CN113567684B - IFNg recognition method based on aptamer probe and IFNg detection kit - Google Patents
IFNg recognition method based on aptamer probe and IFNg detection kit Download PDFInfo
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- CN113567684B CN113567684B CN202111127051.7A CN202111127051A CN113567684B CN 113567684 B CN113567684 B CN 113567684B CN 202111127051 A CN202111127051 A CN 202111127051A CN 113567684 B CN113567684 B CN 113567684B
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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
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/555—Interferons [IFN]
- G01N2333/57—IFN-gamma
Abstract
The invention provides an IFNg recognition method based on an aptamer probe, which enables IFNg in a sample to have immunoreaction with the 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 IFNg, and the aptamer including the sequence shown in SEQ ID NO.1 is used as an aptamer probe for targeting IFNg, so that IFNg antigen protein can be sensitively, accurately and quickly identified and stably combined with the IFNg antigen protein, and false positive can be avoided.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an IFNg identification method based on a nucleic acid aptamer probe and a kit for detecting IFNg.
Background
Gamma interferon (IFNg) is an immunocompetent protein with multiple functions, and has the functions of resisting virus, resisting tumor, regulating immunity, controlling apoptosis and the like. The role of interferons and related cytokines has been a focus of biomedical research. However, interferons are biologically very potent in vivo but in very low amounts, only a few picograms per milliliter, or even lower, in normal human blood. Therefore, it is very important to research and invent a monitoring means with high efficiency, convenience, accuracy and low cost.
At present, the common identification method of IFNg is enzyme-linked immunoassay (ELISA), and the basic principle of this method is: 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 an IFNg recognition method based on an aptamer probe and a kit for detecting IFNg, so as to improve the sensitivity and accuracy of IFNg detection.
According to one aspect of the present invention, there is provided a method for IFNg recognition based on aptamer probes, comprising: and (3) carrying out immunoreaction on the IFNg in the sample and the nucleic acid aptamer probe, wherein the nucleotide sequence of the nucleic acid aptamer probe comprises a sequence shown in SEQ ID NO. 1.
Preferably, the IFNg 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 IFNg are washed away, the aptamer probes which participate in the construction of the sandwich structure are subjected to PCR amplification, and the IFNg in the sample is characterized by using the PCR amplification result.
According to another aspect of the present invention, there is provided a kit for detecting IFNg, which is provided with a solid support and an aptamer probe, wherein the surface of the solid support is coated with a capture antibody targeting IFNg, and the nucleotide sequence of the aptamer probe includes the 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 solution = 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 IFNg recognition and detection, compared with protein antibody targeting IFNg, 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 development, it was found that a significant portion of aptamers targeting IFNg would bind not only to the antigenic protein, but also to the capture antibody or to the solid support used to immobilize the capture antibody, giving 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 IFNg, and the aptamers including the sequence shown in SEQ ID NO.1 serving as aptamer probes for targeting IFNg can sensitively, accurately and quickly recognize and stably combine with the IFNG antigen protein, so that the occurrence of false positive can be avoided.
Preferably, the primer comprises an upstream primer and a downstream primer, 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.
Optionally, the prepared materials further comprise IFNg protein standard, substrate chromogenic solution, stop solution and streptavidin marked by 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
Obtaining of IFNg 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 IFNg antigen, running the thus-formed mixed solution on CE (high performance capillary electrophoresis-laser induced fluorescence detection), separating and collecting nucleic acid molecules (DNA-protein complexes) bound to IFNg 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. Eventually finding an aptamer that binds IFNg.
The sequence of the aptamer obtained in this example was:
ACAGGCTAGCGATTGGTGGAATCTTGTGATTCGACGATAAATGTTAGGGGGTTGGTTTTGGGTTGGGCATCCTGGCTTACCTCCTATCGATCATCCA （SEQ ID NO.1），
the two-stage mechanism is shown in figure 1.
Example 2
1. Kit for establishing and detecting IFNg
The kit for detecting IFNg 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-IFNg monoclonal antibody, and the cell is sealed by using 0.1mol/L phosphate buffer solution with pH =7.2 and containing 5% skimmed milk powder as sealing solution;
aptamer probe: the nucleotide sequence of the aptamer probe is SEQ ID NO. 1;
capture antibody dilution: 15 mM phosphate buffer containing 0.5% casein, 2-4% sucrose, 150 mM NaCl, 0.2% Tween 20, pH = 7.4;
sample diluent: 15 mM phosphate buffer, pH =6.5, containing 2-4% sucrose, 150 mM NaCl, 0.2% Tween 20;
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 ACAGGCTAGCGATTGGTGGAA (SEQ ID NO. 2), and the sequence of the downstream primer is TGGATGATCGATAGGAGGTAAGC (SEQ ID NO. 3);
SYBR Green Mix
IFNg protein standard solution.
2. Use of a kit for detecting IFNg
The kit for detecting IFNg provided by the embodiment is used for carrying out the quantitative detection of IFNg, and the method comprises the following steps:
the method comprises the following steps: and respectively adding the IFNg protein standard substance and the sample to be detected which are subjected to gradient dilution into the micropores of the ELISA plate, adding 100 mu L of sample into each hole, repeating the sample adding process for two times, 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 IFNg protein standard product to carry out PCR amplification, wherein the total volume of the reaction system is 50 mu L, and the amplification conditions are as follows:
3 mins at 94 ℃, then circularly amplifying for 30 times, 10 s at 94 ℃ and 25 s 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 IFNg protein in the sample to carry out PCR amplification, wherein the total volume of a reaction system is 50 mu L, and the amplification conditions are as follows:
3 mins at 94 ℃, then circularly amplifying for 30 times, 10 s at 94 ℃ and 25 s 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 1000 pg/ml standard solution, the 1000 pg/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, 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.
From this, it was calculated that the minimum detection limit of IFN-. gamma.is usually 3 pg/mL, however, a quantitative range of levels as low as 1pg/mL can be detected. Serum spiking tests showed recovery of 94.3%, ranging from 88.4% to 102.1% all samples had intrallate CVs of less than 10% and Interplate CVs of less than 15%. The current ELISA KIT for IFN-gamma detection has a minimum detection limit of 15 pg/mL, such as RayBio Human IFN-gamma ELISA KIT (https:// www.raybiotech.com/Human-IFN-gamma-ELISA-KIT-for-serum-cell-culture-super-and-urea /). Therefore, the IFN-gamma recognition method provided by the invention can effectively improve the detection sensitivity of IFN-gamma, and the kit based on the recognition method can reach the lowest detection limit which is obviously lower than that of the existing kit aiming at IFN-gamma 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> IFNg recognition method based on aptamer probe and kit for detecting IFNg
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 97
<212> DNA
<213> Artificial sequence
<400> 1
acaggctagc gattggtgga atcttgtgat tcgacgataa atgttagggg gttggttttg 60
ggttgggcat cctggcttac ctcctatcga tcatcca 97
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence
<400> 2
acaggctagc gattggtgga a 21
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
<400> 3
tggatgatcg ataggaggta agc 23
Claims (9)
1. An IFNg identification method based on a nucleic acid aptamer probe, which is characterized in that: and (3) immunoreacting the IFNg 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 IFNg recognition method according to claim 1, wherein: and (3) respectively and sequentially carrying out immunoreaction on the IFNg in the sample with an antibody and the aptamer probe to form an antibody-protein-aptamer sandwich structure.
3. The method for IFNg recognition based on aptamer probes according to claim 2, wherein: after the sandwich structure is formed, washing away the excessive aptamer probe which is not combined with the IFNg, carrying out PCR amplification on the aptamer probe which is involved in the construction of the sandwich structure, and characterizing the IFNg in the sample by using the PCR amplification result.
4. A kit for detecting IFNg, characterized in that: 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 IFNg, and the nucleotide sequence of the nucleic acid aptamer probe comprises a sequence shown in SEQ ID NO. 1.
5. The kit for detecting IFNg 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 IFNg of claim 5, wherein: the wash contained 0.05% sodium azide.
7. The kit for detecting IFNg of claim 6, wherein: the pH of the wash = 7.2.
8. The kit for detecting IFNg 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 IFNg of claim 8, wherein: the primer comprises an upstream primer and a downstream primer, 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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