CN106399315A - Sequences and application of oligonucleotide aptamer for specific recognition of H1N1-type and H3N2-type influenza A virus - Google Patents
Sequences and application of oligonucleotide aptamer for specific recognition of H1N1-type and H3N2-type influenza A virus Download PDFInfo
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Abstract
The invention discloses A8 and A20 sequences of an oligonucleotide aptamer and truncated sequences of the A8 and A20 sequences for specific recognition of H1N1-type and H3N2-type influenza A virus, and an application of the modified sequences to recognition of H1N1-type and H3N2-type influenza A virus. The invention relates to an application of the A8 and A20 sequences and the truncated sequences of the A8 and A20 sequences to the aspect of a biological medicine. The A8 and A20 sequences and the truncated sequences of the A8 and A20 sequences are taken as a constituent part of a kit or a detection index and are used for patients who are infected with the influenza A virus or for auxiliary diagnosis of the patients and detection of H1N1-type and H3N2-type influenza A virus. The A8 and A20 sequences and the truncated sequences of the A8 and A20 sequences are combined with nanogold technology to be taken as rapid visual detection method and detection kid of H1N1-type and H3N2-type influenza A virus.
Description
Technical field
The invention belongs to molecular biosciences medicine technology field is and in particular to specific recognition Influenza virus H1N1
Oligonucleotides aglucon A8, A20 of type and H3N2 type and its all truncate after sequence and modification, Yi Jixiu
Application in terms of identification Influenza virus H1N1 type and H3N2 type for the sequence after decorations.The present invention relates to
A8, A20 and its application in terms of biological medicine for all truncated sequences.A8, A20 and its all truncate sequence
Row as the part of kit or Testing index, for infect first stream Virus patients or auxiliary examine
Break and Influenza virus H1N1, the detection of H3N2 type.A8, A20 are combined with its all truncated sequence and receive
Rice technology for gold is as H1N1, H3N2 type Flu-A quick visualization detection method and detection kit.
Background technology
The aglucon phyletic evolution technology of index concentration, abbreviation SELEX (Systematic Evolution of Ligands
By EXponential Enrichment) technology is the high flux life nearly more than ten years risen and be developed rapidly
Thing library screening technology.Apply jumbo random oligonucleotide library (ssDNA library and RNA library),
In conjunction with PCR Amplification Technologies, with the oligonucleotides of exponential enrichment and target molecule specific bond, through anti-
Multiple in-vitro screening, amplification, the final oligonucleotides aglucon (aptamer) obtaining is based on space structure and target
Molecule is in the combination of high specific and high-affinity.Due to aptamer have accurate identification, non-immunogenicity,
The advantages of easily external synthesis is with modifying, is also called " artificial substituting antibody ", preclinical medicine, clinical diagnosis with
The aspects such as new drug development have broad application prospects.The composition target SELEX technology of especially rising in recent years,
So that screening is carried out to unknown target molecule being possibly realized, and by introducing abatement screening step, it is obtained in that
In the compound target of two groups of specific recognition there is the aptamer of molecule in difference, and can be using this aglucon in turn to difference
Different target molecule is studied, and this is just exploitation novel molecular probe and identification biomarker, especially swollen
Tumor markers molecule opens new approach.
Influenza virus can be divided three classes according to the antigenicity of its nucleoprotein：The first and second the third, or ABC type.H1N1、
H3N2 type first stream virus is the influenza virus of relatively common human infection, respectively once in Spain and Hong Kong
Influenza epidemic situation is caused to break out.The glycoprotein on influenza A virus surface: hemagglutinin (HA) and neuraminidase (NA)
Often occur antigenic drift and antigen dress to change, so that the effect of influenza vaccines was lost efficacy, thus causing influenza epidemic disease again
The outburst of feelings.Therefore, research can distinguish the aptamers of Alphavirus, can be the inspection of influenza A virus
Survey and treatment provides help.
The present invention is to obtain oligonucleotides aglucon A8, A20 by SELEX technology.Identified, two sequences
Row can specific recognition H1N1, H3N2 type first stream virus, and do not combine other correlated virus, compare core
Acid sequence is all no combined with above-mentioned virus.Truncated on this basis, find truncated sequence A8S, A8S8,
A8SSS, A20S are consistent with A8, A20 characteristic with A20S8 sequence, and adhesion is higher.
By association colloid technology for gold it is achieved that the quick visualization for detection H1N1, H3N2 type detects.
So that oligonucleotides aglucon mark collaurum has speed faster, detection target takes shorter this labeling method
Advantage.The effective detection to H1N1, H3N2 type first stream virus is can achieve in one minute.
Content of the invention
It is an object of the invention to provide oligonucleotides A8, A20 and its all truncated sequence as H1N1,
The specific aptamers of H3N2 type first stream virus, and dividing as detection H1N1, H3N2 type first stream virus
Sub- mark.
The present invention also aims to providing a kind of non-diagnostic purpose H1N1, H3N2 type first stream Viral diagnosis side
Method (ELISA method).Also include A8, A20 and its all truncated sequence as the part of kit
Or Testing index is used for the quick visualization to H1N1, H3N2 type first stream virus for the aptamers association colloid gold
Detection.
Oligonucleotide sequence A8, A20 and its truncated sequence A8S, A8S8, A8SSS, A20S and A20S8
SEQ ID No.1 in sequence such as sequence table, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4,
SEQ ID No.5, SEQ ID No.6, shown in SEQ ID No.7.
Brief description
Fig. 1 is A8, A20 oligonucleotide sequence and H1N1, H3N2 obtaining after SELEX screening
Type first stream virus, and the adhesion comparison diagram of second stream, parainfluenza 123 type.
Fig. 2 is oligonucleotide sequence A8, A20 and its truncated sequence A8S, A8S8, A20S and A20S8
The sequence adhesion comparing result viral with H1N1, H3N2 type first stream.
Fig. 3 is oligonucleotide sequence A8, A20 and its truncated sequence A8S8, A20S and A20S8 sequence
Measure Dependence Results with the KD value of H1N1, H3N2 type first stream virus.
Fig. 4 is the result that association colloid gold quick visualization detects H1N1, H3N2 type first stream virus.
Specific embodiment
The present invention will be further described with specific embodiment below in conjunction with the accompanying drawings.
Following examples unaccounted experimental procedure reference《Molecular Cloning:A Laboratory guide》The third edition.
Embodiment 1 A8, A20 oligonucleotide sequence and H1N1, H3N2 type first stream virus, and second stream,
The adhesion contrast of parainfluenza 123 type
(1) Bio-A8, Bio-A20 sequence of synthesizing biotinylated mark.
(2) influenza virus is mixed in respectively in the carbonate buffer solution of pH 9.7, adds according to 100 μ l/ holes
Enter in enzyme bracing, 4 DEG C are coated overnight.
(3) abandon and be coated liquid, every hole adds the confining liquid of 100 μ l, 30min closed by 37 DEG C of incubators.
(4) Bio-A8, Bio-A20 sequence is dissolved in incubation buffer (1 × PBS-5mmolMgCl2),
It is immediately placed on sufficiently cool on ice after 100 DEG C of denaturation 5min.
(5) sequence after degenerative treatments adds in enzyme bracing, makes nucleotide sequence common with coated virus
37 DEG C of incubation, 30min.
(6) discard in the hole liquid, every hole is washed with the cleaning solution of 200 μ l, repeated washing 5 times, last
After secondary washing, in the hole liquid is blotted completely.
(7) every hole adds 100 μ l according to 1: the 300 HRP enzyme having diluted, 37 DEG C of incubation 30min, abandons
Remove in the hole liquid, wash plate 5 times, method is ibid.
(8) every hole adds 100 μ l TMB chromogenic substrates, 37 DEG C of lucifuges colour developings, when there being obvious color change
When, plus 100 μ l terminate liquids, enzyme connection instrument OD450nm wavelength readings.
The calculating implementation of the adhesion contrast of truncated sequence and KD value is identical with this method.
Fig. 1 result shows, A8, A20 sequence and H1N1, H3N2 type first stream virus have specific binding,
And with second stream, parainfluenza 1,2,3 type no combines.(B5 sequence is negative control)
Fig. 2 result shows, the truncated sequence of truncated sequence A8S, A8S8 and A20 sequence of A8 sequence
A20S, A20S8 all have specific binding with H1N1, H3N2 type first stream virus.(B5 sequence is negative right
According to)
Fig. 3 result shows, the input of the different oligonucleotide sequences knot viral with H1N1, H3N2 type first stream
Graph of a relation with joint efforts, calculates the balance dissociation that A8 sequence is combined with H1N1, H3N2 type first stream virus
Constant KD value, about 23.98 ± 6.387nmol/L, 21.08 ± 5.112nmol/L；A8S8 and H1N1,
The equilibrium dissociation constant KD value that H3N2 type first stream virus combines, about 4.746 ± 2.860nmol/L, 11.34
±7.686nmol/L；The equilibrium dissociation constant KD that A20 sequence is combined with H1N1, H3N2 type first stream virus
Value, about 42.44 ± 15.36nmol/L, 30.65 ± 11.32nmol/L；A20S and H1N1, H3N2 type first
The equilibrium dissociation constant KD value that stream virus combines, about 5.559 ± 4.141nmol/L, 5.838 ± 3.937
nmol/L；The equilibrium dissociation constant KD value that A20S8 is combined with H1N1, H3N2 type first stream virus, about
7.657±3.397nmol/L、6.175±3.018nmol/L.
Embodiment 2 association colloid gold quick visualization detection H1N1, H3N2 first stream virus
(1) 25 μ L colloidal gold solutions are mixed with the 75 μ L aqueous solution, be transferred to suitable pH.
(2) by the oligonucleotide sequence truncating A8SSS degenerative treatments, it is subsequently added in colloidal gold solution
It is marked.
(3) target H1N1, H3N2 type first stream virus and H7N9, second stream and unrelated protein are added.
(4) after in incubation 1min, add developer, naked eyes contrast color change can get testing result.
Fig. 4 result shows, A is control wells, and B is H1N1 type first stream virus hole, and C is H3N2 type first stream virus
Hole, D is H7N9 type first stream virus hole, and E is second stream virus hole, and F is unrelated protein hole.Oligonucleotides sequence
The colloidal gold solution of row mark effective detection can go out H1N1, H3N2 type first stream virus, and to other targets
No cross reaction.
Claims (4)
1. oligonucleotides aglucon A8, A20 of a kind of specific recognition H1N1, H3N2 type first stream virus and its all sequence truncating it is characterised in that
Described nucleotide sequence is as shown in SEQ ID No.1-SEQ ID No.7 in sequence table.
2., according to method described in claim 1, oligonucleotides aptamers A8, A20 and its all truncated sequence can be iii vitro chemical synthesis,
Can be by PCR or the preparation of other molecular biology methods.
3. according to method described in claim 1,5 ' ends of oligonucleotides aptamers A8, A20 and its all truncated sequence or 3 ' ends can through FITC,
Biotin, digoxin etc. mark.
4., according to method described in claim 1, oligonucleotides aptamers A8, A20 and its all truncated sequence are in H1N1, H3N2 type first stream virus
Detection in application, and oligonucleotides aptamers A8, A20 and its all truncated sequences are used as first stream virus detection kit component institute
The first stream virus detection kit constituting.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109666765A (en) * | 2019-01-25 | 2019-04-23 | 华侨大学 | A kind of kit of quick diagnosis H1N1 influenza virus |
| CN110297028A (en) * | 2018-03-21 | 2019-10-01 | 首都师范大学 | A kind of electrochemical sensor detecting H1N1 influenza virus and its preparation and detection method |
| CN110592280A (en) * | 2019-09-09 | 2019-12-20 | 华侨大学 | Kit and detection method for rapidly diagnosing H1N1 influenza virus based on double-aptamer RCA technology |
| CN111693712A (en) * | 2020-04-02 | 2020-09-22 | 苏州大学 | Method for detecting new coronavirus SARS-CoV-2N protein by adopting aptamer |
| CN112067802A (en) * | 2019-05-25 | 2020-12-11 | 首都师范大学 | H1N1 influenza virus detection method and kit thereof |
| CN114854760A (en) * | 2022-06-27 | 2022-08-05 | 华侨大学 | DNA aptamer of mouse antibody and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101818207A (en) * | 2009-10-26 | 2010-09-01 | 中华人民共和国珠海出入境检验检疫局 | Detection method and detection kit of influenza A virus, H1N1 and H3N2 subtype influenza virus |
| CN102121009A (en) * | 2010-12-09 | 2011-07-13 | 中国科学院化学研究所 | Nucleic acid aptamer specifically combined with hepatitis C virus core protein and application thereof |
| CN102768280A (en) * | 2011-05-06 | 2012-11-07 | 中国科学院上海生命科学研究院 | Influenza A virus subtype detection antigen chip, its preparation method and application thereof |
-
2015
- 2015-07-31 CN CN201510460657.0A patent/CN106399315A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101818207A (en) * | 2009-10-26 | 2010-09-01 | 中华人民共和国珠海出入境检验检疫局 | Detection method and detection kit of influenza A virus, H1N1 and H3N2 subtype influenza virus |
| CN102121009A (en) * | 2010-12-09 | 2011-07-13 | 中国科学院化学研究所 | Nucleic acid aptamer specifically combined with hepatitis C virus core protein and application thereof |
| CN102768280A (en) * | 2011-05-06 | 2012-11-07 | 中国科学院上海生命科学研究院 | Influenza A virus subtype detection antigen chip, its preparation method and application thereof |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110297028A (en) * | 2018-03-21 | 2019-10-01 | 首都师范大学 | A kind of electrochemical sensor detecting H1N1 influenza virus and its preparation and detection method |
| CN110297028B (en) * | 2018-03-21 | 2021-08-17 | 首都师范大学 | Electrochemical sensor for detecting H1N1 influenza virus and preparation and detection methods thereof |
| CN109666765A (en) * | 2019-01-25 | 2019-04-23 | 华侨大学 | A kind of kit of quick diagnosis H1N1 influenza virus |
| CN112067802A (en) * | 2019-05-25 | 2020-12-11 | 首都师范大学 | H1N1 influenza virus detection method and kit thereof |
| CN112067802B (en) * | 2019-05-25 | 2022-09-30 | 首都师范大学 | H1N1 influenza virus detection kit |
| CN110592280A (en) * | 2019-09-09 | 2019-12-20 | 华侨大学 | Kit and detection method for rapidly diagnosing H1N1 influenza virus based on double-aptamer RCA technology |
| CN111693712A (en) * | 2020-04-02 | 2020-09-22 | 苏州大学 | Method for detecting new coronavirus SARS-CoV-2N protein by adopting aptamer |
| CN111693712B (en) * | 2020-04-02 | 2023-05-02 | 苏州大学 | Method for detecting SARS-CoV-2N protein of new coronavirus by using nucleic acid aptamer |
| CN114854760A (en) * | 2022-06-27 | 2022-08-05 | 华侨大学 | DNA aptamer of mouse antibody and application thereof |
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