CN106018838A - Kit used for detecting ophthalmic diseases, and detection method thereof - Google Patents

Kit used for detecting ophthalmic diseases, and detection method thereof Download PDF

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CN106018838A
CN106018838A CN201610637743.9A CN201610637743A CN106018838A CN 106018838 A CN106018838 A CN 106018838A CN 201610637743 A CN201610637743 A CN 201610637743A CN 106018838 A CN106018838 A CN 106018838A
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leptin
aptamer
seq
dna
ophthalmic diseases
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李倩
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • 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
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    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N2333/575—Hormones
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/046—Thyroid disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology

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Abstract

The invention discloses a kit including an aptamer specifically bound with a human leptin protein. The aptamer has good affinity ability to the human leptin. The aptamer being able to capture the human leptin in a solution is used to make the corresponding kit used for screening the ophthalmic diseases. The kit has the advantages of high sensitivity, low cost, easiness in production, and easiness in preservation.

Description

A kind of test kit for detecting ophthalmic diseases and detection method thereof
Technical field
The present invention relates to a kind of test kit for detecting ophthalmic diseases and detection method thereof.
Background technology
Graves disease (Graves'disease) is a kind of commonly encountered diseases, and women sickness rate is 1/1000 person/year. In addition to hyperthyroidism, 25-50% suffers from the individual PD of Graves disease to involving clinically Eyes, i.e. thyroid eye diseas.Graves' ophthalmopathy (Graves'Ophthalmopathy, GO or TAO) is first The canonic form of shape gland oculopathy.Although it is uncomfortable that some GO patients only suffer from slight eyes, but 3-5% suffers from Have an intense pain and inflammation is with diplopia or even visual loss.The brightest due to primary disease reason at present, clinical mainly to swash In element, immunosuppressant, growth hormone analogs, antithyroid drug, socket of the eye, radiation, orbital decompression etc. are combined It is main for closing treatment, but for various reasons, effect is the most unsatisfactory, there is no any medicine energy up to now Effectively treat TAO, and obtain long-term remission.Therefore, the medicine of research treatment thyroid-associated ophthalmopathy is urgently to solve Technical problem certainly.And detect the direction that described disease this area especially is actively studied.
Recent studies indicate that, human leptin protein (rh-leptin) becomes fiber to fat before TAO patient's eye socket Cell proliferation, divide a word with a hyphen at the end of a line and in cell differentiation procedure lipid within endothelial cells formed there is important effect.Therefore, inspection Survey the expression of human leptin protein, be used directly for identifying thyroid-associated ophthalmopathy.But traditional detection The expression of human leptin protein has operation complexity, time-consuming shortcoming.
Aptamer (Aptamer, also known as aptamers, aptamer) is can high-affinity, the combination of high specific Certain biological leather El target strand widow's nucleic acid molecules (ssDNA or ssRNA).Aptamer is rich by index Collection Fas lignand system evolution technology (Systematlc Evolutlon of Llgands by Exponentlal Enrlchment, SELEX) from the DNA/RNA library of synthetic screening obtain can high degree of specificity knot Close the single stranded DNA/RNA of target molecules.Reported the target of aptamer include metal ion, organic molecule, Polypeptide, protein, cell even tissue etc..The molecular recognition function of aptamer is similar with antibody, have with The target identification ability that antibody molecule is the most higher, but there is compared with antibody the most excellent characteristic, as Molecular weight is little, can manufacture, not easy in inactivation, non-immunogenicity, be readily synthesized with labelling, quickly penetrate Between tissue, good dynamic metabolism, different batches, product does not haves difference and has fine chemically stable Property, in fields such as biological detection, medical diagnosis on disease treatments, there is important application prospect.
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 leptin.
The aptamer that the present invention provides, is the single stranded DNA shown in sequence 1-15 of sequence table.
Described aptamer and leptin 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 being deleted part or increases the nucleotide of partial complementarity, obtain fits with described nucleic acid Body has the derivant of the aptamer of identical function；
B) described aptamer carrying out nucleotide replacement or part is modified, obtain has with described aptamer The derivant of the aptamer of identical function；
C) transforming the skeleton of described aptamer as phosphorothioate backbone, obtain has with described aptamer There is the derivant of the aptamer of identical function；
D) aptamer is transform peptide nucleic acid(PNA) as, obtain with described aptamer have identical function nucleic acid fit The derivant of body；
E) after described aptamer being connected upper fluorescence, radioactivity and therapeutic substance, that obtain with described nucleic acid The derivant of the fit aptamer with identical function.
Described aptamer can be used for the test kit of preparation detection leptin.
Utilize the aptamer of the present invention, the leptin in blood can be captured, thus be correlated with Thyreoidine Eye disease screening.The aptamer of the present invention, part is utilized to replace monoclonal antibody capture leptin to detect, 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.In following embodiment Experimental technique, if no special instructions, is conventional method.
Embodiment 1, the screening of aptamer and preparation
Two ends comprise about 20 nucleotide, centre includes that the random nucleic acid library of 39 nucleotide is as follows in design:
5’-TAGACTATCATGTGACTT(N39)GAGTGCTCGATGCTACTAG-3’；N39 represents 39 Individual random nucleotide.
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 pure through PAGE Change.The RNA molecule being combined with film is removed, then with 2ug through anti-sieve of nitrocellulose filter in 75 μ g RNA libraries Leptin albumen (utilizes method disclosed in CN100366738C, express the destination protein obtained), 37 DEG C Hatching 30min, reactant liquor filters through nitrocellulose filter, washs filter membrane；Then filter membrane is shredded, be placed in and wash In de-buffer (6mol/L carbamide, 0.55mol/L ammonium acetate, 1.5mmol/L EDTA, 0.15%SDS) Boil 5min, centrifugal, take supernatant, dehydrated alcohol precipitation RNA, and be redissolved in 20 μ l DEPC 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 with this double-stranded DNA for template in vitro transcription Adaptive word bank, screening carries out 12 altogether and takes turns.Having obtained 15 aptamers, its sequence is respectively SEQ ID NO:1-15 Shown in.Particular sequence is as follows:
Leptin-1:
TAGACTATCATGTGACTTCCTTAACTATATACAATTATGTCATATCTTACAAGTACTGAGTGCTC GATGCTACTAG(SEQ ID NO:1)
Leptin-2:
TAGACTATCATGTGACTTTACAAGATACATTCATCCCGCAACACACTCAACCAACTCGAGTGCTC GATGCTACTAG(SEQ ID NO:2)
Leptin-3:
TAGACTATCATGTGACTTTCTACCTAATAATAACCTATACCTCCCTCAGTAATCACAGAGTGCTC GATGCTACTAG(SEQ ID NO:3)
Leptin-4:
TAGACTATCATGTGACTTACCTATTAACTTGTACTCCTACAAACCCAGAACAACACAGAGTGCTC GATGCTACTAG(SEQ ID NO:4)
Leptin-5:
TAGACTATCATGTGACTTTCACTGTTAACACAGATAATTAACATAACAACGTCTATAGAGTGCTC GATGCTACTAG(SEQ ID NO:5)
Leptin-6:
TAGACTATCATGTGACTTAAACATCCTCCCTATACCTATCCGTATACCGCTTCCTCTGAGTGCTC GATGCTACTAG(SEQ ID NO:6)
Leptin-7:
TAGACTATCATGTGACTTAACACCGCCTAATATTAATATCCCCTTCTTCATAGATCAGAGTGCTC GATGCTACTAG(SEQ ID NO:7)
Leptin-8:
TAGACTATCATGTGACTTCCGACTTCATACATACTCCTCTTCATCCTGTAATCAACCGAGTGCTC GATGCTACTAG(SEQ ID NO:8)
Leptin-9:
TAGACTATCATGTGACTTATATATCCTCCACTTAGATAACAATCTTCAGACACTCCAGAGTGCTC GATGCTACTAG(SEQ ID NO:9)
Leptin-10:
TAGACTATCATGTGACTTCCTCCTTCTTCCTCCGATTCATCCTCAATAGATACTATTGAGTGCTC GATGCTACTAG(SEQ ID NO:10)
Leptin-11:
TAGACTATCATGTGACTTTTCACGCTCAATCATAAATCTTAGAATCCAATAATTACCGAGTGCTC GATGCTACTAG(SEQ ID NO:11)
Leptin-12:
TAGACTATCATGTGACTTCCGCCACTCCTCATCACATCGCCACACCCAAATTCACAAGAGTGCTC GATGCTACTAG(SEQ ID NO:12)
Leptin-13:
TAGACTATCATGTGACTTCGCTCCTCTCTTGTTTACTACACATATCTTGTTATCTTAGAGTGCTC GATGCTACTAG(SEQ ID NO:13)
Leptin-14:
TAGACTATCATGTGACTTACCTATTCCGCCCTCACCTTCCTCTACTAGATCATAACTGAGTGCTC GATGCTACTAG(SEQ ID NO:14)
Leptin-15:
TAGACTATCATGTGACTTATAATTCTATATATATCGCACTACATCAAACGCCATAAAGAGTGCTC GATGCTACTAG(SEQ ID NO:15)
The performance measurement of embodiment 2 protein binding aptamer
RNA aptamer is taken respectively 2.0 μ g, digests lh with calf intestinal alkaline phosphatase (CIP) 37 DEG C, Purification reclaims dephosphorylized RNA；By T4 polynucleotide kinase labelling [γ-32P] ATP in dephosphorylation RNA molecule end.The radiolabeled RNA aptamer of 10nmol respectively with variable concentrations (1-200nM) Leptin 37 DEG C hatch 30min, each group reactant liquor filters through nitrocellulose filter, washs filter membrane, dry Dry filter membrane, liquid scintillation counter measures the exit dose of residual on filter membrane, and same sample is parallel does twice mensuration.Calculate Each aptamer and the dissociation constant of leptin.Result is as follows:
Title Dissociation constant Kd (unit nM)
leptin-1 10.9
leptin-2 10.8
leptin-3 9.9
leptin-4 10.3
leptin-5 10.5
leptin-6 9.6
leptin-7 9.8
leptin-8 10.0
leptin-9 10.3
leptin-10 9.9
leptin-11 9.8
leptin-12 10.1
leptin-13 10.6
leptin-14 9.7
leptin-15 9.7
PBS blank Without binding ability
Aptamer specificity analyses and stability analysis described in embodiment 3
It is respectively adopted human albumin, immune globulin, vibrio cholera VgrG3C albumen, large intestine bar Bacterial outer membrane protein A, Lp-PLA2 albumen, leptin, carry out specific detection, Jing Guojie with 14 aptamers Closing test to find, these aptamers do not combine with these albumen, and be only combined holding with leptin higher Specificity.
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 two weeks, be not degraded.
The diagnosis of aptamer disease described in embodiment 4
By 14 aptamers, blood with 12 graves' ophthalmopathy patients and normal person mixes respectively 30min, is separated by biotin, the content of quantitative analysis leptin therein albumen, is found by analysis, In 12 graves' ophthalmopathy patients, the content of leptin albumen dramatically increases 2 times relative to normal person.
These are only the preferred embodiments of the present invention, be not limited to the present invention, for the skill of this area For art personnel, 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 Qian
＜ 120 ＞ mono-kind is for detecting test kit and the detection method thereof of ophthalmic diseases
〈160〉15
〈210〉1
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-1:
TAGACTATCATGTGACTTCCTTAACTATATACAATTATGTCATATCTTACAAGTACTGAGTGCTCGATGCTACTAG
〈210〉2
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-2:
TAGACTATCATGTGACTTTACAAGATACATTCATCCCGCAACACACTCAACCAACT CGAGTGCTCGATGCTACTAG(SEQ ID NO:2)
〈210〉3
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-3:
TAGACTATCATGTGACTTTCTACCTAATAATAACCTATACCTCCCTCAGTAATCAC AGAGTGCTCGATGCTACTAG(SEQ ID NO:3)
〈210〉4
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-4:
TAGACTATCATGTGACTTACCTATTAACTTGTACTCCTACAAACCCAGAACAACAC AGAGTGCTCGATGCTACTAG(SEQ ID NO:4)
〈210〉5
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-5:
TAGACTATCATGTGACTTTCACTGTTAACACAGATAATTAACATAACAACGTCTAT AGAGTGCTCGATGCTACTAG(SEQ ID NO:5)
〈210〉6
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-6:
TAGACTATCATGTGACTTAAACATCCTCCCTATACCTATCCGTATACCGCTTCCTC TGAGTGCTCGATGCTACTAG(SEQ ID NO:6)
〈210〉7
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-7:
TAGACTATCATGTGACTTAACACCGCCTAATATTAATATCCCCTTCTTCATAGATC AGAGTGCTCGATGCTACTAG(SEQ ID NO:7)
〈210〉8
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-8:
TAGACTATCATGTGACTTCCGACTTCATACATACTCCTCTTCATCCTGTAATCAAC CGAGTGCTCGATGCTACTAG(SEQ ID NO:8)
〈210〉9
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-9:
TAGACTATCATGTGACTTATATATCCTCCACTTAGATAACAATCTTCAGACACTCC AGAGTGCTCGATGCTACTAG(SEQ ID NO:9)
〈210〉10
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-10:
TAGACTATCATGTGACTTCCTCCTTCTTCCTCCGATTCATCCTCAATAGATACTAT TGAGTGCTCGATGCTACTAG(SEQ ID NO:10)
〈210〉11
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-11:
TAGACTATCATGTGACTTTTCACGCTCAATCATAAATCTTAGAATCCAATAATTAC CGAGTGCTCGATGCTACTAG(SEQ ID NO:11)
〈210〉12
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-12:
TAGACTATCATGTGACTTCCGCCACTCCTCATCACATCGCCACACCCAAATTCACA AGAGTGCTCGATGCTACTAG(SEQ ID NO:12)
〈210〉13
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-13:
TAGACTATCATGTGACTTCGCTCCTCTCTTGTTTACTACACATATCTTGTTATCTT AGAGTGCTCGATGCTACTAG(SEQ ID NO:13)
〈210〉14
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-14:
TAGACTATCATGTGACTTACCTATTCCGCCCTCACCTTCCTCTACTAGATCATAAC TGAGTGCTCGATGCTACTAG(SEQ ID NO:14)
〈210〉15
〈211〉76
〈212〉DNA
＜ 213 ＞ artificial sequence
＜ 400 ＞ leptin-15:
TAGACTATCATGTGACTTATAATTCTATATATATCGCACTACATCAAACGCCATAA AGAGTGCTCGATGCTACTAG(SEQ ID NO:15)

Claims (4)

1., for a test kit for ophthalmic diseases detection, it contains aptamer.
2. test kit as claimed in claim 1, it is characterised in that: described aptamer is described in SEQ ID No:10.
3. the method detecting ophthalmic diseases, it is characterised in that utilize the test kit described in claim 1.
4. the test kit as described in claim 1-2, it is characterised in that: described ophthalmic diseases is graves' ophthalmopathy.
CN201610637743.9A 2015-11-01 2015-11-01 Kit used for detecting ophthalmic diseases, and detection method thereof Pending CN106018838A (en)

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CN201610638455.5A Pending CN106290905A (en) 2015-11-01 2015-11-01 A kind of test kit for detecting ophthalmic diseases and detection method thereof
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CN201610637997.0A Pending CN106053850A (en) 2015-11-01 2015-11-01 Kit and method for detection of eye diseases
CN201610637702.XA Pending CN106018837A (en) 2015-11-01 2015-11-01 Kit used for detecting ophthalmic diseases, and detection method thereof
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CN201610638657.XA Pending CN106018842A (en) 2015-11-01 2015-11-01 Kit used for detecting ophthalmic diseases, and detection method thereof
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CN201610638455.5A Pending CN106290905A (en) 2015-11-01 2015-11-01 A kind of test kit for detecting ophthalmic diseases and detection method thereof
CN201610638695.5A Withdrawn CN106290906A (en) 2015-11-01 2015-11-01 A kind of test kit for detecting ophthalmic diseases and detection method thereof
CN201610637997.0A Pending CN106053850A (en) 2015-11-01 2015-11-01 Kit and method for detection of eye diseases
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CN201610638149.1A Pending CN106018840A (en) 2015-11-01 2015-11-01 Kit used for detecting ophthalmic diseases, and detection method thereof
CN201610638033.8A Pending CN106018839A (en) 2015-11-01 2015-11-01 Kit used for detecting ophthalmic diseases, and detection method thereof
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CN201610638150.4A Pending CN106248967A (en) 2015-11-01 2015-11-01 A kind of test kit for detecting ophthalmic diseases and detection method thereof

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