CN113945720A - PDGF-BB recognition method based on aptamer probe and PDGF-BB detection kit - Google Patents
PDGF-BB recognition method based on aptamer probe and PDGF-BB detection kit Download PDFInfo
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Abstract
The invention provides a PDGF-BB recognition method based on a nucleic acid aptamer probe, which comprises the following steps: immunoreacting PDGF-BB in the sample with an aptamer probe, wherein the nucleotide sequence of the aptamer probe comprises a sequence shown in SEQ ID NO. 1. The aptamer with the nucleotide sequence including the sequence shown in SEQ ID NO.1 shows outstanding specificity and affinity to PDGF-BB, and the aptamer including the sequence shown in SEQ ID NO.1 is used as a PDGF-BB targeting aptamer probe, so that PDGF-BB antigen protein can be sensitively, accurately and quickly identified and stably combined with the PDGF-BB antigen protein, and false positive can be avoided.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a PDGF-BB recognition method based on a nucleic acid aptamer probe and a PDGF-BB detection kit.
Background
Platelet-derived growth factor (PDGF) is a potent mitogen and an important growth factor that promotes tumor angiogenesis and can be produced by a variety of blood cells, tissue cells and some tumor cells. The present study of PDGF expression and detection in tumor cells has become one of the hot spots in the field of biomedical research.
PDGF-BB is the most active subtype of platelet-derived growth factor in the PDGF family. At present, the PDGF-BB is generally recognized by enzyme-linked immunosorbent assay (ELISA), and the basic principle of the method is as follows: the antigen or antibody is bound to the surface of a solid phase carrier and the immunocompetence is maintained. ② the antigen or antibody is connected with certain enzyme to form enzyme-labeled antigen or antibody, and the enzyme-labeled antigen or antibody not only retains its immunological activity, but also retains the activity of enzyme. In the measurement, the specimen to be tested (the antibody or antigen to be measured therein) and the enzyme-labeled antigen or antibody are reacted with the antigen or antibody on the surface of the solid carrier in a different step. The antigen-antibody complex formed on the solid phase carrier is separated from other substances by washing, and finally the enzyme quantity bound on the solid phase carrier is in a certain proportion to the quantity of the detected substance in the specimen. After the substrate of the enzyme reaction is added, the substrate is catalyzed by the enzyme to be changed into a colored product, and the amount of the product is directly related to the amount of the detected substance in the sample, so that qualitative or quantitative analysis can be carried out according to the existence of the color reaction.
Aptamers are artificially synthesized nucleic acids that are capable of specifically binding to target molecules, with stable secondary structures. The pattern of the aptamer recognition molecule is similar to that of an antibody, but compared with a protein antibody, the aptamer has more advantages, such as no limit of immune conditions and immunogenicity, capability of in vitro artificial synthesis, simple and rapid preparation, reversible denaturation and renaturation, capability of modification, easiness for long-term storage, room-temperature transportation and the like. More importantly, the target molecules of the aptamer are wider and can be proteins, nucleic acids, small peptides, amino acids, organic matters, even metal ions and the like. In addition, aptamers also have the following advantages, such as: stable chemical property, no immunogenicity or toxicity reported to the present, high affinity, strong specificity and easy modification. These properties make the aptamers widely used in the biomedical research field, and become indispensable powerful tools.
Disclosure of Invention
The invention aims to provide a PDGF-BB recognition method based on an aptamer probe and a PDGF-BB detection kit, so as to improve the sensitivity and accuracy of PDGF-BB detection.
According to one aspect of the present invention, there is provided a method for aptamer probe based PDGF-BB recognition, comprising: immunoreacting PDGF-BB in the sample with an aptamer probe, wherein the nucleotide sequence of the aptamer probe comprises a sequence shown in SEQ ID NO. 1.
Preferably, PDGF-BB in the sample is sequentially immunoreactive with the antibody and the aptamer probe to form an antibody-protein-aptamer sandwich structure.
Preferably, after the sandwich structure is formed, excess aptamer probes which are not combined with the PDGF-BB are washed away, the aptamer probes which participate in the construction of the sandwich structure are subjected to PCR amplification, and the PDGF-BB in the sample is characterized by using the PCR amplification result.
According to another aspect of the invention, a kit for detecting PDGF-BB is provided, wherein the prepared materials comprise a solid phase carrier and an aptamer probe, the surface of the solid phase carrier is coated with a capture antibody targeting PDGF-BB, and the nucleotide sequence of the aptamer probe comprises a sequence shown in SEQ ID NO. 1.
Preferably, the prepared material also comprises a washing solution, and the washing solution is a phosphate buffer solution containing 0.5% of Tween 20 and 0.1 mol/L.
Preferably, the wash solution contains 0.05% sodium azide.
Preferably, the pH of the wash liquor is 7.2.
Preferably, the prepared materials further comprise Taq-DNA polymerase, PCR reaction buffer, dNTP and a primer, wherein the primer is complementary with a part of the nucleotide sequence of the aptamer.
The invention adopts aptamer as probe to be applied to PDGF-BB recognition and detection, and compared with PDGF-BB targeted protein antibody, the aptamer probe is based on the structural characteristics of DNA: the modified polycarbonate resin has the excellent performances of simple and rapid preparation, small molecular weight, stable chemical property and easy modification; the kit is suitable for PCR amplification, and qualitative and quantitative detection is carried out on the antigen protein by PCR amplification, so that the detection scheme is more diverse and convenient than the traditional detection scheme for detecting the antibody by adopting proteins. During the development process, it was found that a considerable portion of aptamers targeting PDGF-BB bound not only to the antigenic protein but also to the capture antibody or to the solid support for immobilization of the capture antibody, with false positives. However, in the screened aptamers, the aptamers with nucleotide sequences including the sequence shown in SEQ ID NO.1 show outstanding specificity and affinity to PDGF-BB, and the aptamers including the sequence shown in SEQ ID NO.1 are used as a PDGF-BB targeting aptamer probe, so that PDGF-BB antigen protein can be sensitively, accurately and quickly identified and stably combined with the PDGF-BB antigen protein, and false positive can be avoided.
Preferably, the primers comprise an upstream primer and a downstream primer, the sequence of the upstream primer is shown as SEQ ID NO.2, and the sequence of the downstream primer is shown as SEQ ID NO.3
Optionally, the prepared materials also comprise PDGF-BB protein standard, a substrate chromogenic solution, a stop solution and streptavidin marked by a chromogenic substrate, and the aptamer probe is a nucleotide sequence marked by biotin.
Drawings
FIG. 1 is a secondary structural diagram of an aptamer obtained in example 1;
FIG. 2 is a graphical representation of an experiment in which a standard curve was constructed in example 2;
FIG. 3 is a graph showing Ct values corresponding to the gradient concentration standard sample and the control sample in example 2;
FIG. 4 is a standard graph constructed in example 2.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Example 1
Acquisition of PDGF-B aptamers
The screening of aptamers was carried out by the capillary electrophoresis SELEX technique (Systematic Evolution of Ligands by electrophoresis) as follows:
mixing nucleic acid with PDGF-B antigen, running the mixed solution on CE (high performance capillary electrophoresis-laser induced fluorescence detection), and separating and collecting nucleic acid molecules (DNA-protein complex) combined with PDGF-B antigen; allowing the DNA-protein complex to pass through the capillary at different rates, identifying the DNA-protein complex by using a fluorescence detection technology when the DNA-protein complex passes through a window in the capillary, collecting DNA by a complex peak and carrying out PCR amplification; we then generated ssDNA using Lambda exonuclease; the above process was repeated 3-10 times. Negative selection was performed in a similar manner using non-specific proteins, but DNA that did not bind to the antigen was collected. After selection, DNA is sequenced using NGS to identify individual sequences. Finally finding the aptamer binding PDGF-B.
The sequence of the aptamer obtained in this example was:
TCCCACGCATTCTCCACATCATAAGCTGAGCATCTTAGATCCCCGTCAA GGGCAGCGTAA CCT TTCTGTCCTTCCGTCAC(SEQ ID NO.1)，
the two-stage mechanism is shown in figure 1.
Example 2
1. Kit for establishing and detecting PDGF-BB
The reagent kit for detecting PDGF-BB is prepared from the following materials:
enzyme label plate: the surface of the cell is coated with a capture antibody, the capture antibody is an anti-PDGF-BB monoclonal antibody, and the cell is sealed by using a pH 7.2 and 0.1mol/L phosphate buffer solution containing 5% of skimmed milk powder as a sealing solution;
aptamer probe: the nucleotide sequence of the aptamer probe is SEQ ID NO. 1;
capture antibody dilution: 15mM phosphate buffer containing 0.5% casein, 2-4% sucrose, 150mM NaCl, 0.2% Tween 20, with pH 7.4;
sample diluent: 15mM phosphate buffer containing 2-4% sucrose, 150mM NaCl, 0.2% Tween 20, pH 6.5;
washing liquid: 0.1M phosphate buffer, pH 7.2, containing 0.5% tween 20, 0.05% sodium azide.
Taq-DNA polymerase;
PCR reaction buffer solution;
dNTP；
primer: the sequence of the upstream primer is TCCCACGCATTCTCCACATCATA (SEQ ID NO.2), and the sequence of the downstream primer is GTGACGGAAGGACAGAAAGGTT (SEQ ID NO. 2);
SYBR Green Mix
PDGF-BB protein standard solution.
2. Use of a kit for detecting PDGF-BB
The PDGF-BB detection kit provided by the embodiment is used for carrying out quantitative detection on PDGF-BB, and the method comprises the following steps:
the method comprises the following steps: and respectively adding a PDGF-BB protein standard substance and a sample to be detected which are subjected to gradient dilution into micropores of the ELISA plate, adding 100 mu L of the sample into each hole, repeating the steps for each sample, reacting for 40 minutes at 37 ℃, and washing the ELISA plate.
Step two: adding the aptamer probe into the micropores of the ELISA plate, incubating for 40 minutes, and washing the ELISA plate.
And (3) washing the ELISA plate for 5 times by using the configured washing liquid on a plate washing machine for 10 minutes. In other embodiments, the washing liquid configured in the ELISA kit can be used to perform plate washing operations commonly used in the art according to practical situations, and is not limited to plate washing with a plate washing machine.
Step three: PCR amplification of standards
(1) Taking 5 mu L of aptamer probe combined with the PDGF-BB protein standard product to carry out PCR amplification, wherein the total volume of a reaction system is 50 mu L, and the amplification conditions are as follows: 3mins at 94 ℃, then circularly amplifying for 30 times, 10s at 94 ℃ and 25s at 55 ℃;
(2) adding 5 mu L of PCR reaction solution after 1 st amplification into a new system, and repeating the 1 st PCR amplification operation;
(3) recovering the aptamer probe.
Step four: PCR amplification of samples
(1) Taking 5 mu L of aptamer probe combined with PDGF-BB protein in the sample to carry out PCR amplification, wherein the total volume of the reaction system is 50 mu L, and the amplification conditions are as follows: 3mins at 94 ℃, then circularly amplifying for 30 times, 10s at 94 ℃ and 25s at 55 ℃;
(2) adding 5 mu L of PCR reaction solution after 1 st amplification into a new system, and repeating the 1 st PCR amplification operation;
(3) recovering the aptamer probe.
The standard curve was constructed as follows:
(1) the standard sample containing the antigen with the concentration gradient and the control sample containing no antigen are set, the standard sample is set in a way that 40 mu L of VEGF standard substance is added into a clean centrifuge tube containing 360 mu L of diluent to prepare 1000pg/ml standard solution, the 1000pg/ml standard solution is diluted to further prepare standard samples with other concentrations, and finally, the same amount of the accounting aptamer (SEQ ID NO.1) is respectively added into the standard sample and the control sample.
(2) Collecting data using qPCR, characterizing the amount of antigen contained in each sample by outputting a Ct value, wherein a lower Ct value indicates a higher antigen concentration; when the DNA is amplified, an additional fluorescent signal is generated, each cycle results in approximately doubling of the DNA, and the Ct value represents the number of cycles required for the sample to pass the fluorescence threshold; thus, higher levels of DNA (directly related to the amount of antigen in the sample) result in lower Ct values.
(3) Calculating the average Ct of each group of triplicate standard samples and control samples; subtracting the Ct value of each sample from the control to obtain the Ct value difference between the control and the sample; the Ct values of the standard sample and the control sample respectively corresponding to the gradient concentrations obtained according to the above steps are shown in fig. 3, and in fig. 3, the intensity of the ordinate is the Ct value corresponding to the sample. The concentration is used as an x coordinate, and the Ct value difference is used as a y coordinate to fit a standard curve, so that the linear relation obtained by fitting is shown in FIG. 4.
Therefore, the minimum detection limit of PDGF-BB is calculated to be 0.2pg/mL, so that the IFN-gamma recognition method provided by the invention can effectively improve the detection sensitivity of PDGF-BB, and the kit based on the recognition method can reach the minimum detection limit which is obviously lower than that of the existing kit aiming at PDGF-BB detection.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the present invention.
SEQUENCE LISTING
<110> Rebo ao (Guangzhou) Biotechnology Ltd
<120> PDGF-BB recognition method based on aptamer probe and PDGF-BB detection kit
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 80
<212> DNA
<213> Artificial sequence
<400> 1
tcccacgcat tctccacatc ataagctgag catcttagat ccccgtcaag ggcagcgtaa 60
cctttctgtc cttccgtcac 80
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence
<400> 2
tcccacgcat tctccacatc ata 23
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
gtgacggaag gacagaaagg tt 22
Claims (9)
1. A method for PDGF-BB recognition based on aptamer probes, characterized in that: immunoreacting PDGF-BB in the sample with the aptamer probe, wherein the nucleotide sequence of the aptamer probe comprises a sequence shown in SEQ ID NO. 1.
2. The aptamer probe-based PDGF-BB recognition method of claim 1, wherein: and respectively and sequentially carrying out immunoreaction on the PDGF-BB in the sample with an antibody and the aptamer probe to form an antibody-protein-aptamer sandwich structure.
3. The aptamer probe-based PDGF-BB recognition method of claim 2, wherein: after the sandwich structure is formed, washing away excessive aptamer probes which are not combined with PDGF-BB, carrying out PCR amplification on the aptamer probes which participate in the construction of the sandwich structure, and characterizing the PDGF-BB in the sample by using the PCR amplification result.
4. A kit for detecting PDGF-BB comprising: the prepared material comprises a solid phase carrier and a nucleic acid aptamer probe, wherein the surface of the solid phase carrier is coated with a capture antibody targeting PDGF-BB, and the nucleotide sequence of the nucleic acid aptamer probe comprises a sequence shown in SEQ ID NO. 1.
5. The kit for detecting PDGF-BB of claim 4, wherein: the prepared material also comprises a washing solution, wherein the washing solution is a phosphate buffer solution containing 0.5% of Tween 20 and 0.1 mol/L.
6. The kit for detecting PDGF-BB of claim 5, wherein: the wash contained 0.05% sodium azide.
7. The kit for detecting PDGF-BB of claim 6, wherein: the pH of the wash was 7.2.
8. The kit for detecting PDGF-BB of claim 5, wherein: the prepared material also comprises Taq-DNA polymerase, PCR reaction buffer solution, dNTP and a primer, wherein the primer is complementary with a part of nucleotide sequence of the aptamer.
9. The kit for detecting PDGF-BB of claim 8, wherein: the sequence of the upstream primer is shown as SEQ ID NO.2, and the sequence of the downstream primer is shown as SEQ ID NO. 3.
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