CN105823894A - Kit for specific detection of endometrial cancer - Google Patents
Kit for specific detection of endometrial cancer Download PDFInfo
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- CN105823894A CN105823894A CN201610369948.3A CN201610369948A CN105823894A CN 105823894 A CN105823894 A CN 105823894A CN 201610369948 A CN201610369948 A CN 201610369948A CN 105823894 A CN105823894 A CN 105823894A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57442—Specifically defined cancers of the uterus and endometrial
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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Abstract
The invention discloses a kit for specifically detecting an endometrial cancer .Aptamer has good affinity with human Cyclophilin A protein .By means of the aptamer, Cyclophilin A protein in the blood can be trapped, the endometrial cancer is detected through changes of the content of the aptamer, and the aptamer is prepared into the corresponding kit to be used for endometrial cancer screening .The kit has the advantages of being high in sensitivity and low in cost.
Description
Technical field
The present invention relates to a kind of test kit for detecting carcinoma of endometrium and detection method thereof.
Background technology
Carcinoma of endometrium, as one of big malignant tumor of female genital tract three, accounts for the 20-30% of female genital tract malignant tumor, has occupied female genital tract malignant tumor in some American-European countries the first, serious threat WomanHealth.Along with social development and the prolongation of average human life, endometrial carcinomas sickness rate rises and year by year with rejuvenation trend.If carcinoma of endometrium can accomplish early diagnosis, rational therapy, its prognosis is preferable.The main clinical manifestation of carcinoma of endometrium is irregular colporrhagia, and different from other tumors such as ovarian cancers, clinical diagnosis relies primarily on diagnostic curettage, but age of sucking or the patient of infiltrative type carcinoma of endometrium, its Uterus wall is weak, is likely to result in perforation, damages to patient during dilatation and curettage of uterine.
CyclophilinA albumen (CYPA), also known as cyclophilin A (GeneBankNo.NP_066953), is positioned at kytoplasm and karyon, has peptide acyl prolyl cis-trans isomerase activity, participates in protein folding, assembles and transport；Be combined with ciclosporin A intracellular, suppress T cell activation approach, show immunosuppressive action, additionally participate in oxidative stress induction and cholesterol metabolism process, and play the function of cytokine.Prior art is reported that carrys out early diagnosis carcinoma of endometrium by detection human Cyclophilin A expression.Therefore, detection human Cyclophilin A becomes particularly important.
Aptamer (Aptamer, also known as aptamers, aptamer) is strand widow's nucleic acid molecules (ssDNA or ssRNA) of certain biomass of combination of energy high-affinity, high specific.Aptamer be screened from the DNA/RNA library of synthetic by index concentration Fas lignand system evolution technology (Systemat1cEvolut1onofL1gandsbyExponent1alenr1chment, SELEX) obtain can combine the single stranded DNA/RNA of target molecules by high degree of specificity.Report that the target of aptamer includes metal ion, organic molecule, polypeptide, protein, cell even tissue etc..The molecular recognition function of aptamer is similar with antibody, has the target identification ability the most higher with antibody molecule.
Summary of the invention
It is an object of the invention to provide aptamer and the test kit thereof of a kind of specific bond CYPA.
The aptamer that the present invention provides, is the single stranded DNA shown in sequence 1-14 of sequence table.
Described aptamer and CYPA albumen have preferable affinity.
Also described aptamer can be modified or transformed, obtain the derivant of described aptamer.
The derivant of described aptamer can be following any one:
A) described aptamer is deleted part or increases the nucleotide of partial complementarity, the derivant of the aptamer with described aptamer with identical function obtained；
B) described aptamer is carried out nucleotide replacement or part is modified, the derivant of the aptamer with described aptamer with identical function obtained；
C) it transform the skeleton of described aptamer as phosphorothioate backbone, the derivant of the aptamer with described aptamer with identical function obtained；
D) aptamer is transform peptide nucleic acid(PNA) as, the derivant of the aptamer with described aptamer with identical function obtained；
E) after described aptamer being connected upper fluorescence, radioactivity and therapeutic substance, the derivant of the aptamer with described aptamer with identical function obtained.
Described aptamer can be used for the test kit of preparation detection CYPA.
Utilize the aptamer of the present invention, in can capturing in saliva in CYPA, thus for correlator endometrial carcinoma examination.Utilize the aptamer of the present invention, there is highly sensitive, low cost, the easy advantage prepared, easily preserve.The present invention has the highest using value.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.
The acquisition of embodiment 1CYPA albumen
CYPA gene shown in Genbank:NP_066953 is expressed by the eukaryotic expression mode that this area is conventional, it is thus achieved that corresponding desired polypeptides albumen.
The screening of embodiment 2 aptamer and preparation
Design two ends comprise about 20 nucleotide, centre includes that the random nucleic acid library of a nucleotide is as follows:
5‘-ATCGTCACCATGGATACATG(N39)TTGCACAATTGGACAGTTAC-3’；N39 represents 39 random nucleotides.
Being double-stranded DNA by single-stranded DNA banks amplification, product is through 2% agarose gel electrophoresis and cuts glue recovery purification；With reclaim double-stranded DNA as template, in vitro transcription goes out single stranded RNA random library, and transcription product is through PAGE purification.The RNA molecule being combined with film is removed, through anti-sieve of nitrocellulose filter, in 75 μ gRNA libraries, then with 2ugCYPA albumen, hatches 30min for 37 DEG C, and reactant liquor filters through nitrocellulose filter, washs filter membrane；Then filter membrane is shredded, be placed in elution buffer (6mol/L carbamide, 0.55mol/L ammonium acetate, l.5mmol/LEDTA, 0.15%SDS) and boil 5min, centrifugal, take supernatant, dehydrated alcohol precipitation RNA, and be redissolved in 20 μ 1DEPC water；With RNA for template RT-PCR amplifying doulbe-chain DNA, in vitro transcription goes out RNA library and screens for next round；Often in wheel screening process, RT-PCR obtains double-stranded DNA library, goes out RNA aptamer storehouse with this double-stranded DNA for template in vitro transcription, and screening carries out 11 altogether and takes turns.Having obtained 14 aptamers, its sequence is respectively shown in SEQIDNO:1-14.Particular sequence is as follows:
CYPA-1:ATCGTCACCATGGATACATGACTTAACCACGCTCCATTCTATCAATTAACCCGCACTAATTGCACAATTGGACAGTTAC
CYPA-2:ATCGTCACCATGGATACATGATATATTAACCCTCACTAATCTCCTCATCCTACTATAAGTTGCACAATTGGACAGTTAC
CYPA-3:ATCGTCACCATGGATACATGCACTATATTCACGCTAAACCTTTCTTCCTCGCATACCTATTGCACAATTGGACAGTTAC
CYPA-4:ATCGTCACCATGGATACATGCTATTCCCTTTCCGCTACAATCCAATAATCACGCAACACTTGCACAATTGGACAGTTAC
CYPA-5:ATCGTCACCATGGATACATGTTATTCCTGTCTTCACACCGACTTCACATATATCTTCAATTGCACAATTGGACAGTTAC
CYPA-6:ATCGTCACCATGGATACATGCTACCGCTTCCACTCTCCTTATCCATCTTACATTCCCTCTTGCACAATTGGACAGTTAC
CYPA-7:ATCGTCACCATGGATACATGCATACTACAAACCTCCTATAATCTGTTATCAACACTAATTTGCACAATTGGACAGTTAC
CYPA-8:ATCGTCACCATGGATACATGTATTTTCTTCCACTCCCATACTTAGAATAAGAATCCACTTTGCACAATTGGACAGTTAC
CYPA-9:ATCGTCACCATGGATACATGTATGACAATTATTGCCCACTAGATACTTCCACGCAAATCTTGCACAATTGGACAGTTAC
CYPA-10:ATCGTCACCATGGATACATGACTTATCATATCCACCTTATCACATCATCCCCACTTCCTTTGCACAATTGGACAGTTAC
CYPA-11:ATCGTCACCATGGATACATGTCGTTCTCACGAATTCACACACTAACTCCCACACATACATTGCACAATTGGACAGTTAC
CYPA-12:ATCGTCACCATGGATACATGCACTATATCTTACTATCATTTAATCCTTTCACTCCCAACTTGCACAATTGGACAGTTAC
CYPA-13:ATCGTCACCATGGATACATGTCACTCCGCCGCACCACTAATTCACGCTAAACTTATCTTTTGCACAATTGGACAGTTAC
CYPA-14:ATCGTCACCATGGATACATGATTCTCTCCGAACTTCTACATATAAAACCAAGAATTCTTTTGCACAATTGGACAGTTAC
The performance measurement of embodiment 3 protein binding aptamer
Aptamer taking 2.0 μ g respectively, digests lh with calf intestinal alkaline phosphatase (CIP) 37 DEG C, purification reclaims dephosphorylized RNA；By T4 polynucleotide kinase labelling [γ-32P] ATP in dephosphorylized RNA molecule end.The radiolabeled aptamer of 10nmol with variable concentrations (1-200nM) CYPA37 DEG C respectively hatches 30min, each group reactant liquor filters through nitrocellulose filter, washs filter membrane, is dried filter membrane, liquid scintillation counter measures the exit dose of residual on filter membrane, and same sample is parallel does twice mensuration.Calculate the dissociation constant of each aptamer and destination protein.Result is as follows:
| Title | Dissociation constant Kd (unit nM) |
| CYPA-1 | 11.3 |
| CYPA-2 | 12.5 |
| CYPA-3 | 13.0 |
| CYPA-4 | 12.8 |
| CYPA-5 | 12.7 |
| CYPA-6 | 11.9 |
| CYPA-7 | 11.4 |
| CYPA-8 | 12.4 |
| CYPA-9 | 12.7 |
| CYPA-10 | 12.0 |
| CYPA-11 | 13.1 |
| CYPA-12 | 13.0 |
| CYPA-13 | 12.6 |
| CYPA-14 | 12.8 |
| PBS blank | Without binding ability |
Aptamer specificity analyses and stability analysis described in embodiment 4
It is respectively adopted human albumin, immune globulin, pg120 albumen, escherichia coli outer membrane protein A, COCH albumen, CYPA albumen, specific detection is carried out with 14 aptamers, finding through binding tests, these aptamers do not combine with these albumen, and only keep higher specificity with CYPA protein binding.
By described aptamer, take 0.2ug, be respectively placed in the serum of room temperature, aqueous solution, place two weeks.Detected by RT-PCR, find its Stability Analysis of Structures of placement of three weeks, be not degraded.
The diagnosis of aptamer disease described in embodiment 5
Take 8 endometrial carcinomas and the blood of 4 normal persons, use normal saline dilution, it is thus achieved that target sample.
By 14 markd aptamers of coupling respectively with the secretions mixing 30min of 8 patients and 4 normal persons, separated by biotin, the content of quantitative analysis CYPA therein albumen, found by analysis, in 8 endometrial carcinomas, the content of CYPA albumen dramatically increases, and has exceeded the threshold value of regulation.Reach the diagnostic criteria of carcinoma of endometrium.As can be seen here, its diagnosis effect is preferable.
These are only the preferred embodiments of the present invention; it is not limited to the present invention; for a person skilled in the art, all any modification, equivalent substitution and improvement etc. done within the spirit and principles in the present invention, should be included within the scope of the present invention.
Sequence table
＜ 110 ＞ Li Jing
The test kit of a ＜ 120 ＞ carcinoma of endometrium specific detection
〈160〉14
〈210〉1
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-1
ATCGTCACCATGGATACATGACTTAACCACGCTCCATTCTATCAATTAACCCGCACTAATTGCACAATTGGACAGTTAC
〈210〉2
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-2
ATCGTCACCATGGATACATGATATATTAACCCTCACTAATCTCCTCATCCTACTATAAGTTGCACAATTGGACAGTTAC
〈210〉3
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-3
ATCGTCACCATGGATACATGCACTATATTCACGCTAAACCTTTCTTCCTCGCATACCTATTGCACAATTGGACAGTTAC
〈210〉4
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-4
ATCGTCACCATGGATACATGCTATTCCCTTTCCGCTACAATCCAATAATCACGCAACACTTGCACAATTGGACAGTTAC
〈210〉5
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-5
ATCGTCACCATGGATACATGTTATTCCTGTCTTCACACCGACTTCACATATATCTTCAATTGCACAATTGGACAGTTAC
〈210〉6
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-6
ATCGTCACCATGGATACATGCTACCGCTTCCACTCTCCTTATCCATCTTACATTCCCTCTTGCACAATTGGACAGTTAC
〈210〉7
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-7
ATCGTCACCATGGATACATGCATACTACAAACCTCCTATAATCTGTTATCAACACTAATTTGCACAATTGGACAGTTAC
〈210〉8
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-8
ATCGTCACCATGGATACATGTATTTTCTTCCACTCCCATACTTAGAATAAGAATCCACTTTGCACAATTGGACAGTTAC
〈210〉9
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-9
ATCGTCACCATGGATACATGTATGACAATTATTGCCCACTAGATACTTCCACGCAAATCTTGCACAATTGGACAGTTAC
〈210〉10
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-10
ATCGTCACCATGGATACATGACTTATCATATCCACCTTATCACATCATCCCCACTTCCTTTGCACAATTGGACAGTTAC
〈210〉11
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-11
ATCGTCACCATGGATACATGTCGTTCTCACGAATTCACACACTAACTCCCACACATACATTGCACAATTGGACAGTTAC
〈210〉12
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-12
ATCGTCACCATGGATACATGCACTATATCTTACTATCATTTAATCCTTTCACTCCCAACTTGCACAATTGGACAGTTAC
〈210〉13
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-13
ATCGTCACCATGGATACATGTCACTCCGCCGCACCACTAATTCACGCTAAACTTATCTTTTGCACAATTGGACAGTTAC
〈210〉14
〈211〉79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-14
ATCGTCACCATGGATACATGATTCTCTCCGAACTTCTACATATAAAACCAAGAATTCTTTTGCACAATTGGACAGTTAC
Claims (3)
1., for a test kit for carcinoma of endometrium detection, it contains the aptamer that energy specificity is combined with CYPA protein-specific.
2. test kit as claimed in claim 1, it is characterised in that: the sequence of described aptamer is as described in SEQIDNo:9.
3. the method detecting carcinoma of endometrium, it is characterised in that utilize the test kit described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610369948.3A CN105823894A (en) | 2015-11-22 | 2015-11-22 | Kit for specific detection of endometrial cancer |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610369948.3A CN105823894A (en) | 2015-11-22 | 2015-11-22 | Kit for specific detection of endometrial cancer |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510807002.6A Division CN105223358B (en) | 2015-11-22 | 2015-11-22 | A kind of test kit of carcinoma of endometrium specific detection |
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| CN105823894A true CN105823894A (en) | 2016-08-03 |
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| CN201610369948.3A Pending CN105823894A (en) | 2015-11-22 | 2015-11-22 | Kit for specific detection of endometrial cancer |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101261279A (en) * | 2008-04-11 | 2008-09-10 | 四川大学 | Endometrium cancer diagnose reagent, reagent kit and controlling medicament |
| WO2012049108A1 (en) * | 2010-10-12 | 2012-04-19 | Consiglio Nazionale Delle Ricerche | Axl receptor tyrosine kinase aptamer inhibitor for use in therapy |
| US20130078645A1 (en) * | 2010-01-29 | 2013-03-28 | Psynova Neurotech Ltd. | Biomarkers |
| CN104830867A (en) * | 2015-06-07 | 2015-08-12 | 杨洋 | Aptamer capable of being specifically combined with DKK1 protein in cancer cells |
-
2015
- 2015-11-22 CN CN201610369948.3A patent/CN105823894A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101261279A (en) * | 2008-04-11 | 2008-09-10 | 四川大学 | Endometrium cancer diagnose reagent, reagent kit and controlling medicament |
| US20130078645A1 (en) * | 2010-01-29 | 2013-03-28 | Psynova Neurotech Ltd. | Biomarkers |
| WO2012049108A1 (en) * | 2010-10-12 | 2012-04-19 | Consiglio Nazionale Delle Ricerche | Axl receptor tyrosine kinase aptamer inhibitor for use in therapy |
| CN104830867A (en) * | 2015-06-07 | 2015-08-12 | 杨洋 | Aptamer capable of being specifically combined with DKK1 protein in cancer cells |
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