CN104388434B - A kind of nucleic acid aptamer of combination CT-HRP method and its application - Google Patents
A kind of nucleic acid aptamer of combination CT-HRP method and its application Download PDFInfo
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- CN104388434B CN104388434B CN201410588833.4A CN201410588833A CN104388434B CN 104388434 B CN104388434 B CN 104388434B CN 201410588833 A CN201410588833 A CN 201410588833A CN 104388434 B CN104388434 B CN 104388434B
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- aptamer
- ssdna
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Abstract
The present invention relates to a kind of oligonucleotide aptamer of energy specific bond CT-HRP method and application.The aptamer is the single strand dna of a 81 base length, with the nucleotide sequence described in SEQ ID No.1 in sequence table.The aptamer is obtained using phyletic evolution index concentration technology screening, available for the detection reagent for preparing cholera enterotoxin, is had the advantages that stable, easy, quick, economical.
Description
Technical field
The invention belongs to field of biological detection, and in particular to a kind of specific high-affinity combination cholera enterotoxin B of energy is sub-
The nucleic acid aptamer of unit, and its application in cholera enterotoxin detection.
Background technology
Cholera (Cholera) is a kind of severe intestinal infectious disease caused by O1 groups and the infection of O139 group cholera vibrios, is
With fall ill it is anxious, propagate it is fast, involve that scope is wide, one of international quarantine infectious disease for being characterized of being very popular can be caused.China is cholera
Class A Notifiable disease is classified as, is the infectious disease of frontier port emphasis quarantine.Comma bacillus is mainly drawn by its toxin secreted
Play the infected's acutely clinical symptoms such as diarrhoea and vomiting.Cholera enterotoxin (cholera enterotoxin, CT) is comma bacillus
Topmost one kind in exotoxin, is also the major virulent factor for causing cholera.It is by an A subunits (cholera
Enterotoxin subunitA, CT-A) and 5 B subunits (cholera enterotoxin subunit B, CT-B) groups
Into.B subunits are non-toxin proteins, are the parts that toxin is combined with host cell receptor, and mediation CT-A enters cell.Cholera intestines
It is popular with important clinic and epidemiological significance that the detection pair of toxin determines whether comma bacillus causes.
Traditional comma bacillus method, which needs first to separate, to be further cultured for, and is then identified with classical method, time-consuming, insensitive to be
These method common problems.Immunological method simply, conveniently, rapidly, specificity preferably, but still have cross reaction ratio
The weak point such as more serious, false positive is more, sensitivity is relatively low.PCR (PCR) though technology have it is accurate, sensitive,
Quick the characteristics of, but its operation in detection number more sample is more numerous and diverse, therefore in PCR (PCR) skill
Many novel polymeric PCR (PCR) technologies are derived on the basis of art again, such as multiple PCR technique, though but multiplex PCR
Simplify the operation of PCR experiment, but be due to the technology need it is several to primer while expanding, it is easy to produce non-specificity bar
Band or false positive, influence testing result.And not yet have the report detected specifically designed for cholera enterotoxin at present.
Nucleic acid aptamer (aptamer) refers to the aglucon that can be combined with target molecule with high-affinity, is by a kind of modularization
Phyletic evolution technology (the systematic evolution of ligands by of technology-index concentration aglucon
Exponential enrichment, SELEX), pass through from the single-stranded random oligonucleotide library of artificial synthesized Large Copacity
The process " with target molecule effect-screening, enrichment " repeatedly, what is filtered out combines to target molecule with high specific and high associativity
Oligonucleotide molecules, can be described as aptamers again.Because nucleic acid aptamer is functionally similar with conventional antibodies, combined with target substance
Specificity and affinity be even better than antibody, add that its target molecule is wide, stability is high, the efficiency high of attack target molecule, easily change
The features such as producing and repairing decorations, it has also become the powerful of molecular recognition, can be in sides such as basic research, clinical quick diagnosis, medicament research and developments
Face is able to extensive exploitation application.
The present invention is using the B subunits of cholera enterotoxin as target protein, and being obtained using SELEX technology screenings can be special with target protein
The aptamer of different combination, available for detection cholera enterotoxin.Because the nucleic acid aptamer deposited in single stranded DNA form can be using chemistry
Method is synthesized in vitro, with low cost, and property is stable, high with target protein affinity, can directly be applied in detection method, is had
Have the advantages that easy to operate quick.
The content of the invention
The invention aims to overcome the deficiencies in the prior art, there is provided a kind of easy quick special cholera enterotoxin
Detection method.
In order to solve the above technical problems, present disclosure includes：
A kind of single stranded oligonucleotide aptamer of specific bond CT-HRP method, its nucleotide sequence such as sequence
In table described in SEQ ID No.1.
Further, present invention also includes the nucleic acid aptamer answering in cholera enterotoxin detection reagent is prepared
With.
With it is existing have technology compared with, aptamer of the invention is single strand dna, and the antibody than protide is more stablized；
The aptamer can be special combination CT-HRP method, using the machine of automation carry out it is external artificial synthesized and
Mark, the acquisition of Relative antibody is more easy to be quick, cheap, has a good application prospect.
In order to more fully understand and implement, the invention will now be described in detail with reference to the accompanying drawings.
Brief description of the drawings
Fig. 1 is that asymmetric PCR expands the 12nd electrophoretogram for taking turns SELEX screening acquisitions ssDNA.Wherein, M is DNA points of 20bp
Son amount mark.It is single-stranded DNA product at 81nt.Swimming lane 1- swimming lanes 5 are managed for parallel amplification 5.
Fig. 2 is the secondary structure of the aptamer.
Fig. 3 is detection aptamer and the film cross experiment figure of CT-HRP method compatibility.The moon that No. 1 lattice are BSA
Property control, No. 2 lattice are blank control；3rd, 4,5, the point sample amount of No. 6 lattice CT-HRP methods be respectively：0.05、0.1、
0.2、0.5μg。
Embodiment
A kind of screening for the single-strand DNA aptamer that can combine CT-HRP method of embodiment 1
Using SELEX technologies, follow these steps to carry out：
1st, the synthesis of ssDNA random libraries and primer
It is 81nt for the SELEX ssDNA libraries total lengths screened, both end of which is the fixed sequence program that primer is combined, in
Between 35 nucleotides be random sequence.
Library sequence is：
5’-CCATGCACTGGTAGTCTGTCGAAGT-(N35)-AGATAGCTGAATGTCCGCCTA-3’。
Amplification library primer sequence be respectively：
Sense primer：5’-CCATGCACTGGTAGTCTGTCGAAGT-3’；
Anti-sense primer：5’-TAGGCGGACATTCAGCTATCT-3’；
Biotin mark sense primer：5’-Biotin-CCATGCACTGGTAGTCTGTCGAAGT-3’
SsDNA pool and primer are synthesized by precious bioengineering (Dalian) Co., Ltd, PAGE purifying.
2nd, the SELEX screening processes of CT-HRP method aptamer
(1) coating and closing：By CT-HRP method (being purchased from Sigma Co., USA) coating buffer solution
(0.05mol/L NaHCO3, pH9.6) and after dissolving, a certain amount of experimental port for adding enzyme-linked microwell plate is taken, 4 DEG C of coatings are stayed overnight, use
PBS-T buffer solutions washing after, then with 3% BSA in 37 DEG C closing 2h.
(2) processing of ssDNA random libraries：Be arranged in parallel blank control wells on microwell plate, first with 3% BSA in 37
DEG C Seal treatment 2h.With combination buffer (20mmol/L Hepes, 5mmol/L KCl, 120mmol/L NaCL, lmmol/
LMgCl2, lmmol/L CaCl2, PH7.35) and the random ssDNA storehouses of dissolving, it is added to the blank control for using 3%BSA Seal treatments
Hole, 37 DEG C combine 1h, it is therefore an objective to which counter-selection removes the ssDNA combined with BSA.
(3) ssDNA is combined：SsDNA after counter-selection is added to the coated screen holes of cholera enterotoxin albumen, allowed
SsDNA, in 37 DEG C of hatching combination 1h, is at war with instead with toxin protein while also adding yeast tRNA (final concentration of 10mg/ml)
Should be to improve specificity.
(4) wash：Washed 3 times, washed away uncombined with dcq buffer liquid (combination buffer+0.05%Tween20)
ssDNA。
(5) elute and purify：Add 200 μ l elution buffers (20mmol/L TrisHCl, 4mol/L guanidinium isothiocyanates,
1mmol/L DTT, pH8.3) 10min is acted in 80 DEG C, the lower and protein bound ssDNA of cholera enterotoxin is eluted, through phenol, chloroform
SsDNA is dissolved in TE buffer solutions by extracting, ethanol precipitation.
(6) the positive ssDNA that directly asymmetric PCR amplification screening is obtained：Ratio between upstream and downstream primer is
100∶1.Using 25 μ l reaction systems, wherein ddH2O15.25 μ l, 10 × PCRBuffer2.5 μ l, dNTP (2.5mM) 2.0 μ l,
The μ l of sense primer (100 μM) 1.0,1.0 μ l, Taq archaeal dna polymerase (5U/ μ l) of anti-sense primer (1 μM) 0.125,3 μ of μ l, ssDNA
l.PCR cycle parameter is：94℃ 3min；(94 DEG C of 30S, 60 DEG C of 30S, 72 DEG C of 30S) × 40 circulations；72℃ 7min.
(7) PCR primer electrophoresis cuts glue purification：All PCR primers are subjected to electroresis appraisal with 3% Ago-Gel, cut
Single-stranded ssDNA amplified band, is carried out with pillar DNA glue reclaims kit (Beijing day bounties Gene Tech. Company Limited product)
Reclaim.Reclaim the ssDNA obtained and determine concentration with ultraviolet specrophotometer, -20 DEG C of preservations remain next round screening use.
(8) repeat step (1)-(7), screening 12 is taken turns, and the 12nd wheel SELEX screenings obtain ssDNA electrophoretogram as shown.The
1-12 wheel toxin protein package amount (pmol) be respectively：200、180、150、120、100、80、70、50、40、30、20、10；
The amount (pmol) in ssDNA libraries is respectively：2000、1800、1500、1200、1000、1000、800、800、500、500、500、
500.By gradually stepping up the preciseness of screening, that is, the relative scale of the usage amount of toxin protein, increase library and albumen is reduced,
Screened in favor of the aptamer of high-affinity from library.The ssDNA and cholera enterotoxin B for determining often wheel screening acquisition are sub-
The Percentage bound of unit (using the methods described of embodiment 2).Stop screening when Percentage bound is without significant change, expanded by symmetrical PCR
Increase and connect carrier T after obtaining dsDNA, recovery purifying, screened through blue hickie, 30 clones are selected at random and hand over Invitrogen (Shanghai)
Trade Co., Ltd is sequenced.Sequencing results are analyzed ssDNA oligonucleotides with molecular biology software DNAMAN6.0 and fitted
The homology and secondary structure of gamete.
(9) according to affinity height and the energy level of secondary structure, 1 sequence is selected from the aptamer that colony screening is obtained
It is further to be applied.Its nucleotides sequence is classified as shown in sequence table SEQ ID No.1, and its secondary structure figure is shown in Fig. 2.
The ssDNA libraries that embodiment 2 is screened and the measure of destination protein Percentage bound
(1) asymmetric PCR amplification is carried out using the sense primer and common anti-sense primer of biotin labeling and obtains 5' ends mark
Remember and glue purification is cut after the ssDNA libraries of biotin, electrophoresis, regulation concentration to 1.5pmol/ μ l.
(2) 96 hole elisa Plates are coated with CT-HRP method albumen, per hole 200ng, while the blank pair that be arranged in parallel
According to hole.
After (3) 95 DEG C of high-temperature denatured 5min, add the mark ssDNA libraries often taken turns and tied to reacting hole with toxin protein
Reaction is closed, 37 DEG C of incubation 2h abandon liquid in hole.
(4) washed 3 times with lavation buffer solution (PBS+0.05%Tween20, PBS-T, pH7.4), each 3min, Ran Houjia
Entering the strepavidin-horseradish peroxidase conjugate of Fresh, (HRP-labeled Streptavidin, 1: 1000 is dilute
Release) 200 μ l, 37 DEG C are incubated 30min, and PBS-T is washed 3 times,
(5) the μ L room temperatures lucifuge of TMB (tetramethyl benzidine) nitrite ion 100 colour developing 15min is added, 50 μ L 0.5M is added
H2SO4 terminating reactions, the OD values in 15min in ELIASA under measure 450nm wavelength.
(6) ssDNA libraries and the Percentage bound of destination protein CT-HRP method the results are shown in Table 1 after often wheel screening.Table
1
| Number of screening round | Blank well | Test hole | Number of screening round | Blank well | Test hole |
| 1 | 0.012 | 0.051 | 7 | 0.011 | 0.450 |
| 2 | 0.011 | 0.058 | 8 | 0.014 | 0.465 |
| 3 | 0.013 | 0.082 | 9 | 0.013 | 0.539 |
| 4 | 0.013 | 0.096 | 10 | 0.011 | 0.608 |
| 5 | 0.012 | 0.105 | 11 | 0.013 | 0.599 |
| 6 | 0.014 | 0.263 | 12 | 0.012 | 0.612 |
Embodiment 3 detects CT-HRP method using the nucleic acid aptamer of biotin modification with membrane hybridization
The method hybridized using film determines the affinity characteristic of aptamer and CT-HRP method.First by SEQ ID
Aptamer described in No.1 is recombined and in 5 ' the upper biotins of end mark, with coating buffer solution (0.05mol/L NaHCO3,
PH9.6 1 μM is configured to after) dissolving；1 1.6 × 3.8cm of clip nitrocellulose filter bar, with seal oil painting into 6 lattice；Numbering
1-6, wherein the negative control that No. 1 lattice are BSA (0.5 μ g), No. 2 lattice are blank control (being free of cholera enterotoxin albumen)；3rd,
4th, 5, the point sample amount of No. 6 lattice CT-HRP methods is respectively：0.05th, 0.1,0.2,0.5 μ g, point sample volume is 1.5 μ L,
Treat that it absorbs complete；Excellent film bar will be put to be placed in a square small magazine, 2ml confining liquids (10% skimmed milk power) are added
Seal treatment is carried out in room temperature 2 hours；Then washed with PBS 2 times, then 1.5ml aptamer solution be added in box, at room temperature with
Film bar carries out hybridization reaction 1 hour；With PBS-T buffer solution for cleaning 2 times, every time 2 minutes；Then the strepto- parent of Fresh is added
Close the μ l of element-horseradish peroxidase conjugate (HRP-labeled Streptavidin, 1: 1000 dilution) 500,37 DEG C of incubations
15min；PBS-T buffer solution for cleaning films bar is used again 2 times, every time 2 minutes；2ml nitrite ions lucifuge is added after cleaning to develop the color 10 minutes
(nitrite ion faces used time preparation, TMB containing 0.1mg/ml, 30% H2O20.2 μ l, 0.1M sodium citrate), finally add 100 μ L
0.5M H2SO4 terminating reactions simultaneously observe result.As a result as shown in figure 3, negative control point (No. 1 lattice) and blank control point (2
Number lattice) immaculate is produced, and blue spot is presented at CT-HRP method point sample, concentration is higher, and spot is deeper.The experiment
Showing the aptamer of the present invention can be specifically bound with CT-HRP method, available for the detection of cholera enterotoxin,
Available for the detection reagent for preparing cholera enterotoxin, have the advantages that it is stable, easy, quick, economical, with good market should
With value.
The preferred embodiments of the present invention are these are only, are not intended to limit the invention, for those skilled in the art
For member, any modification, equivalent substitution and improvements done within the spirit and principles of the invention etc. should be included in this
Within the protection domain of invention.
Claims (2)
1. a kind of single stranded oligonucleotide aptamer of specific bond CT-HRP method, such as sequence table of its nucleotide sequence
Described in middle SEQ ID No.1.
2. application of the single stranded oligonucleotide aptamer in cholera enterotoxin detection reagent is prepared described in claim 1.
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