CN111500583A - Aptamer for specifically recognizing bovine pregnancy-associated glycoprotein 4 and application thereof - Google Patents
Aptamer for specifically recognizing bovine pregnancy-associated glycoprotein 4 and application thereof Download PDFInfo
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Abstract
The invention provides a nucleic acid aptamer specifically binding to bovine pregnancy related glycoprotein 4, wherein the sequence of the nucleic acid aptamer comprises any one of SEQ ID No.1 sequences, and the nucleic acid aptamer can also be various similar sequences with higher homology or derivatives obtained from the sequences of the invention.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a nucleic acid aptamer for specifically recognizing bovine pregnancy related glycoprotein 4 (bPAG 4), and particularly relates to application of the nucleic acid aptamer in pregnancy related glycoprotein (bPAG) detection and early pregnancy diagnosis of livestock.
Background
Bovine pregnancy-associated glycoproteins (bpags) belong to the aspartic protease family and have more than 50% of the same amino acid sequence as pepsin, cathepsin D, cathepsin E. bPAG is widely available, at least 22 bPAG proteins are included, and are produced by placental trophoblast cell expression (Xie et al, 1994; Hughes et al, 2000), and are introduced into maternal blood after embryo implantation, and the expression and secretion during the whole gestation period are space-time specific (Green et al, 2000; Wooding et al, 2005; Telugu et al, 2009), and are often used as markers for early pregnancy diagnosis of livestock (Zoli et al, 1992; Friedrich et al, 2010; Reese et al, 2018).
Currently, the detection of PAG in blood or milk samples based on immunoassay has become the most widely used early pregnancy detection method internationally (Dufour et al, 2017; Kaya et al, 2016; Commun et al, 2016). Early pregnancy diagnosis can be performed on livestock 28d after insemination, and the diagnosis accuracy reaches more than 90% (Ricci et al 2015; Zhangchun et al 2015; Karen et al 2015). Currently, commercial PAG detection kits are applied to production, for example, PAG in bovine serum or EDTA plasma can be detected by a rapid visual detection kit for cattle developed by Idexx (Idexx) in America based on the enzyme-linked immunosorbent assay principle, but the rapid detection kit sold in the domestic market is quite high in price, and large-scale popularization and use in China are limited. Therefore, in view of the problems of long antibody preparation period, high cost and the like in the current immunoassay method, a novel recognition molecule is urgently needed to be developed so as to overcome the defects of the antibody in preparation and use, and the method has great significance to the livestock early pregnancy detection technology.
In recent years, aptamers (aptamers) have become a focus of research as novel recognition molecules, and essentially a single-stranded oligonucleotide is folded into a secondary or tertiary structure such as a hairpin, stem-loop, pseudoknot, or G-quadruplex, and interacts with a target molecule through hydrogen bonds, van der waals forces, and the like to form a stable complex, and the diversity of spatial structures thereof can be bound to almost all kinds of target molecules (cytokines, proteins, biotoxins, metal ions, small molecular substances, cells, microorganisms, and the like). Compared with the traditional antibody, the antibody has wide application range; high affinity and high specificity, and is not limited by immune conditions and immunogenicity; the preparation is simple and can be artificially synthesized in vitro; the denaturation and the renaturation are reversible, and the stability is high; easy to transform, mark and store. Therefore, the aptamer is widely applied as an ideal molecular probe in the fields of analysis and detection, disease diagnosis, treatment and the like.
At present, no research report of the livestock pregnancy related glycoprotein aptamer is found.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the defects of the prior art, the invention provides an aptamer specifically binding to bPAG4 protein and application thereof, and the aptamer has high binding capacity to bPAG4 protein and can selectively recognize bPAG protein family members.
The technical scheme is as follows: an aptamer specifically recognizing bovine pregnancy related glycoprotein 4 (bPAG 4), wherein the aptamer sequence comprises a DNA sequence shown in SEQ ID No.1, and the DNA sequence shown in SEQ ID No.1 is
5’-TTGAAGTGACTCCGCACTGGGTGGGTGGGAGGGTCGTGCGGCTGGTCATAGCAGGT-3’。
Preferably, the aptamer sequence may be modified, including phosphorylation, methylation, amination, carboxylation, sulfhydrylation, or isotopolylation, provided that the aptamer sequence so modified has desirable properties.
Preferably, the aptamer sequence may be linked to a fluorescent label, a radioactive substance, biotin, streptavidin, digoxigenin, a nano-luminescent material, or an enzyme, provided that the aptamer sequence thus modified has desirable properties.
An aptamer that specifically recognizes bovine pregnancy associated glycoprotein 4 (bPAG 4), the aptamer sequence comprising any of three sequences:
(1) a DNA sequence which has more than 60% of homology with the DNA sequence shown in SEQ ID No.1 and can specifically bind bPAG, preferably, the homology can be more than 70%, more than 80%, more than 90%, or more than 99%;
(2) a DNA sequence which hybridizes with the DNA sequence shown in SEQ ID No.1 under strict conditions;
(3) RNA sequence transcribed from the DNA sequence shown in SEQ ID No. 1.
An aptamer derivative that specifically recognizes bovine pregnancy-associated glycoprotein 4 (bPAG 4), the aptamer derivative comprising:
(1) deleting, adding or replacing one or more bases in any one of the nucleic acid aptamer sequences shown in SEQ ID number 1 to obtain a nucleic acid aptamer derivative with the same function as the nucleic acid aptamer;
(2) modifying a molecular skeleton of any one of the aptamer sequences shown in SEQ ID number 1 to obtain an aptamer derivative with the same function as the aptamer;
(3) The peptide nucleic acid coded by the aptamer shown in SEQ ID number 1 is used for obtaining the aptamer derivative with the same function as the aptamer.
The application of the aptamer or the aptamer derivative in preparation of detecting, separating and purifying bovine pregnancy related glycoprotein.
The application of the aptamer or the aptamer derivative in a bovine early pregnancy diagnosis product.
Has the advantages that: the invention has the following beneficial effects:
(1) by utilizing an exponential enrichment ligand phylogenetic technology (SE L EX), magnetic beads are used as a separation medium, bPAG4 is used as target protein, an aptamer which is high in affinity and high in specific binding with a target is obtained through 9 rounds of screening, the aptamer can be specifically bound with bPAG4 protein, the affinity is extremely high, the dissociation constant reaches 11.7nM, and bPAG protein family members can be selectively identified, but other proteins are hardly bound;
(2) the aptamer obtained by screening has good affinity and specificity, can be artificially synthesized, has low cost and short production period, and is easy for chemical modification;
(3) the aptamer can be prepared into molecular probes, detection reagents and the like for bPAG protein detection, separation and purification and early pregnancy diagnosis of cattle.
Description of the drawings:
FIG. 1 is a graph showing the binding capacity of ssDNA library to bPAG4 protein for each round of screening;
FIG. 2 is a polyacrylamide gel electrophoresis image of ssDNA libraries obtained from each round of screening;
FIG. 3 is a graph showing data on affinity detection of aptamers to bPAG4 protein according to the screening of the present invention;
FIG. 4 is a secondary structural diagram of the aptamer selected according to the present invention;
FIG. 5 shows the result of the aptamer-specific assay of the present invention.
Detailed Description
The present invention is further described in the following examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the scope of the present invention.
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are all conventional biochemical reagents, and are commercially available, unless otherwise specified.
Example 1: selection of bPAG4 nucleic acid aptamers
1. Synthesis of random single-stranded dna (ssdna) library and primers:
random single-stranded dna (ssdna) library:
5 '-CTACGGTGCCTTGAAGTGAC-N36-CATAGCAGGTCACTTCCAGG-3', wherein N36 represents 36 random nucleotides, which library was synthesized by Biotechnology engineering (Shanghai) GmbH;
an upstream primer: 5 '-FAM-CTACGGTGCCTTGAAGTGAC-3',
a downstream primer: 5 '-20A-spacer 18-CCTGGAAGTGACCTGCTATG-3',
among the downstream primers, 20A indicated a polyA tail composed of 20 adenylate (A), and Spacer18 indicated an 18-atom hexaethyleneglycol intermediate arm, which was synthesized by Cisco Biotech, Inc., of Nanjing King Kingsry.
The random single-stranded DNA library, 5 '-end primer and 3' -end primer were dissolved in DPBS buffer (NaCl: 8 g/L: 0.2 g/L, Na2HPO 4: 1.15 g/L2, pH 4: 0.2 g/L, pH 7.4) and stored at-20 ℃ for further use.
2. Magnetic bead-bPAG 4 protein (MB-bPAG 4) coupling
Washing carboxylated magnetic beads with 50 mu L and 10mM PBS (ph 7.4), adding a mixed solution of 0.1M NHS +0.4M EDC (v/v, 1: 1) and 50 mu L into the magnetic beads, carrying out a shaking reaction for 20 min, carrying out magnetic separation after activation, washing the PBS, adding NaAC (10 mM, pH 5.0) and bPAG4 protein (0.5 mg/M L), carrying out a shaking reaction for 60min, carrying out coupling, carrying out magnetic separation, washing the PBS, adding 100 mu L ethanolamine (1.0M, pH8.5) into the PBS, carrying out a shaking reaction for 20 min, carrying out magnetic separation, washing the PBS, suspending the magnetic beads in the PBS, storing the magnetic beads at 4 ℃ for later use, carrying out coupling of magnetic bead-bovine serum albumin (MB-BSA) in the same manner as the above, using MB-bPAG4 as a positive screening target and using MB-BSA as a reverse screening target.
3. Screening
3.1 incubation and separation, dissolving 1OD random single-stranded DNA library with PBS to make the concentration 5 mu M, denaturing at 95 ℃ for 10 minutes in a PCR instrument, carrying out ice-water bath for 5 minutes, instantly separating, keeping at room temperature, adding the denatured 200 mu L library into MB-BSA magnetic beads, carrying out shake incubation for 60 minutes, carrying out magnetic separation, adding the supernatant into MB-PAG4, carrying out shake incubation for 60 minutes, carrying out magnetic separation, discarding the supernatant, adding PBS, boiling for 10 minutes in boiling water, carrying out magnetic separation, and collecting the supernatant.
3.2 PCR amplification, namely adding the library obtained in the step 3.1 into 2 m L PCR mix, mixing uniformly, subpackaging into 100 mu L tubes, and carrying out PCR amplification, wherein the PCR amplification procedure comprises the steps of 1 min at 95 ℃, 1 min at 60 ℃, 1 min at 72 ℃, 25 cycles, 5min at 72 ℃ and 2 min at 25 ℃.
3.3 PCR product concentration: collecting PCR product, adding 5 times volume of n-butanol, vortex mixing, centrifuging at 7500 rpm for 5min, discarding the upper n-butanol layer, and the lower layer is amplified dsDNA product.
3.4 preparation of FAM-labeled Single-stranded DNA (Long-short-stranded method) concentrated PCR product was added to an equal volume of 2 × TBE/urea denaturation buffer, denatured at 95 ℃ for 10min in a PCR instrument, and then added to a well of 8% polyacrylamide gel (PAGE), electrophoresed at 300V until bromophenol blue reached the bottom of gel, separating single-stranded DNA with PolyA from FAM-labeled single-stranded DNA, removing PAGE gel, cutting the band with fluorescence with a clean blade under an ultraviolet lamp, loading into a 0.5 m L gel centrifuge tube, placing into a2 m L centrifuge tube, centrifuging at 14000 rpm for 1 min, discarding the gel centrifuge tube, adding PBS, boiling the photometer for 10min, transferring the supernatant into a 15 m L centrifuge tube, concentrating single-stranded DNA according to step (3), loading into a 3KD dialysis bag, dialyzing overnight at 4 ℃ in PBS, and determining the concentration of nucleic acid as an initial library for the next round of screening after measuring the concentration of nucleic acid by NanoDrop-2000c amount spectrophotometry.
4. Multiple rounds of screening
The ssDNA obtained in step 3.4 above was used as the starting library instead of the random nucleic acid library in step 3.1 and the screening was repeated for 9 rounds. The binding capacity of the ssDNA library obtained from each round to the target protein bPAG4 was tested in a Thermo Scientific ™ Varioskan ™ Flash multi-functional microplate detector during the screening procedure.
5. Binding assays to target proteins for each round of ssDNA libraries
MB-bPAG4 was prepared according to step 2, FAM-labeled ssDNA library obtained in each round was diluted to a concentration of 0.5. mu.M with PBS buffer, denatured at 90 ℃ for 10min, iced water-washed for 5min, then incubated with MB-bPAG4 for 60min, magnetic separated, washed 2 times with PBS buffer, 200. mu. L resuspended in PBS buffer, and binding to the target was detected using Thermo Scientific, Varioskan, Flash MultiProlate microplate detector (. lamda.Ex =492nm,. lamda.Em =518 nm). The results are shown in FIG. 1, indicating that aptamers were enriched when fluorescence did not increase any more, and screening was stopped.8% polyacrylamide gel electrophoresis and Bio-Rad gel imager were performed on each round of ssDNA library, and the results are shown in FIG. 2, with a single 1-9 round ssDNA electrophoresis band at 76 bp.
6. Cloning and sequencing
The aptamer-enriched pool obtained from the 9 th round of screening was amplified with unmodified primers, and the PCR product was purified and cloned with pGEM-T vector (Takara Biotech. Co., &lTtT translation = L "&gTt L &lTt/T &gTt td) and transformed into E.ColiDH5 α, through blue-white screening, randomly selecting 30 positive clones to be sent to the company of Biotechnology engineering (Shanghai) GmbH for synthesis sequencing, adopting G L USTA L X software to carry out homology analysis on the aptamer sequences successfully sequenced, dividing the aptamer sequences into 5 families, selecting 1 sequence from each family as a representative based on the principle of lowest free energy, sending to Shanghai for synthesis, marking 5'FAM groups, and further sending to the Shanghai for synthesis and marking 5' FAM groupsAffinity and specificity analysis.
Example 2 affinity assay of aptamers
Several aptamers synthesized in example 1 were taken, and each aptamer was prepared in a series of aptamer solutions with gradient concentrations (0 nM, 100 nM, 200 nM, 400 nM, 800 nM, 1600 nM, 3200 nM, 6400 nM) in 10mM PBS; the binding of each aptamer to MB-bPAG4 was analyzed sequentially as in step 4, and the dissociation constant Kd value for each aptamer was calculated by fitting the binding curve using GraphPad prism7.0 software. FIG. 3 is a curve of the saturation binding of the aptamer of SEQ ID No.1 with the target protein bPAG4, the aptamer having high affinity for bPAG4 protein with a dissociation constant of 11.7 nM. The aptamer sequence shown in SEQ ID No.1 was analyzed for secondary structure prediction using online MFold, and the results are shown in FIG. 4.
Example 3: specific analysis of nucleic acid aptamers
MB-bPAG1, MB-bPAG4, MB-bPAG9, MB-BSA, and MB-OVA were prepared according to step 2, the aptamer sequence shown in SEQ ID No.1 was diluted to a concentration of 0.5. mu.M with 10mM PBS, and binding of the aptamer to each protein-coupled magnetic bead was analyzed according to step 4, with the magnetic beads to which no protein was linked being negative controls. The detection result is shown in figure 5, the aptamer shown in SEQ ID No.1 preferentially binds to the target protein bPAG4, and the aptamer also has higher binding capacity with the family protein of the same type, but does not bind with other BSA and OVA proteins.
The above examples are only specific embodiments of the present invention, but should not be construed as limiting the scope of the present invention. Any person skilled in the art should also be able to cover the technical scope of the present invention by the equivalent or change of the technical solution and the inventive concept of the present invention.
SEQUENCE LISTING
<110> academy of agricultural reclamation of Sinkiang
<120> aptamer capable of specifically recognizing bovine pregnancy-associated glycoprotein 4 and application thereof
<130>2020.03.03
<160>1
<170>PatentIn version 3.3
<210>1
<211>56
<212>DNA
<213> Artificial Synthesis (Artificial Synthesis)
<400>1
ttgaagtgac tccgcactgg gtgggtggga gggtcgtgcg gctggtcata gcaggt 56
Claims (8)
1. The aptamer capable of specifically recognizing the bovine pregnancy related glycoprotein 4 is characterized in that the sequence of the aptamer comprises a DNA sequence shown as SEQ ID No.1, wherein the DNA sequence shown as the SEQ ID No.1 is
5’-TTGAAGTGACTCCGCACTGGGTGGGTGGGAGGGTCGTGCGGCTGGTCATAGCAGGT-3’。
2. The aptamer specifically recognizing bovine pregnancy-associated glycoprotein 4 according to claim 1, which is characterized in that: the aptamer sequence may be modified, including phosphorylation, methylation, amination, carboxylation, sulfhydrylation, or isotopolyization.
3. The aptamer specifically recognizing bovine pregnancy-associated glycoprotein 4 according to claim 1, which is characterized in that: the nucleic acid aptamer sequence can be connected with a fluorescent marker, a radioactive substance, biotin, streptavidin, digoxigenin, a nano luminescent material and an enzyme.
4. A nucleic acid aptamer specifically recognizing bovine pregnancy related glycoprotein 4, wherein the nucleic acid aptamer sequence comprises any one of the following three sequences:
(1) a DNA sequence which has more than 60 percent of homology with the DNA sequence shown in SEQ ID No.1 and can be specifically combined with bPAG;
(2) a DNA sequence which hybridizes with the DNA sequence shown in SEQ ID No.1 under strict conditions;
(3) RNA sequence transcribed from the DNA sequence shown in SEQ ID No. 1.
5. A nucleic acid aptamer derivative specifically recognizing bovine pregnancy related glycoprotein 4, characterized in that: the aptamer derivative comprises a framework modification or base modification of the aptamer sequence of any one of claims 1 to 4, and the aptamer derivative has the same function as the aptamer.
6. The aptamer derivative according to claim 5, wherein the aptamer derivative specifically recognizes bovine pregnancy-associated glycoprotein 4 is characterized in that: the base alteration includes substitution, deletion or addition.
7. Use of the aptamer according to any one of claims 1 to 4 or the aptamer derivative according to any one of claims 5 to 6 for the preparation of a reagent for detecting, isolating and purifying bovine pregnancy related glycoprotein.
8. Use of the aptamer according to any one of claims 1 to 4 or the aptamer derivative according to any one of claims 5 to 6 in a diagnostic product for early pregnancy in cattle.
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Cited By (2)
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| CN112322625A (en) * | 2020-11-11 | 2021-02-05 | 长江师范学院 | Broad-spectrum nucleic acid aptamer for specifically recognizing related glycoprotein of cow and sheep pregnancy and application thereof |
| CN114438090A (en) * | 2021-11-07 | 2022-05-06 | 吉林大学重庆研究院 | Aptamer specifically binding to Brucella outer membrane protein Omp31 and application thereof |
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| CN101918445A (en) * | 2007-12-13 | 2010-12-15 | 孟山都技术公司 | The composition and the method that are used for diagnosis of early gestation |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112322625A (en) * | 2020-11-11 | 2021-02-05 | 长江师范学院 | Broad-spectrum nucleic acid aptamer for specifically recognizing related glycoprotein of cow and sheep pregnancy and application thereof |
| CN112322625B (en) * | 2020-11-11 | 2023-07-18 | 长江师范学院 | Broad-spectrum nucleic acid aptamer for specifically recognizing cow and sheep pregnancy-related glycoprotein and application thereof |
| CN114438090A (en) * | 2021-11-07 | 2022-05-06 | 吉林大学重庆研究院 | Aptamer specifically binding to Brucella outer membrane protein Omp31 and application thereof |
| CN114438090B (en) * | 2021-11-07 | 2023-08-04 | 吉林大学重庆研究院 | Specific binding Brucella outer membrane protein Omp31 nucleic acid aptamer and application thereof |
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