CN113092209B - Method for enriching and identifying molecular markers in disease sample to be detected - Google Patents

Abstract

The application discloses a method for identifying molecular markers in a sample to be detected by utilizing a deoxyribozyme probe, which utilizes the interaction between the deoxyribozyme probe and the molecular markers, combines a protein purification technology and a magnetic separation technology to specifically capture and enrich the molecular markers in a certain component, and then elutes target proteins through complementary displacement chains for subsequent identity identification. For Triple Negative Breast Cancer (TNBC) and any other disease model with undefined markers, researchers can simply and rapidly identify the markers by using the method of the application. Therefore, the application provides a novel method for simply and rapidly determining the marker based on the probe enrichment and probe detection aiming at the research object of unknown target molecules.

Description

Method for enriching and identifying molecular markers in disease sample to be detected

Technical Field

The application belongs to the technical field of molecular marker identification, and particularly relates to a method for enriching and identifying triple negative breast cancer molecular markers by using a deoxyribozyme probe.

Background

Triple-negative breast cancer (TNBC for short) is a type of breast cancer in which estrogen receptor, progestogen receptor and human EGF receptor 2 are all expressed as negative, and accounts for 15% -20% of the total breast cancer. TNBC has the characteristics of younger patients, strong tumor invasiveness, poor prognosis of radiotherapy and chemotherapy, distant metastasis in early disease stage, and the like, and is the breast cancer which is the most malignant at present. Because of the lack of definite markers (i.e., therapeutic targets), existing endocrine and targeted therapies have poor effects on TNBC, and conventional chemoradiotherapy can only benefit part of patients. Currently, screening studies on tumor (such as TNBC) markers mostly use multiple sets of methods of combination of proteome, genome, transcriptome, epigenetic group, metabolome and the like, and further analyze and identify tumor markers through molecular action mechanism studies. This approach often produces many suspected, false positive/negative candidate targets, and it is difficult to accurately ascertain the identity of the marker in the short term.

Deoxyribozymes (deoxyribozymes) are a class of single-stranded DNA with catalytic functions, with efficient catalytic activity and structural recognition capability, and are generally evolved from random single-stranded DNA libraries by the exponential enrichment ligand system evolution technique (Systematic Evolution of Ligands by Exponential Enrichment, SELEX). For unknown targets such as TNBC, a differential screening method of "counter-cancellation" may be used, i.e., positive screening with target cells and negative screening with control cells, to obtain a deoxyribose nucleic acid probe. The method has been used by researchers to obtain a deoxyribose nucleic acid probe which specifically recognizes the TNBC cell line MDA-MB-231 lysate and cleaves at a specific RNA base site, but the identity of the target molecule interacting with the probe is not clear. Meanwhile, the existing researchers use a deoxyribose enzyme probe as a detection means to determine target molecules in the clostridium difficile Clostridium difficile metabolite, which interact with the probe; however, the study has been mainly directed to the gradual narrowing of the range of possible target molecules by means of bioinformatic analysis, which is still an indirect approach. Therefore, there is a need to develop new methods for rapidly, simply and definitely interacting with deoxyribose nucleic acid probes to provide new ideas for subsequent accurate treatment.

Disclosure of Invention

In order to overcome the technical problems, the application provides a method for simply and rapidly identifying molecular markers in a disease sample to be detected by taking a deoxyribozyme probe as a gripper, which utilizes the interaction between the deoxyribozyme probe and the molecular markers, combines a protein purification technology and a magnetic separation technology to specifically capture and enrich the molecular markers in a certain component, and then elutes target proteins through complementary displacement chains for subsequent identity identification. For any other disease model with undefined markers, the molecular markers can be identified simply and rapidly by using the method of the application.

The application provides a method for identifying a molecular marker in a sample to be detected by utilizing a deoxyribozyme probe, which is characterized by comprising the following steps of,

and a, performing proteinase K and heating treatment on the extracellular metabolites, then mixing and incubating with a deoxyribozyme probe, and judging the nature of the molecular marker according to the existence of a deoxyribozyme probe cleavage signal. If the deoxyribozyme probe is not cleaved after heating and proteinase K treatment, the nature of the molecular marker can be judged to be protein, namely target protein.

Step b, concentrating and purifying extracellular metabolites to be screened by using an AKTApure system, and separating the extracellular metabolites into different components; the AKTApure system is a set of flexible and controllable liquid chromatography system, and can be used for rapidly purifying target substances such as proteins, polypeptides, nucleic acids and the like at the level from micrograms to grams. Different chromatographic columns are selected, and the mixture can be divided into different components according to different properties (such as size, electronegativity, affinity and the like). The AKTApure system also plays a role in concentration, and can concentrate hundreds of milliliters to liters of extracellular metabolites to a certain component of milliliters, thereby facilitating the subsequent enrichment of target proteins with extremely low abundance.

Step c, mutating the deoxyribozyme probe capable of recognizing the molecular marker to obtain a mutated deoxyribozyme probe;

step d, modifying the mutated deoxyribozyme probe on the surface of the magnetic bead to obtain the magnetic bead probe;

step e, incubating the magnetic bead probes with different components respectively, performing specific capture and enrichment on molecular markers, and then performing magnetic separation and washing to obtain washed incubation solution;

and f, adding a displacement chain into the washed incubation solution, and eluting the target protein.

In certain embodiments, step e is preceded by an assessment of whether the extracellular metabolite components contain the protein of interest.

In some embodiments, the target protein is evaluated by mixing and incubating the deoxyribozyme probe with each component, and selecting one or more groups with strongest cleavage signals of the deoxyribozyme probe for subsequent specific capture and enrichment.

In certain embodiments, the molecular markers are of the molecular type nucleic acids, proteins, small molecules, and the like.

In certain embodiments, the molecular marker is apolipoprotein a.

In certain embodiments, the test sample is one or more of a cell lysate, an extracellular metabolite, a disease-like tissue, a bodily fluid, and a serum.

In certain embodiments, the test disease sample is a TNBC cell line.

In certain embodiments, the test sample is an Extracellular Metabolite (EMs) of the TNBC cell line MDA-MB-231.

In certain embodiments, the deoxyribose nucleic acid probe is a specific induction TNBC deoxyribose nucleic acid probe with the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 4

In certain embodiments, the mutation is the addition of 10T bases and a biotin molecule at the 5' end of the deoxyribose nucleic acid probe. In certain embodiments, the mutation is a mutation of base rA in the deoxyribose nucleic acid probe to an a base. And modifying the probe mutant on the surface of the magnetic bead by utilizing the strong affinity between biotin and streptavidin to obtain the magnetic bead probe. Mutations of the probe include: the 5 'end is prolonged by 10T bases, and two biotin molecules (biotins) and rA bases are modified at the 5' end to be mutated into A bases. Wherein the first two mutations ensure that the probe mutant can be firmly modified on the streptavidin magnetic beads. Mutation of the rA base to the A base renders the probe incapable of cleavage, retaining only the ability to interact with the target molecule. The mutation can not only achieve the purpose of enriching target protein, but also greatly reduce the cost of probe synthesis.

In certain embodiments, the substitution strand is a single strand of DNA that is capable of being complementary to the 3' end of the mutation probe. The displacement strand is a segment of 71nt DNA single chain and is completely complementary and paired with 71nt base at the 3' end of the deoxyribozyme probe mutant, so that the interaction between the deoxyribozyme probe mutant and the molecular marker can be destroyed, and the molecular marker is displaced from the deoxyribozyme probe mutant, thereby achieving the aim of eluting the target protein.

In certain embodiments, the method further comprises the step of identifying the protein of interest after eluting the protein.

Compared with the prior art, the application has the following technical effects:

1) The application relies on the extremely high sensitivity and good specificity of the deoxyribozyme probe, uses the AKTApure system and the magnetic bead probe to carry out specific purification, enrichment and elution on target proteins in EMs, uses the deoxyribozyme probe to monitor the steps in real time, and successfully identifies the target proteins interacted with the deoxyribozyme probe as human apolipoprotein A. In the process of identifying the target protein, the method of probe enrichment and probe detection is adopted in the whole process, so that the apolipoprotein A is a component special for the MDA-MB-231 EMs.

2) The application uses the deoxyribozyme probe as a 'gripper', and uses the means of AKTApure system purification and concentration, magnetic bead probe specific enrichment, capturing, elution, mass spectrometry and the like to successfully identify the target protein interacted with the deoxyribozyme probe as the apolipoprotein A.

3) The method is not only suitable for researching TNBC on the cellular level, but also can popularize a researched object into clinical samples such as tissues, body fluids and the like. Furthermore, for any other disease model with unknown markers, the markers can be identified simply and rapidly by using the method of the application. Therefore, aiming at the disease model of unknown target molecules, the application provides a novel method for simply and rapidly determining molecular markers by taking a deoxyribozyme probe as a 'gripper'.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1 shows the EMs of the AKTApure system for the isolation of MDA-MB-231, wherein (a) is a plot of the cell supernatant analysis for the collection of different fractions F1-F8; FIG. 1b is a dPAGE gel of different components after purification of the 4# probe detection AKTApure system, wherein C is a negative control, W is a flow-through solution, 1-8 correspond to different components F1-F8 respectively, and P is a positive control.

FIG. 2a is a schematic flow chart of a modified magnetic bead (i.e., magnetic bead probe) of a 4# probe mutant for enriching a target protein in an F6 component; FIG. 2b is a graph of a cleavage gel for detecting the target protein by a bead method, wherein C is a negative control, 1 is a supernatant obtained by magnetic separation after the target protein is enriched by the bead probe, 2 is a supernatant obtained by washing the bead probe twice, 3 is a solution obtained by eluting the target protein by a displacement strand, 4 is a three-time target protein eluent concentrated 10 times, and P is a positive control; FIG. 2c shows the result of SDS-PAGE to verify that the magnetic bead probe can enrich and elute target protein, M is a protein gradient ruler, 1 is a component F6 before magnetic bead enrichment, and 2 is a target protein concentrated solution obtained after magnetic bead probe enrichment and elution.

FIG. 3 is a graph of the identification result of a target protein, wherein FIG. 3a is the result of mass spectrometry; FIG. 3b is a Western Blot validation of the target protein apolipoprotein A, wherein M is a protein gradient ruler, 1 is apolipoprotein A commercial protein, 2 is a 10-fold concentrated F6 fraction; FIG. 3C is a graph of the gel of the probe detection of EMs over-expressing apolipoprotein A, wherein P is a positive control, C1 is a negative control, A is the EMs over-expressing apolipoprotein A by HEK293T cells, four replicates, M is the EMs of HEK293T cells transfected with empty plasmid, two replicates, L is the EMs of HEK293T cells containing only transfection reagent, two replicates, C2 is the EMs of untreated HEK 293T; FIG. 3d is a graph of a detection gel of the co-incubation of the 4# probe with apolipoprotein A at different dilution factors, wherein C is a negative control, P is a positive control, and 1-100 fold corresponds to apolipoprotein A at different dilution factors.

FIG. 4 shows the results of the verification of the function of apolipoprotein A. FIG. 4a is a Western Blot and fluorescent quantitative PCR method demonstrating successful construction of an apolipoprotein A overexpressing and knockdown cell line; FIG. 4b is a graph showing the effect of over-expression and knock-down of apolipoprotein A on TNBC cell proliferation phenotype using the CCK8 method; FIGS. 4c and 4d are graphs showing the effect of transwell assay on TNBC cell migration phenotype of knockdown and over-expression of apolipoprotein A.

Detailed Description

Embodiments of the technical scheme of the present application will be described in detail below. The following examples are only for more clearly illustrating the technical aspects of the present application, and thus are merely examples, which should not be construed as limiting the scope of the present application. It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this application belongs.

Example 1 determination of the type of target molecule

The 4# and 6# probes were dissolved in 10. Mu.L of 2 Xbuffer 1 and 20. Mu.L of buffer 2 at a probe concentration of 1. Mu.M, heated in a metal bath at 70℃for 5min, and returned to room temperature. The probe solutions described above were divided into two groups: one set of 5. Mu.L of EMs of untreated MDA-MB-231 and one set of 5. Mu.L of EMs of proteinase K or heat (75 ℃ C., 15 min) treated MDA-MB-231 were incubated at room temperature for 30min, and 8% dPAGE characterizes cleavage signals of the 4# and 6# probes. The results show that proteinase K and heat treated EMs failed to trigger cleavage of the 4# and 6# probes; the nature of the target molecule that indicates specific interaction with the deoxyribose nucleic acid probe is a protein, i.e., the target protein.

EXAMPLE 2 AKTApure System separation of EMs concentrating MDA-MB-231

In this example, 8 flasks of MDA-MB-231 were incubated with T175 flasks, and 50mL of EMs were collected per flask, for a total of 400mL. 400mL of EMs were concentrated to 30mL using a 15mL loading, 10KD pore size, ultrafiltration tube at 4000rpm at 4℃for 20min. The concentrated EMs were diluted to 100mL with buffer A and filtered through a 0.22 μm membrane to give an AKTApure system loading solution. A HiTrap Heparin Sepharose FF chromatographic column was mounted, and pump A was purged with buffer A, pump B was purged with buffer B, and the flow rate of the chromatographic column was 20mL/min. The AKTApure system separation and concentration mainly comprises four steps of loading, cleaning, gradient elution and component collection. Loading: when the detection value of UV280 (ultraviolet absorption detection) is close to 0 and tends to be stable, inserting a hose connected with the buffer solution A into the sample solution, enabling the sample solution to be purified and separated to flow through a chromatographic column, setting the flow rate to be 5mL/min, and setting the pre-column pressure to be 0.5MPa; as loading proceeds, the UV280 detection value increases, indicating adsorption of protein on the column. Cleaning: the column was washed with about 40mL of buffer A until the UV280 detection value became 0, and the component having a weak binding ability to hepatin, i.e., the flow-through (W), was washed off, and the flow rate was set at 5mL/min and the column front pressure was 0.5MPa. Gradient elution: gradient elution is carried out on the protein adsorbed on the chromatographic column by using a buffer B, and the salt concentration of the solution in the chromatographic column gradually increases along with the increase of the buffer B, which is shown by the increase of a conductivity value (Cond); thus, at different salt concentrations, different proteins are eluted. And (3) collecting components: when the Cond value starts to increase, the eluate is automatically collected in centrifuge tubes, 2mL per tube, until the solution concentration in the column reaches 1M (i.e. 100% buffer B). In this example 27 tubes were collected together, as in fig. 1a.

The collected 27-tube eluate was separated into different fractions, in this example into 8 fractions, i.e., F1-F8. The flow-through W and F1-F8 fractions were concentrated 10-fold using a 15mL ultrafiltration tube with a pore size of 10K. The probe No. 4 was dissolved in 55. Mu.L of 2 Xbuffer 1 and 110. Mu.L of buffer 2 at a probe concentration of 1. Mu.M, and the mixture was heated in a metal bath at 70℃for 5 minutes, and then returned to room temperature.

The solution of the 4# deoxyribose nucleic acid probe (5 '-GTAGCCTTCGCAT-R-TGAGACATCGCAACCGTGACGCAGGTTGCGATGTCATAATAGCGGAGGTAAAGCGTGATGCCATACGACACTGCATAGGTTGGGCGCGAAGGCTACATCACGCTAC-3' wherein R represents a single inserted RNA base A (rA))) (SEQ ID NO: 1)) was aliquoted into 11 parts, 2 of which were each associated with 5. Mu.L of ddH ₂ O was co-incubated with EMs of unpurified MDA-MB-231 as negative and positive controls; the remaining 9 parts were incubated with 5. Mu.L of the concentrated W, F1-F8 fractions, respectively. After 1h of reaction at room temperature, the amount of cleavage signal of the deoxynucleic acid probe was characterized by 8% dPAGE to determine the amount of the target protein in a certain fraction, as shown in FIG. 1 b. In this example, the F6 component triggered the strongest cleavage signal of the probe, and thus the target protein in the F6 component was subsequently eluted by enrichment with the magnetic bead probe.

In this example, the EMs of MDA-MB-231 were purified and concentrated using the AKTApure system, and separated into different fractions. The AKTApure system is a set of flexible and controllable liquid chromatography system, and can be used for rapidly purifying target substances such as proteins, polypeptides, nucleic acids and the like at the level from micrograms to grams. Different chromatographic columns are selected, and the mixture can be divided into different components according to different properties (such as size, electronegativity, affinity and the like). In this example, hiTrap Heparin Sepharose FF chromatographic column (heparin agarose pre-packed column) was used to separate the EMs of MDA-MB-231 into different components according to their affinity to heparin, as shown in FIG. 1a. Incubating the 4# probe with different components respectively, judging whether a certain component contains or contains target proteins which can interact with the deoxyribozyme probe according to the presence or the quantity of cleavage signals in the dPAGE gel, and selecting a certain component with the strongest cleavage signals for subsequent purification and enrichment as shown in figure 1 b. It is worth mentioning that the AKTApure system also plays a role in concentration, and can concentrate hundreds of milliliters to liters of EMs to a component of milliliters, thereby facilitating the subsequent enrichment of target proteins with extremely low abundance.

EXAMPLE 3 enrichment and elution of target proteins by magnetic bead probes

The magnetic beads used in this example were modified with streptavidin to a size of 1 μm and a concentration of 10mg/mL, and the free biotin binding amount was 2500pmol. The steps of enriching the magnetic bead probe and eluting the target protein mainly comprise the steps of modifying the 4# probe mutant on the magnetic bead, incubating the magnetic bead probe and the F6 component together, cleaning the magnetic bead probe, replacing a strand to elute the target protein, and the like, as shown in fig. 2a. Step 1: 100. Mu.L of magnetic beads were washed 3 times with 200. Mu.L of binding/washing buffer (B & W buffer), and after magnetic separation 500. Mu.L of binding/washing buffer and 100pmol of the No. 4 probe mutant were added, incubated at room temperature and gently spun for 30min. After magnetic separation, the supernatant was discarded, and 500. Mu.L of binding/washing buffer was added for washing 2 times to wash away unbound 4# probe mutants, thus obtaining 4# probe mutant modified magnetic beads, abbreviated as magnetic bead probes. Step 2: the magnetic bead probe was washed 3 times with 500. Mu.L of reaction buffer, the washing liquid was removed after magnetic separation, 500. Mu.L of reaction buffer and 30. Mu.LF 6 fraction were added, incubated at room temperature and gently rotated for 30min. After magnetic separation, the co-incubation supernatant was retained. Step 3: the magnetic bead probe was washed 2 times by adding 500. Mu.L of reaction buffer, and the washing liquid was retained twice after magnetic separation. Step 4: mu.L of reaction buffer and 120pmol of displaced strand were added, incubated at room temperature and gently spun for 30min. After magnetic separation, the eluent was retained. In order to obtain more target proteins at one time, the steps 2-4 can be repeated twice, namely, the component F6 is subjected to three times of enrichment and elution. And finally, mixing the three target protein eluents, and concentrating the three target protein eluents by using an ultrafiltration tube with the loading capacity of 1.5mL and the aperture of 10K for 10 times to obtain the concentrated target protein eluent.

The 4# probe mutant sequence is as follows, underlined bases are the mutant region:

5′-Biotin-TT-Biotin-TTTTTTTTGATGTAGCCTTCGCAT-A-TGAGACATCGCAACCGTGACGCAGGTTGCGATGTCATAATAGCGGAGGTAAAGCGTGATGCCATACGACACTGCATAGGTTGGGCGCGAAGGCTACATCACGCTAC-3′(SEQ ID NO:2)

the substitution strand sequence is as follows:

5′-GTAGCGTGATGTAGCCTTCGCGCCCAACCTATGCAGTGTCGTATGGCATCACGCTTTACCTCCGCTATTAT-3′(SEQ ID NO:3)

the 6# probe was dissolved in 75. Mu.L of 2 Xbuffer 1 and 150. Mu.L of buffer 2 at a probe concentration of 1. Mu.M, heated in a metal bath at 70℃for 5min, and returned to room temperature. A solution of 6# deoxyribozyme probe (5 '-GTAGCCTTCGCAT-R-TGAGACATCGCAACCGTGACGCAGGTTGCGATGTCATAATAGCGGAGAGCTGCGGAGATGTATGCCGGGTCGAACGTGTGGCGGACGAAGGCTACATCACGCTAC-3', wherein R represents a single inserted RNA base A (rA) (SEQ ID NO: 4)) was divided equally into 15 parts, 2 of which were respectively mixed with 5. Mu.L of ddH ₂ O was co-incubated with EMs of unpurified MDA-MB-231 as negative and positive controls; the remaining 13 parts were incubated with 5. Mu.L of the above-mentioned magnetic bead probe, respectively, of the supernatant (3 times), of the washing solution (3 times. 2 times), of the target protein eluent (3 times) and of the concentrated target protein eluent. The reaction was stopped after 1h at room temperature, and the amount of cleavage signal of 6# probe was characterized by 8% dPAGE to determine whether the magnetic bead probe successfully enriched and eluted the target protein, as shown in FIG. 2 b. In this example, after three times of enrichment and elution of the magnetic bead probe, the target protein was successfully enriched: the concentrated target protein eluent can trigger the obvious cleavage of the 6# probe.

The eluted target protein concentrate was enriched with the F6 fraction and the magnetic bead probe by 12% SDS-PAGE. As shown in FIG. 2c, lane 1 is an F6 fraction comprising multiple protein bands; lane 2 is the eluted target protein concentrate, with a single protein band around 40 kD. Under the condition of equal loading amount, the F6 component can not see the protein with about 40kD, which indicates that the magnetic bead probe can effectively enrich the target protein with low abundance. In addition, the target protein is obtained by displacement strand elution after binding to the 4# probe mutant, and is specifically enriched elution, i.e., the target protein is a component specific to the EMs of MDA-MD-231.

Example 4 identification of target proteins

Mass spectrometry analysis of the target protein yielded candidate proteins, as shown in figure 3a. The identity of the apolipoprotein A was verified by Western Blot, over-expressed and purified, respectively.

First, the F6 fraction was concentrated 20-fold with a ultrafiltration tube having a pore size of 10K and a loading of 1.5 mL. mu.L of the concentrated F6 fraction and 2. Mu.L of recombinant human apolipoprotein A (commercial) were loaded and run on 12% SDS-PAGE. After membrane transfer, sealing, primary antibody incubation, secondary antibody incubation and chemiluminescence development, western Blot characterization is obtained. As shown in FIG. 3b, lane 1 is commercial recombinant human apolipoprotein A and lane 2 is the F6 fraction after concentration; the results indicate that the F6 component contains apolipoprotein A.

Next, a recombinant expression plasmid containing His tag and apolipoprotein a gene was constructed. HEK293T cells were cultured, plated in 6 well plates, approximately 6X 10 per well ⁵ Individual cells (50-60% confluence). Reference to the specification, lipofectamine ^TM Reagents 3000 recombinant plasmids were transfected into HEK293T cells, i.e. over-expression group (group a), and control group (group M, L, C) was set, as specified in the table below. After 36h of transfection, the medium was replaced with serum-free DMEM medium, and after 24h the cell supernatants of each group, i.e. the EMs of each group, were collected.

The probe No. 4 was dissolved in 60. Mu.L of 2 Xbuffer 1 and 120. Mu.L of buffer 2 at a probe concentration of 1. Mu.M, and the mixture was heated in a metal bath at 70℃for 5 minutes, and then returned to room temperature. The above 4# probe solution was equally divided into 12 parts, 2 of which were respectively mixed with 5. Mu.L of ddH ₂ O was co-incubated with EMs of unpurified MDA-MB-231 as negative and positive controls; the remaining 10 parts were incubated with 5 μl of EMs of group a, group M, group L and group C, respectively. After 1h reaction at room temperature, the cleavage signal of the # 4 probe was characterized by 8% dPAGE. As shown in fig. 3C, only the EMs of group a could trigger cleavage of the 4# probe, while none of group M, group L and group C had a distinct cleavage signal.

Further, the present example uses Ni-NTA agarose resin to purify the over-expressed, his-tagged human apolipoprotein A. The probe No. 4 was dissolved in 140. Mu.L of buffer 1 and 140. Mu.L of buffer 2 at a probe concentration of 1. Mu.M, and the mixture was heated in a metal bath at 70℃for 5 minutes, and then returned to room temperature. The above 4# probe solution was equally divided into 14 parts, 2 of which were respectively mixed with 2. Mu.L of ddH ₂ O was co-incubated with EMs of unpurified MDA-MB-231 as negative and positive controls; the remaining 12 parts were incubated with 2. Mu.L of apolipoprotein A at different dilutions, respectively. After 1h of reaction at room temperatureThe cleavage signal of the 4# probe was characterized by 8% dPAGE. As shown in fig. 3d, the cleavage signal of the 4# probe gradually decreased with increasing dilution, indicating that the cleavage signal of the 4# probe is proportional to the concentration of apolipoprotein a. In conclusion, apolipoprotein a is the target protein specifically sensed by the deoxyribose nucleic acid probe.

EXAMPLE 5 construction of Apolipoprotein A knockdown and TNBC over-expression cell lines

1. Knockdown cell line construction: after digestion, the cells were plated in six well plates with a cell number of 0.25X10 ⁶ The medium used was 2mL antibiotic-free DMEM medium (containing 10% fetal bovine serum) per well. Adding 10 mu L of siRNA into 120 mu L of opti-MEM, and uniformly mixing; another 12. Mu.L lipomax was added to 120. Mu.L opti-MEM, and after mixing well, the mixture was added to siRNA diluent, and after stirring well, it was allowed to stand for 15 minutes. The mixture was added dropwise to 6-well plate cells, and mixed well cross-wise. After 8 hours, the cell state was observed and the solution was changed. After 36 hours of intervention, RNA of the cells was extracted, gDNA was removed, and after reverse transcription, the expression level of apolipoprotein A mRNA was detected by RT-PCR, thereby verifying the knockdown efficiency of siRNA. Primer sequences for RT-PCR were as follows:

forward primer: 5'-GCAAGGACAGAGGTTCAGGAT-3' (SEQ ID NO: 5)

Reverse primer: 5'-AGTCCTCTGTGGCAGCAAAT-3' (SEQ ID NO: 6).

2. And (3) construction of an overexpression line: after digesting the cells, they were spread in 6cm dishes with a cell number of 0.6X10 ⁶ The medium used was 4mL DMEM whole medium (containing 10% fetal bovine serum and penicillin, streptomycin) per dish. After 1 day, cells were attached, washed twice with PBS buffer, and 4mL of antibiotic-free medium was added. Adding 4 μg of plasmid into 250 μl of opti-MEM, and mixing well; 10. Mu.L of lipo2000 was added to 250. Mu.L of opti-MEM, and after mixing, the mixture was added with a plasmid diluent, and after stirring, the mixture was allowed to stand for 15 minutes. Subsequently, the mixture was added dropwise to the cells, and the cross was mixed well. After 8 hours, the cell state was observed and the solution was changed. After 36 hours of intervention, cellular proteins were extracted, and after concentration was detected, the expression level of apolipoprotein A was detected by Western Blot.

The results are shown in FIG. 4a, and Western Blot and fluorescent quantitative PCR methods verify successful construction of apolipoprotein A over-expressed and knocked down cell lines.

Example 6 ability to detect proliferation of cells

After 36 hours of intervention, cells were digested with pancreatin and plated in 96-well plates at 2500 cells/well using 0.2mL DMEM whole medium (containing 10% fetal bovine serum and penicillin, streptomycin). After 24 hours, the medium was poured out, 10. Mu.L of CCK-8 reagent and 90. Mu.L of DMEM whole medium were added to each well, and after placing in the incubator for 90 minutes, the absorbance of each well at 450nm was measured with a microplate reader as data on day 1. Absorbance was measured in the same manner on days 2, 3 and 4, and a corresponding growth curve was made based on the results.

Example 7 ability to detect cell migration

After 36 hours of intervention, cells were digested with pancreatin, centrifuged to discard the supernatant, washed twice with PBS, resuspended in serum-free DMEM medium and the cell number was controlled at 0.25X10 ⁶ And each milliliter. 600. Mu.L of DMEM whole medium (containing 10% fetal calf serum and penicillin, streptomycin) was added to the lower chamber. The cells were wetted with serum-free DMEM medium and subsequently placed in culture plates. 200. Mu.L of the cell suspension was added to the upper chamber. After 8 hours, the Transwell chamber was removed, the medium in the wells was discarded, washed twice with calcium-free PBS, fixed with formaldehyde for 30min, air-dried, stained with 0.1% crystal violet, and cells were observed under a microscope and counted.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application, and are intended to be included within the scope of the appended claims and description.

SEQUENCE LISTING

<110> Hospital for affiliated tumors at university of double denier

<120> a method for enriching and identifying molecular markers in a sample to be tested

<130> 2021

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 120

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<221> RNA base A (rA)

<222> (14)..(14)

<223> r represents RNA base A (rA)

<400> 1

gtagccttcg catrtgagac atcgcaaccg tgacgcaggt tgcgatgtca taatagcgga 60

ggtaaagcgt gatgccatac gacactgcat aggttgggcg cgaaggctac atcacgctac 120

<210> 2

<211> 131

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

ttttttttga tgtagccttc gcatatgaga catcgcaacc gtgacgcagg ttgcgatgtc 60

ataatagcgg aggtaaagcg tgatgccata cgacactgca taggttgggc gcgaaggcta 120

catcacgcta c 131

<210> 3

<211> 71

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

gtagcgtgat gtagccttcg cgcccaacct atgcagtgtc gtatggcatc acgctttacc 60

tccgctatta t 71

<210> 4

<211> 119

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<221> RNA base A (rA)

<222> (1)..(14)

<223> r represents RNA base A (rA)

<400> 4

gtagccttcg catrtgagac atcgcaaccg tgacgcaggt tgcgatgtca taatagcgga 60

gagctgcgga gatgtatgcc gggtcgaacg tgtggcggac gaaggctaca tcacgctac 119

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

gcaaggacag aggttcagga t 21

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

agtcctctgt ggcagcaaat 20

Claims (5)

1. A method for identifying molecular markers in a sample to be detected by using a deoxyribozyme probe is characterized in that the sample to be detected is extracellular metabolites (extracellular mixtures, EMs) of a TNBC cell line MDA-MB-231,

step a, detecting proteinase K and the extracellular metabolite after heating treatment by a deoxyribozyme probe, and determining the molecular type of a molecular marker;

step b, concentrating and purifying extracellular metabolites to be screened by using an AKTApure system, and separating the extracellular metabolites into different components;

step c, mutating a deoxyribozyme probe capable of recognizing the molecular marker to obtain a mutated deoxyribozyme probe, wherein the deoxyribozyme probe is a deoxyribozyme probe for specifically sensing TNBC, the sequence of the deoxyribozyme probe is shown as SEQ ID NO. 1 or SEQ ID NO. 4, 10T bases and biotin molecules are added at the 5' end of the deoxyribozyme probe, and a base rA in the deoxyribozyme probe is mutated into an A base;

step d, modifying the mutated deoxyribozyme probe on the surface of the magnetic bead to obtain the magnetic bead probe;

step e, incubating the magnetic bead probes with different components respectively, performing specific capture and enrichment on molecular markers, and then performing magnetic separation and washing to obtain washed incubation solution;

and f, adding a substitution strand into the washed incubation solution, and eluting the molecular marker, wherein the substitution strand is a DNA single strand which can be complementary with the 3' -end of the mutation probe.

2. The method of claim 1, further comprising, prior to step e, assessing whether each component of the extracellular metabolite contains a molecular marker.

3. The method according to claim 2, wherein the molecular marker is evaluated by using the deoxyribose nucleic acid probe to perform mixed incubation with each component, and selecting one or more groups with strongest cleavage signals of the deoxyribose nucleic acid probe for subsequent specific capture and enrichment.

4. The method of claim 1, wherein the molecular markers are nucleic acids, proteins, small molecule compounds.

5. The method of claim 1, wherein the molecular marker is apolipoprotein a.