CN111500583B - 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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- CN111500583B CN111500583B CN202010143889.4A CN202010143889A CN111500583B CN 111500583 B CN111500583 B CN 111500583B CN 202010143889 A CN202010143889 A CN 202010143889A CN 111500583 B CN111500583 B CN 111500583B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
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. The nucleic acid aptamers of the present invention may also be various analogous sequences having high homology or derivatives derived from the sequences of the present invention. The invention also provides the application of the aptamer. The aptamer is obtained by screening through a magnetic bead-SELEX technology, can be combined with bPAG4 target protein, can also be selectively combined with bPAG congeneric protein, does not generate specific combination with other proteins, can be used for capturing bPAG protein from a complex system, and is suitable for detection, separation and purification of bPAG protein and rapid diagnosis of early pregnancy of cattle.
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 exponential enrichment ligand phylogenetic evolution technology (SELEX), magnetic beads are used as a separation medium, bPAG4 is used as a 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, KCl: 0.2 g/L, Na2HPO 4: 1.15 g/L, KH2PH 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 by 50 mu L with 10mM PBS (ph 7.4), adding 50 mu L of mixed solution of 0.1M NHS +0.4M EDC (v/v, 1: 1) 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 bPAG4 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-bPAG4 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 1OD random single-stranded DNA library was dissolved in PBS to a concentration of 5. mu.M, denatured in a PCR apparatus at 95 ℃ for 10 minutes, subjected to ice-water bath for 5 minutes, and instantaneously isolated at room temperature for use. The denatured 200. mu.L library was added to MB-BSA magnetic beads and incubated with shaking for 60 min. Magnetic separating, adding the supernatant into MB-PAG4, shaking and incubating for 60min, 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 5min, 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 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 Strand method): adding the concentrated PCR product into an equal volume of 2 XTBE/urea denaturation buffer solution, denaturing at 95 ℃ for 10min in a PCR instrument, adding the mixture into a loading hole of 8% polyacrylamide gel (PAGE), and carrying out electrophoresis at 300V until bromophenol blue reaches the bottom of the gel, so that single-stranded DNA with polyA is separated from single-stranded DNA marked by FAM. Taking off the PAGE gel, cutting off a strip with fluorescence under an ultraviolet lamp by using a clean blade, putting the strip into a 0.5 mL gel-breaking centrifuge tube, putting the gel-breaking centrifuge tube into a2 mL centrifuge tube, centrifuging at 14000 rpm for 1 min, removing the gel-breaking centrifuge tube, adding PBS (phosphate buffer solution), boiling in boiling water for 10min, transferring the supernatant into a 15 mL centrifuge tube, and concentrating the single-stranded DNA according to the step (3). Then, the cells were packed in a dialysis bag of 3KD and dialyzed overnight at 4 ℃ in PBS, and the nucleic acid concentration was measured by a NanoDrop-2000c ultramicrospectrophotometer and used as the starting library for the next round of screening.
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 as per 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, subjected to ice-water bath for 5min, then incubated with MB-bPAG4 for 60min, magnetic separated, washed 2 times with PBS buffer, 200. mu.L of PBS buffer was used to resuspend the magnetic beads, and binding of ssDNA library to the target was detected using Thermo Scientific Varioskan Flash Multi-plate Detector (. lamda.Ex =492nm,. lamda.Em =518 nm). The results are shown in FIG. 1, when the fluorescence value is no longer increased, indicating that the aptamer has been enriched, the screening is stopped. Then, each round of ssDNA library was subjected to 8% polyacrylamide gel electrophoresis and a Bio-Rad gel imager, and the result is shown in FIG. 2, where a single 1-9 round of objective ssDNA electrophoresis band is present 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, cloned with pGEM-T vector (Takara biotech, co., Ltd) and transformed into E.ColiDH5 α. Through blue-white screening, 30 positive clones are randomly selected and sent to a company of Biotechnology engineering (Shanghai) GmbH for synthesis sequencing, and the nucleic acid aptamer sequence successfully sequenced is subjected to homology analysis by adopting GLUSTALX software and is divided into 5 families. Based on the principle of lowest free energy, 1 sequence from each family was selected as a representative, sent to Shanghai for biosynthesis and labeling of the 5' FAM group, and further analyzed for affinity and specificity.
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 of each aptamer was calculated by fitting the binding curve using GraphPad Prism 7.0 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 (5)
1. The aptamer specifically recognizing the bovine pregnancy related glycoprotein 4 is characterized in that the sequence of the aptamer consists of a DNA sequence shown in SEQ ID No.1, wherein the DNA sequence shown in 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 is modified, the modification being phosphorylation, 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 is connected with a fluorescent marker, a radioactive substance, biotin, streptavidin, digoxigenin or a nano luminescent material.
4. Use of the nucleic acid aptamer of claim 1 in the preparation of a product for detecting, isolating and purifying bovine pregnancy-associated glycoprotein.
5. Use of the nucleic acid aptamer of claim 1 in the preparation of a diagnostic product for early pregnancy in cattle.
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| 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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