CN106367420A - Single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in candida albicans and screening method of single-stranded DNA aptamer and reagent - Google Patents
Single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in candida albicans and screening method of single-stranded DNA aptamer and reagent Download PDFInfo
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Abstract
The invention relates to a single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in candida albicans and a screening method of the single-stranded DNA aptamer and a reagent. The nucleotide sequence of the single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in the candida albicans is shown as SEQ ID NO.1 or SEQ ID NO.2. According to the single-stranded DNA aptamer specifically bonded with (1,3)-beta-D-glucose in the candida albicans, invasive fungal infection can be differentiated and diagnosed, a new idea is provided for differentiation and diagnosis of invasive fungal infection, and a good application prospect is achieved.
Description
Technical field
The present invention relates to molecular microbiology and infection immunity field are and in particular to specifically bind in Candida albicans
(1,3) the single-stranded dna aptamer of-β-d- glucosan and its screening technique and reagent.
Background technology
Deep fungal infection (invasive fungal infection, ifi) be pathological form funguses invade subcutaneous tissue,
Fungal infectious disease caused by mucosa and internal organs.Antibiotic and the lack of standardization of immunosuppressant use and aggressive at present
The widely using of medical procedure, in hospital, the infection rate of deep fungal assumes rising trend, and its high fatality rate also causes medical matters people
The great attention of member.And early diagnosiss and treatment are the effective means reducing deep fungal fatality rate.
Fungal culture is still currently the goldstandard of diagnosis deep fungal infection, difficulty and funguses but deep fungal is drawn materials
Cultivation cycle is longer etc., and factor is all unfavorable for its quick diagnosis；And widely used g test still suffers from some disadvantages at present
End: the acquisition quality of specimen requires higher, some medicines use, strepticemia etc., easily causes false positive.Therefore clinic is urgently
Treat that one kind specificity can differentiate fungal infectious and operate easy new method.
And Candida albicans are the clinical most commonly seen funguses leading to deep infection.And in fungal cell wall glucosan is about
Account for the 60% of fungal cell wall dry weight, and (1,3)-β-d- glucosan accounts for the 84% of glucosan total amount.And in fungal cell wall
(1,3)-β-d- glucosan is the poor polysaccharose substance of water solublity, and sugar antigen immunogenicity is poor, and Antibody preparation is more
Difficulty is it is difficult to be detected with conventional immunological method.
Content of the invention
It is an object of the present invention to proposing a kind of specific binding white that can differentiate, diagnose deep fungal infection
The single-stranded dna aptamer of (1,3)-β-d- glucosan in candidiasises.
Single-stranded dna adaptation according to (1,3)-β-d- glucosan in a kind of specific binding Candida albicans of the present invention
Son, in this specific binding Candida albicans, the nucleotides sequence of the single-stranded dna aptamer of (1,3)-β-d- glucosan is classified as seq
Shown in id no.1 or seq id no.2.
The single-stranded dna of (1,3)-β-d- glucosan in a kind of specific binding Candida albicans according to embodiments of the present invention
Aptamer, it is possible to authenticate, diagnosis deep fungal infection, be that the Differential Diagnosiss of deep fungal infection provide new thinking, have
Good application prospect.
In addition, (1,3)-β-d- glucosan in a kind of specific binding Candida albicans according to the above embodiment of the present invention
Single-stranded dna aptamer, can also have as follows add technical characteristic:
Further, in this specific binding Candida albicans the single-stranded dna aptamer of (1,3)-β-d- glucosan core
Nucleotide sequence is seq id no.1 or nucleotide sequence shown in seq id no.2 through replacement, deletion and/or insertion one or several
The sequence with seq id no.1 or the equal function of seq id no.2 that individual nucleotide is formed.
Further, in this specific binding Candida albicans the single-stranded dna aptamer of (1,3)-β-d- glucosan core
Nucleotide sequence is arranged as core with seq id no.1 or nucleotides sequence shown in seq id no.2, and extends to both sides.
Another object of the present invention is to proposing one kind containing (1,3)-β-d- in above-mentioned specific binding Candida albicans
The reagent of the single-stranded dna aptamer of glucosan.
Another object of the present invention is in a kind of specific binding Candida albicans as described in power 1 for the screening of proposition (1,
3) method of-β-d- glucosan single-stranded dna aptamer.
One kind according to the present invention screens (1,3)-β-d- glucosan single-stranded dna in above-mentioned specific binding Candida albicans
The method of aptamer, comprises the steps: s101: synthesis specific primer, and by the initial single-stranded dna of specific primer synthesis
Library；S102: described initial single-stranded dna library is carried out asymmetric pcr amplification, to obtain single-stranded dna library and double-strand dna literary composition
Storehouse；S103: extract Candida albicans endobacillary (1,3)-β-d- glucosan as Screening target；S104: with described (1,3)-β-
D- glucosan, as Screening target, carries out gradient screening by selex technology, to obtain specifically binding in Candida albicans
(1,3) the single-stranded dna aptamer of-β-d- glucosan；Wherein, described specific primer is: 5 '-gcggaattcgaacagtccga
Gcc-3 ' and ' 5-gcgggatcctatgacgcattgaccc-3 ', described initial single-stranded dna library is: 5 '-
gcggaattcgaacagtccgagcc-n60- gggtcaatgcgtcata-3 ', the n in described initial single-stranded dna library60Represent
60 random nucleotides.
Further, in s103 step, extract (1,3)-β-d- glucosan conduct that molecular weight is 1.3kd～1.5kd
Screening target.
Further, in step s102, carry out asymmetric pcr amplification procedure and include single-stranded dna library degeneration,
The annealing in single-stranded dna library and the extension in single-stranded dna library, and will be predetermined secondary for the annealing process circulation in described single-stranded dna library
Number, to obtain double-strand dna library, and with described double-strand dna library as template, amplifies the single-stranded dna library of next round screening,
Wherein, in each wheel amplification procedure, the cycle-index of the annealing process in described single-stranded dna library is all different.
Further, in step s104, the detailed process of described gradient screening is: takes in the 1st wheel the～the 3 wheel screening
(1,3)-β-d- glucosan of 1 μ g/ml as Screening target, take in the 4th wheel the～the 6 wheel screening (1,3)-β of 0.1 μ g/ml-
D- glucosan, as Screening target, takes (1,3)-β-d- glucosan of 0.01 μ g/ml as sieve in the 7th wheel the～the 9 wheel screening
Select target, take (1,3)-β-d- glucosan of 0.001 μ g/ml as Screening target in the 10th wheel the～the 12 wheel screening, and from
6th wheel screening starts reversely to be screened to the 12nd wheel screening by the blood plasma of healthy human body and polystyrene micropore.
Further, after the 12nd wheel screening, reclaim double-strand dna, and with ecori and bamhi, described double-strand dna is entered
Row double digestion, to obtain digestion products.
The additional aspect of the present invention and advantage will be set forth in part in the description, and partly will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description
Fig. 1 a is to identify schematic diagram through staining for glycogen after (1,3)-β-d- glucosan row sds-page after enzymolysis；
Fig. 1 b is (1,3)-β-d- glucosan traveling wave long scan identification after enzymolysis；
Fig. 1 c and Fig. 1 d is (1,3)-β-d- glucosan after gas chromatography identification enzymolysis；
Fig. 2 is the amplification in double-strand dna library；
Fig. 3 is that 12 wheel pcr expand single-stranded library；
Fig. 4 a is the comparison of different adaptation sublibrary relative binding capacity；
Fig. 4 b is the comparison of 30 monoclonal aptamer relative binding capacity in the 12nd wheel adaptation sublibrary；
Fig. 5 a is the secondary structure of aptamer ad1；
Fig. 5 b is the secondary structure of aptamer au1；
Fig. 5 c is that aptamer au1 and ad1 detects to the competition of target epitope；
Fig. 6 is (1,3)-β-d- glucosan in double aptamer sandwich assay detection deep fungal patients blood plasma；
Fig. 7 is the roc curve of double aptamer sandwich assay detection methods.
Specific embodiment
The present invention is expanded on further with reference to experimental data and specific embodiment.It should be noted that these embodiments
It is only used for explaining the present invention rather than limit the scope of the present invention.
Embodiment 1: the selection of material
Reagent and instrument:
Candida albicans atcc10231 is purchased from Wuhan University's culture classical collection center；Candida albicans bacterium culture medium is purchased from green grass or young crops
Dao Haibo biotech company；Puc19 plasmid, dh5 α strain are frozen by this laboratory；The main agents building for clone
Bamh i, ecor i, t4dna ligase are purchased from fermentas company；It is biological that albumen marker (cat:sm1861) is purchased from precious Ke
Company；Dna marker is purchased from Dongsheng biotech firm；Axyprep plasmid dna small volume of reagent box is purchased from axygen company；Dna coagulates
Glue reclaim test kit is purchased from axyprep company；Pcr mix is purchased from tiangen company；Length scanning instrument (Beckman-Kurt)；
Nucleic acid-protein detector (German eppendorf company)；Ultraviolet transilluminator (uvp company of the bioimaging system U.S.)；Platform
Formula centrifuge, pcr instrument (German eppendorf company)；Single-stranded dna library and primer are synthesized by invitrogen company；Chemistry
It is pure, purchased from traditional Chinese medicines chemical industry that reagent is analysis.
The structure in random single chain dna (ssdna) library and primer synthesis:
The ssdna library that this library is 99 bases for length, two ends are fixed sequence program, and middle 60 nucleotide are stochastic ordering
Row, storage capacity is about 1014～1015.The restriction enzyme site of i containing ecor and bamh i respectively in two primers, random ssdna literary composition
Storehouse and primer are synthesized by invitrogen company.Random ssdna library: 5 '-gcggaattcgaacagtccgagcc-n60-
gggtcaatgcgtcata-3’.Primer 1:5 '-gcggaattc(dashed part is ecori enzyme action position to gaacagtccgagcc-3 '
Point)；Primer 2: ' 5-gcgggatccTatgacgcattgaccc-3 ' (dashed part is bamhi restriction enzyme site).
Specimen origin:
Deep fungal infection blood plasma is provided by Wuhan City medical treatment center and Central-South hospital, Healthy Human Serum specimen by
Central-South Hospital Physical Examination center provides.Wherein deep fungal infection diagnosing patient standard: 1) sputum or blood cultivation fungal culture are
Positive；2) g test is positive；3) there is remarkable result using after antifungal drug, at least conform to above two.Experiment collects 40 altogether
Make a definite diagnosis respiratory system deep fungal infection patients blood plasma (17 tumor patients, 11 HIV sufferers, 9 severe burn patients,
Thoracic sugery postoperative patient 3), the fungemia patient after 12 detained central venous catheters, and fungal species divide be white
Color candidiasises；Collect simple respiratory system, blood circulation, 28 ((Staphylococcus aureus of urinary system bacterial infection patients simultaneously
Bacterium, Pseudomonas aeruginosas, Klebsiella Pneumoniae, escherichia coli) continuous three times, every minor tick one day is simultaneously using fungi culture medium
Identification all no funguses growths) and 34 human normal plasmas are as a control group.
Embodiment 2: extracting method
(1,3) extraction of-β-d- glucosan and identification:
Collect Candida albicans thalline on solid medium, be centrifuged lyophilization after adding a small amount of pbs washing, take 5 grams to do
Dry thalline adds resuspended reaction 3 hours in 90 DEG C of water-baths of 100ml 1m sodium hydroxide；After centrifugation, pure water precipitates 3 times,
Processed 3 hours at 37 DEG C with 4% acetum again, centrifuged deposit thing pure water 3 times, then with absolute ethanol washing 3
Secondary, after ether dehydration, nitrogen dries up and obtains final product product, and after being identified by gas chromatography, freezen protective is standby.Gathered with (1,3)-β-Portugal
Carbohydrase it is carried out enzymolysis increase its water solublity, cut glue reclaim molecular weight in 1.4kd about low polymerization degree water solublity (1,
3)-β-d- glucosan is as Screening target.
Single-stranded dna (ssdna) library and the amplification of double-strand dna (dsdna):
The ssdna amplified library of synthesis is dsdna library: 95 DEG C of denaturations 5 minutes, then carry out 95 DEG C, 36s, 60 DEG C,
36s, 72 DEG C, 30s, totally 15 circulations, 72 DEG C extend 5 minutes, and row 3% agarose gel electrophoresiies are identified；And to expand gained
Dsdna library is template, the asymmetric ssdna library amplifying next round screening of forward primer p1.Reaction condition: 95 DEG C
5min, 95 DEG C of 36s, 60 DEG C of 36s, 72 DEG C of 30s, period often takes turns different, then extension 7min at 72 DEG C.Produce to pcr
The nacl of the 3m of 1/10 volume pre-cooling is added in thing, after mixing, the more anhydrous second adding the ice pre-cooling of 2 times of volumes in system
Alcohol, after fully mixing, is placed in -80 DEG C and precipitates 4 hours, low-temperature centrifugation obtains.
Selex screens:
Take 1 μ g/ml solubility (1,3)-β-d- glucosan 50 μ l to be coated in 96 orifice plates, in 37 DEG C of environment, add salmon essence
Dna closes 2h, adds the single-stranded library containing 1000pm to be dissolved in 50 μ l screening buffer (20mm hepes (ph=after pbs washing
7.4), 120mm nacl, 5mm kcl, 1mm cacl2, 1mm mgcl2·6h2O), 37 DEG C are incubated 1 hour；Centrifugation discards liquid,
After addition screening buffer solution 5 times, add 100 μ l sterilizing distilled waters, heat 5 minutes in 95 DEG C；Drawing supernatant in hole is mould
Plate is conventional pcr, as the dsdna library of next round screening.In order to improve the affinity of screening aptamer, using gradient screening
Mode carry out, that is, as target, the 4th to the 6th takes turns with 0.1 μ g/ the 1st to the 3rd wheel (1,3)-β-d- glucosan of 1 μ g/ml
Ml, the 7th to the 9th takes turns with 0.01 μ g/ml；10th to the 12nd wheel use 0.001 μ g/ml, and from the 6th take turns screening begin through health
Human plasma and polystyrene micropore are reversely screened.Reclaim the 12nd wheel dsdna storehouse and puc19 plasmid ecor i and bamh i
Carry out double digestion, purification reclaims digestion products, is attached reaction using t4 ligase and is placed in 16 DEG C, act on 16 hours.To connect
Thing of practicing midwifery converts to dh5 α, picking monoclonal, extracts plasmid and passes through elona Direct Determination relatively after carrying out enzyme action identification
Adhesion size, and choose the two aptamer sequencings of relative binding capacity highest, as couple candidate detection instrument, use simultaneously
Mfold predicts its secondary structure, and verifies whether it combines same site using competition law.
Detection to clinical blood specimen:
96 microwell plate ultra-vioket radiation 2 hours, are coated aptamer ad1 in 96 microwell plates, every hole 40pmol, 37 DEG C of incubations 1 are little
When；10 μ g/ml salmon essence dna closed porosity plates, 37 DEG C are incubated 1 hour；37 DEG C of test plasma is added to be incubated 1 hour；Add
The aptamer au1 of biotin labeling, every hole 40pmol, 37 DEG C are incubated 1 hour, are eventually adding the chain that horseradish peroxidase couples
Mould Avidin, 37 DEG C of incubation 30min, addition tmb chromogenic substrate, 1 minute about, 2m h2so4Pass through microplate reader after terminating reaction
Reading.
Statistical analysis
Data of the present invention is represented using mean ± sd, and using sas 8.0 software, data is processed.Comparing between group makes
With t inspection, with p < 0.05 for having significant difference.
Embodiment 3: result
(1,3) extraction of-β-d- glucosan, purification and identification:
Extraction (1,3)-β-d- glucosan from Candida albicans, supernatant after processing through (1,3)-β-d- glucanase, OK
Through staining for glycogen after sds-page electrophoresis, find to be concentrated mainly on three regions, < near 1.7kd, 4.6kd, and 10kd～
Between 15kd, and assume continuous distribution, as shown in Figure 1a, arrow show the target substance of this research；This test is mainly led to
Cross cut glue reclaim molecular weight < low polymerization degree (1,3)-β-d- glucosan of 1.7kd be Screening target；Reclaim gained low polymerization degree
(1,3)-β-d- glucosan scans through 190nm～900nm continuous wavelength, visible obvious absorption peaks at 196nm, and 260nm
With equal no obvious absorption peaks at 280nm, institute's extract matter is pointed out to be more pure carbohydrate, as shown in Figure 1 b, arrow institute
It is shown as maximum absorption peak at 196nm；It is followed successively by from left to right shown in arrow in Fig. 1 c: rhamnose, glucose, arabinose, sweet
Dew sugar, galactose and xylose standard product, in Fig. 1 d, arrow show the monosaccharide after hydrolysis.To regenerant metallic substance pass through acidolysis,
Reduction, acylated derivative rear circulation of qi promoting phase chromatography detection, only have single peak value (remaining is miscellaneous peak) in retention time 5.896min, such as
Shown in Fig. 1 d, according to the comparison of Fig. 1 c Plays product gas chromatograph results, dextrose standard sample retention time is 6.07min, two
Person is close, and its fine difference the monosaccharide conformation after derivative may be led to the difference of dextrose standard sample monomer conformation for hydrolysis.
The pcr amplification of dna library and screening product:
The ssdna random library that the initial ssdna random library of chemosynthesis obtains through asymmetric pcr amplification, result shows
Show dsdna storehouse clip size near dna marker molecule 100bp, be consistent with expected clip size (108bp), as Fig. 2 a institute
Show, 1 swimming lane is no template control, m is dna marker, 2 swimming lanes are double-strand dna library.Obtained by the screening of selex triage techniqueses
The 12 wheel ssdna adaptation word banks of (1,3)-β-d- glucosan must be specifically bound, by the dna of each wheel single-stranded adaptation word bank row 3%
Agarose gel electrophoresiies are identified, result shows, between 150bp～200bp, the 9th～12 takes turns front 8 wheel ssdna library sizes
Ssdna aptamer leads to size to change as shown in Figure 2 b because of structure change reason, the 1st to the 12nd wheel ssdna literary composition
Storehouse, m is dna marker, and l is initial ssdna library, and 1～12 swimming lane is respectively the 1st and takes turns to the screening ssdna literary composition of the 12nd wheel
Storehouse.
Each wheel adaptation word bank and the detection of the single aptamer affinity of different clone:
It is coated water solublity (1,3)-β-d- glucosan, tied therewith using the ssdna library of biotinylated each wheel respectively
Close, according to a450nmCompare the size of relative binding capacity, data display is with the increase of screening wheel number, the aptamer of same substance amount
Binding ability is significantly raised, but significantly reduces in the 6th wheel, with the result adding the anti-sieve of blood plasma, but subsequently may still hold rising
Trend, in 11th round, its adhesion reaches peak, and the 12nd wheel tends to plateau, and as shown in fig. 4 a, wherein, b is blank right
According to l is start library.By the 12nd wheel dsdna library clone to puc19 plasmid, after picking monoclonal, measure monoclonal aptamer
With the relative binding capacity of target substance, data display o.11 aptamer (being named as ad1) (is named as with No. 20 aptamer
Au1) there is higher adhesion, as shown in Figure 4 b, wherein, b is blank, c is unrelated aptamer comparison.
Screening aptamer secondary structure and the checking of binding site:
Using http://www.bioinfo.rpi.edu/applications/mfold/ to two aptamers that are sequenced to obtain
Carry out secondary structure to be predicted, as shown in figure 5a and 5b；Simultaneously using two ssdna aptamers and water solublity (1,3)-β-d-
Glucosan competition binding, the addition of data display au1 aptamer combines target no obvious inhibiting effect to ad1 aptamer, points out
The non-binding same site of two aptamers, as shown in Figure 5 c, wherein, bio- represents biotin labeling；Ctrl is unrelated aptamer,
Blank is blank.
Aptamer sandwich assay detection plasma specimen:
It is coated ad1 aptamer, as detection aptamer, result shows that the method can be good at distinguishing not to au1 aptamer
With position deep fungal infection and bacterium infection crowd and healthy population (p < 0.01), as shown in Figure 6；To 62 non-deep fungals
Infection group membership and 52 deep fungal infection group patient's testing results are analyzed using roc curve it is known that this research is set up
Its sensitivity of elona sandwich assay be: 92.31% (95%ci:81.46%-97.86%), specificity is: 91.94%,
(95%ci:82.17%-97.33%), show preferable differentiation effect, as shown in Figure 7.
Conclusion:
Deep fungal sickness rate rises year by year in recent years, and quick and specifically detection fungal infection method is true for reducing
Bacterium fatality rate is of great importance.Existing deep fungal infection diagnostic method is based on g experiment, and comes with some shortcomings, and basic unit
Hospital is still leading with experience.After deep fungal infection, body it is carried out in immunologic process produce different polymerization degree (1,
3)-β-d- glucosan, and itself also there is certain density (1,3)-β-d- Portugal in some gram negative bacterias and body and gather
Sugar.And g test cannot be distinguished by small difference in this kind of material, therefore the method is also affected by multiple factors.The present invention passes through
Extract albicans cell wall specific (1,3)-β-d- glucosan, and this polysaccharide in-vitro simulated is gathered by (1,3)-β-d- Portugal
The process of carbohydrase digestion, thus more really using Candida albicans this polysaccharide distinctive as Screening target.In view of saccharide resists
Former immunogenicity is poor, and therefore the present invention, can be thin with Candida albicans using this technology screening according to the feature of selex technology
The ssdna aptamer of cell wall glucan binding domian, and in the 6th wheel using human plasma as anti-sieve material, through the positive sieve of 12 wheels
The reverse screenings of choosing and 6 wheels, thus eliminate the dry of the interference of (1,3)-β-d- glucan in human plasma and detection equipment
Disturb, obtain relative affinity and higher aptamer ad1 and au1 of specificity.And its secondary structure is carried out point by mfold
Analysis, thus it is speculated that it may be incorporated into different target spots, subsequently and by competion experiment checking draws this conclusion.
The present invention has been carried out to deep fungal infection patients blood plasma and human normal plasma just using double aptamer sandwich assays
Step detection, result shows that the method can significantly distinguish deep fungal infection.This assay is analyzed, its sensitivity
It is superior to existing g experiment and morphological examination with specificity.As shown by data the method has to the detection of deep fungal infection blood plasma
Good separating capacity.And nucleic acid aptamer is as the new identification molecule of a class, it is relatively low that it has a molecular weight, can be by chemistry
Synthetically prepared and structure of modification, and the several functions labelling such as fluorescence, biotin can be carried out, chemical stability is good, can be reversible
Degeneration and renaturation, can at normal temperatures storage and transport the advantages of.These advantages make aptamer have in clinical diagnosises and treatment
There is good application prospect.
And the present invention is detected only for respiratory system and blood circulation candida albicans infection patient, and its
The detection work of deep fungal infection patient other body fluid and other kind funguses and the knot of this aptamer and biosensor
The work such as raising sensitivity will be carried out further to close expectation.
In sum, the present invention successfully screens high-affinity with selex technology and high specific combines white and reads
The ssdna aptamer of pearl bacterium cell wall glucan, is that the Differential Diagnosiss of deep fungal infection provide new thinking, has good
Application prospect.
In describing the invention, term " first ", " second " are only used for describing purpose, and it is not intended that indicating or dark
Show relative importance or the implicit quantity indicating indicated technical characteristic.Thus, " first ", the feature of " second " are defined
Can express or implicitly include one or more this feature.In describing the invention, " multiple " are meant that two
More than individual, unless otherwise expressly limited specifically.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy describing with reference to this embodiment or example
Point is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.And, the specific features of description, structure, material or feature can be in office
Combine in an appropriate manner in one or more embodiments or example.Additionally, in the case of not conflicting, the skill of this area
The feature of the different embodiments described in this specification or example and different embodiment or example can be tied by art personnel
Close and combine.
Although embodiments of the invention have been shown and described above it is to be understood that above-described embodiment is example
Property it is impossible to be interpreted as limitation of the present invention, those of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (9)
1. a kind of single-stranded dna aptamer specifically binding (1,3)-β-d- glucosan in Candida albicans is it is characterised in that be somebody's turn to do
In specific binding Candida albicans, the nucleotides sequence of the single-stranded dna aptamer of (1,3)-β-d- glucosan is classified as seq id
Shown in no.1 or seq id no.2.
2. a kind of single-stranded dna aptamer specifically binding (1,3)-β-d- glucosan in Candida albicans is it is characterised in that be somebody's turn to do
In specific binding Candida albicans, the nucleotides sequence of the single-stranded dna aptamer of (1,3)-β-d- glucosan is classified as seq id
Nucleotide sequence shown in no.1 or seq id no.2 is through having that the one or several nucleotide of replacement, deletion and/or insertion is formed
Seq id no.1 or the sequence of the equal function of seq id no.2.
3. a kind of single-stranded dna aptamer specifically binding (1,3)-β-d- glucosan in Candida albicans is it is characterised in that be somebody's turn to do
In specific binding Candida albicans, the nucleotide sequence of the single-stranded dna aptamer of (1,3)-β-d- glucosan is with seq id
Nucleotides sequence shown in no.1 or seq id no.2 is classified as core, and extends to both sides.
4. a kind of poly- containing (1,3)-β-d- Portugal in the specific binding Candida albicans as described in any one of claims 1 to 3
The reagent of the single-stranded dna aptamer of sugar.
5. (1,3)-β-d- glucosan single-stranded dna aptamer in a kind of specific binding Candida albicans as described in power 1 for screening
Method it is characterised in that comprising the steps:
S101: synthesis specific primer, and by the initial single-stranded dna library of specific primer synthesis；
S102: described initial single-stranded dna library is carried out asymmetric pcr amplification, to obtain single-stranded dna library and double-strand dna literary composition
Storehouse；
S103: extract Candida albicans endobacillary (1,3)-β-d- glucosan as Screening target；
S104: gradient screening, as Screening target, is carried out by selex technology, to obtain with described (1,3)-β-d- glucosan
The single-stranded dna aptamer of (1,3)-β-d- glucosan in specific binding Candida albicans；
Wherein, described specific primer is: 5 '-gcggaattcgaacagtccgagcc-3 ' and ' 5-
Gcgggatcctatgacgcattgaccc-3 ', described initial single-stranded dna library is: 5 '-
gcggaattcgaacagtccgagcc-n60- gggtcaatgcgtcata-3 ', the n in described initial single-stranded dna library60Represent
60 random nucleotides.
6. method according to claim 5 is it is characterised in that in s103 step, extraction molecular weight be 1.3kd～
(1,3)-β-d- glucosan of 1.5kd is as Screening target.
7. method according to claim 5 is it is characterised in that in step s102, carrying out asymmetric pcr amplification procedure
Include the extension of the degeneration, the annealing in single-stranded dna library and single-stranded dna library in single-stranded dna library, and by described single-stranded dna
The annealing process circulation pre-determined number in library, to obtain double-strand dna library, and with described double-strand dna library as template, amplifies
The single-stranded dna library of next round screening, wherein, in each wheel amplification procedure, the circulation of the annealing process in described single-stranded dna library
Number of times is all different.
8. method according to claim 5 is it is characterised in that in step s104, the detailed process of described gradient screening
For: (1, the 3)-β-d- glucosan taking 1 μ g/ml in the 1st wheel the～the 3 wheel screening, as Screening target, is taken turns in the 4th wheel～the 6
(1, the 3)-β-d- glucosan taking 0.1 μ g/ml during screening, as Screening target, takes 0.01 μ g/ in the 7th wheel the～the 9 wheel screening
(1,3)-β-d- glucosan of ml as Screening target, take in the 10th wheel the～the 12 wheel screening (1,3)-β of 0.001 μ g/ml-
D- glucosan is as Screening target, and starts to the 12nd wheel screening to pass through blood plasma and the polyphenyl second of healthy human body from the 6th wheel screening
Alkene micropore is reversely screened.
9. method according to claim 8 is it is characterised in that after the 12nd wheel screening, reclaiming double-strand dna, being used in combination
Ecori and bamhi carries out double digestion to described double-strand dna, to obtain digestion products.
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| CN108663509A (en) * | 2017-06-16 | 2018-10-16 | 江苏诺鬲生物科技有限公司 | A kind of immune colour reagent of label fungi |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108663509A (en) * | 2017-06-16 | 2018-10-16 | 江苏诺鬲生物科技有限公司 | A kind of immune colour reagent of label fungi |
| CN108467866A (en) * | 2017-12-18 | 2018-08-31 | 广州医科大学附属第医院 | A kind of aptamer and its application with the specific binding of (1,3)-callose |
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