CN111334511A - Aptamer for specifically recognizing bovine pregnancy-associated glycoprotein and application thereof - Google Patents
Aptamer for specifically recognizing bovine pregnancy-associated glycoprotein and application thereof Download PDFInfo
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Abstract
The invention discloses a nucleic acid aptamer for specifically recognizing bovine pregnancy associated glycoprotein (bPAG) and application thereof. The sequence of the aptamer is a DNA fragment shown in the sequence of SEQ ID No.1 or a DNA fragment shown in the sequence of SEQ ID No.2, and a derivative of the aptamer is also disclosed, and the aptamer sequence is subjected to skeleton modification or base modification to obtain the derivative of the aptamer with the same function as the aptamer. The aptamer is obtained by screening through a magnetic bead-SELEX technology, can be specifically combined with a bPAG protein family, is not specifically combined with other proteins, is easy to synthesize and modify, can be used for capturing bPAG protein from a complex system, is suitable for detection, separation and purification of bPAG protein and rapid diagnosis of early pregnancy of livestock, and has a wide application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a nucleic acid aptamer for specifically recognizing bovine pregnancy-associated glycoprotein, and particularly relates to application of the nucleic acid aptamer in pregnancy-associated glycoprotein 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 28 days 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 enzyme-linked immunosorbent assay kit developed by Idexx (America) company, but the price of the rapid ELISA kit sold in domestic markets is quite high, and large-scale popularization and use in China are limited. In view of the problems of long antibody preparation time, high cost and the like in the existing immunoassay, a novel recognition molecule is developed to replace an antibody, and the method has great significance for early pregnancy detection of livestock.
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
Aiming at the technical problems in the background art, the invention provides a nucleic acid aptamer capable of specifically recognizing bPAG and application thereof, which have high binding capacity to bPAG protein and can specifically recognize bPAG protein family.
In order to solve the technical problems, the invention adopts the following technical scheme:
an aptamer specifically recognizing bovine pregnancy related glycoprotein, the sequence of the aptamer comprises a DNA fragment shown as SEQ ID No.1 sequence or a DNA fragment shown as SEQ ID No.2 sequence, wherein,
SEQ ID No.1 sequence:
5’-TTGAAGTGACTCCCACCCACCGTCCACCGTATTTCACCGTCCATTGCATAGCAGGT-3’；
SEQ ID No.2 sequence:
5-TTGAAGTGACGCCAGGGTGGGGGGGTGGGTGTTGGCGTACACTTCGCATAGCAGGT-3’。
preferably, the aptamer sequence may be modified, the modification including phosphorylation, methylation, amination, carboxylation, sulfhydrylation, or isotopolylation.
Preferably, the sequence of the nucleic acid aptamer can be connected with a fluorescent marker, a radioactive substance, biotin, streptavidin, digoxigenin, a nano luminescent material and an enzyme.
As a general technical concept, the present invention provides an aptamer specifically recognizing bovine pregnancy-associated glycoprotein, the nucleotide sequence of the aptamer comprising any one of the following three sequences:
(1) a DNA sequence having 60% or more homology with the DNA sequence of the aptamer according to claim 1 and capable of specifically binding to bovine pregnancy-associated glycoprotein;
(2) a DNA sequence capable of hybridizing with the DNA sequence of the aptamer according to claim 1 under stringent conditions; or
(3) An RNA sequence transcribed from the DNA sequence of the aptamer according to claim 1.
As a general technical concept, the invention also provides a nucleic acid aptamer derivative for specifically recognizing the bovine pregnancy related glycoprotein, wherein the nucleic acid aptamer derivative comprises the nucleic acid aptamer sequence which is subjected to skeleton modification or base modification to obtain the nucleic acid aptamer derivative with the same function as the nucleic acid aptamer.
Preferably, the base is engineered as a substitution, deletion or addition.
The application of the aptamer specifically recognizing bPAG or the aptamer derivative specifically recognizing bPAG in the preparation of products for detecting, separating and purifying bPAG.
The application of the aptamer specifically recognizing bPAG or the aptamer derivative specifically recognizing bPAG in the livestock early pregnancy diagnosis product.
Has the advantages that:
the method utilizes exponential enrichment ligand phylogenetic evolution technology (SELEX), takes magnetic beads as a separation medium, takes bPAG9 as a target protein, obtains two aptamers specifically combined with the target through 7 rounds of screening, has extremely high affinity, has dissociation constant reaching nM level, can specifically combine with bPAG protein family, and has almost no combination with other proteins. The aptamer obtained by screening has good affinity and specificity, can be artificially synthesized, has low cost and short production period, is easy to chemically modify, and can be prepared into molecular probes, detection reagents and the like for detection, separation and purification of bPAG protein and early pregnancy diagnosis of livestock.
Drawings
FIG. 1 is a polyacrylamide gel electrophoresis image of ssDNA libraries obtained from each round of screening.
FIG. 2 is a secondary structure diagram of the aptamer of the invention, wherein, the sequence 1 is the aptamer with the sequence shown in SEQ ID No.1, and the sequence 2 is the aptamer with the sequence shown in SEQ ID No. 2.
FIG. 3 is a graph showing data on affinity detection of the aptamer of the present invention to bPAG9 protein.
FIG. 4 shows the results of experiments on the binding specificity of aptamers of the present invention to bPAG protein family.
FIG. 5 is a graph comparing the results of experiments on the binding specificity of aptamers to bPAG proteins, BSA and OVA according to example 3 of the present invention.
Detailed Description
The invention is further illustrated by the following specific examples, which are not intended to limit the scope of the invention.
Example 1: screening of bPAG 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.
Random single-stranded DNA library, 5 '-end primer and 3' -end primer were treated with PBS buffer (PBS, NaCl 8 g/L, KCl 0.2 g/L, Na)2HPO4：1.15 g/L，KH2PH4: 0.2 g/L; pH 7.4) and stored at-20 ℃ for further use.
2. Magnetic bead-bPAG 9 protein (MB-bPAG 9) coupling:
taking 50 mu L of carboxylated magnetic beads, washing with PBS, taking 50 mu L of mixed solution (v/v, 1: 1) of 0.1M NHS +0.4M EDC, adding into the magnetic beads, and carrying out shake reaction for 20 min; after activation, magnetic separation, PBS washing, adding NaAC (10 mM, pH5.0) and bPAG9 protein (0.5 mg/mL), and shaking for 60 min; after the coupling is finished, carrying out magnetic separation, washing by PBS, adding 100 mu L of ethanolamine (1.0M, pH8.5), and carrying out shaking reaction for 20 min; magnetic separation, PBS washing, magnetic bead heavy suspension in PBS, 4 degrees C storage for use. The magnetic bead-bovine serum albumin (MB-BSA) coupling was performed as described above. MB-bPAG9 was used as the positive screen target, and MB-BSA was used as the negative screen target.
3. Screening:
3.1 incubation and isolation: the 1 OD random single-stranded DNA library was diluted and dissolved with PBS to a concentration of 5. mu.M, denatured in a PCR instrument at 95 ℃ for 10 minutes, ice-water-bath for 5 min, and immediately isolated at room temperature for use. Adding the denatured 200 mu L library into MB-BSA magnetic beads, and performing shake incubation for 60 min; magnetic separating, adding the supernatant into MB-bPAG9, shaking and incubating for 60 min, magnetic separating, discarding the supernatant, adding PBS, boiling for 10min, magnetic separating, and collecting the supernatant.
3.2 PCR amplification: adding the library obtained in the step 3.1 into 2 mL of PCR mix, mixing uniformly, subpackaging into 100 mu L of each tube, and carrying out PCR amplification. PCR amplification procedure: 95 ℃ for 1 min, 60 ℃ for 1 min, 72 ℃ for 1 min, 25 cycles, 72 ℃ for 5 min, 25 ℃ for 2 min.
3.3 PCR product concentration: collecting PCR product, adding 5 times volume of n-butanol, vortex mixing, centrifuging at 7500 rpm for 5 min, 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-chain 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 to separate single-stranded DNA with PolyA from FAM-labeled single-stranded DNA, the PAGE gel was removed, the band with fluorescence was cut off with a clean blade under an ultraviolet lamp, and put into a 0.5 mL gel-breaking centrifuge tube, 2 mL centrifuge tube, centrifuge at 14000 rpm for 1 min, discard the gel centrifuge tube, PBS was added, boiled in boiling water for 10min, the supernatant was transferred to a 15 mL centrifuge tube, the single-stranded DNA was concentrated according to step (3), then put into a PBS 3 KD dialysis bag, dialyzed overnight at 4 ℃ in a PBS, and the concentration of nucleic acid was determined as an initial library for the next round of screening.
4. Surface Plasmon Resonance (SPR) measures the binding of ssDNA libraries to target proteins in each round:
the CM5 chip was washed with 50 mM NaOH aqueous solution at a flow rate of 10. mu.L/min for 180s, the channel was activated with 0.1M NHS +0.4MEDC mixed reagent (v/v, 1: 1) at the same flow rate for 600s, followed by injecting bPAG9 protein (30. mu.g/ml) solution into the chip at a flow rate of 10. mu.L/min, and then injecting 1M ethanolamine solution into the chip at a flow rate of 10. mu.L/min to block the chip. The resulting ssDNA library was then diluted with PBS to a concentration of 0.5. mu.M, injected at a flow rate of 30. mu.L/min for 180s, and unbound ssDNA was washed with PBS at a flow rate of 10. mu.L/min for 30 min. When the affinity no longer increased (see FIG. 1), the screening was stopped. The result of each round of ssDNA library detected by 8% polyacrylamide gel electrophoresis and Bio-Rad gel imager is shown in FIG. 2, and there is a single 1-7 round of objective ssDNA electrophoresis band under 80 bp.
5. High-throughput sequencing:
PCR amplification was performed on each round of ssDNA library according to step 3.2, the PCR products were concentrated and purified using UNIQ-10 oligonucleotide purification kit (Shanghai Producer, SK1144), the concentration was measured by NanoDrop 2000c ultramicro spectrophotometry, and finally the purified samples were sent to Beijing Nuo Segetascience technologies GmbH for high throughput sequencing. Then, the first 300 sequences are given according to the repetition rate from high to low, homology comparison is carried out by using CLUSTALX software, a plurality of sequences with high enrichment degree in each family are selected and sent to the company of biological engineering (Shanghai) GmbH for synthesis, the method in the step 4 is adopted to detect the affinity of each aptamer, and the aptamer with high affinity is selected from the sequences to carry out further dissociation constant determination on the bPAG9 protein.
Example 2: SPR measurement of dissociation constants of aptamer and bPAG9 protein
Taking a plurality of ssDNAs (single stranded DNAs) synthesized in example 1, namely aptamers, and preparing a series of aptamer solutions with gradient concentrations by using PBS (phosphate buffer solution); coupling bPAG9 protein on a chip according to the step 4, then sequentially injecting aptamer solutions with different concentrations into the chip coupled with the bPAG9, detecting the affinity of each aptamer and the bPAG9 protein, and finding that the aptamers with the sequences shown in SEQ ID No.1 and SEQ ID No.2 have high affinity with the bPAG9 protein, and dissociation constants obtained by SPR detection are in nM level (shown in figure 3); wherein, the dissociation constant of the aptamer with the sequence 1 as shown in SEQ ID No.1 to bPAG9 protein is 1.04 nM, and the dissociation constant of the aptamer with the sequence 2 as shown in SEQ ID No.2 to bPAG9 protein is 2.40 nM. The aptamer sequences shown in SEQ ID No.1 and SEQ ID No.1 were subjected to secondary structure prediction analysis using online MFold, and the results are shown in FIG. 4.
Example 3: SPR detection of binding specificity of aptamers to bPAG proteins
Four proteins of the same type, bPAG4, bPAG6, bPAG9 and bPAG16, as well as BSA and OVA proteins, were diluted with sodium acetate solution (pH5.0) to a concentration of 50. mu.g/mL, and the respective proteins were coupled to different channels of the chip in accordance with step 4, and the aptamer sequences shown in SEQ ID No.1 and SEQ ID No.2 were each formulated with PBS to a concentration of 500 nM, and the binding of each aptamer to the respective proteins was determined in accordance with step 4. The detection results are shown in FIG. 5, and the aptamers shown in SEQ ID No.1 and SEQ ID No.2 can be combined with b-PAG 4, b-PAG 6, b-PAG 9 and b-PAG 16 in the bPAG protein family, but not combined with 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> nucleic acid aptamer for specifically recognizing bovine pregnancy-associated glycoprotein and application thereof
<130>2020.03.03
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>56
<212>DNA
<213> Artificial Synthesis (Artificial sequence)
<400>1
ttgaagtgac tcccacccac cgtccaccgt atttcaccgt ccattgcata gcaggt 56
<210>2
<211>56
<212>DNA
<213> Artificial Synthesis (Artificial sequence)
<400>2
ttgaagtgac gccagggtgg gggggtgggt gttggcgtac acttcgcata gcaggt 56
Claims (8)
1. An aptamer that specifically recognizes a bovine pregnancy-associated glycoprotein, characterized in that: the sequence of the aptamer comprises the DNA fragment shown in the sequence of SEQ ID No.1 or the DNA fragment shown in the sequence of SEQ ID No.2, wherein,
SEQ ID No.1 sequence:
5’-TTGAAGTGACTCCCACCCACCGTCCACCGTATTTCACCGTCCATTGCATAGCAGGT-3’；
SEQ ID No.2 sequence:
5-TTGAAGTGACGCCAGGGTGGGGGGGTGGGTGTTGGCGTACACTTCGCATAGCAGGT-3’。
2. the aptamer specific for recognizing bovine pregnancy-associated glycoprotein according to claim 1, wherein the aptamer sequence can be modified, and the modification comprises phosphorylation, methylation, amination, carboxylation, sulfhydrylation or isotopolylation.
3. The aptamer capable of specifically recognizing the bovine pregnancy related glycoprotein according to claim 1, wherein the sequence of the aptamer can be linked with a fluorescent marker, a radioactive substance, biotin, streptavidin, digoxigenin, a nano luminescent material and an enzyme.
4. The aptamer specifically recognizing bovine pregnancy-associated glycoprotein according to claim 1, wherein the aptamer comprises any one of the following three sequences:
(1) a DNA sequence having 60% or more homology with the DNA sequence of the aptamer according to claim 1 and capable of specifically binding to bovine pregnancy-associated glycoprotein;
(2) a DNA sequence capable of hybridizing with the DNA sequence of the aptamer according to claim 1 under stringent conditions; or
(3) An RNA sequence transcribed from the DNA sequence of the aptamer according to claim 1.
5. A aptamer derivative specifically recognizing bovine pregnancy-associated glycoprotein, wherein the aptamer derivative comprises a aptamer derivative which is subjected to backbone modification or base modification by the aptamer sequence of any one of claims 1 to 4 to obtain the same function as the aptamer.
6. The aptamer derivative specifically recognizing bovine pregnancy-associated glycoprotein according to claim 5, wherein the base is modified to be a substitution, a deletion or an addition.
7. Use of the aptamer specifically recognizing bovine pregnancy related glycoprotein according to any one of claims 1 to 4 or the aptamer derivative specifically recognizing bovine pregnancy related glycoprotein according to any one of claims 5 to 6 in the preparation of products for detecting, separating and purifying bovine pregnancy related glycoprotein.
8. Use of the aptamer specifically recognizing bovine pregnancy related glycoprotein according to any one of claims 1 to 4 or the aptamer derivative specifically recognizing bovine pregnancy related glycoprotein according to any one of claims 5 to 6 in a product for diagnosing early pregnancy in livestock.
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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 |
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| CN103038645A (en) * | 2010-04-13 | 2013-04-10 | 普罗诺塔股份有限公司 | Biomarkers for hypertensive disorders of pregnancy |
| CN103014000A (en) * | 2012-12-26 | 2013-04-03 | 江南大学 | A set of oligonucleotide aptamers capable of specifically recognizing streptococcus agalactiae |
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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 |
| 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 |
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