleotides.
[0028] An experimental synthesis of a full-length 86-bp random ssDNA initial library (AGATAGTTACAGTCCACAGGAGC-N40-CTGAAGTCTCCAGATGAACGTG) was carried out. The two ends of the random ssDNA library are fixed primer sequences for PCR amplification of the library, and the middle is a random sequence of 40 bases.
[0029] The specific steps of the first round of screening are as follows:
[0030] Take 10 μL of a 100 μmol·L -1 random ssDNA library, heat it at 95 °C for 10 min, and then place it on ice for 10 min. Take 1 mL of Bacillus licheniformis bacterial solution, centrifuge to collect the bacteria, and mix it with the ssDNA library. Incubate at 37 °C and 100 r·min -1 for 2 h, and then wash away the ssDNA that does not bind to the bacteria or binds weakly. Resuspend the ssDNA-Bacillus licheniformis complex in 100 μL of ddH₂O, heat-denature it at 95 °C for 10 min, and then centrifuge at 4 °C and 12,000 r·min -1 for 20 min. The supernatant obtained is the dissociation product of the ssDNA that binds tightly to the bacteria.
[0031] S4. The specific steps of the 2nd - 9th round of screening process are as follows:
[0032] Take the supernatant of the 1st round as the template, and use primers: a biotin-labeled nucleotide sequence as shown in SEQ ID NO.4 (5’Biotin-AGATAGTTACAGTCCACAGGAGC) and an unlabeled nucleotide sequence as shown in SEQ ID NO.5 (5’CTGAAGTCTCCAGAT GAACGTG) for PCR amplification and enrichment to prepare the secondary library for the 2nd round of screening. The amplification program is pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing temperature at 57.6 °C (see Table 1 specifically), 30 s, 21 cycles, and extension at 72 °C for 10 min.
[0033] Prepare the ssDNA secondary library using streptavidin magnetic beads. Vigorously mix 250 μL of the PCR product (Biotin-dsDNA) and magnetic beads at a concentration of 2 ng / mL, incubate at 30 °C for 30 min, and then incubate at 4 °C for 2 h. Separate the magnetic beads, denature them with alkali at 37 °C for 2 h to make the ssDNA without labeled Biotin bound to the magnetic beads fall off, collect the supernatant, and measure the specific content of the ssDNA with a ultra-micro spectrophotometer to obtain the secondary library for the next round of screening.
[0034] S5. Starting from the 10th round, use Bacillus subtilis, Lactobacillus plantarum, Lactobacillus paracasei, Alcaligenes faecalis, Vibrio alginolyticus, and Vibrio splendidus to perform reverse screening on the obtained secondary library.
[0035] Among them, all strains are fermented in LB medium under the conditions of 37 °C, 220 rpm, and 12 h.
[0036] During reverse screening, first incubate the secondary library with the reverse-screening bacteria for different times, centrifuge to take the supernatant, and then incubate it with the target bacteria for different times.
[0037] S6. The screening was carried out for a total of 19 rounds, and the screening conditions for each round are shown in Table 1.
[0038] Table 1. Screening conditions
[0039]
[0040] S7. Determine the aptamer sequence, and the specific method is as follows:
[0041] The product obtained from the 19th round of screening was amplified and purified, then ligated into the pMD18-T vector by TA ligation, and transferred into the cloning host Escherichia coli DH5α. Randomly select multiple single colonies for sequencing. Use the Mfold software to simulate the secondary structures of these 23 aptamer sequences respectively. The lower the free energy (low ΔG value) of the ssDNA sequence, the more stable its secondary structure, and it is more conducive to the stable binding to Bacillus licheniformis. The results are shown in detail in Table 2 and Table 3.
[0042] Table 2. Sequence information and free energy of the selected nucleic acid aptamers
[0043]
[0044] Table 3. Dissociation constants of the selected nucleic acid aptamers binding to Bacillus licheniformis
[0045]
[0046]
[0047] S8. Screen nucleic acid aptamers with high affinity and targeted recognition of Bacillus licheniformis, and the specific method is as follows:
[0048] Use primers (the sequence of 5’FAM-AGATAGTTACAGTCCACAGGAGC is as shown in SEQ ID NO.4) and (the sequence of 5’Biotin-CTGAAGTCTCCAGAT GAACGTG is as shown in SEQ ID NO.5) to perform PCR amplification on the obtained 23 nucleic acid aptamers respectively. Obtain the FAM-ssDNA library by streptavidin magnetic bead method and determine its concentration. Take FAM-ssDNA solutions with different concentration gradients (0 - 112 nmol·L -1 -1) and a quantified amount of Bacillus licheniformis and incubate at 37℃, 180 r·min
[0049] Y = B max × X / (K d + X)
[0050] Calculate the Kd value of each nucleic acid aptamer binding to the target bacterium, where Bmax is the maximum fluorescence intensity in the system, X is the concentration of the aptamer, and Y is the fluorescence intensity. The results are shown in Table 3. The nucleic acid aptamers with stable secondary structures and high affinity for binding to Bacillus licheniformis are Apt-1, Apt-2, and Apt-3; the nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3. Among them, the affinity of Apt-3 targeting Bacillus licheniformis is significantly the best.
[0051] Through the Mfold program (http: / / mfold.rna.albany.edu / ?q=mfold), the secondary structures of the nucleic acid aptamers Apt-1, Apt-2, and Apt-3 were predicted, and the results are as Figure 2 shown.
[0052] The above-described embodiments are only the preferred embodiments of the present invention, and not all the feasible embodiments of the present invention. For those of ordinary skill in the art, any obvious changes made without departing from the principles and spirit of the present invention should be considered to be included within the scope of the claims of the present invention.
Claims
1. A nucleic acid aptamer that specifically recognizes the probiotic Bacillus licheniformis for aquaculture, characterized in that, Nucleic acid aptamer Apt-1; the nucleotide sequence is as shown in SEQ ID NO.
1.
2. A nucleic acid aptamer that specifically recognizes the probiotic Bacillus licheniformis for aquaculture, characterized in that, Nucleic acid aptamer Apt-2; the nucleotide sequence is as shown in SEQ ID NO.
2.
3. A nucleic acid aptamer that specifically recognizes the probiotic Bacillus licheniformis for aquaculture, characterized in that, Nucleic acid aptamer Apt-3; the nucleotide sequence is as shown in SEQ ID NO.
3.
4. A method for screening a nucleic acid aptamer as described in any one of claims 1-3, characterized in that, Construct a random library; prepare a biotin-labeled secondary library based on specific primer pairs; conduct screening: use Bacillus licheniformis as the positive screening strain, and Bacillus subtilis, Lactobacillus plantarum, Lactobacillus paracasei, Alcaligenes faecalis, Vibrio alginolyticus, and Vibrio splendidus as the negative screening strains. The 1st - 9th rounds of screening are positive screening, the 9th - 16th rounds are negative screening, and the 17th - 19th rounds are positive screening.
5. Use of the nucleic acid aptamer according to any one of claims 1 - 3 for targeted recognition of Bacillus licheniformis.