CN106645050A - Aptamer used for detecting lactoferrin content, and application thereof - Google Patents
Aptamer used for detecting lactoferrin content, and application thereof Download PDFInfo
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- CN106645050A CN106645050A CN201610894671.6A CN201610894671A CN106645050A CN 106645050 A CN106645050 A CN 106645050A CN 201610894671 A CN201610894671 A CN 201610894671A CN 106645050 A CN106645050 A CN 106645050A
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- 102000010445 Lactoferrin Human genes 0.000 title claims abstract description 63
- 108010063045 Lactoferrin Proteins 0.000 title claims abstract description 63
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 title claims abstract description 63
- 229940078795 lactoferrin Drugs 0.000 title claims abstract description 63
- 235000021242 lactoferrin Nutrition 0.000 title claims abstract description 63
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 38
- 238000002875 fluorescence polarization Methods 0.000 claims abstract description 31
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 15
- 230000005284 excitation Effects 0.000 claims description 11
- 235000018102 proteins Nutrition 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 11
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- 238000011088 calibration curve Methods 0.000 claims description 6
- 102000008192 Lactoglobulins Human genes 0.000 claims description 4
- 108010060630 Lactoglobulins Proteins 0.000 claims description 4
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- 238000012360 testing method Methods 0.000 claims description 3
- 230000006978 adaptation Effects 0.000 claims description 2
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- XLJMAIOERFSOGZ-UHFFFAOYSA-N cyanic acid Chemical compound OC#N XLJMAIOERFSOGZ-UHFFFAOYSA-N 0.000 claims 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims 1
- 239000005864 Sulphur Substances 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 28
- 235000013336 milk Nutrition 0.000 abstract description 14
- 239000008267 milk Substances 0.000 abstract description 14
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- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 6
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 6
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 6
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
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- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6445—Measuring fluorescence polarisation
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- Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses an aptamer used for detecting lactoferrin content, and the nucleotide sequence of the aptamer is shown as SEQ ID NO.5-65. The invention also discloses a method for detecting the lactoferrin by the aptamer. According to the method, a fluorescently-labeled aptamer is taken as a probe molecule, and through the specific binding of the aptamer and the lactoferrin, a fluorescence polarization signal is changed. The fluorescence polarization signal and lactoferrin concentration have a linear relationship so as to realize the quantitative detection of the lactoferrin. The method has the advantages of high sensitivity and good specificity, and a detection linear range is 0.78-50 mu g/mL. According to the method, the measurement, such as the measurement of the lactoferrin in milk, of a target object in a complex sample can be realized. In addition, according to the method, a BioTeK microplate reader is used for directly scanning a 96 pore plate to achieve the requirement of quick and high flux measurement, and a plurality of samples can be simultaneously effectively measured.
Description
Technical field
The invention belongs to technical field of biological, and in particular to one kind is examined using fluorescence polarization method to lactoferrin
The aptamers of survey and its application.
Background technology
Fluorescence polarization technology can be based on the molecular mass of fluorophor as a kind of simple and reliable signal transduction means
Change provide with regard to molecularly oriented, migration and mechanism information.Fluorescence Polarization assay has many advantages：The
One, it is that a kind of homogeneous analysis method and sensitivity are high, without the need for the complex operations such as separating, as a result favorable reproducibility；Second, during response
Between it is fast, can be with real-time monitoring intermolecular interaction；3rd, can be with the Instrument crosslinking such as ELIASA, it is possible to achieve automation is high
Flux is determined；4th, fluorescence polarization depends on the ratio of the fluorescence intensity in two vertical direction, with fluorescence absolute intensity without
Close, fluorescence polarization value is insensitive to the fluctuation of fluorescence and photobleaching, so as to become the homogeneous determination target in complex biological sample
The preferable analysis method of molecule.Therefore, fluorescence polarization technology receives extensive research concern in fluorescence sense application.
Lactoferrin is a kind of functional protein of great broad mass market prospect, and it is primarily present in the milk of mammal
In, its content is closely bound up with the kind of animal, with human milk, cow's milk as Typical Representative.Research shows that lactoferrin has various
BA.It has participate in iron metabolism, antitumor, anti-oxidant, antibacterial, blocking oxygen radical, immunological regulation and anti-inflammatory,
Promote cell to increase, reduce the several functions such as interior fat.And due to lactoferrin be it is a kind of can by baby intake, safe pole
High natural milk composition, it is approved for food additives in Japan, is included into it is generally acknowledged that among safe material in the U.S..Plus
It, the extensive biological function of lactoferrin is paid much attention to by domestic and international food relevant enterprise deeply, China GB 14880-
2012《Food enrichment uses standard》In, lactoferrin be also listed in a kind of novel nourishing hardening agent and define its
Content standard in related dairy products.Thus, can set up fast and accurately lactoferrin detection technique for food security come
Say most important.At present, being adapted to the detection technique of lactoferrin in different type biological sample mainly has AAS, efficiently
Liquid chromatography, enzyme linked immunosorbent assay, high-effective affinity chromatography method, high performance capillary electrophoresis method and surface plasma resonance
The detection techniques such as technology, but still there is the spies such as pretreatment process is complicated, the actual sample rate of recovery is low, stability is poor in these methods
Point, hence set up out it is a kind of can realize comprehensively it is accurate, quick, without the need for sample is carried out pre-treatment full-automatic lactoferrin inspection
Survey method is particularly significant and urgent.
The content of the invention
The technical problem to be solved is to provide a kind of aptamers for detecting lactoferrin content.
The technical problem also to be solved of the invention is to provide application of the above-mentioned aptamers in detection lactoferrin.
To solve above-mentioned technical problem, the technical solution used in the present invention is as follows：
A kind of aptamers for detecting lactoferrin content, the nucleotide sequence such as SEQ ID NO.5 of the aptamers
Shown in～65, the preferred SEQ ID NO.6 of described aptamers, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.35.
Application of the above-mentioned aptamers for detecting lactoferrin content in detection lactoferrin.
A kind of method that fluorescence polarization method measures negative proteins content, comprises the steps：
(1) calibration curve is drawn：Add in the adaptation liquid solution of 10 μ L, 40nM～4000nM marked by fluorescein isothiocyanate
Enter the negative proteins standard sample of 100 μ L variable concentrations, mix, the nucleotides sequence of the aptamers be classified as SEQ ID NO.1～
Any one in 61, reacts 5～120min at 25 DEG C～40 DEG C, detected with ELIASA, and excitation wavelength is 480nm, launch wavelength
For 528nm, calibration curve is drawn；
(2) sample detection：The aptamers of marked by fluorescein isothiocyanate are mixed with testing sample, according to step (1)
Condition is detected.
Wherein, the concentration of the aptamers of the marked by fluorescein isothiocyanate is 40nM～4000nM；
Wherein, the concentration of the testing sample is 0.78～50 μ g/mL.
Wherein, described negative proteins be lactoferrin, ALA, beta lactoglobulin, BSA, preferred lactoferrin.
Beneficial effect：
The present invention makes fluorescence inclined by lactoferrin with the aptamers of FITC marks as probe with the specific binding of probe
The signal that shakes changes, and lactoferrin concentration logarithm value linearly changes with fluorescence polarization signal, so as to realize to newborn iron egg
White quantitative determination.This kind of fluorescence polarization method sensitivity is high, and anti-interference is very strong, can be prevented effectively from false positive signal, real
Now to the measurement of object in complex sample, the measurement of lactoferrin such as in milk, the range of linearity of the method detection is 0.78
～50 μ g/mL.
Description of the drawings
Fig. 1 fluorescence polarization method reaction time optimizes figure.
Fig. 2 variable concentrations lactoferrin and fluorescence polarization signal graph of a relation.
Fig. 3 fluorescence polarization methods are to lactoferrin specific detection figure.
Fig. 4 fluorescence polarization methods detect linear graph to lactoferrin.
Other aptamers of Fig. 5 detect linear graph to lactoferrin fluorescence polarization.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, and should not also without limitation on sheet described in detail in claims
Invention.
Embodiment 1：Lactoferrin aptamers are screened
Lactoferrin aptamers screening concrete steps, including chip preparations, positive and negative sieved journey, PCR expand etc. detailed mistake
Journey, it is described in detail below, wherein：
Library：5′-TAMRA-GACAGGCAGGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3′
The forward direction primer of TARMA modifications：5′-TARMA-GACAGGCAGGACACCGTAAC-3′
Biotinylated backward primer：5′Biotin-GAAGAGGAGGGAGGTAGCAG-3′
(1) prepared by chip：Fabrication techniques microfluidic channel template of the profit mask sharp first in combination with chemical attack
Grinding tool is manufactured as PDMS passages；After weigh mass ratio for 10：Aggressiveness is sufficiently mixed with curing agent before 1 PDMS, after vacuumizing
Be poured onto it is cured in microfluidic channel template after obtain PDMS microfluidic channels；It is in glass substrate point concentration using point sample instrument
The lactoferrin and negative proteins microarray of 5mg/mL, and to the simultaneously plasma treatment of the glass substrate after PDMS and point sample, it
After be brought into close contact collectively as screening chip, for it is ensuing often wheel screening；Screening prepares：The screening that step (1) is obtained
Chip is put in constant water bath box incubation albumen 2h at 37 DEG C；20mg/mlBSA and 0.01mM random sequence short chains are passed through again
SsDNA (20nt) closes 1h under the conditions of 37 DEG C；Afterwards with 150 μ L, 1 × PBST solution cleans positive and negative sieve passage；
(2) first round screening：By 0.5nmol, 125 μ L primary libraries heat 5min at 95 DEG C, and freeze on ice immediately
10s；With syringe pump library is input into 2.5 μ L/min flow velocitys again and just sieve passage, under normal temperature condition 50min is reacted；Afterwards with
15 μ L/min flow velocitys are passed through 150 μ L, and 1 × PBS cushioning liquid just sieve unconjugated ssDNA sequences in passage to remove；Gently tear
Fall PDMS layer, using Luxscan～10K/A microarray scanners chip is scanned；Finally heat 5min at 95 DEG C with DPEC water
The ssDNA sequences that wash-out lactoferrin is combined, it is 69 μ L that resulting solution High Purity Nitrogen under the conditions of 50 DEG C is dried up to volume；
(3) PCR amplification procedures：Step (2) resulting solution is divided into into 3 parts of same solutions that volume is 23 μ L, per part according to
25 μ L of secondary addition, 2 × Taq polymerase, 1 μ L, the forward direction primer and 1 μ L of 20 μM of TAMRA marks, 20 μM are biotinylated backward
Primer, is well mixed laggard performing PCR amplification；The Thermal Cycling of PCR is as follows：94℃5min；94 DEG C of 30s of circulation, 60.5 DEG C
30s, 72 DEG C of 30s processes 10 are taken turns, 72 DEG C of 5min terminating reactions；Carry out again as template after 10 times of the product dilution that the step is obtained
Once expand：Take PCR primer and 25 μ L, 2 × SYBR after 5 μ L dilutionsPremix Ex TaqTMEnzyme, 1 μ L, 20 μM of TAMRA marks
Forward direction primer, 1 μ L, 20 μM of biotinylated backward primers and 18 μ L DPEC water are sufficiently mixed uniformly, and PCR amplifications are often entered
Row takes 10 μ L samples and detects its fluorescence signal with BioTek into microwell plate once taking turns, and fluorescence signal soprano is used as optimal wheel
Number, resultant product expands the wheel number；
(4) isolate and purify：The PCR primer that step (4) is obtained fully is mixed with 600 μ L Promega magnetic beads, in shaking table
Supernatant is removed after upper quick shake 1h；25 μ L are added, 50mM NaOH vortexs 5min dissociate the double-strand connected on magnetic bead；
Draw supernatant liquor and sequentially add 12.5 μ L 100mM HCl, 25 μ L H2O, 62.5 μ L2 × PBSM neutralizations, the solution of gained
Used as the secondary library of next round screening, its content is about 40pmol；
(5) second wheel screenings：Library is input into into negative sieve passage with 2.5 μ L/min flow velocitys with pump, is reacted under normal temperature condition
50min；With pump library is input into 2.5 μ L/min flow velocitys again and just sieve passage, under normal temperature condition 50min is reacted；Then with 15 μ
L/min flow velocitys are passed through the removing of 150 μ 1 × PBS of L cushioning liquid and are just sieving unreacted chain in passage；PDMS layer is gently torn, is used
Luxscan～10K/A microarray scanners scan chip；Finally the combination of 5min wash-out proteins is heated at 95 DEG C with seedless water
Chain, it is 92 μ L that resulting solution is dried up to volume at 60 DEG C, gives over to PCR amplifications；
(6) repeat step (4), (5), (6) are to the 8th wheel screening；
(7) the five, the six, seven wheel pcr amplification products of screening are delivered to into Shanghai life work sequencing, often wheel randomly selects 120
Sequencing, then secondary structure analysis are carried out to the chain for obtaining with IDT softwares, finally obtain optimal aptamers.
Embodiment 2：Fluorescence polarization method is detected to lactoferrin standard sample：
(1) in 10 μ L, the aptamers N2 (base sequence of 250nM FITC (fluorescein isothiocynate) marks：
AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT 100 μ L concentration) are added to be 25 μ g/ in solution
The lactoferrin standard sample of mL, fully mixes.
(2) mixed liquor of step (1) is placed in 96 microwell plates and reacts 15min under the conditions of 37 DEG C, use BioTeK ELIASAs
Directly scan, excitation wavelength is 480nm, and launch wavelength is 528nm.
Embodiment 3：The fluorescence polarization method reaction time optimizes
(1) in 10 μ L, the aptamers N2 (base sequence of 250nM FITC (fluorescein isothiocynate) marks：
AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT 100 μ L concentration) are added to be 25 μ g/ in solution
The lactoferrin standard sample of mL, fully mixes.
(2) mixed liquor of step (1) is placed in 96 microwell plates under the conditions of 37 DEG C react respectively 5min, 10min, 15min,
20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, are directly scanned with BioTeK ELIASAs,
Excitation wavelength is 480nm, and launch wavelength is 528nm.
Fig. 1 is fluorescence polarization signal and fluorescence polarization signal figure in blank solution under the conditions of different time.
Embodiment 4：Variable concentrations lactoferrin and fluorescence polarization signal relation
(1) in 10 μ L, the aptamers N2 (base sequence of 250nM FITC (fluorescein isothiocynate) marks：
CGGTGCATCTATGGCTACTAGCTTTTCCTGCCTATACTAC it is 0.19 μ g/mL that 100 μ L concentration) are separately added in solution,
The μ g/mL of 0.39 μ g/mL 0.78,1.56 μ g/mL, 3.12 μ g/mL, 6.25 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL
Lactoferrin standard sample, fully mixes.
(2) mixed liquor of step (1) is placed in 96 microwell plates and reacts 15min under the conditions of 37 DEG C, use BioTeK ELIASAs
Directly scan, excitation wavelength is 480nm, and launch wavelength is 528nm.
(3) by analysis, it is found that analysis of fluorescence polarization signal linearly changes with the logarithm value of lactoferrin concentration, obtain
Calibration curve, by calibration curve the concentration of lactoferrin in sample is calculated.
Fig. 2 is the fluorescence polarization value figure for adding variable concentrations lactoferrin, and figure d is canonical plotting (R=0.988).
Embodiment 5：Fluorescence polarization method is to lactoferrin specific detection
(1) in five part of 10 μ L, the aptamers N2 (base sequence of 250nM FITC (fluorescein isothiocynate) marks：
CGGTGCATCTATGGCTACTAGCTTTTCCTGCCTATACTAC) the newborn iron egg of 100 μ L25 μ g/mL will be added respectively in solution
In vain, the μ g/mL ALAs of 100 μ L 500, the μ g/mL beta lactoglobulins of 100 μ L 500, the μ g/mL BSA of 100 μ L 500 and
100 μ L PBS solutions, fully mix.
(2) mixed liquor of step (1) is placed in 96 microwell plates and reacts 15min under the conditions of 37 DEG C, use BioTeK ELIASAs
Directly scan, excitation wavelength is 480nm, and launch wavelength is 528nm.
Fig. 3 be this kind of fluorescence polarization method to lactoferrin, ALA, beta lactoglobulin, BSA specific detection
Figure.
Embodiment 6：The method of detection lactoferrin content
(1) in 10 μ L, the aptamers N2 (base sequence of 250nM FITC (fluorescein isothiocynate) marks：
AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT the ox of 25-100 times of dilution) is added in solution
Milk sample, fully mixes.
(2) mixed liquor of step (1) is placed in 96 microwell plates and reacts 15min under the conditions of 37 DEG C, use BioTeK ELIASAs
Directly scan, excitation wavelength is 480nm, and launch wavelength is 528nm.
(3) contrast standard curve obtains the concentration of lactoferrin in milk sample, is specifically shown in Fig. 4.
Embodiment 7：The method of detection lactoferrin content
(1) in 10 μ L, the aptamers N6 (base sequence of 250nM FITC (fluorescein isothiocynate) marks：
AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT the ox of 25-100 times of dilution) is added in solution
Milk sample, fully mixes.
(2) mixed liquor of step (1) is placed in 96 microwell plates and reacts 15min under the conditions of 37 DEG C, use BioTeK ELIASAs
Directly scan, excitation wavelength is 480nm, and launch wavelength is 528nm.
(3) contrast standard curve obtains the concentration of lactoferrin in milk sample, is specifically shown in Fig. 5 (a).
Embodiment 8：The method of detection lactoferrin content
(1) in 10 μ L, the aptamers N14 (base sequence of 250nM FITC (fluorescein isothiocynate) marks：
AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT the ox of 25-100 times of dilution) is added in solution
Milk sample, fully mixes.
(2) mixed liquor of step (1) is placed in 96 microwell plates and reacts 15min under the conditions of 37 DEG C, use BioTeK ELIASAs
Directly scan, excitation wavelength is 480nm, and launch wavelength is 528nm.
(3) contrast standard curve obtains the concentration of lactoferrin in milk sample, is specifically shown in Fig. 5 (b).
Embodiment 9：The method of detection lactoferrin content
(1) in 10 μ L, the aptamers N16 (base sequence of 250nM FITC (fluorescein isothiocynate) marks：
AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT the ox of 25-100 times of dilution) is added in solution
Milk sample, fully mixes.
(2) mixed liquor of step (1) is placed in 96 microwell plates and reacts 15min under the conditions of 37 DEG C, use BioTeK ELIASAs
Directly scan, excitation wavelength is 480nm, and launch wavelength is 528nm.
(3) contrast standard curve obtains the concentration of lactoferrin in milk sample, is specifically shown in Fig. 5 (c).
Embodiment 10：The method of detection lactoferrin content
(1) in 10 μ L, the aptamers N31 (base sequence of 250nM FITC (fluorescein isothiocynate) marks：
AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT the ox of 25-100 times of dilution) is added in solution
Milk sample, fully mixes.
(2) mixed liquor of step (1) is placed in 96 microwell plates and reacts 15min under the conditions of 37 DEG C, use BioTeK ELIASAs
Directly scan, excitation wavelength is 480nm, and launch wavelength is 528nm.
(3) contrast standard curve obtains the concentration of lactoferrin in milk sample, is specifically shown in Fig. 5 (d).
The present invention is applied to fluorescence polarization method in the analysis of lactoferrin detection, when the concentration of lactoferrin is 0.78
Between the μ g/mL of μ g/mL～50, there is good correlation between fluorescence polarization signal intensity and the logarithm of lactoferrin concentration,
Coefficient correlation is 0.988.Meanwhile, present invention energy fast high-flux detection lactoferrin not only can avoid false positive signal
The interference of external environment is produced and can prevent, signals collecting mode is simple and quick.
Claims (6)
1. a kind of aptamers for detecting lactoferrin content, it is characterised in that the nucleotide sequence of the aptamers such as SEQ
Shown in ID NO.5～65.
2. it is used to detect application of the aptamers of lactoferrin content in detection lactoferrin described in claim 1.
3. a kind of method that fluorescence polarization method measures negative proteins content, it is characterised in that comprise the steps：
(1) calibration curve is drawn：To add in the adaptation liquid solution of 10 μ L, 40nM～4000nM marked by fluorescein isothiocyanate
The negative proteins standard sample of 100 μ L variable concentrations, mixes, and the nucleotides sequence of the aptamers is classified as SEQ ID NO.1～61
In any one, at 25 DEG C～40 DEG C react 5～120min, with ELIASA detect, excitation wavelength is 480nm, and launch wavelength is
528nm, draws calibration curve；
(2) sample detection：The aptamers of marked by fluorescein isothiocyanate are mixed with testing sample, according to the condition of step (1)
Detection.
4. the method that fluorescence polarization method according to claim 3 measures negative proteins content, it is characterised in that the different sulphur
The concentration of the fluorescein-labeled aptamers of cyanic acid is 40nM～4000nM.
5. the method that fluorescence polarization method according to claim 3 measures negative proteins content, it is characterised in that described to be measured
The concentration of sample is 0.78～50 μ g/mL.
6. the method that fluorescence polarization method according to claim 3 measures negative proteins content, it is characterised in that described the moon
Property albumen be lactoferrin, ALA, beta lactoglobulin, BSA.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107607722A (en) * | 2017-07-21 | 2018-01-19 | 南京医科大学 | A kind of method for quantitatively detecting beta lactoglobulin in milk powder |
| CN109706153A (en) * | 2018-12-27 | 2019-05-03 | 南京祥中生物科技有限公司 | For detecting the ELONA aptamers and its application of lactoferrin |
| CN110554080A (en) * | 2019-09-29 | 2019-12-10 | 陕西科技大学 | Gel electrophoresis detection method and kit for lactoferrin content |
| CN112114074A (en) * | 2020-08-14 | 2020-12-22 | 北京农业质量标准与检测技术研究中心 | Lactoferrin aptamer affinity column and preparation method and application thereof |
| WO2021159650A1 (en) * | 2020-02-15 | 2021-08-19 | 江南大学 | Nucleic acid aptamer specifically recognizing beta-lactoglobulin and use thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004277312A (en) * | 2003-03-14 | 2004-10-07 | National Institute Of Advanced Industrial & Technology | Fluorescent labeled steroid compound and method for producing the same |
| CN101832935A (en) * | 2009-03-10 | 2010-09-15 | 苏州市长三角系统生物交叉科学研究院有限公司 | Target molecule detection method based on nanometer-gold and nucleic acid structure and kit thereof |
| CN103045600A (en) * | 2011-10-11 | 2013-04-17 | 上海市肺科医院 | Serum IgG antibody aptamer of tuberculosis patients and preparation method thereof |
| CN103525927A (en) * | 2013-10-11 | 2014-01-22 | 南京师范大学 | Method for detecting ochratoxin A |
| CN104152552A (en) * | 2014-07-18 | 2014-11-19 | 山东大学 | Colocalization trigger chain type hybridization reaction based aptamer sensor for detecting adenosine |
-
2016
- 2016-10-13 CN CN201610894671.6A patent/CN106645050B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004277312A (en) * | 2003-03-14 | 2004-10-07 | National Institute Of Advanced Industrial & Technology | Fluorescent labeled steroid compound and method for producing the same |
| CN101832935A (en) * | 2009-03-10 | 2010-09-15 | 苏州市长三角系统生物交叉科学研究院有限公司 | Target molecule detection method based on nanometer-gold and nucleic acid structure and kit thereof |
| CN103045600A (en) * | 2011-10-11 | 2013-04-17 | 上海市肺科医院 | Serum IgG antibody aptamer of tuberculosis patients and preparation method thereof |
| CN103525927A (en) * | 2013-10-11 | 2014-01-22 | 南京师范大学 | Method for detecting ochratoxin A |
| CN104152552A (en) * | 2014-07-18 | 2014-11-19 | 山东大学 | Colocalization trigger chain type hybridization reaction based aptamer sensor for detecting adenosine |
Non-Patent Citations (2)
| Title |
|---|
| HONG-QI WANG等: "Fluorescence protection assay: a novel homogeneous assay platform toward development of aptamer sensors for protein detection", 《NUCLEIC ACIDS RESEARCH》 * |
| 何为等: "蛋白质微阵列芯片技术及其在抗体筛选中的应用", 《分析化学(FENX IHUAXUE) 研究报告》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107607722A (en) * | 2017-07-21 | 2018-01-19 | 南京医科大学 | A kind of method for quantitatively detecting beta lactoglobulin in milk powder |
| CN107607722B (en) * | 2017-07-21 | 2019-07-26 | 南京医科大学 | A kind of method of beta lactoglobulin in quantitative detection milk powder |
| CN109706153A (en) * | 2018-12-27 | 2019-05-03 | 南京祥中生物科技有限公司 | For detecting the ELONA aptamers and its application of lactoferrin |
| CN110554080A (en) * | 2019-09-29 | 2019-12-10 | 陕西科技大学 | Gel electrophoresis detection method and kit for lactoferrin content |
| WO2021159650A1 (en) * | 2020-02-15 | 2021-08-19 | 江南大学 | Nucleic acid aptamer specifically recognizing beta-lactoglobulin and use thereof |
| CN112114074A (en) * | 2020-08-14 | 2020-12-22 | 北京农业质量标准与检测技术研究中心 | Lactoferrin aptamer affinity column and preparation method and application thereof |
| CN112114074B (en) * | 2020-08-14 | 2022-06-28 | 北京农业质量标准与检测技术研究中心 | Lactoferrin aptamer affinity column and preparation method and application thereof |
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