CN109371031A - A kind of screening technique specifically binding bovine serum albumin(BSA) aptamer - Google Patents
A kind of screening technique specifically binding bovine serum albumin(BSA) aptamer Download PDFInfo
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- CN109371031A CN109371031A CN201811403943.3A CN201811403943A CN109371031A CN 109371031 A CN109371031 A CN 109371031A CN 201811403943 A CN201811403943 A CN 201811403943A CN 109371031 A CN109371031 A CN 109371031A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
- G01N2333/765—Serum albumin, e.g. HSA
Abstract
It is a kind of specifically bind bovine serum albumin(BSA) aptamer screening technique the present invention relates to chemical analysis technology fields.The bovine serum albumin(BSA) aptamer screening technique combined in the present invention based on SELEX technology and agarose gel electrophoresis is compared with traditional screening technique, with the advantage that easy to operate, screening process is at low cost, the screening period is shorter.The aptamer stability that the present invention filters out is strong, synthesis is convenient and is easy to mark various report molecules, can long-term preservation use.Aptamer obtained in the present invention can be specifically bound with bovine serum albumin(BSA), and affinity is high, be the sequence for the related bovine serum albumin(BSA) aptamer that first success is screened so far.The aptamer modified different report molecule is utilized in the present invention, can construct various biosensors, for various analysis detections such as food and drugs.
Description
Technical field
The present invention relates to chemical analysis technology field, specifically a kind of specific binding bovine serum albumin(BSA) nucleic acid is suitable
The screening technique of ligand.
Background technique
Aptamer (also known as aptamers, aptamer) is (systematic that evolved by the Fas lignand system of index concentration
Evolution of ligands by exponential enrichment, SELEX) technology screening obtain can be with target point
The a bit of ssDNA or RNA of son specific binding.Aptamer has many advantages, such as that target molecule is extensive, affinity is strong, easily modification, is dividing
Sub- chemistry, food safety, clinical diagnosis and treatment etc. are widely used.
SELEX is the principle using combinatorial chemistry, constructs an artificial synthesized random oligonucleotide library, warp in vitro
The specific binding of sequence and target in library is crossed, a kind of new technology of target nucleic acid aptamers is screened.Due in random library
Containing the different single strand oligonucleotide acid fragment of a large amount of primary structures, when they meet with target molecule in the solution, can be formed not
Same space structure filters out energy and its by the matched mode of conformation for target molecule due to the diversity of its space structure
Nucleic acid sequence with high-affinity and specificity.
Bovine serum albumin(BSA) is one of cow's serum globulin, also known as fifth component.Albumin in blood mainly rises
Maintain osmotic pressure effect, PH buffer function, carrier function and trophism.In animal cell non-serum culture, white egg is added
It is white to play the role of biology and mechanical protection and carrier function.Bovine serum albumin(BSA) molecular weight is 66.446kDa, and isoelectric point is
4.7.Bovine serum albumin(BSA) is mostly used in Biochemical Research, genetic engineering and medical research, medicines and health protection food etc..Due to human seralbumin
The medical value of albumen is very high, and the source of goods on the market is very nervous, and it is very high to make the value tested of human serum albumins, and cow's serum
The amino acid sequence of albumin and the similitude of human serum albumins are very high, bovine serum albumin(BSA) can be used as previous experiments
Substitute.Not yet there is the screening of bovine serum albumin(BSA) aptamer to report in scientific research at present, and tradition SELEX screening process
It takes time and effort, the stronger aptamers of specificity can be just obtained through multi-turns screen.This experiment is established based on SELEX technology and agar
The bovine serum albumin(BSA) aptamer screening technique that sugared gel electrophoresis combines.
Summary of the invention
The purpose of the present invention is aiming at the shortcomings in the prior art, provide a kind of specific binding bovine serum albumin(BSA) nucleic acid
The screening technique of aptamers.
Above-mentioned purpose is achieved through the following technical solutions:
1. synthesizing original ssDNA pool and primer shown in following sequence:
Original ssDNA pool:
5'-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3'；
Upper primer: 5 '-FAM-AGCAGCACAGAGGTCAGATG-3 '；
Lower primer: 5 '-biotin-TTCACGGTAGCACGCATAGG-3 '.
2 establish the bovine serum albumin(BSA) aptamer screening technique based on agarose gel electrophoresis
Screen used in overall process: the group of combination buffer becomes 20mM Tris-HCL, 1M NaCl, 1mM EDTA, pH
7.8。
The bovine serum albumin(BSA) aptamer screening technique step based on agarose gel electrophoresis:
The screening of 2.1 first round
2.1.1 single-stranded random library preparation
The original ssDNA pool of 1-2OD uses after being subsequently placed at cooled on ice to room temperature in 95 DEG C of water-bath 5min.
2.1.2 bovine serum albumin(BSA) (BSA) solution prepares
500ng/ml BSA solution for standby is prepared with distilled water.
2.1.3 Ago-Gel and immobilized BSA albumen are prepared
The Ago-Gel solution (i.e. every 100mlTAE buffer contains 2g agarose) of configuration 2% adds in micro-wave oven
The BSA solution of the 500ng/mL made in advance, is added to not cold by 2% agarose solution 1-2min of heat at once at this time by 95-98 DEG C
But it is formed in the agarose solution of gel, the BSA liquor capacity ratio of agarose solution dosage and addition is 3:2, after mixing well
It is transferred to plastic plate and is stored at room temperature 30min or more and be cooled into Ago-Gel.
2.1.4 agarose gel electrophoresis
The Ago-Gel of the immobilized BSA albumen of step 2.1.3 is put into electrophoresis tank, 3-5 μ l DNA is separately added into
Marker as instruction and 50-300nmol original random single chain library after start electrophoresis, the dosage in original random single chain library
It is 1:1200, voltage 150-200V, electrophoresis time 25-35min with BSA liquor capacity ratio fixed in gel.
2.1.5 gel extraction
It carries out gel extraction band after electrophoresis at 300-600bp by multi-functional gel imager, is cut according to DNA
The specification that plastic recovery kit provides extracts template of the DNA as next step PCR amplification in gel.
2.1.6 using DNA obtained by 2.1.5 as template, downstream primer and upstream primer carry out PCR amplification.
2.2 second wheel screenings
2.2.1 prepare single stranded DNA: take Streptavidin MagneSphere with combination buffer wash three times (Streptavidin MagneSphere and
Combination buffer dosage volume ratio is 1:2), it is added in the product of 2.1.6PCR amplification, shaking captures 30-60min, magnetic
Supernatant is separated, is washed three times (Streptavidin MagneSphere and combination buffer dosage volume ratio be 1:2), is added with combination buffer
The sodium hydroxide solution reaction 5min (Streptavidin MagneSphere and sodium hydroxide solution dosage volume ratio be 8:5) of 0.2mol/L,
Magneto separate takes supernatant, and the hydrochloric acid being added afterwards is adjusted to 7 with wide pH value test paper, purifying, freeze-drying.The library secondary ssDNA is used as next
Wheel screening.
2.2.2 single-stranded random library prepares
The secondary library ssDNA that step 2.2.1 is obtained makes after being subsequently placed at cooled on ice to room temperature in 95 DEG C of water-bath 5min
With.
2.2.3 bovine serum albumin(BSA) (BSA) solution prepares
500ng/ml BSA solution for standby is prepared with distilled water.
2.2.4 Ago-Gel and immobilized BSA albumen are prepared
Operating procedure is the same as step 2.1.3
2.2.5 agarose gel electrophoresis
The Ago-Gel of the immobilized BSA albumen of step 2.2.4 is put into electrophoresis tank, 3-5 μ l DNA is separately added into
Start electrophoresis after the secondary library that marker prepares as instruction and step 2.2.2, is fixed in the dosage and gel of secondary library
BSA liquor capacity ratio be 1:1200 voltage 150-200V, electrophoresis time 25-35min.
2.2.6 gel extraction
It carries out gel extraction band after electrophoresis at 300-600bp by multi-functional gel imager, is cut according to DNA
The specification that plastic recovery kit provides extracts template of the DNA as next step PCR amplification in gel.
2.2.7 using DNA obtained by 2.2.6 as template, downstream primer and upstream primer carry out PCR amplification.
2.3 recycle 5-8 times according to step 2.2, the aptamer that enrichment can be specifically bound with bovine serum albumin(BSA).
3. obtaining bovine serum albumin(BSA) aptamer: every wheel screens the obtained secondary library ssDNA and passes through 5 ' end marks
After remembering fluorophor in conjunction with target, the secondary library ssDNA and target binding capability are surveyed with microplate reader.Until fluorescence intensity reaches
Maximum saturation state obtains bovine serum albumin(BSA) aptamer, such as Fig. 3 at this time.
4. cloning and sequencing: by last wheel screening obtained aptamer be sent to Shanghai Sheng Gong Technology Co., Ltd. into
Row cloning and sequencing to get arrive the resulting nucleic acid aptamer sequence of above-mentioned screening, i.e. 5 '-AGCAGCACAGAGGTCAGATGGTATC
GAGCGCAGGGGCGCCTTTG TTAATGATTACGGGCGCCTATGCGTGCTACCGTGAA-3’。
5. affinity analysis: aptamer being configured to a series of concentration with combination buffer.Each concentration gradient core
It is multiple to be added into bovine serum albumin(BSA)-magnetic bead by 90 DEG C of water-bath 10min, 4 DEG C of 15min, room temperature 5min activation for sour aptamers
It closes in object, 37 DEG C of incubation 30-60min, aptamer dosage and bovine serum albumin(BSA)-bead complexes dosage volume ratio are 1:
5.Magneto separate collects the supernatant being not bonded on bovine serum albumin(BSA)-bead complexes, with ultramicron ultraviolet specrophotometer
Unbonded aptamer concentration is surveyed, and calculates the aptamer concentration in conjunction with albumen composition.It is adapted to nucleic acid
Bulk concentration is abscissa, and light absorption value is ordinate, draws and combines saturation curve, and acquiring Kd value is 69.44.As shown in Fig. 2, explanation
The binding ability of aptamer and bovine serum albumin(BSA) is very strong.
The invention has the advantages that
(1) the bovine serum albumin(BSA) nucleic acid adaptation combined in the present invention based on SELEX technology and agarose gel electrophoresis
Body screening technique is compared with traditional screening technique, with the advantage that easy to operate, screening process is at low cost, the screening period is shorter.
(2) the aptamer stability that the present invention filters out is strong, synthesis is convenient and is easy to mark various report molecules, can
Long-term preservation uses.
(3) aptamer obtained in the present invention, can specifically bind with bovine serum albumin(BSA), and affinity is high, is
The sequence for the related bovine serum albumin(BSA) aptamer that first success is screened so far.
(4) the aptamer modified different report molecule is utilized in the present invention, can construct various biosensors,
For various analysis detections such as food and drug.
Detailed description of the invention
Fig. 1: the secondary structure of bovine serum albumin(BSA) aptamer.
Fig. 2: the kd value nonlinear fitting of aptamer.
Fig. 3: secondary library and the fluorescence intensity of target binding capability reach saturation state
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1: original ssDNA pool and primer shown in following sequence are synthesized
Original ssDNA pool:
5'-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3'；
Upper primer: 5 '-FAM-AGCAGCACAGAGGTCAGATG-3 '；
Lower primer: 5 '-biotin-TTCACGGTAGCACGCATAGG-3 '.
Embodiment 2: the bovine serum albumin(BSA) aptamer screening technique based on agarose gel electrophoresis is established
Screen used in overall process: the group of combination buffer becomes 20mM Tris-HCL, 1M NaCl, 1mM EDTA, pH
7.8。
50 × TAE buffer: trishydroxymethylaminomethane (Tris) 12.1g, disodium ethylene diamine tetraacetate (EDTA- is weighed
2Na·2H2O) 1.86g, glacial acetic acid 2.855mL are settled to 50mL with tri-distilled water, and when use dilutes 50 times.
10 × tbe buffer liquid: trishydroxymethylaminomethane (Tris) 5.4g, disodium ethylene diamine tetraacetate (EDTA- is weighed
2Na·2H2O) 0.338g, boric acid 2.75g are settled to 50mL with tri-distilled water, and when use dilutes 10 times.
2% Ago-Gel of 10mL: weighing agarose powder 0.2g, and 200mL50 × TAE and 9.8mL tri-distilled water is added,
Microwave heating is dissolved after mixing, is added after micro Gelred dyestuff mixes that be transferred to gel slab to be cooled.
30% acrylamide (PAG): weighing acrylamide/methylene diacrylamide (19:1) 3g, and 10mL tri-distilled water is added
Dissolution.
20% ammonium persulfate (AP): weighing ammonium persulfate 1.0g, and the dissolution of 5mL tri-distilled water is added.
7% polyacrylamide gel: 30%PAG 8.15mL, 200 μ l of 10 × TBE 1.75mL, 20%AP, tetramethyl are measured
Base ethylenediamine (TEMED) 30 μ l, tri-distilled water 25.2mL are transferred to Vertial electrophorestic tank after sufficient vortex mixing, place loading and comb rear chamber
Temperature standing 30 minutes or more.
The bovine serum albumin(BSA) aptamer screening technique step based on agarose gel electrophoresis:
The screening of 2.1 first round
2.1.1 single-stranded random library preparation
It takes the original ssDNA pool of 1OD in 95 DEG C of water-bath 5min, is used after being subsequently placed at cooled on ice to room temperature.
2.1.2 bovine serum albumin(BSA) (BSA) solution prepares
500ng/ml BSA solution for standby is prepared with distilled water.
2.1.3 Ago-Gel and immobilized BSA albumen are prepared
The Ago-Gel solution (i.e. every 100mlTAE buffer contains 2g agarose) of configuration 2% adds in micro-wave oven
The 2% agarose solution 1min of hot 90ml, the BSA solution 60ml of the 500ng/mL made in advance is added at once at this time by 95 DEG C
Into the uncolled agarose solution for forming gel, it is transferred to plastic plate after mixing well it is stored at room temperature 30min or more and be cooled into
Ago-Gel.
2.1.4 agarose gel electrophoresis
The immobilized BSA albumen Ago-Gel of step 2.1.3 is put into electrophoresis tank, 5 μ l DNA marker work is separately added into
To start electrophoresis, voltage 200V, electrophoresis time 25min behind the original random single chain library of instruction and 50 μ l 200nmol.
2.1.5 gel extraction
After electrophoresis by multi-functional gel imager 300-600bp (molecular weight after DNA and protein binding between
300-600bp) place carries out gel extraction band, and the DNA in gel is extracted according to the specification that DNA gel extraction kit provides
Template as next step PCR amplification.
2.1.6 using DNA obtained by 2.1.5 as template, downstream primer and upstream primer carry out PCR amplification.
2.2 second wheel screenings
2.2.1 it prepares single stranded DNA: taking the Streptavidin MagneSphere of 200 μ l, wash three times (400 μ every time with combination buffer
L), it is added in the product of 2.1.6PCR amplification, shaking captures 30min, and Magneto separate removes supernatant, washes three with combination buffer
All over (400 μ l every time), the sodium hydroxide solution that 125 μ l, 0.2mol/L are added reacts 5min, and Magneto separate takes supernatant, is added afterwards
Hydrochloric acid (0.2mol/L) is adjusted to 7 with wide pH value test paper, purifying, freeze-drying.It is screened as next round in the library secondary ssDNA.
2.2.2 single-stranded random library prepares
The secondary library ssDNA that step 2.2.1 is obtained makes after being subsequently placed at cooled on ice to room temperature in 95 DEG C of water-bath 5min
With.
2.2.3 bovine serum albumin(BSA) (BSA) solution prepares
500ng/ml BSA solution for standby is prepared with distilled water.
2.2.4 Ago-Gel and immobilized BSA albumen are prepared
The Ago-Gel solution (i.e. every 100mlTAE buffer contains 2g agarose) of configuration 2% adds in micro-wave oven
The 2% agarose solution 1min of hot 90ml, the BSA solution 60ml of the 500ng/mL made in advance is added at once at this time by 95 DEG C
Into the uncolled agarose solution for forming gel, it is transferred to plastic plate after mixing well it is stored at room temperature 30min or more and be cooled into
Ago-Gel.
2.2.5 agarose gel electrophoresis
The Ago-Gel of the immobilized BSA albumen of step 2.2.4 is put into electrophoresis tank, 5 μ l DNA marker are separately added into
Start electrophoresis, voltage 200V, electrophoresis time 25min after the 50 μ l secondary libraries prepared as instruction and step 2.2.2.
2.2.6 gel extraction
After electrophoresis by multi-functional gel imager 300-600bp (molecular weight after DNA and protein binding between
300-600bp) place carries out gel extraction band, and the DNA in gel is extracted according to the specification that DNA gel extraction kit provides
Template as next step PCR amplification.
2.2.7 using DNA obtained by 2.2.6 as template, downstream primer and upstream primer carry out PCR amplification.
2.3 recycle 5-8 times according to step 2.2, the aptamer that enrichment can be specifically bound with bovine serum albumin(BSA).
Embodiment 3:PCR expands library system
PCR system includes: 15 μ l, Takara Premix TaqTM of template, 25 μ l μ l, and 1.25 μ l of upstream primer, downstream is drawn
1.25 7.5 μ l of μ l, ddH2O of object
Embodiment 4: bovine serum albumin(BSA) aptamer is obtained
After the obtained secondary library ssDNA of every wheel screening passes through 5 ' end mark fluorescent groups in conjunction with target, with enzyme mark
(absorbing wavelength 488,525) received wave is a length of to survey the secondary library ssDNA and target binding capability to instrument.Until fluorescence intensity reaches
Maximum saturation state obtains bovine serum albumin(BSA) aptamer, such as Fig. 3 at this time.
Embodiment 5: cloning and sequencing
Last obtained aptamer of wheel screening is sent to Shanghai Sheng Gong Technology Co., Ltd. to be sequenced to get arriving
Nucleic acid aptamer sequence described in above-mentioned screening.That is 5 '-AGCAGCACAGAGGTCAGATGGTATCGAGCGCAGGGGCGCCTT
TGTTAATGATTACGGGCGCCTATGCGTGCTACCGTGAA-3'.Fig. 1 is the second level knot of bovine serum albumin(BSA) aptamer
Composition
Embodiment 6: affinity analysis
500 μ l bovine serum albumin(BSA)s-bead complexes are prepared according to carboxyl magnetic bead and albumen coupling specification.It is slow with combining
Aptamer is configured to a series of concentration (i.e. 0,0.1,0.2,0.4,0.6,0.8,1.0 μM) by fliud flushing.Each concentration gradient
100 μ l are taken, 90 DEG C of water-bath 10min, 4 DEG C of 15min, room temperature 5min activation are added into 500 μ l bovine serum albumin(BSA)s-magnetic bead
In compound, 37 DEG C of incubation 30min.Magneto separate collects the supernatant being not bonded on bovine serum albumin(BSA)-bead complexes, uses
Ultramicron ultraviolet specrophotometer surveys unbonded nucleic acid concentration, and calculates in conjunction with bovine serum albumin(BSA)-bead complexes
It is adapted to bulk concentration.Using aptamer concentration as abscissa, light absorption value is ordinate, passes through equation Y=Bmax × X ÷ (Kd+X)
It carries out curve fitting, wherein Y is the light absorption value of Aptamer；Bmax is maximum light absorption value；X is Aptamer concentration.It draws and combines
Saturation curve, acquiring Kd value is 69.44.As shown in Fig. 2, illustrating the binding ability of aptamer and bovine serum albumin(BSA) very
By force.
Sequence table
<110>Beijing University of Chemical Technology
<120>a kind of screening technique for specifically binding bovine serum albumin(BSA) aptamer
<141> 2018-11-23
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 80
<212> DNA
<213>a kind of specific binding bovine serum albumin(BSA) aptamer (2 Ambystoma laterale x
Ambystoma jeffersonianum)
<400> 1
agcagcacag aggtcagatg gtatcgagcg caggggcgcc tttgttaatg attacgggcg 60
cctatgcgtg ctaccgtgaa 80
Claims (3)
1. a kind of screening technique for specifically binding bovine serum albumin(BSA) aptamer, it is characterised in that the following steps are included:
1) synthesizes original ssDNA pool and primer shown in following sequence:
Original ssDNA pool:
5'-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3'；
Upper primer: 5 '-FAM-AGCAGCACAGAGGTCAGATG-3 '；
Lower primer: 5 '-biotin-TTCACGGTAGCACGCATAGG-3 '；
2) it establishes used in the bovine serum albumin(BSA) aptamer screening technique screening overall process based on agarose gel electrophoresis
The group of combination buffer becomes 20mM Tris-HCL, 1M NaCl, 1mM EDTA, pH 7.8；
The bovine serum albumin(BSA) aptamer screening technique step based on agarose gel electrophoresis:
The screening of 2.1 first round
2.1.1 single-stranded random library preparation
The original ssDNA pool of 1-2OD uses after being subsequently placed at cooled on ice to room temperature in 95 DEG C of water-bath 5min；
2.1.2 bovine serum albumin(BSA) BSA solution prepares
500ng/ml BSA solution for standby is prepared with distilled water；
2.1.3 Ago-Gel and immobilized BSA albumen are prepared
Ago-Gel solution, that is, every 100mlTAE buffer of configuration 2% contains 2g agarose, and 2% fine jade is heated in micro-wave oven
Lipolysaccharide solution 1-2min, is added to uncolled formation for the BSA solution of the 500ng/mL made in advance at once at this time by 95-98 DEG C
In the agarose solution of gel, the BSA liquor capacity ratio of agarose solution and addition is 3:2, is transferred to plastic plate after mixing well
It is stored at room temperature 30min or more and is cooled into Ago-Gel；
2.1.4 agarose gel electrophoresis
The Ago-Gel of the immobilized BSA albumen of step 2.1.3 is put into electrophoresis tank, 3-5 μ l DNA marker work is separately added into
For indicate and the original random single chain library of 50-300nmol after start electrophoresis, in the dosage and gel in original random single chain library
Fixed BSA liquor capacity ratio is 1:1200, voltage 150-200V, electrophoresis time 25-35min；
2.1.5 gel extraction
It carries out gel extraction band after electrophoresis at 300-600bp by multi-functional gel imager, cuts glue according to DNA and return
Receive template of the DNA in the specification extraction gel that kit provides as next step PCR amplification；
2.1.6 using DNA obtained by 2.1.5 as template, downstream primer and upstream primer carry out PCR amplification；
2.2 second wheel screenings
2.2.1 it prepares single stranded DNA: Streptavidin MagneSphere being taken to be washed three times with combination buffer, be added to 2.1.6PCR amplification
In product, shaking captures 30-60min, and Magneto separate removes supernatant, washed three times with combination buffer, the hydrogen of 0.2mol/L is added
Sodium hydroxide solution reacts 5min；Streptavidin MagneSphere and sodium hydroxide solution dosage volume ratio are 8:5；Magneto separate takes supernatant,
The hydrochloric acid being added afterwards is adjusted to 7 with wide pH value test paper, and purifying, freeze-drying obtain the secondary library ssDNA and screen as next round；
2.2.2 single-stranded random library prepares
The secondary library ssDNA that step 2.2.1 is obtained uses after being subsequently placed at cooled on ice to room temperature in 95 DEG C of water-bath 5min；
2.2.3 bovine serum albumin(BSA) BSA solution prepares
500ng/ml BSA solution for standby is prepared with distilled water；
2.2.4 Ago-Gel and immobilized BSA albumen are prepared
Operating procedure is the same as step 2.1.3
2.2.5 agarose gel electrophoresis
The Ago-Gel of the immobilized BSA albumen of step 2.2.4 is put into electrophoresis tank, 3-5 μ l DNA marker work is separately added into
To indicate and starting electrophoresis, the BSA solution fixed in the dosage and gel of secondary library after the secondary library of step 2.2.2 preparation
Volume ratio is 1:1200, voltage 150-200V, electrophoresis time 25-35min；
2.2.6 gel extraction
It carries out gel extraction band after electrophoresis at 300-600bp by multi-functional gel imager, cuts glue according to DNA and return
Receive template of the DNA in the specification extraction gel that kit provides as next step PCR amplification；
2.2.7 using DNA obtained by 2.2.6 as template, downstream primer and upstream primer carry out PCR amplification；
2.3 recycle 5-8 times according to step 2.2, the aptamer that enrichment can be specifically bound with bovine serum albumin(BSA)；
3) obtains bovine serum albumin(BSA) aptamer: it is glimmering by 5 ' end labels that every wheel screens the obtained secondary library ssDNA
After light group is in conjunction with target, the secondary library ssDNA and target binding capability are surveyed with microplate reader；Until fluorescence intensity reaches maximum
Saturation state obtains bovine serum albumin(BSA) aptamer at this time.
2. a kind of specific binding bovine serum albumin(BSA) nucleic acid aptamer sequence is 5 '-AGCAGCACAGAGGTCAGATGGTATC
GAGCGCAGGGGCGCCTTTGTTAATGATTACGGGCGCCTATGCGTGCTACCGTGAA-3’。
3. according to the method described in claim 1, it is characterized by: the group of combination buffer used in screening overall process becomes
20mM Tris-HCL, 1M NaCl, 1mM EDTA, pH 7.8.
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| CN110317812A (en) * | 2019-04-16 | 2019-10-11 | 中国科学院青岛生物能源与过程研究所 | One group of Nattokinase aptamer and its screening technique |
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