CN105886512B - Oligonucleotide aptamer group for high-specificity recognition of clenbuterol hydrochloride, salbutamol and ractopamine - Google Patents
Oligonucleotide aptamer group for high-specificity recognition of clenbuterol hydrochloride, salbutamol and ractopamine Download PDFInfo
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Abstract
The invention provides a group of oligonucleotide aptamers Apt-1, Apt-2 and Apt-3 capable of simultaneously identifying clenbuterol hydrochloride and salbutamine, an oligonucleotide aptamer CLB-2 capable of identifying clenbuterol hydrochloride with high specificity, an oligonucleotide aptamer SAL-5 capable of identifying salbutamol with high specificity, and two oligonucleotide aptamers RAC-5 and RAC-6 capable of identifying ractopamine with high specificity. Based on Fe3O4The SELEX technology for separating magnetic nanoparticles fixes a random oligonucleotide library on avidin-coated magnetic nanoparticles through a complementary strand labeled by biotinylation, and finally obtains oligonucleotide aptamers with high specific affinity through 16 rounds of screening. The aptamer has wide application prospect, can be applied to the detection of clenbuterol hydrochloride, salbutamol and ractopamine in food by marking functional groups or fluorescent dyes, and provides a new choice for the existing detection method depending on antibodies.
Description
Technical Field
The invention relates to the field of food safety biotechnology, in particular to a method for respectively screening a group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, a group of oligonucleotide aptamers for identifying salbutamol and a group of oligonucleotide aptamers for identifying ractopamine by utilizing SELEX technology (exponential enrichment ligand system evolution technology), and provides scientific basis and theoretical basis for application of detecting clenbuterol in food based on the oligonucleotide aptamers.
Background
Clenbuterol is used as an artificially synthesized β -adrenoceptor activator, can reduce the fat content of animals, increase the lean meat percentage, promote the growth of animals and reduce the feed consumption, so the clenbuterol is used for livestock production.
At present, methods for determining clenbuterol hydrochloride, salbutamol and ractopamine mainly comprise an instrumental analysis method and an immunological method. Instrumental methods including High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), micellar Capillary Electrochromatography (CE), Thin Layer Chromatography (TLC), etc., have been successfully used for the detection of clenbuterol. Although the instrumental analysis method has the advantages of good reproducibility, low detection limit and high sensitivity, the detection equipment is expensive, the sample treatment is complex, and the aim of field detection is difficult to achieve. The immunological method relies on the detection method of the specific combination of the antigen and the antibody, and has the advantages of simple operation, high sensitivity, strong specificity, no need of large instruments and the like. However, clenbuterol hydrochloride, salbutamol and ractopamine are small molecular haptens, have no immunogenicity, and can stimulate animals to secrete antibodies only after being combined with macromolecular carrier proteins such as BSA (bovine serum albumin) and the like to prepare complete antigens, so that the preparation of the antibodies is time-consuming, tedious and expensive, and the prepared antibodies are also easily influenced by environmental factors such as temperature and the like.
The oligonucleotide aptamer is obtained by screening a random oligonucleotide single-stranded library synthesized in vitro by a SELEX technology, and can be used for preparing a short single-stranded DNA sequence with high specific affinity with a target in a specific structure. Oligonucleotide aptamers have been widely used in various fields such as target detection, enzyme inhibition, receptor modulation, and drug delivery. The oligonucleotide aptamer has great advantages compared with an antibody, except that the oligonucleotide aptamer has high specific affinity, the oligonucleotide aptamer is completely screened in vitro, the screening period is short, the synthesis is convenient, the cost is low, some functional groups and reporter molecules are easy to mark, the denaturation and the renaturation are reversible, the speed is high, the stability is good, the influence of environmental conditions is small, and the oligonucleotide aptamer can be stored for a long time. With the continuous improvement of SELEX technology, aptamers of various targets, such as small molecular substances (organic dyes, metals, drugs, amino acids, nucleotides, peptides, etc.), proteins (including enzymes, antibodies, gene regulatory factors, and lectins), tumor cells, viruses, and pathogenic bacteria, have been screened out. However, no research report on the preparation method of oligonucleotide aptamers containing clenbuterol hydrochloride, salbutamol and ractopamine exists at present. The invention uses the common illegal additives of clenbuterol hydrochloride and salbutamol in food or feedAmine alcohol, ractopamine as target, and Fe immobilized with random oligonucleotide library3O4And (2) incubating the magnetic nanoparticles, and screening to obtain a group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, a group of oligonucleotide aptamers for identifying salbutamol and a group of oligonucleotide aptamers for identifying ractopamine, wherein the prepared oligonucleotide aptamers have high stability, are convenient to synthesize, are easy to mark functional groups and reporter molecules, and can be widely applied to rapid detection of clenbuterol in food and feed.
Disclosure of Invention
The invention aims to provide a group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, a group of oligonucleotide aptamers for identifying salbutamol and a group of oligonucleotide aptamers for identifying ractopamine, and lays a good foundation for developing a novel separation, enrichment or analysis and detection tool for clenbuterol.
The invention also aims to provide a method for preparing the clenbuterol oligonucleotide aptamer, which can conveniently and accurately obtain the clenbuterol hydrochloride, salbutamol and ractopamine oligonucleotide aptamer with high specific affinity and remarkable effect.
The method of the invention utilizes a Fe-based catalyst3O4SELEX technology for magnetic nanoparticle separation, random oligonucleotide library is fixed on avidin coated Fe through biotinylation labeled complementary strand3O4Magnetic nanoparticles. And incubating with the fixed library by taking clenbuterol hydrochloride, salbutamol and ractopamine as targets. After 16 rounds of SELEX repeated screening, cloning and sequencing are carried out on the enrichment library, and the affinity and the specificity of a representative sequence are analyzed, so that a group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, a group of oligonucleotide aptamers for identifying salbutamol and a group of oligonucleotide aptamers for identifying ractopamine are finally obtained.
Drawings
FIG. 1 is based on Fe3O4Schematic diagram of SELEX technology for magnetic nanoparticle separation.
FIG. 2 is a simulated secondary structure diagram of sequences Apt1, Apt2, Apt-3, CLB-2, SAL-5, RAC-5, RAC-6.
FIG. 3 is a graph of the saturation binding of the aptamers of the sequences Apt1, Apt2, Apt-3, CLB-2, SAL-5, RAC-5, RAC-6 oligonucleotides.
FIG. 4 shows the results of the specificity test of the oligonucleotide aptamers of the sequences Apt1, Apt2, Apt-3, CLB-2, SAL-5, RAC-5, RAC-6.
The specific implementation mode is as follows:
the invention is further described below with reference to the drawings and examples of the specification, but the invention is not limited thereto.
Example 1: SELEX screening of clenbuterol hydrochloride, salbutamol, ractopamine specific binding oligonucleotide aptamers 1, in vitro chemical synthesis of the initial random oligonucleotide (ssDNA) library and primers (completed by Integrated dna technologies, usa) the sequence is as follows:
5 '-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3' (40N represents 40 random nucleotides);
an upstream primer: : 5'-AGCAGCACAGAGGTCAGATG-3'
5' phosphorylated downstream primer: 5 '-P-TTCACGGTAGCACGCATAGG-3'
Biotinylated library complementary short strand P1: 5 '-Bio-AGCACGCATAGG-3'
Both random ssDNA libraries and primers were made up into 100. mu.M stock in TE buffer and stored at-20 ℃ until use.
2. Immobilization and incubation of random ssDNA libraries
First round screening reaction system 600. mu.L, 1nmol ssDNA library and short chain P1 added BB buffer (50mM Tris-HCl,5mM KCl,100mM NaCl,1mM MgCl) at 1:2 molar ratio2pH 7.4), heating at 95 deg.C for 10min, and transferring to 37 deg.C for hybridization and complementation for 3 h. The complementary hybrid strand was then reacted with washed 600. mu.g avidin-coated magnetic beads for 6h at 37 ℃ and 130rpm to immobilize the ssDNA library on the beads by specific binding of avidin and biotin. The ssDNA-immobilized magnetic beads were washed several times with BB buffer to remove non-specifically bound ssDNA. A solution of 600 μ lsssdna immobilized magnetic beads was mixed with a mixed target (clenbuterol hydrochloride, salbutamol, ractopamine,initial concentrations of 0.1mM each) were incubated at 37 ℃ for 2h and ssDNA specifically affinity for the target was dissociated from the beads. Under the action of an external magnetic field, ssDNA with specific affinity and a target are left in a supernatant and used as a template for PCR amplification.
3. PCR amplification and ssDNA Single Strand preparation
Taking the supernatants of the incubation systems of the negative control group and the experimental group as templates for PCR amplification, wherein the 50 mu L PCR amplification system is as follows: mu.L of template, 1. mu.L of forward primer (10. mu. mol/L), 1. mu.L of phosphorylated reverse primer (10. mu. mol/L), 1. mu.L of dNTPmix (5mmol/L), 0.5. mu.L of Taq DNA polymerase (5U/. mu.L), 5. mu.L of 10 XPCR amplification buffer, 36.5. mu.L of ultrapure water. The thermal cycle parameters were: denaturation at 94 ℃ for 5min, followed by denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s, for 20 cycles, followed by extension at 72 ℃ for 2min, and finally cooling at 4 ℃. And (3) separating the PCR product by 8% polyacrylamide gel electrophoresis, staining by Gelred, and then placing in a Bio-Rad gel imager for photographing to verify whether the dsDNA size of the PCR product is 80bp and whether the band is single. The PCR product was purified using a PCR product purification kit. And (3) measuring the concentration of the purified PCR product by using an ND-1000 micro ultraviolet visible spectrophotometer, and calculating the addition amount of the Lambda exonuclease to ensure that the Lambda exonuclease can completely cut the reverse DNA chain modified by the phosphate group at the 5' end so as to obtain the single-stranded DNA screened in the next round. The purified PCR product was digested with Lambda exonuclease and 1/10 volumes of digestion buffer at 37 ℃ for 1h, and then quenched at 75 ℃ for 10min to terminate the reaction. And (3) separating the enzyme digestion product at 250V for 20min by adopting 8% denatured polyacrylamide gel (containing 7M urea) electrophoresis, dyeing the gel in Gelred dyeing solution for 15min, and then placing the gel under a Bio-Rad gel imager for imaging and photographing to determine whether the enzyme digestion is complete and the size of a single-chain product strip is correct. The cleaved product was transferred to a 1.5ml centrifuge tube and an equal volume of phenol was added: chloroform: isoamyl alcohol (V: V: V: 25: 24:1), mixed uniformly for 30s as white milky liquid, centrifuged at 4 deg.C and 2000rpm for 5min and 4 deg.C and 8000rpm for 1min, the supernatant was carefully removed and transferred to a centrifuge tube. Then, an equal volume of chloroform isoamyl alcohol (V: V-24: 1) was added thereto, mixed well, centrifuged under the same centrifugation conditions as above, and the supernatant was collected. Adding 1/10 volumes of 3M NaAC into the supernatant, fully mixing uniformly, adding 2 volumes of absolute ethyl alcohol, mixing uniformly, standing at-20 ℃ for precipitation overnight. After precipitation, the solution was centrifuged at 4 ℃ and 14000rpm for 15min to remove the supernatant, 200. mu.L of 70% ethanol was added, the precipitate was washed upside down and washed, then centrifuged at 14000rpm for 15min at 4 ℃ to remove the supernatant and the white solid precipitate was removed, the solution was dried in an oven at 50 ℃ and dissolved in 200. mu.L of TE buffer, and the concentration of purified ssDNA was measured with ND-1000 micro ultraviolet-visible spectrophotometer.
4. Circular screening
The second to eleventh rounds of screening were carried out according to the first round, and the screening reaction system was 300. mu.L. To increase the screening pressure and increase the affinity of the screening oligonucleotide aptamers, 100pmol ssDNA library was added to the immobilization system and gradually decreased to 20pmol with increasing number of screening rounds, the concentration of the target mixture added to the incubation system was gradually decreased from 0.1mM to 1. mu.M, and the incubation time was gradually decreased from 2h to 1 h. In order to improve the specificity of the oligonucleotide aptamer, the inverse screening is performed every other round from the third round, and inverse screening substances of epinephrine hydrochloride, dopamine hydrochloride, norepinephrine bitartrate, isoproterenol sulfate and avidin are added into the ssDNA library immobilized magnetic beads, incubated and cleaned, and then incubated and combined with the target. And simultaneously, from the twelfth round to the sixteenth round, the ssDNA library is respectively and independently incubated with clenbuterol hydrochloride, salbutamol and ractopamine, and the other two molecules are added into a reverse sieve system to obtain oligonucleotide aptamers which are respectively specifically combined with the clenbuterol hydrochloride, the salbutamol and the ractopamine.
5. Clone sequencing and sequence analysis
After sixteen rounds of screening, PCR products screened respectively aiming at three targets of clenbuterol hydrochloride, ractopamine and salbutamol are sent to Shanghai Biotechnology Limited company for DNA sequence determination. For each target, 40 sequences were obtained for each assay, and the homology information and secondary Structure of the 40 sequences were analyzed using DNAMAN and RNA Structure 4.6 software, respectively. Combining with the analysis result of software, the sequence is divided into 8 families aiming at the target clenbuterol hydrochloride, and 8 candidate oligonucleotide aptamers with stable structures and lower free energy levels are selected from each family. The sequence is divided into 10 families aiming at the target salbutamol, and 10 candidate oligonucleotide aptamers with stable structures and lower free energy levels are selected from each family. The sequence is divided into 9 families aiming at the target ractopamine, and 9 oligonucleotide aptamers with stable structures and low free energy levels are selected from 1 family. As a result, Apt-1, Apt-2 and Apt-3 are found to be simultaneously present in candidate oligonucleotide aptamer sequences of clenbuterol hydrochloride and salbutamol. The selected candidate oligonucleotide aptamers are synthesized into 5' end labeled FAM sequences by Shanghai Bioengineering technology service, Inc. for affinity and specificity analysis.
6. Affinity and specificity analysis of three clenbuterol oligonucleotide aptamers
6.1 affinity assay
Based on the characteristic that graphene oxide has the capability of adsorbing single-stranded DNA, the oligonucleotide aptamer affinity verification method is constructed. The clenbuterol target (1 μ M) with fixed concentration is respectively incubated with a series of candidate oligonucleotide aptamers with different concentrations (10, 25, 50, 75, 100, 150, 200nM) corresponding to the clenbuterol target, the total volume is 300 μ L, the clenbuterol target is incubated for 2h at 37 ℃ in a dark place, and BB buffer solution is used for replacing the target as a negative control group. After incubation and combination, adding GO with the optimal dosage ratio to adsorb the aptamer which is not combined with the target, after centrifugal operation, adopting an F-7000 fluorescence photometer to measure the fluorescence intensity emitted by 520nm under 490nm excitation of the supernatant, setting the experiment for three times of parallel repetition, and adopting light-shielding treatment. Taking the fluorescence intensity of the experimental group relative to the negative control group as the ordinate, taking the aptamer concentration as the abscissa, and adopting GraphPad Prism 5.0 software to perform nonlinear regression fitting to calculate the dissociation constant K of the aptamerdAnd drawing a binding saturation curve, thereby obtaining the oligonucleotide aptamer with better affinity with clenbuterol hydrochloride, salbutamol and ractopamine, namely the oligonucleotide aptamer with lower dissociation constant. (see Table 1).
TABLE 1 dissociation constant K of high affinity oligonucleotide aptamersdValue of
FIG. 3 is a graph of the saturation binding of sequences Apt1, Apt2, Apt-3, CLB-2, SAL-5, RAC-5 and RAC-6 to the corresponding targets.
6.2 specific assay
According to the analysis result of 5.1, the Apt-1, Apt-2, Apt-3 and CLB-2 sequences with better affinity with clenbuterol hydrochloride, the Apt-1, Apt-2, Apt-3 and SAL-5 sequences with better affinity with salbutamol, and the RAC-5 and RAC-6 sequences with better affinity with ractopamine are obtained, wherein the Apt-1, Apt-2 and Apt-3 have high affinity with clenbuterol hydrochloride and salbutamol. Thus, the oligonucleotide aptamers Apt-1, Apt-2, Apt-3, CLB-2, SAL-5, RAC-6 were specifically analyzed. The binding force of oligonucleotide aptamers of clenbuterol hydrochloride, ractopamine and salbutamol to the anti-sieve substances (epinephrine hydrochloride, dopamine hydrochloride, norepinephrine bitartrate, isoproterenol sulfate and avidin) is analyzed through a specificity experiment. The anti-screening material was incubated with 200nM oligonucleotide aptamer at 37 ℃ for 2h in the dark and BB buffer was used instead of the target as a negative control. And after incubation and combination, adding GO with the optimal dosage ratio to adsorb oligonucleotide aptamers which are not combined with the target, after centrifugal operation, measuring the fluorescence intensity emitted by 520nm under 490nm excitation of supernatant by using an F-7000 fluorescence photometer, and performing parallel repetition for three times in the experiment, wherein the experiment is performed in a dark treatment manner. The results show that the capabilities of Apt-1, Apt-2, Apt-3 and CLB-2 combined with clenbuterol hydrochloride, Apt-1, Apt-2, Apt-3 and SAL-5 combined with salbutamol and the capabilities of RAC-5 and RAC-6 combined with ractopamine are all stronger than those of other anti-screening substances, and the specificity test results are shown in figure 3. Thus, by being based on Fe3O4SELEX technology for separating magnetic nanoparticles is used for preparing a group of oligonucleotide aptamers Apt-1, Apt-2 and Apt-3 for simultaneously identifying clenbuterol hydrochloride and salbutamol, one oligonucleotide aptamer CLB-2 for high-specificity identification of clenbuterol hydrochloride, one oligonucleotide aptamer SAL-5 for high-specificity identification of salbutamol and two high-specificity identificationsOligonucleotide aptamers RAC-5 and RAC-6 of alloractopamine lay a good foundation for developing novel separation, enrichment or analysis and detection tools of clenbuterol. Provides an important basis for the detection of clenbuterol hydrochloride, salbutamol and ractopamine in food or feed.
The present invention includes, but is not limited to, the above embodiments, and any equivalents and modifications within the spirit and principle of the present invention are deemed to be within the scope of the present invention.
Claims (3)
1. A group of oligonucleotide aptamers for recognizing clenbuterol hydrochloride, salbutamol and ractopamine with high specificity comprises: three oligonucleotide aptamers Apt-1, Apt-2 and Apt-3 capable of simultaneously identifying clenbuterol hydrochloride and salbutamol are respectively shown as sequences 1-3 in a sequence table; an oligonucleotide aptamer CLB-2 capable of recognizing clenbuterol hydrochloride with high specificity, the sequence of which is shown as sequence 4 in the sequence table; an oligonucleotide aptamer SAL-5 capable of recognizing salbutamol with high specificity, the sequence of which is shown as sequence 5 in the sequence table; two oligonucleotide aptamers RAC-5 and RAC-6 capable of recognizing ractopamine with high specificity have sequences shown as 6-7 in a sequence table respectively.
2. The oligonucleotide aptamer according to claim 1, characterized in that its 5 'or 3' end can be chemically modified with FITC, amino, biotin or thiol.
3. The use of the oligonucleotide aptamer according to claim 1 for separation, enrichment and analytical detection of clenbuterol hydrochloride, salbutamol and ractopamine in food or feed.
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