CN105802972A - Aptamer and kit for diagnosing fetus premature delivery in gynaecology and obstetrics department - Google Patents
Aptamer and kit for diagnosing fetus premature delivery in gynaecology and obstetrics department Download PDFInfo
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Abstract
The invention relates to an aptamer capable of being specifically bound with fFN (fetal fibroneetin). The aptamer has a higher binding characteristic and stability and is obtained in the method as follows: the aptamer capable of being specifically bound with fFN is screened from a random library with fFN as a target with a new combinatorial chemistry technique SELEX. The invention further relates to a kit for identifying fetus premature delivery. The kit comprises the aptamer capable of quickly identifying fetus premature delivery and has very high application value.
Description
Technical field
The present invention relates to biological technical field, particularly to a kind of aptamer for diagnosing for fetus premature labor in department of obstetrics and gynecology and test kit thereof.
Background technology
Fetal fibronectin (fetalFibroneetin, fFN) is a kind of glycoprotein, and molecular weight is about 500KD, has molecular forms 20 kinds different.FFN results from chorion trophocyte, is mainly distributed on amniotic fluid, placenta tissue and fine hair decidua interface, adhering to each other and protecting important role in Placenta Hominis and uterine decidua.Lot of documents is reported, in pregnant early stage, along with pregnant capsule is planted and is attached to endometrium, fFN may occur in which in cervical secretions, but after pregnant 20 weeks, the fusion of chorion and decidua prevents the release of fFN, so anemia of pregnant woman is extremely low at the content in 22-35 period fFN of pregnant week normally.Only when chorion separate with decidua, the extracellular matrix at chorion and decidua interface suffer the degraded of mechanical damage or proteolytic enzyme time, fFN is just found in cervicovaginal secretions.Therefore, in normal pregnancy, only containing a small amount of fFN in amniotic fluid in late period and cervicovaginal secretions；When premature labor has uterine contraction, chorion-decidua interfacial fracture, fFN penetrates in amniotic fluid and cervix uteri, there will be excessive fFN in cervicovaginal secretions.Cervix uteri, intravaginal secretions fFN positive prompting Placenta Hominis be damaged sticking of uterine decidua, indication lower uterine segment decidua separates with fetal membrane, and fFN leaks into intravaginal through cervix uteri, and is considered to be a high-risk index just before giving birth starting of non-full-term pregnancy.Therefore, between pregnant 22-35 week, in cervicovaginal secretions the level of fFN with whether occur premature labor to have good dependency, fFN detection can as an important objective index of prediction premature labor.
Fetal fibronectin (fFN) detection is U.S. FDA approval, for having the anemia of pregnant woman of premature labor symptom and having the premature delivery risk of high risk factor anemia of pregnant woman to assess, for 22-30 pregnant week asymptomatic anemia of pregnant woman routine screening and have premature labor symptom anemia of pregnant woman to check in 24-35 pregnant week.FFN detection is the project being conventionally used for premature labor diagnosis that AAOG (AC0G) is recommended.
Compared with traditional method, before pregnant 32 weeks, fetal fibronectin (fFN) detection is reliable, it is possible to independent prediction premature labor index.For the anemia of pregnant woman in pregnant 22-35 week, fFN detection can help to determine that anemia of pregnant woman is the need of carrying out Drug therapy, lying up, if can work on.For there is premature labor symptom and fFN testing result is negative anemia of pregnant woman, in ensuing 7 days, will not there is premature labor in the anemia of pregnant woman of 99.5%.
China just has a Premature Birth in about 1 second；Premature labor is neonatal death and the first cause disabled, and premature infant can show as hearing disability, mental retardation, cerebral palsy, poor growth and some other health problem.How to predict and early diagnosis premature labor, thus positive treatment, reduce premature labor and the generation of corresponding complication, be one of the vital task of antenatal care.
With marginal value 50ng/ml for criterion, fFN management is used to there is the patient of preterm delivery risk, the number (more than 50%) that the premature labor of minimizing is in hospital.
Existing fFN reagent is prepared by gold colloidal method, and gold colloidal forms red granules, judges that marginal value judges disease event by naked eyes, owing to naked eyes judge to bring very big subjectivity, causes that clinical trial is easily caused doctor-patient dispute.
SELEX technology is a kind of new combinatorial chemistry technique grown up early 1990s.Utilize this technology can screen the aptamer that specificity is affine with target material height from random single chain oligonucleotide library.Its basic ideas are one single stranded oligonucleotide storehouses of iii vitro chemical synthesis, mix with target material, form target material-nucleic acid complexes, the unconjugated nucleic acid of eluting, separate the nucleic acid molecules being combined with target material, and carry out pcr amplification with this nucleic acid molecules for template, enter back into the screening of lower whorl.By the screening repeated and amplification, some are not combined with target material or have low-affinity with target material, the nucleic acid molecules of middle affinity is washed away, and with leather G material have the nucleic acid molecules of strong affinity from very big with hangar point still out, and purity carrying out and increase with SELEX process, finally occupy the great majority (> about 90% in storehouse).First using this technology screening to after the specific nucleic acid aptamers of specific adsorption phage T4DNA polymerase and organic dye molecule from Tuerk and Ellington etc., through the development of more than ten years, SELEX technology has become as a kind of important grinds the means of making internal disorder or usurp and instrument.Therefore, adopting selex technology, screening specifically has comparatively important meaning in conjunction with the aptamers of fFN.
Summary of the invention
Technical scheme is realized by following steps:
It is an object of the invention to provide the nucleic acid aptamer sequence of a kind of fFN.
In the present invention, the aptamer (sequence 1-24) of described fFN can specific bond fFN.
The present invention further objective is that the purposes providing described nucleic acid aptamer sequence.According to this sequence is applied in the present invention, can be further used for preparing the test kit of specific binding fFN.
From the random oligo DNA library of external synthesis, (5 '-CTGACCATGCATGAACGGCA----N35----ACGTGACCAATGTGTTACAC-3 '), wherein N35 is 35 random oligonucleotides；Therefrom filter out and the aptamer of fFN specific bond；The sequence primer P1:CTGACCATGCATGAACGGCA that will filter out；Primer P2:GTGTAACACATTGGTCACGT, carries out expanding and carry out TA and is cloned into pMD19-T carrier (purchased from the rich photo bio company in Shanghai), convert DH5a antibacterial (purchased from Beijing Tian Gen biotech firm)；Choose white colony and carry out after PCR determines positive colony, extracting plasmid sequencing reaction, upper sequencer.
The present invention adopts in-vitro screening (SELEX) technology of aptamer, with fFN for just sieving target, and the aptamer of screening and ICAM-1 specific bond, prepare the sequence with specific bond fFN, called after aptamers fFNap-1～24 in the present invention.Sequence is as follows:
FFNap-1:CTGACCATGCATGAACGGCAAATAAAATCTTCAACACTCCACCAACCT CTTCTACGTGTAACACATTGGTCACGT
FFNap-2:CTGACCATGCATGAACGGCATCAAAACTCTATACATATTACTCATTCC CAATATTGTGTAACACATTGGTCACGT
FFNap-3:CTGACCATGCATGAACGGCACCACCAACCTCTTCTACTACATATATTC AAAACTCGTGTAACACATTGGTCACGT
FFNap-4:CTGACCATGCATGAACGGCACTAATACATTTTACTCCAATATTCATAA TACCATAGTGTAACACATTGGTCACGT
FFNap-5:CTGACCATGCATGAACGGCACAACATCTACCTACCCAAAAATAATTAT CCAATTCGTGTAACACATTGGTCACGT
FFNap-6:CTGACCATGCATGAACGGCAAATTCATTTCATTAACATTCCACATCTA TAAAATAGTGTAACACATTGGTCACGT
FFNap-7:CTGACCATGCATGAACGGCACTTCCAAAAAAACAACATAACATTCACA AAATACTGTGTAACACATTGGTCACGT
FFNap-8:CTGACCATGCATGAACGGCAACTCAAATTCATCCTTATCCCTCCATCA CTACCATGTGTAACACATTGGTCACGT
FFNap-9:CTGACCATGCATGAACGGCATTCCCCCCCTCATCACATCCCACACCCA AATTCACGTGTAACACATTGGTCACGT
FFNap-10:CTGACCATGCATGAACGGCACTACCAATCTCTTCCCCCCTCCACACC TCTCCCCTGTGTAACACATTGGTCACGT
FFNap-11:CTGACCATGCATGAACGGCATATCCTAACATTACACTTTCTTATCCT CATCCATTGTGTAACACATTGGTCACGT
FFNap-12:CTGACCATGCATGAACGGCATTCCTCCCCAAACAAACTTTTTTACCC TTCACATCGTGTAACACATTGGTCACGT
FFNap-13:CTGACCATGCATGAACGGCATACCTCAAAACTATTATATTCCTTTAT ACTATAATGTGTAACACATTGGTCACGT
FFNap-14:CTGACCATGCATGAACGGCATCTCTTCTATTCTTACAACTCCCATAC TTACCACTGTGTAACACATTGGTCACGT
FFNap-15:CTGACCATGCATGAACGGCAATACATTAACACTCCACTAACTCAAAT CTACTAAAGTGTAACACATTGGTCACGT
FFNap-16:CTGACCATGCATGAACGGCACATACCACTAACTTAATCCTCATTTCT CAATTCCCGTGTAACACATTGGTCACGT
FFNap-17:CTGACCATGCATGAACGGCACTACACTATACCACATACTAACCACCT CCATTCTAGTGTAACACATTGGTCACGT
FFNap-18:CTGACCATGCATGAACGGCAACTAATTCCTCATCCTACTATAACCTT TTAACCCTGTGTAACACATTGGTCACGT
FFNap-19:CTGACCATGCATGAACGGCACCACTCCCTTATCATCTTACATTCCCC CTCAATTCGTGTAACACATTGGTCACGT
FFNap-20:CTGACCATGCATGAACGGCAACCTCTATAATCTTTATCAACACTAAT TCACTAATGTGTAACACATTGGTCACGT
FFNap-21:CTGACCATGCATGAACGGCATAAAATCCACTTTCCTCCTACATATAA CAAACTTTGTGTAACACATTGGTCACGT
FFNap-22:CTGACCATGCATGAACGGCATCATCTAACATTCCCCCTTTACTATCC CCATCATCGTGTAACACATTGGTCACGT
FFNap-23:CTGACCATGCATGAACGGCATACATACCAAACATATCATCTCCCCAA TATCCATTGTGTAACACATTGGTCACGT
FFNap-24:CTGACCATGCATGAACGGCATTCCAAAATCTCTCTCCCATATTCCAC ATCTCCATGTGTAACACATTGGTCACGT
The aptamer of the present invention may be used for building test kit, and this test kit may be used for specific separation and detection by quantitative fFN, has separating effect fast, and efficiency is high, saves the time, saves effect of cost.There is very wide application prospect.
Beneficial effects of the present invention: (1) obtain a kind of can differential high efficient in conjunction with the aptamer of fFN.This aptamer can manually be prepared in a large number, and method is simple, with low cost.(2) based on nucleic acid aptamer, can be prepared as the test kit into specific detection fFN.
Detailed description of the invention
Embodiment 1: aptamer screens
From the random oligo DNA library of external synthesis, 5 '-CTGACCATGCATGAACGGCA----N35----ACGTGACCAATGTGTTACAC-3 '.
The primer:
F:5 ,-CTGACCATGCATGAACGGCA-3
R:5 ,-GTGTAACACATTGGTCACGT-3
Being double-stranded DNA by single-stranded DNA banks amplification, product is through 2% agarose gel electrophoresis and cuts glue recovery purification；With the double-stranded DNA of recovery for 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 80 μ gRNA libraries, then hatches 40min with 10ugfFN protein 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, 0.7mol/L ammonium acetate, l.6mmol/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 14 altogether and takes turns.Last takes turns the aptamer that obtains of screening carry out expanding and carry out TA and be cloned into pMD19-T carrier (purchased from the rich photo bio company in Shanghai), convert DH5a antibacterial (purchased from Beijing Tian Gen biotech firm)；Choose white colony and carry out after PCR determines positive colony, extracting plasmid sequencing reaction, upper sequencer.Mensuration obtains sequence such as shown in SEQIDNO:1-24.
The fFN of embodiment 2 specificity high-affinity is in conjunction with the acquisition of aptamer
RNA aptamer taking 1.5 μ g respectively, digests 1h 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 RNA aptamer of 10nmol hatches 30min with the fFN protein 37 DEG C of variable concentrations (1-200nM) respectively, each group reaction liquid filters through nitrocellulose filter, washing filter membrane, dry 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 fFN albumen.Result is as follows:
Aptamer specificity analyses and stability analysis described in embodiment 3
As can be seen from the above results, 24 aptamers of the present invention have very strong binding characteristic, can in conjunction with fFN albumen also without the aptamer of described binding characteristic in prior art.
Embodiment 4 degrading activity analysis
Being respectively adopted human albumin, RASAL1 albumen, IgG, hemoglobin and 24 aptamers carry out specific detection, find through binding tests, and these aptamers do not combine with these albumen, and only keep higher specificity with fFN protein binding.
By described aptamer, take 0.3ug, be respectively placed in the serum of room temperature, aqueous solution, place surrounding.Detected by RT-PCR, it has been found that its Stability Analysis of Structures of the placement of surrounding, it does not have be degraded.
Embodiment 6 clinical trial analysis
24 aptamers are respectively used to the clinical sample of the same gravid woman's vaginal secretions of detection, include fetus premature labor group and normal healthy controls group, wherein fetus premature labor group is 50 examples, and normal healthy controls group is 50 examples, and vaginal secretions when puerpera's clinic is started is as sample to be checked.Experimental subject screening standard:
The anemia of pregnant woman in pregnant 22-35 week, takes its vaginal secretions and adds PBS solution and frozen stock solution storage.50 examples of the final premature labor of this anemia of pregnant woman incorporate fetus premature labor group into, and the anemia of pregnant woman of normal mature production incorporates normal healthy controls group into.
100 groups of gravid woman's vaginal secretionies are joined in sterile centrifugation tube, adds PBS solution concussion and dissolve.Coupling have 24 groups of aptamers of magnetic bead and the vaginal secretions solution mixing of 100 groups hatch 20 minutes respectively, then Magneto separate, the fFN albumen of corresponding separation can be obtained, find by detecting, in 50 example fetus premature labor groups, 24 groups of aptamers all detect the fFN albumen of rather high concentration, and are not detected by the fFN albumen of high concentration in healthy group, as can be seen here, it is possible to achieve the Detection results of 100%.There is high accuracy.
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention are also not restricted to the described embodiments; the change made under other any spirit without departing from the present invention and principle, modification, replacement, combination, simplification; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Sequence table
< 110 > Yang Ling swallow
< 120 > mono-kind is used for aptamer and the test kit thereof that in department of obstetrics and gynecology, fetus premature labor diagnoses
〈210〉1
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-1
CTGACCATGCATGAACGGCAAATAAAATCTTCAACACTCCACCAACCTCTTCTACGTGTAACACATTGGTCACGT
〈210〉2
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-2
CTGACCATGCATGAACGGCATCAAAACTCTATACATATTACTCATTCCCAATATTGTGTAACACATTGGTCACGT
〈210〉3
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-3
CTGACCATGCATGAACGGCACCACCAACCTCTTCTACTACATATATTCAAAACTCGTGTAACACATTGGTCACGT
〈210〉4
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-4
CTGACCATGCATGAACGGCACTAATACATTTTACTCCAATATTCATAATACCATAGTGTAACACATTGGTCACGT
〈210〉5
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-5
CTGACCATGCATGAACGGCACAACATCTACCTACCCAAAAATAATTATCCAATTCGTGTAACACATTGGTCACGT
〈210〉6
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-6
CTGACCATGCATGAACGGCAAATTCATTTCATTAACATTCCACATCTATAAAATAGTGTAACACATTGGTCACGT
〈210〉7
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-7
CTGACCATGCATGAACGGCACTTCCAAAAAAACAACATAACATTCACAAAATACTGTGTAACACATTGGTCACGT
〈210〉8
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-8
CTGACCATGCATGAACGGCAACTCAAATTCATCCTTATCCCTCCATCACTACCATGTGTAACACATTGGTCACGT
〈210〉9
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-9
CTGACCATGCATGAACGGCATTCCCCCCCTCATCACATCCCACACCCAAATTCACGTGTAACACATTGGTCACGT
〈210〉10
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-10
CTGACCATGCATGAACGGCACTACCAATCTCTTCCCCCCTCCACACCTCTCCCCTGTGTAACACATTGGTCACGT
〈210〉11
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-11
CTGACCATGCATGAACGGCATATCCTAACATTACACTTTCTTATCCTCATCCATTGTGTAACACATTGGTCACGT
〈210〉12
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-12
CTGACCATGCATGAACGGCATTCCTCCCCAAACAAACTTTTTTACCCTTCACATCGTGTAACACATTGGTCACGT
〈210〉13
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-13
CTGACCATGCATGAACGGCATACCTCAAAACTATTATATTCCTTTATACTATAATGTGTAACACATTGGTCACGT
〈210〉14
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-14
CTGACCATGCATGAACGGCATCTCTTCTATTCTTACAACTCCCATACTTACCACTGTGTAACACATTGGTCACGT
〈210〉15
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-15
CTGACCATGCATGAACGGCAATACATTAACACTCCACTAACTCAAATCTACTAAAGTGTAACACATTGGTCACGT
〈210〉16
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-16
CTGACCATGCATGAACGGCACATACCACTAACTTAATCCTCATTTCTCAATTCCCGTGTAACACATTGGTCACGT
〈210〉17
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-17
CTGACCATGCATGAACGGCACTACACTATACCACATACTAACCACCTCCATTCTAGTGTAACACATTGGTCACGT
〈210〉18
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-18
CTGACCATGCATGAACGGCAACTAATTCCTCATCCTACTATAACCTTTTAACCCTGTGTAACACATTGGTCACGT
〈210〉19
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-19
CTGACCATGCATGAACGGCACCACTCCCTTATCATCTTACATTCCCCCTCAATTCGTGTAACACATTGGTCACGT
〈210〉20
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-20
CTGACCATGCATGAACGGCAACCTCTATAATCTTTATCAACACTAATTCACTAATGTGTAACACATTGGTCACGT
〈210〉21
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-21
CTGACCATGCATGAACGGCATAAAATCCACTTTCCTCCTACATATAACAAACTTTGTGTAACACATTGGTCACGT
〈210〉22
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-22
CTGACCATGCATGAACGGCATCATCTAACATTCCCCCTTTACTATCCCCATCATCGTGTAACACATTGGTCACGT
〈210〉23
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-23
CTGACCATGCATGAACGGCATACATACCAAACATATCATCTCCCCAATATCCATTGTGTAACACATTGGTCACGT
〈210〉24
〈211〉75
〈212〉DNA
< 213 > artificial sequence
〈400〉fFNap-24
CTGACCATGCATGAACGGCATTCCAAAATCTCTCTCCCATATTCCACATCTCCATGTGTAACACATTGGTCACGT
Claims (6)
1. one kind fit, it is characterised in that: can specificity check fetus premature labor.
2. fit as claimed in claim 1, it is characterised in that: it realizes checking fetus premature labor by specific binding fFN.
3. fit as claimed in claim 2, it is characterised in that: its sequence is for including shown in SEQIDNo.1-24 any bar sequence, or the replacement of one or several nucleotide carried out on the basis of this sequence, and retains its activity.
4. the fit application at detection fFN described in claim 1-3.
5. the application in the fit test kit for preparing detection fetus premature labor described in claim 1-3.
6. detecting a test kit for fetus premature labor, it is fit that it includes described in any one of claim 1-3.
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| CN101509917A (en) * | 2007-10-18 | 2009-08-19 | 塞尔维马克二世有限责任公司 | Methods and kits for predicting risk for preterm labor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101509917A (en) * | 2007-10-18 | 2009-08-19 | 塞尔维马克二世有限责任公司 | Methods and kits for predicting risk for preterm labor |
Non-Patent Citations (3)
| Title |
|---|
| 别荔 等: ""100 例胎儿纤维连接蛋白检测在早产预测中的临床应用价值"", 《宁夏医学杂志》 * |
| 张敏青 等: ""观察胎儿纤维连接蛋白检测在早产预测中的临床应用效果"", 《中国实用医药》 * |
| 费嘉: "《小核酸药物开发技术》", 31 August 2011, 军事医学科学出版社 * |
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