CN100500865C - Method for detecting CatD protein by identifying a plurality of epitopes synchronously - Google Patents
Method for detecting CatD protein by identifying a plurality of epitopes synchronously Download PDFInfo
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- CN100500865C CN100500865C CNB2006100259115A CN200610025911A CN100500865C CN 100500865 C CN100500865 C CN 100500865C CN B2006100259115 A CNB2006100259115 A CN B2006100259115A CN 200610025911 A CN200610025911 A CN 200610025911A CN 100500865 C CN100500865 C CN 100500865C
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- oligonucleotide
- catd
- adaptive son
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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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
Abstract
The invention adopts oligonucleotide aptamers for identifying special tripeptide sequence KAI, GEL, DGI and LAS, extanding oligonucleotide aptamers by Structure-switch and phosphorylation decorating 5' end thereof, synthetising four connexons; connecting oligonucleotide aptamers combinated with target albumen through proximity dependent DNA Ligation technique as chain ringlike DNA; amplifying detecting signals via rolling-circle DNA duplicate method; the technique can detect target albumen sensitively, the least detecting concentration can get nM level; this method with flexibility can adjust according needed numbers of ligand; can detect albumen composite, provide sensitive detecting method for chip technique using oligonucleotide aptamers as ligand.
Description
Technical field
The present invention relates to the investigative technique of proteomics, the adaptive son of oligonucleotide (Nucleic Acid Aptamer) that is specifically related to the identification of protein epi-position is used for discerning simultaneously the method that a plurality of epi-positions detect protein or albumen composition.
Background technology
Protein expression can exist with monomer, also can exist with dimer, the mixture that also has more complicated polyprotein to form, detection to these albumen and even mixture is very important, particularly in protein science research, we not only will know for which type of protein expression, and we also will know proteic state simultaneously, comprises and modifying and mixture.Therefore to proteic detection particularly the various status detection behind the protein translation be vital.Research to protein science needs high-throughput techniques and sensitive detection means, nearest two dimensional electrophoresis technology and be that the protein chip of part provides important instrument for correlative study with antibody, but because limitation separately needs sensitiveer convenient mode detect albumen.Duplicate the whole bag of tricks that comes out with principle design such as proximity dependent DNA ligation and can detect SNP and albumen etc. to roll circular DNA, but some shortcomings are all arranged.
In Protein Detection; the method of proximity dependent DNA ligation can detect trace of albumin; but because the last quantitative methods that it adopted is the method for quantitative fluorescent PCR; because causing carrying out PCR, the GC too high levels of the non-specific or oligonucleotide aptamer of pcr amplification reacts the auxiliary segment of for this reason just having to prolong through regular meeting.This in addition method can only detect at the identical epi-position of proteic binary complex of the same race.The main drawback of certain this method is the existence of ligation of having powerful connections, and need carry out loaded down with trivial details optimization to its reaction conditions and disturb to reduce as far as possible.This method at most also can only detect two epi-positions simultaneously, and therefore the research for protein science needs method more flexibly.
Summary of the invention
Purpose of the present invention: be intended to propose a kind of by discerning the method that a plurality of albumen epi-positions detect specific albumen or albumen composition simultaneously, thereby overcome the defective of the above-mentioned method of protein detection of having come out.
The adaptive son of a plurality of oligonucleotides of this application is discerned the method for protein that contains corresponding sequence simultaneously: utilize the principle of proximitydependent DNA Ligation to connect into the strand cyclic DNA with target in the adaptive son of the oligonucleotide of different epi-positions, detect the strand cyclic DNA then.
Its specific practice is: I, the adaptive son of synthetic oligonucleotide is carried out proper extension, and makes sequence and the oligonucleotide aptamer complementation of two arms respectively, again it 5 ' is held and carry out phosphorylation modification; II, in reaction system, add above-mentioned various oligonucleotide aptamers and the protein room temperature was placed 1 hour, add connexon then, ligase enzyme damping fluid and ligase enzyme; III, adopt and to roll circular DNA and duplicate and detect.
Target is in tripeptides KAI, GEL, and the nucleotides sequence of the adaptive son of oligonucleotide of DGI and LAS is classified as:
The adaptive son of GEL-oligonucleotide:
gcgaagcggg?ctgaagtgca?cacagctgga?ggagtattgt?tgggtgctc
The adaptive son of KAI-oligonucleotide:
gcgcagcggg?tggagtgtta?agatgaattg?cggtgtgggc?cggcctctat?tggc
The adaptive son of LAS-oligonucleotide:
acgaagtggg?tgtatagcga?ataatcatta?agaaagggcg?ctgtgttgtg
The adaptive son of DGI-oligonucleotide:
agcctaaaat?attgcttagta?agggtggtct?ggctccgaga?ggggt
The corresponding tripeptide sequence of the adaptive son of oligonucleotide in can recognition protein, and can from the thick lysate of recombination bacillus coli, catch CathepsinD (CatD) albumen that contains corresponding tripeptide sequence with purifying.Be that recombinant expressed CatD albumen contains four epi-positions that can be identified.Also used the CatD albumen (containing three epi-positions that can discern) of posttranslational modification etc. for the specificity of checking experiment.
The use result of this detection method that proposes according to the present invention shows: this technology very sensitive detects target protein, and its detectable level is minimum can to reach the fM level.This method has handiness and can adjust the number of part as required.This technology also can detect albumen composition, to being that the chip technology of part provides the sensitive detection method with the adaptive son of oligonucleotide.
Description of drawings
Fig. 1 is CatD Protein Detection result:
Sample placement location figure in the A quantitative PCR wherein marks two positions 8,9 that signal is arranged in experiment; B is that (sample from right to left is followed successively by: 1 is Marker to 18 hours rear electrophoresis results; 2,3 is BSA; 4,5 is 1.0aM CatD; 6,7 is 100.0aM CatD; 8,9 is 10.0fM CatD); C is the detection by quantitative result data, wherein marks the position 8,9 of two samples that signal is arranged in experiment; D is the result of detection by quantitative result data behind software analysis, wherein marks the position 8,9 of two samples that signal is arranged in experiment.
Fig. 2 is CatD Protein Detection specificity result:
Sample from right to left is followed successively by: 1 is BSA; 2 is 1.0aM CatD; 3 is 100.0aM CatD; 4,5 is 10.0fM CatD; 6 is 10.0nM mCatD; 7 is 1.0nM mCatD; CatD contains four can detect epi-position; MCatD is that maturation protein only contains three detectable epi-positions.
Be described in further detail the present invention below in conjunction with accompanying drawing.
Utilize Structure-switch oligonucleotide aptamer technology to make the combination of aptamer and the corresponding tripeptides of protein and promote coupled reaction:
The main side of Structure-switch signaling oligonucleotide aptamer technology Method is after the oligonucleotide aptamer is carried out the end prolongation, can reach simultaneously with the oligomerization nucleosides The inner annealed combination of acid aptamer, two arms can not be combined with connexon coupled reaction was taken place this moment, There is not signal to produce. When the target protein of oligonucleotide aptamer occurs, target protein and oligomerization The nucleotide aptamer combination, thus thereby causing two arms to be combined with connexon promotes to connect instead Should. Be conducive to like this reduction of background coupled reaction.
(1) according to the synthetic structure-switch oligomerization of oligonucleotide aptamer sequence Nucleotide aptamer and corresponding connexon.
GEL：
acttcagccc ttttttttaa tcacttatgc gagcgggct gaagtgcaca cagctggagg
agtattgttg ggtgctcttt ctcctccagc
KAI：
ttaacactcc ttttttttat cttgcgcagc gggtggagtg ttaagatgaa ttgcggtgtg
ggccggcctc tattggcttt cccacaccgc
LAS：
cgctatacac ttttttatca cttatacgaa gtgggtgtat agcgaataat cattaagaaa
gggcgctgtg ttgtgttttt cctttcttaa
DGI：
taagcaatat tttatcactt atccatagcc taaaatattg cttagtaagg gtggtctggc
tccgagaggg?gttgaattct?gagccagacc
connector1：
aaagggctga?agtggtctgg?ctcttt
connector2：
aaaggagtgt?taagctggag?gagttt
connector3：
aaagtgtata?gcggcggtgt?gggttt
connector4：
aaaatattgc?ttattaagaa?aggttt
(2) in the PCR pipe, add four kinds of oligonucleotide aptamer mixed solutions of 1.0uL100pM after, add albumen room temperatures such as the BSA of different concns and CatD respectively and placed 1 hour; Add the damping fluid of the T4 DNA Ligase of four kinds of connexons containing 0.1uL25uM and 1.0U more respectively, room temperature is placed and was carried out ligation in 5 hours; Adopt the phi29 archaeal dna polymerase that single-stranded cyclic DNA is increased in 30 ℃ then; Detect with quantitative PCR and agarose electrophoresis at last.
Present technique can adopt the adaptive son of a plurality of oligonucleotides to detect albumen, because the adaptive son of oligonucleotide has the character that is similar to antibody, and has unapproachable advantage of antibody such as chemical stability etc.Therefore present technique can be used as with the signal detecting method of the adaptive son of oligonucleotide as the chip of protein ligands.Present technique can use the adaptive son of a plurality of oligonucleotides to determine a unique target spot, can increase result's accuracy and reliability like this.Present technique can have very big adjustment leeway simultaneously, also can design the special adaptive sub-portfolio of oligonucleotide at each target spot, and can adjust the number of part as required.Therefore be well-adapted for high-throughout chip technology.
Embodiment
Four adaptive sons of oligonucleotide detect CatD albumen
1, four kinds of oligonucleotide aptamer mixed solutions that in the PCR pipe, add 1.0uL 100pM
2, be that diluent carries out doubling dilution with CatD albumen with 1%BSA, adding concentration then in the difference pipe respectively is 1.0aM, the CatD albumen of 100.0aM and 10.0fM and 1%BSA in contrast, and room temperature was placed 1 hour
3, add contain 0.1uL 25uM four kinds of connexon mixed solutions in the damping fluid of the T4 DNA Ligase that contains 1.0U, this mixed solution is joined behind the reaction tubes room temperature places and carried out ligation in 5 hours
4, every Guan Zhongzai adds 1.0uL 10X phi29 damping fluid, 0.5uL 10mM dNTP, 0.2uL 100X BSA, 0.3uL phi archaeal dna polymerase, 7.5uL ddH
2O, 0.5uL Eva Green, 0.1uL primer1 and 0.1uL primer2.Increased 8-12 hour in 30 ℃
5, began to adopt OPTICON2 continuous fluorescencedetector (MJ Research) record data at the 8th hour four hours, every 2.5 minutes values of reading once, approximately continue four hours, during keep 30 ℃.Increase and adopt 2% agarose electrophoresis to detect after 18 hours.
6, result such as Fig. 1 show: 10.0fM albumen can detect fluorescent signal at 8-12 hour; Reacting 18 hours rear electrophoresis can obtain amplified band (from right to left sample is followed successively by among Figure 1B: 1 is Marker; 2,3 is BSA; 4,5 is 1.0aM CatD; 6,7 is 100.0aM CatD; 8,9 is 10.0fM CatD; Fig. 1 C and D represent corresponding change in fluorescence curve)
Four adaptive sons of oligonucleotide detect CatD albumen
1, four kinds of oligonucleotide aptamer mixed solutions that in the PCR pipe, add 1.0uL 100pM
2, be that diluent carries out doubling dilution with CatD albumen with 1%BSA, adding concentration then in the difference pipe respectively is 1.0aM, the CatD albumen of 100.0aM and 10.0fM and 1%BSA in contrast; Also use the CatD albumen that only contains three epi-positions promptly to add the albumen of 10.0nM and 1.0nM in contrast simultaneously in pipe, room temperature was placed 1 hour
3, add contain 0.1uL 25uM four kinds of connexon mixed solutions in the damping fluid of the T4 DNA Ligase that contains 1.0U, this mixed solution is joined behind the reaction tubes room temperature places and carried out ligation in 7 hours
4, every Guan Zhongzai adds 1.0uL 10X phi29 damping fluid, 0.5uL 10mM dNTP, 0.2uL 100X BSA, 0.3uL phi archaeal dna polymerase, 8.0uL ddH
2O, 0.1uL primerl and 0.1uL primer2 increased 36 hours in 30 ℃
5, adopt 2% agarose electrophoresis to detect
6, result such as Fig. 2 show: the CatD of 100.0aM and 10.0fM albumen can make and roll circular DNA and duplicate the generation signal, and the latter is because protein concentration is more weak than low signal.And the mCatD of high density only contains three and can detect epi-position, can't obtain amplified signal and show that very high (sample from right to left is followed successively by: 1 is BSA for the specificity of this detection method; 2 is 1.0aM CatD; 3 is 100.0aM CatD; 4,5 is 10.0fM CatD; 6 is 10.0nM mCatD; 7 is 1.0nM mCatD; CatD contains four can detect epi-position; MCatD is that maturation protein only contains three detectable epi-positions).
Above-mentioned testing process shows:
By duplicating in conjunction with rolling circular DNA, principles such as structure-switch and proximitydependent DNA ligation adopt a plurality of oligonucleotide aptamers to come recognition protein.This method can detect proteic existence, and high specificity is highly sensitive, detects minimum concentration and can reach the fM level at least.The advantage of this method is to use the adaptive son of oligonucleotide to discern, and can avoid the inconvenience of antibody like this.Roll the method that circular DNA duplicates and be applied to chip field, therefore this detection method not only can be applied to trace of albumin and detects and can also be applied to very easily in the protein chip technology of the adaptive son of oligonucleotide as part.If in conjunction with the adaptive son of oligonucleotide of identification tripeptides then can realize detection to single albumen or albumen composition.
In addition, this technology adopts the oligonucleotide aptamer of a plurality of 5 ' phosphorylations to affact respectively on the different components of albumen composition or on the proteic different epi-positions of the same race, adopts the method for proximity dependent DNA ligation to connect into the strand cyclic DNA then.The oligonucleotide aptamer of a plurality of 5 ' phosphorylations is simultaneously with corresponding target spot effect and promote ligation when having only target protein or albumen composition to occur, like this since the connection of a plurality of DNA to make the interference of background response reduce to minimum, in conjunction with the method for structure-switch, it is minimum that background response is further reduced to simultaneously.
Sequence table
＜110〉East China Normal University
＜120〉a kind of by discerning the method that a plurality of epi-positions detect albumen or albumen composition simultaneously
<160>4
<210>1
<211>49
<212>DNA
＜213〉artificial sequence
<220>
<400>1
<210>2
<211>54
<212>DNA
＜213〉artificial sequence
<220>
<400>2
<210>3
<211>50
<212>DNA
＜213〉artificial sequence
<220>
<400>3
<210>4
<211>45
<212>DNA
＜213〉artificial sequence
<220>
<400>4
Claims (4)
1, a kind ofly detects the proteic method of CatD by discerning a plurality of epi-positions simultaneously, it is characterized in that: utilize the principle of proximitydependent DNA Ligation to connect into the strand cyclic DNA with target in the adaptive son of the oligonucleotide of different epi-positions, detect the strand cyclic DNA then.
2, as claimed in claim 1ly a kind ofly detect the proteic method of CatD by discerning a plurality of epi-positions simultaneously, it is characterized in that: detecting the proteic step of CatD is: A, the adaptive son of synthetic oligonucleotide is prolonged, and makes the sequence of two arms complementary with the adaptive son of oligonucleotide respectively simultaneously, again with its 5 ' hold and carry out phosphorylation modification; B, in reaction system, add above-mentioned various oligonucleotide aptamers and the protein room temperature was placed 1 hour, add connexon then, ligase enzyme damping fluid and ligase enzyme; C, adopt and to roll circular DNA and duplicate and detect.
3, as claimed in claim 1 or 2ly a kind ofly detect the proteic method of CatD, it is characterized in that: according to the principle of Structure-switch two arms are carried out particular design and make that the adaptive son of itself and oligonucleotide is inner complementaryly to be connected background to reduce by discerning a plurality of epi-positions simultaneously.
4, according to claim 1 and 2ly a kind ofly detect the proteic method of CatD by discerning a plurality of epi-positions simultaneously, it is characterized in that: target is in tripeptides KAI, GEL, and the nucleotides sequence of the adaptive son of oligonucleotide of DGI and LAS is classified as:
The adaptive son of GEL-oligonucleotide:
gcgaagcggg?ctgaagtgca?cacagctgga?ggagtattgt?tgggtgctc
The adaptive son of KAI-oligonucleotide:
gcgcagcggg?tggagtgtta?agatgaattg?cggtgtgggc?cggcctctat?tggc
The adaptive son of LAS-oligonucleotide:
acgaagtggg?tgtatagcga?ataatcatta?agaaagggcg?ctgtgttgtg
The adaptive son of DGI-oligonucleotide:
agcctaaaat?attgcttagta?agggtggtct?ggctccgaga?ggggt。
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| CNB2006100259115A CN100500865C (en) | 2006-04-21 | 2006-04-21 | Method for detecting CatD protein by identifying a plurality of epitopes synchronously |
| PCT/US2007/008274 WO2007117444A2 (en) | 2006-03-31 | 2007-03-30 | Protein detection by aptamers |
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| CNB2006100259115A CN100500865C (en) | 2006-04-21 | 2006-04-21 | Method for detecting CatD protein by identifying a plurality of epitopes synchronously |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US10473654B1 (en) | 2016-12-01 | 2019-11-12 | Nautilus Biotechnology, Inc. | Methods of assaying proteins |
| US11203612B2 (en) | 2018-04-04 | 2021-12-21 | Nautilus Biotechnology, Inc. | Methods of generating nanoarrays and microarrays |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9612236B2 (en) * | 2010-06-17 | 2017-04-04 | Koninklijke Philips N.V. | Multi epitope assay |
| CN108795781B (en) * | 2017-05-03 | 2020-06-02 | 中国科学院微生物研究所 | Recombinant bacterium for high-yield trichoderma harzianum α -1, 3-glucanase and application thereof |
| CN108334903B (en) * | 2018-02-06 | 2021-06-11 | 南京航空航天大学 | Instruction SDC vulnerability prediction method based on support vector regression |
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Non-Patent Citations (4)
| Title |
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| Protein detection using proximity-dependent DNA ligationassays.. S Fredriksson, et al.Nature Biotechnology,Vol.20 . 2002 |
| Protein detection using proximity-dependent DNA ligationassays.. S Fredriksson, et al.Nature Biotechnology,Vol.20 . 2002 * |
| Proximity extension of circular DNA aptamers with real-timeprotein detection.. Daniel A. Di Giusto, et al.Nucleic Acids Research.,Vol.33 No.6. 2005 |
| Proximity extension of circular DNA aptamers with real-timeprotein detection.. Daniel A. Di Giusto, et al.Nucleic Acids Research.,Vol.33 No.6. 2005 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10473654B1 (en) | 2016-12-01 | 2019-11-12 | Nautilus Biotechnology, Inc. | Methods of assaying proteins |
| US10921317B2 (en) | 2016-12-01 | 2021-02-16 | Nautilus Biotechnology, Inc. | Methods of assaying proteins |
| US10948488B2 (en) | 2016-12-01 | 2021-03-16 | Nautilus Biotechnology, Inc. | Methods of assaying proteins |
| US11448647B2 (en) | 2016-12-01 | 2022-09-20 | Nautilus Biotechnology, Inc. | Methods of assaying proteins |
| US11549942B2 (en) | 2016-12-01 | 2023-01-10 | Nautilus Biotechnology, Inc. | Methods of assaying proteins |
| US11579144B2 (en) | 2016-12-01 | 2023-02-14 | Nautilus Biotechnology, Inc. | Methods of assaying proteins |
| US11754559B2 (en) | 2016-12-01 | 2023-09-12 | Nautilus Subsidiary, Inc. | Methods of assaying proteins |
| US11768201B1 (en) | 2016-12-01 | 2023-09-26 | Nautilus Subsidiary, Inc. | Methods of assaying proteins |
| US11203612B2 (en) | 2018-04-04 | 2021-12-21 | Nautilus Biotechnology, Inc. | Methods of generating nanoarrays and microarrays |
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