CN113092209B - Method for enriching and identifying molecular markers in disease sample to be detected - Google Patents
Method for enriching and identifying molecular markers in disease sample to be detected Download PDFInfo
- Publication number
- CN113092209B CN113092209B CN202110361337.5A CN202110361337A CN113092209B CN 113092209 B CN113092209 B CN 113092209B CN 202110361337 A CN202110361337 A CN 202110361337A CN 113092209 B CN113092209 B CN 113092209B
- Authority
- CN
- China
- Prior art keywords
- probe
- deoxyribozyme
- molecular markers
- markers
- molecular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 31
- 201000010099 disease Diseases 0.000 title abstract description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title abstract description 10
- 239000000523 sample Substances 0.000 claims abstract description 119
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 66
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 65
- 108091027757 Deoxyribozyme Proteins 0.000 claims abstract description 37
- 238000007885 magnetic separation Methods 0.000 claims abstract description 11
- 230000000295 complement effect Effects 0.000 claims abstract description 5
- 239000011324 bead Substances 0.000 claims description 36
- 102000007592 Apolipoproteins Human genes 0.000 claims description 28
- 108010071619 Apolipoproteins Proteins 0.000 claims description 28
- 108020004414 DNA Proteins 0.000 claims description 23
- 238000003776 cleavage reaction Methods 0.000 claims description 19
- 230000007017 scission Effects 0.000 claims description 19
- 102000053602 DNA Human genes 0.000 claims description 14
- 239000003147 molecular marker Substances 0.000 claims description 14
- 239000002207 metabolite Substances 0.000 claims description 13
- 239000002853 nucleic acid probe Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 10
- 238000011534 incubation Methods 0.000 claims description 9
- 230000035772 mutation Effects 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 7
- 235000020958 biotin Nutrition 0.000 claims description 6
- 108010067770 Endopeptidase K Proteins 0.000 claims description 5
- 229960002685 biotin Drugs 0.000 claims description 5
- 239000011616 biotin Substances 0.000 claims description 5
- 238000006467 substitution reaction Methods 0.000 claims description 5
- 102000039446 nucleic acids Human genes 0.000 claims description 4
- 108020004707 nucleic acids Proteins 0.000 claims description 4
- 150000007523 nucleic acids Chemical class 0.000 claims description 4
- 238000011282 treatment Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 3
- -1 small molecule compounds Chemical class 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 10
- 238000006073 displacement reaction Methods 0.000 abstract description 6
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 230000003993 interaction Effects 0.000 abstract description 4
- 208000022679 triple-negative breast carcinoma Diseases 0.000 abstract description 4
- 239000003550 marker Substances 0.000 abstract description 2
- 238000001742 protein purification Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 18
- 239000000872 buffer Substances 0.000 description 15
- 239000002609 medium Substances 0.000 description 14
- 238000010828 elution Methods 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 239000003480 eluent Substances 0.000 description 8
- 238000011068 loading method Methods 0.000 description 8
- 239000013642 negative control Substances 0.000 description 8
- 239000013641 positive control Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000002156 mixing Methods 0.000 description 7
- 238000003197 gene knockdown Methods 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 238000010276 construction Methods 0.000 description 5
- 239000002184 metal Substances 0.000 description 5
- 230000002018 overexpression Effects 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 239000012124 Opti-MEM Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 229960002897 heparin Drugs 0.000 description 4
- 229920000669 heparin Polymers 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000011535 reaction buffer Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- 206010006187 Breast cancer Diseases 0.000 description 3
- 208000026310 Breast neoplasm Diseases 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 108020004459 Small interfering RNA Proteins 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- 239000012148 binding buffer Substances 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000012292 cell migration Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000013076 target substance Substances 0.000 description 2
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 241000193163 Clostridioides difficile Species 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 101000851176 Homo sapiens Pro-epidermal growth factor Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000001615 biotins Chemical class 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 238000009261 endocrine therapy Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 102000015694 estrogen receptors Human genes 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000000583 progesterone congener Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000011127 radiochemotherapy Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
-
- 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
-
- 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
- G01N2333/775—Apolipopeptides
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
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110361337.5A CN113092209B (en) | 2021-04-02 | 2021-04-02 | Method for enriching and identifying molecular markers in disease sample to be detected |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110361337.5A CN113092209B (en) | 2021-04-02 | 2021-04-02 | Method for enriching and identifying molecular markers in disease sample to be detected |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113092209A CN113092209A (en) | 2021-07-09 |
| CN113092209B true CN113092209B (en) | 2023-10-27 |
Family
ID=76673865
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110361337.5A Active CN113092209B (en) | 2021-04-02 | 2021-04-02 | Method for enriching and identifying molecular markers in disease sample to be detected |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113092209B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102203291A (en) * | 2008-08-28 | 2011-09-28 | 肿瘤疗法科学股份有限公司 | Tbc1d7 as tumor marker and therapeutic target for cancer |
| CN103675294A (en) * | 2013-11-26 | 2014-03-26 | 北京大学 | Novel tumour serum marker and application thereof |
| WO2015084800A1 (en) * | 2013-12-02 | 2015-06-11 | The General Hospital Corporation | Nano-plasmonic sensor for exosome detection |
| WO2018001258A1 (en) * | 2016-06-30 | 2018-01-04 | 厦门艾德生物医药科技股份有限公司 | Probe for nucleic acid enrichment and capture, and design method thereof |
| CN112159835A (en) * | 2020-09-30 | 2021-01-01 | 北京吉检医疗科技有限公司 | Method for capturing and enriching nucleic acid through probe |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140273007A1 (en) * | 2013-03-12 | 2014-09-18 | Dr. Susan Love Research Foundation | Devices and methods for screening nipple aspirate for markers indicative of risk of developing breast cancer |
| US20090117549A1 (en) * | 2006-07-18 | 2009-05-07 | Weihong Tan | Aptamer-based methods for identifying cellular biomarkers |
| JP6286358B2 (en) * | 2011-11-11 | 2018-02-28 | ミレニアム ファーマシューティカルズ， インコーポレイテッドＭｉｌｌｅｎｎｉｕｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． | Biomarkers that respond to proteasome inhibitors |
| DK3201630T3 (en) * | 2014-09-29 | 2019-02-25 | Ventana Med Syst Inc | Multiplex assay for simultaneous detection of TP53 / CEN17 / B cell gene protein and uniquely specific probes for 19Q12, INSR, ATM, DLEU2, TP53 and 13Q12 |
| EP3529374B1 (en) * | 2016-10-21 | 2024-04-03 | Exosome Diagnostics, Inc. | Sequencing and analysis of exosome associated nucleic acids |
-
2021
- 2021-04-02 CN CN202110361337.5A patent/CN113092209B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102203291A (en) * | 2008-08-28 | 2011-09-28 | 肿瘤疗法科学股份有限公司 | Tbc1d7 as tumor marker and therapeutic target for cancer |
| CN103675294A (en) * | 2013-11-26 | 2014-03-26 | 北京大学 | Novel tumour serum marker and application thereof |
| WO2015084800A1 (en) * | 2013-12-02 | 2015-06-11 | The General Hospital Corporation | Nano-plasmonic sensor for exosome detection |
| WO2018001258A1 (en) * | 2016-06-30 | 2018-01-04 | 厦门艾德生物医药科技股份有限公司 | Probe for nucleic acid enrichment and capture, and design method thereof |
| CN112159835A (en) * | 2020-09-30 | 2021-01-01 | 北京吉检医疗科技有限公司 | Method for capturing and enriching nucleic acid through probe |
Non-Patent Citations (4)
| Title |
|---|
| DNA 酶: 筛选、生物传感及展望;佟宗轩 等;高等学校化学学报;第41卷(第11期);第2345-2354页 * |
| Production of single-stranded DNAs by self-cleavage of rolling-circle amplification products;Hongzhou Gu 等;BioTechniques;第54卷(第6期);第337-343页 * |
| Recent advances of DNAzyme-based nanotherapeutic platform in cancer gene therapy;Wendi Huo 等;Biophysics Reports;第6卷(第6期);第256–265页 * |
| 核酸探针及其在生物分析中的应用;张园 等;分析试验室(第9期);第16-26页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113092209A (en) | 2021-07-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xiong et al. | Cancer protein biomarker discovery based on nucleic acid aptamers | |
| Freund et al. | Unbiased identification of trans regulators of ADAR and A-to-I RNA editing | |
| KR101189790B1 (en) | Nucleic Acid Aptamer Capable of Specifically Binding to Pancreatic Cancer Cell or Tissue and Use Thereof | |
| KR20210093266A (en) | Method for Simultaneous Detection of Exosome Membrane Protein and mRNA | |
| Guo et al. | Discussion of the protein characterization techniques used in the identification of membrane protein targets corresponding to tumor cell aptamers | |
| CN102839181B (en) | Nucleic acid aptamer sequence of gastric cancer cells, and uses thereof | |
| CN108872438B (en) | Rapid detection kit for lung cancer marker GK5 in exosome | |
| CN109504775B (en) | Rapid detection kit for exosome lung tumor marker LSM2 | |
| Wei et al. | PDLIM5 identified by label-free quantitative proteomics as a potential novel biomarker of papillary thyroid carcinoma | |
| CN107794268A (en) | Detect the aptamer of people's PDL1 albumen and its prepare the application in detecting preparation | |
| CN109837281B (en) | Aptamer specifically binding to S100P protein and screening, identification and application thereof | |
| CN105755005A (en) | Aptamer for osteosarcoma detection and kit for osteosarcoma detection | |
| CN113092209B (en) | Method for enriching and identifying molecular markers in disease sample to be detected | |
| EP3123175A1 (en) | Method for identifying a biomarker indicative of a reduced drug response using a thermal shift assay | |
| EP3035058B1 (en) | Cancer marker screening method through detection of deglycosylation of glycoprotein and hepatocellular cancer marker | |
| CN114317544B (en) | Aptamer specifically binding to CD133, screening method and application thereof | |
| CN110511935B (en) | Aptamer truncation optimization method based on S1 enzyme cleavage | |
| CN111394458A (en) | Application of PTBP1 as marker for diagnosing and treating osteosarcoma chemotherapy resistance | |
| CN114457040B (en) | RSPO1 activity detection stable transgenic cell strain and construction method and application thereof | |
| CN105288659A (en) | Application of TENM1 gene and its expression product on diagnosis and treatment of papillary adenocarcinoma | |
| CN108660216B (en) | Kit and its detection method based on peripheral blood free nucleotide miRNAs super sensitivity detection device | |
| KR102320436B1 (en) | Biomarker composition for diagnosis of glioblastoma comprising glioblastoma stem cell exosome-derived peptide | |
| Li et al. | CircCDK1 blocking IGF2BP2-mediated m6A modification of CPPED1 promotes laryngeal squamous cell carcinoma metastasis via the PI3K/AKT signal pathway | |
| CN113564172A (en) | Liquid biopsy tumor cell DNA aptamer | |
| CN112322719A (en) | Application of circulating blood exosome miRNA as retinal vein occlusion disease diagnosis marker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |