CN106018842A - 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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CN106018842A
CN106018842A CN201610638657.XA CN201610638657A CN106018842A CN 106018842 A CN106018842 A CN 106018842A CN 201610638657 A CN201610638657 A CN 201610638657A CN 106018842 A CN106018842 A CN 106018842A
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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
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    • G01—MEASURING; TESTING
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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.Except first Beyond shape gland hyperfunctioning, 25-50% suffers from the individual PD of Graves disease to involving eyes clinically, i.e. first shape Gland oculopathy.Graves' ophthalmopathy (Graves'Ophthalmopathy, GO or TAO) is the canonic form of thyroid eye diseas.Though So some GO patients only to suffer from slight eyes uncomfortable, but 3-5% suffers from and has an intense pain and inflammation is with diplopia or even vision Lose.The brightest due to primary disease reason at present, clinical main with hormone, immunosuppressant, growth hormone analogs, antithyroid drug The Comprehensive Treatments such as thing, socket of the eye interior radiation, orbital decompression are main, but for various reasons, effect is the most unsatisfactory, the most still Can effectively treat TAO without any medicine, and obtain long-term remission.Therefore, the medicine of research treatment thyroid-associated ophthalmopathy is Technical problem urgently to be resolved hurrily.And detect the direction that described disease this area especially is actively studied.
Recent studies indicate that, fat fibroblast before TAO patient's eye socket is increased by human leptin protein (rh-leptin) Grow, 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, the table of human leptin protein is detected Reach situation, be used directly for identifying thyroid-associated ophthalmopathy.But the expression of traditional detection human leptin protein has Operation complexity, time-consuming shortcoming.
Aptamer (Aptamer, also known as aptamers, aptamer) is can high-affinity, certain life of combination of high specific Thing leather El target strand widow's nucleic acid molecules (ssDNA or ssRNA).Aptamer is by index concentration Fas lignand system evolution technology (Systemat1c Evolut1on of L1gands by Exponent1al enr1chment, SELEX) is from synthetic What in DNA/RNA library, screening obtained can combine the single stranded DNA/RNA of target molecules by high degree of specificity.Report aptamer Target include metal ion, organic molecule, polypeptide, protein, cell even tissue etc..The molecular recognition merit of aptamer Can be similar with antibody, there is the target identification ability the most higher with antibody molecule, but have the most excellent compared with antibody Good 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 chemical stability, at biology The fields such as detection, medical diagnosis on disease treatment have 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 has with described aptamer There is the derivant of the aptamer of identical function；
B) described aptamer carrying out nucleotide replacement or part is modified, obtain has identical with described aptamer The derivant of the aptamer of function；
C) transforming the skeleton of described aptamer as phosphorothioate backbone, obtain has phase with described aptamer The derivant of the aptamer of congenerous；
D) aptamer transform peptide nucleic acid(PNA) as, obtain has the aptamer of identical function with described aptamer Derivant；
E) after described aptamer being connected upper fluorescence, radioactivity and therapeutic substance, that obtain with described aptamer There is the derivant of the aptamer of 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 sieve for thyroid-associated ophthalmopathy Look into.Utilize the aptamer of the present invention, part to replace monoclonal antibody capture leptin to detect, there is highly sensitive, cost Preparation low, easy, the advantage easily preserved.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.Experiment in following embodiment Method, 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 random nucleoside Acid.
Being double-stranded DNA by single-stranded DNA banks amplification, product is through 2% agarose gel electrophoresis and cuts glue recovery purification；To return The double-stranded DNA received is template, and in vitro transcription goes out single stranded RNA random library, and transcription product is through PAGE purification.75 μ g RNA library warps The anti-sieve of nitrocellulose filter removes the RNA molecule being combined with film, then (utilizes CN100366738C public with 2ug leptin albumen The method opened, expresses the destination protein obtained), hatch 30min for 37 DEG C, 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/L EDTA, 5min is boiled in 0.15%SDS), centrifugal, take supernatant, dehydrated alcohol precipitation RNA, and be redissolved in 20 μ 1DEPC water；With RNA is template RT-PCR amplifying doulbe-chain DNA, and in vitro transcription goes out RNA library and screens for next round；Often RT-in wheel screening process 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 12 altogether and takes turns.Obtain 15 aptamers, its sequence is respectively shown in SEQ ID NO:1-15.Particular sequence is as follows:
Leptin-1:
TAGACTATCATGTGACTTCCTTAACTATATACAATTATGTCATATCTTACAAGTACTGAGTGCTCGATGCTACTAG (SEQ ID NO:1)
Leptin-2:
TAGACTATCATGTGACTTTACAAGATACATTCATCCCGCAACACACTCAACCAACTCGAGTGCTCGATGCTACTAG (SEQ ID NO:2)
Leptin-3:
TAGACTATCATGTGACTTTCTACCTAATAATAACCTATACCTCCCTCAGTAATCACAGAGTGCTCGATGCTACTAG (SEQ ID NO:3)
Leptin-4:
TAGACTATCATGTGACTTACCTATTAACTTGTACTCCTACAAACCCAGAACAACACAGAGTGCTCGATGCTACTAG (SEQ ID NO:4)
Leptin-5:
TAGACTATCATGTGACTTTCACTGTTAACACAGATAATTAACATAACAACGTCTATAGAGTGCTCGATGCTACTAG (SEQ ID NO:5)
Leptin-6:
TAGACTATCATGTGACTTAAACATCCTCCCTATACCTATCCGTATACCGCTTCCTCTGAGTGCTCGATGCTACTAG (SEQ ID NO:6)
Leptin-7:
TAGACTATCATGTGACTTAACACCGCCTAATATTAATATCCCCTTCTTCATAGATCAGAGTGCTCGATGCTACTAG (SEQ ID NO:7)
Leptin-8:
TAGACTATCATGTGACTTCCGACTTCATACATACTCCTCTTCATCCTGTAATCAACCGAGTGCTCGATGCTACTAG (SEQ ID NO:8)
Leptin-9:
TAGACTATCATGTGACTTATATATCCTCCACTTAGATAACAATCTTCAGACACTCCAGAGTGCTCGATGCTACTAG (SEQ ID NO:9)
Leptin-10:
TAGACTATCATGTGACTTCCTCCTTCTTCCTCCGATTCATCCTCAATAGATACTATTGAGTGCTCGATGCTACTAG (SEQ ID NO:10)
Leptin-11:
TAGACTATCATGTGACTTTTCACGCTCAATCATAAATCTTAGAATCCAATAATTACCGAGTGCTCGATGCTACTAG (SEQ ID NO:11)
Leptin-12:
TAGACTATCATGTGACTTCCGCCACTCCTCATCACATCGCCACACCCAAATTCACAAGAGTGCTCGATGCTACTAG (SEQ ID NO:12)
Leptin-13:
TAGACTATCATGTGACTTCGCTCCTCTCTTGTTTACTACACATATCTTGTTATCTTAGAGTGCTCGATGCTACTAG (SEQ ID NO:13)
Leptin-14:
TAGACTATCATGTGACTTACCTATTCCGCCCTCACCTTCCTCTACTAGATCATAACTGAGTGCTCGATGCTACTAG (SEQ ID NO:14)
Leptin-15:
TAGACTATCATGTGACTTATAATTCTATATATATCGCACTACATCAAACGCCATAAAGAGTGCTCGATGCTACTAG (SEQ ID NO:15)
The performance measurement of embodiment 2 protein binding aptamer
RNA aptamer taking 2.0 μ g respectively, digests lh with calf intestinal alkaline phosphatase (CIP) 37 DEG C, purification reclaims dephosphorization The RNA of acidifying；By T4 polynucleotide kinase labelling [γ-32P] ATP in dephosphorylized RNA molecule end.10nmol radiates Property labelling RNA aptamer leptin 37 DEG C with variable concentrations (1-200nM) respectively hatch 30min, each group reactant liquor is through nitre Acid cellulose membrane filtration mistake, washs filter membrane, is dried filter membrane, and liquid scintillation counter measures the exit dose of residual on filter membrane, and same sample is put down Row does twice mensuration.Calculate the dissociation constant of each aptamer and 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, escherichia coli outer membrane protein A, Lp-PLA2 albumen, leptin, carry out specific detection with 14 aptamers, find through binding tests, these aptamers are all Do not combine with these albumen, and be only combined the specificity keeping higher with leptin.
By described aptamer, take 0.2ug, be respectively placed in the serum of room temperature, aqueous solution, place two weeks.Pass through RT- PCR detects, and finds its Stability Analysis of Structures of placement of two weeks, is not degraded.
The diagnosis of aptamer disease described in embodiment 4
By 14 aptamers respectively with the blood mixing 30min of 12 graves' ophthalmopathy patients and normal person, pass through Biotin separates, and 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 those skilled in the art For Yuan, all any modification, equivalent substitution and improvement etc. done within the spirit and principles in the present invention, should be included in this Within the protection domain of 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:8.
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.
CN201610638657.XA 2015-11-01 2015-11-01 Kit used for detecting ophthalmic diseases, and detection method thereof Pending CN106018842A (en)

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CN201610637999.XA Pending CN106290904A (en) 2015-11-01 2015-11-01 A kind of test kit for detecting ophthalmic diseases and detection method thereof
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CN201610637704.9A Pending CN106053849A (en) 2015-11-01 2015-11-01 Kit and method for detection of eye diseases
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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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CN106290905A (en) 2017-01-04

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