CN105483133A - Staphylococcus aureus toxic shock syndrome toxin-1(TSST-1) aptamer T-7 and preparation method and application thereof - Google Patents
Staphylococcus aureus toxic shock syndrome toxin-1(TSST-1) aptamer T-7 and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a staphylococcus aureus toxic shock syndrome toxin-1(TSST-1) aptamer T-7 and a preparation method and application thereof. The aptamer T-7 is of the stem-loop structure as the secondary structure; the stems thereof are composed by pairing of G=C and are connected with loops in different size. In the method, the in-vitro SELEX (Systematic Evolution of Ligands by Exponential Enrichment) selection technology for the aptamer is utilized, carboxyl magnetic beads are employed as solid-phase media, and the TSST-1 is taken as a target; the toxic shock syndrome toxin-1 aptamer which is selected from ssDNA library by means of the TSST-1 carboxyl magnetic beads can be applied to reagent purifying and testing in preparation of the staphylococcus aureus toxic shock syndrome toxin-1 and applied to research and preparation of drugs for treating diseases related to the staphylococcus aureus toxic shock syndrome toxin-1, can be combined with the TSST-1 in a high-affinity and high-specificity way, and can be used as potential antagonist for inhabiting activity of the TSST-1.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of Staphylococcus aureus toxic shock toxin-1 aptamer T-7 and its preparation method and application.
Background technology
Streptococcus aureus produces toxic shock toxin-1 (toxicshocksyndrometoxin-1, TSST-1) and causes toxic shock syndrome (toxicshocksyndrome, TSS).Toxic shock syndrome is got involved because of multiple organ, and to have a fever, ypotension, exfoliative dermexanthesis, diarrhoea, vomiting be principal character, and finally cause shock and multiple organs failure, case fatality rate is high and extremely pay attention to.Domesticly reported that mumps, nasal cavity are postoperative, after serious burn and transfusion to occur together the infection of S. aureus L-forms or the pollution of TSST-1 and toxic shock syndrome, TSS occurs.
TSST-1 is the extracellular toxin produced by phage I group of streptococcus aureus, is a kind of polypeptide protein, belongs to pyrogenicity superantigen family, have very strong superantigen activity.TSST-1 be one by the tightly packed kidney shape molecule of Liang Ge subunit (A, B), its A subunit is the active centre of toxin, it determines the pathogenic and mode of action of toxin, cellulotoxic effect is played by acting on intracellular target spot, B subunit can be combined by the specific receptors on target cell, and it determines that toxin is to the selection affinity of host cell.
TSST-1 is without antigen presenting cell process, directly be attached to the outside of cell surface MHC II (MHC II) quasi-molecule antigen engagement groove, this combination is nonrestrictive, the mixture formed is combined with the β chain V district of t lymphocyte antigen receptor (TCR), activated T lymphocytes makes it activate, breeds, and discharge a large amount of inflammatory cytokines and cause strong immunne response, finally cause the infringement of uncontrolled inflammation and multiple organ.
Aptamers (Aptamer) is a kind of oligonucleotide sequence (DNA or RNA) obtained through in-vitro screening technology.Combinative part is had to the avidity of strict recognition capability and height, this is some secondary structures due to single stranded oligonucleotide, as hair clip, stem ring, false joint, bulge loop, the G-tetramer etc., nucleic acid molecule can be made to form multiple three-dimensional structure, become the basis that aptamers is combined with target material specific region, and both produce efficiently special bonding force mainly through interaction forces such as the accumulation of " false base pair ", hydrogen bond action, electrostatic interaction and form fit.Therefore, an aptamers can distinguish closely related hypotype or different identical molecular conformation states.Oligonucleotide aptamers is by the chemosynthesis of external generation, makes it chemical modification or adds fluorescence dye to control its stability, for laboratory diagnosis or clinical treatment.Aptamers outstanding feature is that its action target spot is extensive, non-immunogenicity, host's adaptability are good, tissue permeability is strong, is therefore a kind of rising drug molecule, can be used for the generation evolution of direct interference disease.Due to aptamer be combined with protein-specific after often can the function of arrestin, and its lacks immunogenicity, and in body, seepage force is strong, is therefore a kind of rising drug molecule, can be used for the generation evolution of direct interference disease.By aptamer and TSST-1 specific binding, and carry out structural analysis, sequence alterations and modify optimizing to it, reach the object that aptamers suppresses TSST-1 function, potential applicability in clinical practice is wide.
Summary of the invention
Staphylococcus aureus toxic shock toxin-1 aptamer T-7 that the object of the present invention is to provide a kind of high-affinity, high specific and its preparation method and application.
Object of the present invention is achieved through the following technical solutions: a kind of Staphylococcus aureus toxic shock toxin-1 aptamer T-7, and the sequence of this aptamer T-7 is as follows:
tgcgtgtgtagtgtgtctgtgggccaggtccctattataaataactcttagggatttggg60
cgg63
The secondary structure of described aptamer T-7 is loop-stem structure, and wherein stem is many is composed of by G ≡ C, and connects ring not of uniform size, and its molecular structure as shown in Figure 1.
The preparation method of described Staphylococcus aureus toxic shock toxin-1 aptamer T-7, is obtained by following method:
Adopt the external SELEX triage techniques of aptamer, utilizing carboxyl magnetic bead as solid-phase media, take TSST-1 as target, is screened obtain toxic shock toxin-1 aptamers by TSST-1 carboxyl magnetic bead from ssDNA library.
Described ssDNA library, two ends are the immobilized primer sequence of 19nt, and middle 25nt is stochastic sequence:
5'-TGCGTGTGTAGTGTGTCTG-N25-CTCTTAGGGATTTGGGCGG-3'。
Screening is taken turns, by cloning and sequencing and sequential analysis through 8.
By measuring combined with fluorescent intensity, compare order-checking aptamers and target in conjunction with situation, find that described aptamers T-7 has stronger bonding force, and by the avidity of fluorescent quantitation analysis aptamers and specificity.
Described Staphylococcus aureus toxic shock toxin-1 aptamer T-7, is characterized in that: carry out the chemically modifieds such as FAM, Biotin, amino to the 5'-of aptamer T-7 end or 3'-end.
The application of described Staphylococcus aureus toxic shock toxin-1 aptamer T-7,
1) application of described Staphylococcus aureus toxic shock toxin-1 aptamer T-7 in the purifying preparing Staphylococcus aureus toxic shock toxin-1 and detection reagent.
2) application of described Staphylococcus aureus toxic shock toxin-1 aptamer T-7 in the medicine of development treatment Staphylococcus aureus toxic shock toxin-1 relative disease.
Compared to prior art, the invention has the advantages that: the present invention by SELEX method screening obtain aptamer T-7 can high-affinity, be combined with TSST-1 with high specificity, wherein Kd value is 103.8nM, therefore this aptamer T-7 can be used as a kind of potential antagonist suppressing TSST-1 activity, and has application prospect in the treatment and diagnosis of infection of staphylococcus aureus.
Accompanying drawing explanation
Fig. 1 is the secondary structure of aptamer T-7.
Fig. 2 is the specificity analyses result of aptamer T-7.
Fig. 3 is the avidity detected result of aptamer T-7.
Fig. 4 is that CCK-8 method measures aptamers T-7 to the effect of the PBMC proliferation activity that TSST-1 induces.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and the specific embodiments, such scheme is described further.But should not be construed as limitation of the present invention.Under the prerequisite not deviating from the present invention's spirit and essence, modification made for the present invention or replacement, all belong to category of the present invention.
A kind of Staphylococcus aureus toxic shock toxin-1 aptamer T-7, the sequence of this aptamer T-7 is as follows:
tgcgtgtgtagtgtgtctgtgggccaggtccctattataaataactcttagggatttggg60
cgg63
As shown in Figure 1: the structure of described aptamer T-7 is: based on loop-stem structure, wherein stem is many is composed of by G ≡ C, and connects ring not of uniform size.
The preparation method of described Staphylococcus aureus toxic shock toxin-1 aptamer T-7, is obtained by following method:
Adopt the external SELEX triage techniques of aptamer, utilizing carboxyl magnetic bead as solid-phase media, take TSST-1 as target, and from ssDNA library, screened the aptamer obtaining TSST-1 by TSST-1 carboxyl magnetic bead, concrete preparation method as described in Example 1.
Embodiment 1 in-vitro screening toxic shock toxin-1 aptamer
1) get magnetic bead DynabeadsM-270CarboxylicAcid NaOH solution and wash 3 times, then use ddH
2o washes 3 times, and the EDC solution adding precooling is put shaking table in room temperature and hatched 30min, abandons supernatant, with the ddH of precooling
2o washes 1 time.
2) with MES solution, TSST-1 is diluted to 60 μ g/100 μ L, the magnetic bead of equal-volume and activation mixes, and room temperature shaker hatches 30min.Tris solution (50mM) washes 3 times.
3) magnetic bead in conjunction with TSST-1 hatches 15min in Tris solution, abandons supernatant, is resuspended in storage liquid stand-by.
4) external synthesizing single-stranded DNA library, two ends are the immobilized primer sequence of 19nt, and middle 25nt is stochastic sequence:
5'-TGCGTGTGTAGTGTGTCTG-N25-CTCTTAGGGATTTGGGCGG-3', storage capacity is 10
15.Primer P1:5'-TGCGTGTGTAGTGTGTCTG-3', primer P2:5'-CCGCCCAAATCCCTAAGAG-3', primer P3:5'-FAM-TGCGTGTGTAGTGTGTCTG-3', primer P4:5'-Bio-CCGCCCAAATCCCTAAGAG-3'.Above nucleotide sequence all entrust biotech company synthesize and through HPLC purifying.
5) with selecting damping fluid (100mMNaCl, 50mMTrisHCl, 5mMKCl, 1mMMgCl
2) ssDNA pool is dissolved in EP pipe, 95 DEG C of sex change 5min, ice bath 10min at being placed at 0 DEG C immediately.
6) ssDNA and bag are mixed by the magnetic bead of TSST-1,37 DEG C of shaking tables hatch 1h, inhale and abandon supernatant.
7) damping fluid (100mMNaCl, 50mMTrisHCl, 5mMKCl, 1mMMgCl is selected with washing
2, 0.02g/LBSA) and wash away unconjugated ssDNA, add ddH
2o, 100 DEG C of heating 5min, be separated supernatant as template, above downstream primer P3 and P4 carries out pcr amplification, and the concentration of upstream and downstream primer P3 and P4 is 10 μMs:
8) get supernatant liquor as template, optimize PCR condition, PCR reaction conditions is: 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, and 56 DEG C of annealing 30s, 72 DEG C extend 40s, and carry out 25 circulations, 72 DEG C extend 5min again.
9) PCR primer purifying: the solution of the DNA fragmentation after amplification TEBuffer is adjusted volume to 100 μ l, adds the BindingBufferI mixing of 10 times of volumes.Transferred to by mixed solution in adsorption column, room temperature places the centrifugal 2min of 2min, 8,000rpm.Outwell the liquid in collection tube, adsorption column is put into same collection tube.500 μ lWashSolution are added, the centrifugal 1min of 10,000rpm in adsorption column.Repeated washing once.Outwell the waste liquid in collection tube, adsorption column is put into same collection tube, the centrifugal 2min of 10,000rpm.Adsorption column is put into clean 1.5ml centrifuge tube, add 50 μ lElutionBuffer in adsorption film central authorities, room temperature leaves standstill 5min, the centrifugal 1min of 12,000rpm.
10) the purifying Bio-dsDNA using P3 and P4 as primer amplification and DynabeadsM-280Streptavidin shaking table are in conjunction with 20min, supernatant is abandoned in suction, add freshly prepared NaOH solution 37 DEG C and hatch 30min, dsDNA alkaline denaturation is made to be ssDNA, again be separated on reset and add wait amount of substance HCl solution neutralize, measure its concentration, obtain next round library.
11) the 8th take turns screening after, screening product P1 and P2 is carried out increase, purifying, carries out TA clone with pEASY-T1SimpleCloningKit.
12) random picking positive colony carries out Zengjing Granule, serves Hai Shenggong biotechnology company limited and carries out sequencing analysis.Use the homology of its primary structure of DNAMAN7.0 software analysis and simulate secondary structure.
13) by measuring combined with fluorescent intensity, compare order-checking aptamers and target in conjunction with situation.We find the stronger bonding force that aptamers T-7 (see Fig. 2) has.
Embodiment 2 fluorescent quantitation detects the specificity of aptamers T-7 and toxic shock toxin-1
1) iii vitro chemical synthesis obtains the fluorescently-labeled aptamers dilution of FAM is 150nM.
2) aptamers 95 DEG C of denaturation reaction 10min, then place cooled on ice 5min rapidly.
3) bag is hatched 1h by candidate's aptamers lucifuge that the magnetic bead of TSST-1 and BSA marks with FAM respectively, using Magnetic particles as blank, 0.1%PBST washes 3 times.
4) analysis of TBS-380 fluorescent quantitation instrument is used to combine the fluorescence intensity of front and back aptamers.
5) each data make parallel three samples.
The fluorescence intensity level of mensuration is made interpretation of result figure by GraphPadPrismv5.0 software, as shown in Figure 2, X-coordinate is respectively blank Magnetic particles, BSA, TSST-1, ordinate zou is average fluorescent strength, respectively organizes fluorescence intensity level visible: the fluorescence intensity that aptamers T-7 and TSST-1 combines is significantly higher than the combination with Magnetic particles and BSA.
Embodiment 3 fluorescent quantitation detects the avidity of aptamers T-7
1) iii vitro chemical synthesis obtains the fluorescently-labeled aptamers of FAM.
2) aptamers of 0nM, 62.5nM, 125nM, 250nM, 500nM gradient concentration and target protein TSST-1 is used to measure dissociation constant Kd.
3) will aptamers 95 DEG C of denaturation reaction 10min of each concentration be prepared, then place cooled on ice 5min rapidly.
4) bag is hatched 1h by candidate's aptamers lucifuge that the magnetic bead of TSST-1 and different concns FAM mark, 0.1%PBST washes 3 times.
5) TBS-380 fluorescent quantitation instrument is used to detect the fluorescence intensity change combining front and back aptamers.
6) each data make parallel three samples.
The fluorescence intensity level of mensuration is made interpretation of result figure by GraphPadPrismv5.0 software, as shown in Figure 3, X-coordinate is the concentration (nmol/L) of aptamers T-7, ordinate zou is average fluorescent strength, and use the dissociation curve of GraphPadPrismv5.0 software matching aptamers T-7, measure its Kd value for 103.8nM.
Embodiment 4 aptamers is to the effect of TSST-1 superantigen activity
1) healthy volunteer's venous blood 2ml is extracted with disposal vacuum anticoagulant blood-collecting pipe.
2) aseptically, anticoagulation is taken out and is placed in 15ml centrifuge tube, then add equivalent D-Hank ' s solution and fully mix.
3) isopyknic human lymphocyte parting liquid is added in 15ml centrifuge tube.
4) slowly add the blood of equivalent 1:1 dilution above parting liquid along tube wall, keep two liquid level interfaces clear.
5) the centrifugal 20min of horizontal 800g.
6) collect interface middle white and be rich in PBMCs layer in another 15ml centrifuge tube.
7) appropriate D-Hank ' s solution mixing washing is added, the centrifugal 20min of 500g.
8) add appropriate D-Hank ' s solution after supernatant discarded again and wash silk ribbon once, the centrifugal 20min of 500g.
9) supernatant discarded, gained cell precipitation is then PBMCs.
10) 1ml is diluted to the RPMl-1640 containing 10% calf serum, by hemocyte instrument counting PBMCs.
11) human PBMC s is inoculated 10
53 multiple holes, in 96 well culture plates, are done in individual/hole (100 μ l).
12) adding TSST-1 final concentration is 250ng/ml, adds different concns aptamers simultaneously and makes its final concentration be 5 μMs, 10 μMs.
13) control wells not adding aptamers is set.
14) 37 DEG C, 5%CO is put
224h is cultivated under saturated humidity.
15) 10 μ lCCK-8 are added.
16) cultivate 4h, application enzyme plate automatic reading instrument measures 450nm absorbancy, and result represents with the average of 3 multiple hole OD values.
Respectively organize OD value by GraphPadPrismv5.0, as shown in Figure 4, X-coordinate is the concentration of aptamers T-7, and ordinate zou is the OD value at 450nm place.When aptamers is 10 μMs, TSST-1 stimulates the proliferation water aobvious reduction (P < 0.05) dawn of PBMCs, having restraining effect to the superantigen activity of TSST-1 during aptamers T-7 tests in vitro, is a kind of potential TSST-1 inhibitor.
The present invention includes but be not limited to above embodiment, every any equivalent replacement of carrying out under the spirit and principles in the present invention or local improvement, all will be considered as within protection scope of the present invention.
<110> Fuzhou General Hospital, Nanjing Military Area, PLA
<120> Staphylococcus aureus toxic shock toxin-1 aptamer T-7 and its preparation method and application
<130>2015
<160>1
<170>PatentInversion3.3
<210>1
<211>63
<212>DNA
<213> synthetic
<400>1
tgcgtgtgtagtgtgtctgtgggccaggtccctattataaataactcttagggatttggg60
cgg63
Claims (7)
1. Staphylococcus aureus toxic shock toxin-1 aptamer T-7, is characterized in that: the sequence of this aptamer T-7 is as follows:
tgcgtgtgtagtgtgtctgtgggccaggtccctattataaataactcttagggatttggg60
cgg63
2. Staphylococcus aureus toxic shock toxin-1 aptamer T-7 according to claim 1, is characterized in that: the secondary structure of described aptamer T-7 is loop-stem structure, and its molecular structure as shown in Figure 1.
3. Staphylococcus aureus toxic shock toxin-1 aptamer T-7 according to claim 1 and 2, is characterized in that: carry out FAM, Biotin, amino chemically modified to the 5'-of aptamer T-7 end or 3'-end.
4. Staphylococcus aureus toxic shock toxin as claimed in any of claims 1 to 3
The preparation method of-1 aptamer T-7, is characterized in that, is obtained by following method:
Adopt the external SELEX triage techniques of aptamer, utilizing carboxyl magnetic bead as solid-phase media, take TSST-1 as target, is screened obtain toxic shock toxin-1 aptamers by TSST-1 carboxyl magnetic bead from ssDNA library.
5. the preparation method of Staphylococcus aureus toxic shock toxin-1 aptamer T-7 according to claim 4, is characterized in that: described ssDNA library, and two ends are the immobilized primer sequence of 19nt, and middle 25nt is stochastic sequence:
5'-TGCGTGTGTAGTGTGTCTG-N25-CTCTTAGGGATTTGGGCGG-3'。
6. the application of Staphylococcus aureus toxic shock toxin-1 aptamer T-7 as claimed in any of claims 1 to 3, is characterized in that: the application in the purifying preparing Staphylococcus aureus toxic shock toxin-1 and detection reagent.
7. the application of Staphylococcus aureus toxic shock toxin-1 aptamer T-7 as claimed in any of claims 1 to 3, is characterized in that: the application in the medicine of development treatment Staphylococcus aureus toxic shock toxin-1 relative disease.
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