CN109251916A - - 1 mutain of Aminopeptidases albumen of people a kind of and its application - Google Patents
- 1 mutain of Aminopeptidases albumen of people a kind of and its application Download PDFInfo
- Publication number
- CN109251916A CN109251916A CN201810959257.8A CN201810959257A CN109251916A CN 109251916 A CN109251916 A CN 109251916A CN 201810959257 A CN201810959257 A CN 201810959257A CN 109251916 A CN109251916 A CN 109251916A
- Authority
- CN
- China
- Prior art keywords
- albumen
- aminopeptidases
- people
- seq
- mutain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/11—Aminopeptidases (3.4.11)
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/20—Dermatological disorders
- G01N2800/205—Scaling palpular diseases, e.g. psoriasis, pytiriasis
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to -1 mutain of Aminopeptidases albumen of people a kind of and its applications, by using a large amount of psoriatics as research case, genetic test is carried out to case and is analyzed, determine the mutain of the Aminopeptidases albumen -1 of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the mutain of the Aminopeptidases albumen -1 of the people, impulse is provided for the gene diagnosis of psoriasis, and certain theoretical basis is provided for the diagnosing and treating of disease.
Description
Technical field
The present invention relates to -1 mutain of Aminopeptidases albumen of a kind of genetic engineering field more particularly to a kind of people and its
Using.
Background technique
Aminopeptidase (Aminopeptidase) belongs to expeptidase, can make amino acid from the N- terminal order of polypeptide chain by
A ground separate out.Aminopeptidases albumen -1 (Aminopeptidase-like 1, NPEPL1) is a kind of 523 peptide ammino acids, is belonged to
In peptase M17 family, wide expression.NPEPL1 can remove unsubstituted -terminal amino acid by catalysis from various peptides
Participate in processing, decomposition and the degradation of intracellular protein.
The mutation of Aminopeptidases albumen -1 of people can cause to seriously affect to human health.To a large amount of psoriatics
Peripheral blood carry out gene sequencing during, it is found that the Aminopeptidases albumen -1 of patient is mutated, therefore, to the ammonia of people
Peptase albuminoid -1 be mutated detection to judge human body whether suffer from related disease have certain impulse.
Summary of the invention
It is an object of that present invention to provide -1 mutain of Aminopeptidases albumen of people a kind of and its applications.
Technical solution of the present invention includes:
In a first aspect, -1 mutain of Aminopeptidases albumen of people a kind of is provided, amino acid sequence such as SEQ ID NO:1
It is shown.
Second aspect provides a kind of encoding gene of -1 mutain of Aminopeptidases albumen of people, and nucleotide sequence is such as
Shown in SEQ ID NO:2.
The third aspect provides a kind of genetic chip, comprising: solid phase carrier and fixed nucleotide probe on this carrier；
Aminopeptidase of the nucleotide probe according to the nucleotide sequence of -1 mutain of Aminopeptidases albumen of people described above, with people
The comparison result of the nucleotide sequence of the normal albumen of albuminoid -1 determines.
Preferably, the nucleotide probe is base sequence shown in SEQ ID NO:3；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:4；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:5.
Fourth aspect provides a kind of reagent, dashes forward in the reagent containing the Aminopeptidases albumen -1 for detecting above-mentioned people
The white primer pair of a kink of preserved egg.
Preferably, the primer pair includes the combination of following any upstream primers Yu following any downstream primers:
Upstream primer is the base sequence as shown in any in SEQ ID NO:8~NO:24；
Downstream primer is the base sequence as shown in any in SEQ ID NO:25~NO:39.
5th aspect, provides a kind of monoclonal antibody of -1 mutain of Aminopeptidases albumen of specific recognition people, by
Mentioned reagent is prepared, can be in conjunction with amino acid sequencespecific shown in SEQ ID NO:1.
6th aspect, provides a kind of for detecting the ELISA reagent of the antibody of -1 mutain of Aminopeptidases albumen of people
Box, the ELISA kit include: the ELISA ELISA Plate for being coated with said monoclonal antibody, ELIAS secondary antibody, test object
- 1 albumen of Aminopeptidases albumen, sample diluting liquid, coating buffer, ELISA ELISA Plate cleaning solution, developing solution and terminate liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeled.
The present invention provides -1 mutain of Aminopeptidases albumen of people a kind of and its applications, and a large amount of psoriatics are made
To study case, genetic test is carried out to case and is analyzed, the mutain of the Aminopeptidases albumen -1 of people is determined, according to this
- 1 mutain of Aminopeptidases albumen of people prepares genetic chip, monoclonal antibody and ELISA kit, is the gene of psoriasis
Diagnosis provides impulse, provides certain theoretical basis for the diagnosing and treating of disease.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention and the accompanying drawings.
Detailed description of the invention
Fig. 1 is a kind of comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 2 is a kind of genetic chip layout that the embodiment of the present invention one provides；
Fig. 3 is a kind of colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 4 is another colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 5 is the standard curve that the offer of the embodiment of the present invention five is protein content；
Fig. 6 is the Western blot testing result schematic diagram that the embodiment of the present invention five provides.
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.
The preparation of embodiment one, genetic chip
It is possible, firstly, to be a kind of " detection side of mutain according to application No. is " 201710429915.8 ", patent name
Detection method described in the Chinese invention patent of method and device " determines the volume of -1 mutain of Aminopeptidases albumen of people
Code gene, nucleotide sequence is as shown in SEQ ID NO:2；Correspondingly, the Aminopeptidases of people are determined according to the encoding gene
- 1 mutain of albumen, amino acid sequence is as shown in SEQ ID NO:1.
Secondly, realizing gene as follows according to -1 mutain of Aminopeptidases albumen and its encoding gene of people
The preparation of chip.
1, the design of nucleotide probe
(1) design of nucleotide probe: nucleotide probe is according to the nucleotide of -1 mutain of Aminopeptidases albumen of people
Sequence determines with the comparison result of the nucleotide sequence of the normal albumen of Aminopeptidases albumen -1 of people, and according to following probe
Design principle, design for people -1 mutain of Aminopeptidases albumen specificity nucleotide probe.
Wherein, the principle of nucleotide probe design is as follows:
1. nucleotide probe Tm value should be close to the average Tm value of whole gene group, 5 DEG C of fluctuation up and down；
2. the duplicate single base of nucleotide probe intramolecular is continuously no more than 4；
3. G+C content is 40%-60%, the specificity that non-specific hybridization guarantees hybridization is reduced；
4. it should be less than 4bp with the bases longs of probe molecule internal stability secondary structure pairing in nucleotide probe sequences,
Can guarantee that hybridization efficiency will not be influenced because of the secondary structure of nucleotide probe internal stability in this way；
5. the similitude through Homology search and other sequences is less than 40%；
6. being continuously no more than 20 bases with the homologous fragment of non-probe alternative sequence by contrast.
Wherein, the corresponding amino acid sequence of -1 mutain of Aminopeptidases albumen of people and the Aminopeptidases albumen -1 of people is being just
The comparison result of the corresponding amino acid sequence of normal albumen is referring to FIG. 1, wherein, the Query sequence in Fig. 1 is the Aminopeptidases of people
The corresponding amino acid sequence of -1 mutain of albumen, Sbjct sequence are the corresponding ammonia of the normal albumen of Aminopeptidases albumen -1 of people
Base acid sequence, the sequence between Query sequence and Sbjct sequence are comparison result, as can be seen from FIG. 1, the Aminopeptidases egg of people
Aminopeptidases albumen -1 normal albumen of white -1 mutain relative to people, inserts a part of amino acid sequence.According to SEQ
The comparison result of nucleotide sequence shown in ID NO:2 and Fig. 1, in order to the ammonia peptide of specific recognition object to be detected
Whether enzyme albumen -1 is mutated, then when choosing nucleotide probe, it can be according to any in following several modes
Kind mode designs nucleotide probe:
1. choosing the insertion nucleotides sequence column-generation nucleotide probe of insertion position；
2. before insertion position, and/or, after insertion position, several nucleotide sequences are respectively selected, by selection
Several consecutive nucleotides and the insertion nucleotide sequence of insertion position generate nucleotide probe jointly, wherein the nucleosides of generation
The sequencing of adjacent nucleotide and its sequence consensus in the corresponding nucleotide sequence of mutain in acid probe；
3. several nucleotide sequences are selected before insertion position, at the starting position of insertion position select it is several
A nucleotide sequence generates nucleotide probe；
4. several nucleotide sequences are selected after insertion position, at the end position of insertion position select it is several
A nucleotide sequence generates nucleotide probe.
In the present embodiment, it the nucleotide sequence according to shown in SEQ ID NO:2 and is set according to above-mentioned nucleotide probe
Principle is counted, at least can be designed that following several preferably nucleotide probes in the manner described above:
The nucleotide probe as shown in SEQ ID NO:3 are as follows: aagagggcac ggaa；
The nucleotide probe as shown in SEQ ID NO:4 are as follows: ggggtactac；
The nucleotide probe as shown in SEQ ID NO:5 are as follows: acctctttgg ga.
(2) above-mentioned designed nucleotide probe the synthesis of nucleotide probe: is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether the Aminopeptidases albumen -1 of detection people mutates
In order to guarantee the quality of test object sample, blank control, positive control and yin need to be also designed on genetic chip
Property control.Wherein, the layout of the genetic chip can be at least a kind of layout as shown in Figure 2.In Fig. 2, is blank pair
According to, zero is negative control,For positive control, ☆ is experimental group.
Wherein, the position of experimental group is the point sample position of nucleotide probe.
Point sample needed for negative internal reference Quality Control probe and positive control for point sample needed for blank control, negative control
Positive internal control Quality Control probe design it is as follows:
Blank control: the blank sampling liquid for being free from any genetic fragment refers to as the contamination monitoring in chip fabrication process
Mark.
Negative internal reference Quality Control probe: being one section does not have other genetic fragments of homology with detection gene, as hybridizing
The monitor control index of non-specific hybridization in journey, the nucleotide number that negative internal reference Quality Control probe includes can be with nucleotide probes
Base number is identical, can also be different.In the embodiment of the present invention, negative internal reference probe sequence needed for genetic chip can be with are as follows:
tgatgctgat aattgcat。
Positive internal control Quality Control probe: being one section of other genetic fragment for having homology with detection gene, in the aminopeptidase of people
In -1 mutain of albuminoid, select sequence corresponding from nucleotide probe different, and can be with of nucleotide probe base
Number is identical, and one section of nucleotide sequence that can also be different from the number of nucleotide probe base is as positive internal control Quality Control probe.
Preferably, nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen.The present invention is implemented
In example, positive internal control probe sequence needed for genetic chip can be with are as follows: gcggctagca gc.
It should be noted that clicking and entering negative internal reference in the deposition process of genetic chip according to the layout of genetic chip and visiting
Needle and positive internal control probe solution；The spotting buffer (10% aqueous trehalose) that reagent used in blank control is 1 times.
The application of embodiment two, genetic chip
1, sample treatment
(1) the blood 1-3mL of acquisition testing object.
(2) the 1.5mL EP pipe for taking DEPC to handle, as detected sample processing tube, in detected sample processing tube
The middle 300 μ L of blood that test object is added, adds 700 μ L of Trizol, mixes well, be placed at room temperature for 10min.
(3) chloroform of 140 μ L is added, covers tightly pipe lid, firmly shakes, be placed at room temperature for 3-5min, be placed in a centrifuge,
12000r/min, 4 DEG C of centrifugation 15min, it is careful to draw supernatant in centrifuge tube, the supernatant of absorption is transferred to clean centrifuge tube
In.
(4) isometric isopropanol is added in the centrifuge tube of supernatant in storing step (3), it is abundant gently overturns centrifuge tube
Liquid is mixed, 10min, 12000r/min, 4 DEG C of centrifugation 15min is placed at room temperature for, carefully sucks all supernatants.
(5) it being cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min, 4 DEG C of centrifugation 15min carefully suck all supernatants,
10 μ L DEPC processing water dissolution is added in the dry 15min in super-clean bench.
(6) products therefrom will affect the labeling effciency and chip hybridization knot of probe if the purity of total serum IgE is not high for RNA
Fruit.So using QIAGENKit purifies total serum IgE.
2, the first chain of cDNA and the second chain one-step synthesis method
(1) 2 μ g RNA is taken to configure following reaction solution in the centrifuge tube of 1.5mL:
| Total serum IgE | The most 6.5 μ L of 2 μ g |
| T7 Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
Wherein, the additional amount X μ L of RNase-free Water is to subtract T7 Promotor according to 11.5 μ L of total volume
The 5 μ L of additional amount of primer, then subtract the additional amount of total serum IgE and calculate and get.
(2) 10min, and ice bath 5min are kept the temperature at 65 DEG C, and 5 × First Strand B μ ffer is preheated at 65 DEG C
5min。
(3) following cDNA synthetic system is configured:
(4) above-mentioned 8.5 μ L is added after mixing after being denaturalized in the RNA of ice bath.
(5) it is centrifuged after being mixed with pipette tips.
(6) 40 DEG C of reaction 2h.
3, aaUTP marks cRNA synthesis
The configuration of NTP:
| 100mM ATP | 250μL |
| 100mM GTP | 250μL |
| 100mM CTP | 250μL |
| 100mM UTP | 187.5μL |
| RNase free H2O | 62.5μL |
| Total volume | 1000μL |
It is spare to be distributed into 10 pipes, note: 40 DEG C of heat preservation 1min before 50%PEG (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcription mix；
(1) Transcription mix is configured
| RNase-free Water | 5.7μL |
| 4×Transcription Buffer | 20μL |
| NTP | 16μL |
| 0.1M DTT | 6μL |
| 50%PEG | 6.4μL |
| aa-UTP(25mM) | 4μL |
| Inorganic Pyrophosphatase | 0.6μL |
| T7 RNA Polymerase | 0.8μL |
| Total volume | 60μL |
(2) 60 μ L Transcription mix are added and mix.
(3) 60 DEG C of hot lid in PCR instrument, 40 DEG C of reaction 2h.
4, cRNA is purified
QIAGEN RNeasy Mini kit purifies cRNA, and specific method can be found in what QIAGEN company provided with kit
Operation manual.
(1) 20 μ L RNase free water are added, 350 μ L Buffer RLT are added and mix well.
(2) 250 μ L dehydrated alcohols are added, Tip mix well.
(3) total 700 solution of the μ L containing total serum IgE are transferred to cover in the RNeasy pillar in 2mL centrifuge tube >=8000g from
Heart 15-30s, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=8000g centrifuge washing 15-30s discards filter
It crosses liquid and discards the casing of filtered solution and 2mL for RNeasy in >=8000g centrifuge washing 2min with 500 μ L Buffer RPE again
Mini pillar is transferred in a new 1.5mL Eppendorf pipe.
(5) water for drawing 30 μ L RNase free stands 1min, >=8000g centrifugation elution 1min.
(6) it is primary that step (5) are repeated.
5, cRNA concentration mensuration
With spectrophotometric analysis cRNA concentration.Need to measure the light absorption value at 260nm and 280nm to determine the dense of sample
Degree and purity, A260/A280 should be purer cRNA (ratio 1.9-2.1 can also) close to 2.0.
6, cRNA fluorescent marks；
(1) it takes above-mentioned 4 μ g of cRNA and is concentrated into 6.6 μ L.
(2) plus 10 μ L DMSO are mixed.
(3) add the sodium bicarbonate (NaHCO that the 0.3M pH of 3.4 μ L is 9.03) and mix.
(4) above-mentioned 20 μ L cRNA mixture is added in fluorescent dye Cy3 and is mixed.25 DEG C of heat preservation 1h.
(5) 25 DEG C of heat preservation 15min after adding the 4M Hydroxylamine of 9 μ L to mix.
7, fluorescent marker cRNA Sample Purification on Single
The process that specific steps are purified with cRNA in the step 4 of the present embodiment, this step are not repeating.
8, cRNA sample fragment and 4 × 44K of chip hybridization microarrays
(1) according to the form below prepares fragmentation mixed liquor and then carries out fragmentation in 60 DEG C of warm bath 30min.
| Cy3 cRNA green fluorescence | 875ng |
| 10×Blocking Agent | 11μL |
| 25×Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ L 2 × GEx Hybridization Buffer are added.
(3) 100 μ L hybridization solutions is taken to be added drop-wise on chip ware, at the same be added dropwise respectively as shown in Figure 2 by chip layout blank,
On negative, positive, experimental group position.It covers chip and is sealed in hybridizing box, 60 DEG C of rollings hybridize 16h.
9, chip washs
Washing lotion 1 (1L) configuration:
| DEPC-H2O | 700mL |
| 20×SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 2 (1L) configuration:
| DEPC-H2O | 997mL |
| 20×SSPE | 3.0mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 3:Stabilization and Drying Solution
(1) it takes out chip and washs 1min in washing lotion 1；
(2) chip is put into washing 1min (37 DEG C) in washing lotion 2 again；
(3) chip is finally washed into 30s in washing lotion 3.
10, chip scanning
Chip is scanned in scanner, -1 albumen of Aminopeptidases albumen of test object is determined according to scanning result
Whether it is mutated.Please refer to Fig. 3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, positive control is green
Color fluorescence shows that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence, then showing to detect
- 1 albumen of Aminopeptidases albumen of object is mutated.In result shown in Fig. 4, negative control is colorless fluorescent, positive right
According to for green fluorescence, showing that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, then table
- 1 albumen of Aminopeptidases albumen of bright test object does not mutate.
Embodiment three, specific recognition people -1 mutain of Aminopeptidases albumen monoclonal antibody preparation
1, the determination of reagent
Contain the primer pair for detecting -1 mutain of Aminopeptidases albumen of above-mentioned people in the reagent.
Wherein, it according to the ORF of the normal albumen of the Aminopeptidases albumen of people -1 (as shown in SEQ ID NO:6), can determine
The corresponding base sequence of ORF of the normal albumen of Aminopeptidases albumen -1 of people out (as shown in SEQ ID NO:7).
It is possible to further according to the base sequence of -1 mutain of Aminopeptidases albumen of people (such as SEQ ID NO:2 institute
Show), and according to the corresponding base sequence of ORF of the normal albumen of Aminopeptidases albumen -1 of the people of people (such as SEQ ID NO:7 institute
Show), design following upstream primer and downstream primer:
Upstream primer (F):
As shown in SEQ ID NO:8: atggtctgcg agcagccgga ggtctttgct
As shown in SEQ ID NO:9: tccgcctgtg ccctggcccg ggccttcccg
As shown in SEQ ID NO:10: ctgttcaccc accgctcagg tgcctctcgg
As shown in SEQ ID NO:11: cgcttggaga agaagacggt caccgtggag
As shown in SEQ ID NO:12: tttttcctgg tgggacaaga caacgggccg
As shown in SEQ ID NO:13: gtggaggtgt ccacattgca gtgcttagcg
As shown in SEQ ID NO:14: aatgccacag acggcgtgcg gctagcagcc
As shown in SEQ ID NO:15: cgcatcgtgg acacaccctg caatgagatg
As shown in SEQ ID NO:16: aacaccgaca ccttcctcga ggagattaac
As shown in SEQ ID NO:17: aaagttggaa aggagctggg gatcatccca
As shown in SEQ ID NO:18: accatcatcc gggatgagga actgaagacg
As shown in SEQ ID NO:19: agaggatttg gaggaatcta tggggttggc
As shown in SEQ ID NO:20: aaagccgccc tgcatccccc agccctggcc
As shown in SEQ ID NO:21: gtcctcagcc acaccccaga tggagccacg
As shown in SEQ ID NO:22: cagaccatcg cctgggtggg caaagg
As shown in SEQ ID NO:23: catcgtctat gacactggag
As shown in SEQ ID NO:24: gcctcagcat caaagggaag
Downstream primer (R):
As shown in SEQ ID NO:25: ttatgaaaac ttcctgacgg cagccccc
As shown in SEQ ID NO:26: acgcgctgcc ttccccacct tgaggtca
As shown in SEQ ID NO:27: agagtggggt ggggttccct gggactac
As shown in SEQ ID NO:28: agctaagggg tggatccagg gagcactc
As shown in SEQ ID NO:29: acctgagccc cggtcagggt ggccatgt
As shown in SEQ ID NO:30: ccaggatgat gtcggccccc aggtcctt
As shown in SEQ ID NO:31: gcaagcatag gacacgccat ctgccagc
As shown in SEQ ID NO:32: accagcctgc cctcggcatc cgtgttgt
As shown in SEQ ID NO:33: tgatttccac cgtcttccct gagtacag
As shown in SEQ ID NO:34: caggtggatg tcatctggcc ttgtcgca
As shown in SEQ ID NO:35: ttgggcccca ccgagttctc agccaagc
As shown in SEQ ID NO:36: agaacacagc gtggaggttg tctttgaa
As shown in SEQ ID NO:37: accctgcttg attgcggctc tgaaggcc
As shown in SEQ ID NO:38: cccaggacgg ccgcagcacc cccgcagt
As shown in SEQ ID NO:39: ctcgcttcat ccccggcatg gtagt
In the present embodiment, the primer of -1 mutain of Aminopeptidases albumen for detecting above-mentioned people contained in reagent
Combination to including: any of the above-described upstream primer Yu any of the above-described downstream primer.
For the primer pair (primer (F) and primer (R)) of each combination producing, following PCR amplification steps are executed.
2, the DNA of test object is that template carries out PCR amplification
| 10×Buffer | 5uL |
| dNTP | 2uL |
| Ex Taq | 1uL |
| ddH2O | 5uL |
| Template DNA | 1uL |
| Primer (F) | 3uL |
| Primer (R) | 3uL |
| Total system | 20uL |
PCR amplification is carried out by template of the DNA of test object, -1 mutating protein gene of Aminopeptidases albumen for obtaining people is complete
Full wafer section, and pMD19-T Vector (Takara company) is connected, it is sequenced.Then it is prepared by special biotech firm anti-
Body is a kind of humanization or Chimeric antibodies.Wherein, the monoclonal antibody prepared can be with amino shown in SEQ ID NO:1
Acid sequence specific binding.The antibody of preparation is measured into content using ELISA method.
Example IV, -1 mutain of Aminopeptidases albumen for detecting people antibody ELISA kit
In the present embodiment, the composition of ELISA kit are as follows: be coated with the ELISA enzyme of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, -1 albumen of Aminopeptidases albumen of test object, sample diluting liquid, coating buffer, ELISA ELISA Plate
Cleaning solution, developing solution and terminate liquid.
Wherein, the parameter of above-mentioned each composition can be at least a kind of following parameter:
ELISA ELISA Plate can be the ELISA enzyme mark in 96 holes；
ELIAS secondary antibody can be the Goat anti-Human IgG of diluted HRP (horseradish peroxidase) label；Wherein, dilution times
Number can be 8000 times.
Coating buffer can be 1 × PBS, pH:7.4；
ELISA ELISA Plate cleaning solution can be 1 × PBS solution containing 0.05%Tween-20；
Developing solution can be 3,3 ', 5,5 '-tetramethyl benzidines；
Terminate liquid can be the sulfuric acid solution of 2mol/L.
Embodiment five
In embodiments of the present invention, using psoriatic as test object, and the silver is detected using ELISA kit and is considered to be worth doing
Whether -1 albumen of Aminopeptidases albumen of patient is mutated, and this method at least may include following one kind:
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plate, it is incubated at room temperature 1-2h.
B, -1 albumen of Aminopeptidases albumen of psoriatic is diluted to various concentration gradient using sample diluting liquid, and
- 1 albumen of Aminopeptidases albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plate, and is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plate cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated for 10-20min, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
H, make standard curve: using standard concentration as abscissa, the light absorption value of standard items measurement is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measurement is effective when > 0.99；Wherein it is possible to use ELISACalc
The Software on Drawing standard curve can know regression coefficient R according to the ELISACalc software2=0.99971, therefore, this survey
It is fixed effective.The wavelength that will test, which substitutes into standard curve for the light absorption value at 450nm, can acquire the ammonia peptide of psoriatic
The concentration of -1 albumen of enzyme albumen.Utilize Aminopeptidases albumen-in the ELISA method detection Sera in Patients with Psoriasis Vulgaris sample of foundation
The content of 1 albumen.And it is identified with Western blot.Referring to FIG. 5, being the standard curve of light absorption value.Wherein, X-axis is
Light absorption value, Y-axis are corresponding concentration.
I, Western blot is identified
1, glue
(1) it cleans glass plate and dries；
(2) reference molecule cloning process prepares SDS-PAGE glue:
A. lower layer's separation gel prepares system (Total Volum:15mL)
| ddH2O | 5.9mL |
| 30% acrylamide mixed liquor | 5.9mL |
| 1.5mol/L Tris(PH 8.8) | 3.8mL |
| 10%SDS | 0.15mL |
| 10% ammonium persulfate | 0.15mL |
| TEMED | 0.006mL |
B. upper layer spacer gel concentration preparation system (Total Volum:8mL)
| ddH2O | 5.5mL |
| 30% acrylamide mixed liquor | 1.3mL |
| 1.0mol/L Tris(PH 6.8) | 1.0mL |
| 10%SDS | 0.08mL |
| 10% ammonium persulfate | 0.08mL |
| TEMED | 0.008mL |
(3) lower layer's separation gel of respective volume is added in each offset plate, adds the sterile ddH of 1mL2O flattens separation
Glue blots remaining moisture in plastic plate with filter paper after gelling to be separated is solid, upper layer is then added, glue is concentrated, after being inserted into comb
Wait upper layer concentration gelling solid.
2, albumen loading electrophoresis:
(1) comb is pulled up, duct label is carried out, 5 × SDS electrophoretic buffer is added, then to being added in protein sample
SDS-PAGE albumen sample-loading buffer (5 ×), boiling water bath heat 3-5min, loading；
(2) electrophoresis first is carried out with 80V voltage, after running concentration glue to band, uses 100V voltage instead and run about 100min.
3, transferring film and incubation:
(1) first pvdf membrane is activated in methyl alcohol about 30s.Respectively by gel, foam-rubber cushion and pvdf membrane in advance in electrotransfer
30min is balanced in buffer.It is clipped by filter paper-glue-film-filter paper sequence, is put into transferring film device according to principle of the black flour to black flour
In, it is added refrigerator (ice bag), about 120min is run with the electric current of 150mA in ice；
(2) it takes the film out, is put into 5% skim milk at once after the completion of, gently room temperature closes 1h on shaking table；
(3) PBST cleans closed film, cleans 3 times, each 10min；
(4) primary antibody for being diluted to corresponding multiple is added, 4 DEG C of shaking tables are incubated overnight；
(5) primary antibody is recycled, PBST is cleaned film 3 times, each 10min；
(6) secondary antibody (being diluted with 3% milk with 1:5000-1:10000) is incubated at room temperature film 2h；
(7) film 3 times are cleaned with PBST, each 10min；
(8) after Pierce ECL Western Blotting Substrate kit A, B liquid mixes in equal volume, dropwise
It is added dropwise on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment are analyzed with Image J.
Wherein, primary antibody is -1 mutain monoclonal antibody of Aminopeptidases albumen of the standby people of corporation, and secondary antibody is horseradish
The Goat anti-Human IgG of peroxidase labelling.
J, Western blot Analysis of test results: showing according to Western blot experimental procedure as a result, such as Fig. 6 institute
Show, wherein the Marker in Fig. 6 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 55KD
There is obvious band, wherein -1 mutant protein molecules amount of Aminopeptidases albumen is about 55KD, shows the ammonia of the psoriatic
- 1 albumen of peptase albuminoid is implicitly present in mutation.
The above is only a preferred embodiment of the present invention, it is not intended to restrict the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Sequence table
<110>Tianjin lakeside Pan Gu's genetic science Development Co., Ltd
<120>-1 mutain of Aminopeptidases albumen of people a kind of and its application
<160> 39
<170> SIPOSequenceListing 1.0
<210> 1
<211> 226
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 1
Ala Asn Ala Thr Asp Gly Val Arg Leu Ala Ala Arg Ile Val Asp Thr
1 5 10 15
Pro Cys Asn Glu Met Asn Thr Asp Thr Phe Leu Glu Glu Ile Asn Lys
20 25 30
Val Gly Lys Glu Leu Gly Ile Ile Pro Thr Ile Ile Arg Asp Glu Glu
35 40 45
Leu Lys Thr Arg Gly Phe Gly Gly Ile Tyr Gly Val Gly Lys Ala Ala
50 55 60
Leu His Pro Pro Ala Leu Ala Val Leu Ser His Thr Pro Asp Gly Ala
65 70 75 80
Thr Gln Thr Ile Ala Trp Val Gly Lys Gly Ile Val Tyr Asp Thr Gly
85 90 95
Gly Leu Ser Ile Lys Gly Lys Arg Ala Arg Lys Arg Ile Gln Trp Leu
100 105 110
Pro Asp Leu Phe Gly Thr Val Ala Ser Ser Thr Gly Gly Thr Thr Met
115 120 125
Pro Gly Met Lys Arg Asp Cys Gly Gly Ala Ala Ala Val Leu Gly Ala
130 135 140
Phe Arg Ala Ala Ile Lys Gln Gly Phe Lys Asp Asn Leu His Ala Val
145 150 155 160
Phe Cys Leu Ala Glu Asn Ser Val Gly Pro Asn Ala Thr Arg Pro Asp
165 170 175
Asp Ile His Leu Leu Tyr Ser Gly Lys Thr Val Glu Ile Asn Asn Thr
180 185 190
Asp Ala Glu Gly Arg Leu Val Leu Ala Asp Gly Val Ser Tyr Ala Cys
195 200 205
Lys Asp Leu Gly Ala Asp Ile Ile Leu Asp Met Ala Thr Leu Thr Gly
210 215 220
Ala Gln
225
<210> 2
<211> 684
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 2
gcgaatgcca cagacggcgt gcggctagca gcccgcatcg tggacacacc ctgcaatgag 60
atgaacaccg acaccttcct cgaggagatt aacaaagttg gaaaggagct ggggatcatc 120
ccaaccatca tccgggatga ggaactgaag acgagaggat ttggaggaat ctatggggtt 180
ggcaaagccg ccctgcatcc cccagccctg gccgtcctca gccacacccc agatggagcc 240
acgcagacca tcgcctgggt gggcaaaggc atcgtctatg acactggagg cctcagcatc 300
aaagggaaga gggcacggaa acgaatacag tggctcccgg acctctttgg gactgtggcc 360
tgatactcca gcacaggggg tactaccatg ccggggatga agcgagactg cgggggtgct 420
gcggccgtcc tgggggcctt cagagccgca atcaagcagg gtttcaaaga caacctccac 480
gctgtgttct gcttggctga gaactcggtg gggcccaatg cgacaaggcc agatgacatc 540
cacctgctgt actcagggaa gacggtggaa atcaacaaca cggatgccga gggcaggctg 600
gtgctggcag atggcgtgtc ctatgcttgc aaggacctgg gggccgacat catcctggac 660
atggccaccc tgaccggggc tcag 684
<210> 3
<211> 14
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aagagggcac ggaa 14
<210> 4
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ggggtactac 10
<210> 5
<211> 12
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
acctctttgg ga 12
<210> 6
<211> 300
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 6
Met Val Cys Glu Gln Pro Glu Val Phe Ala Ser Ala Cys Ala Leu Ala
1 5 10 15
Arg Ala Phe Pro Leu Phe Thr His Arg Ser Gly Ala Ser Arg Arg Leu
20 25 30
Glu Lys Lys Thr Val Thr Val Glu Phe Phe Leu Val Gly Gln Asp Asn
35 40 45
Gly Pro Val Glu Val Ser Thr Leu Gln Cys Leu Ala Asn Ala Thr Asp
50 55 60
Gly Val Arg Leu Ala Ala Arg Ile Val Asp Thr Pro Cys Asn Glu Met
65 70 75 80
Asn Thr Asp Thr Phe Leu Glu Glu Ile Asn Lys Val Gly Lys Glu Leu
85 90 95
Gly Ile Ile Pro Thr Ile Ile Arg Asp Glu Glu Leu Lys Thr Arg Gly
100 105 110
Phe Gly Gly Ile Tyr Gly Val Gly Lys Ala Ala Leu His Pro Pro Ala
115 120 125
Leu Ala Val Leu Ser His Thr Pro Asp Gly Ala Thr Gln Thr Ile Ala
130 135 140
Trp Val Gly Lys Gly Ile Val Tyr Asp Thr Gly Gly Leu Ser Ile Lys
145 150 155 160
Gly Lys Thr Thr Met Pro Gly Met Lys Arg Asp Cys Gly Gly Ala Ala
165 170 175
Ala Val Leu Gly Ala Phe Arg Ala Ala Ile Lys Gln Gly Phe Lys Asp
180 185 190
Asn Leu His Ala Val Phe Cys Leu Ala Glu Asn Ser Val Gly Pro Asn
195 200 205
Ala Thr Arg Pro Asp Asp Ile His Leu Leu Tyr Ser Gly Lys Thr Val
210 215 220
Glu Ile Asn Asn Thr Asp Ala Glu Gly Arg Leu Val Leu Ala Asp Gly
225 230 235 240
Val Ser Tyr Ala Cys Lys Asp Leu Gly Ala Asp Ile Ile Leu Asp Met
245 250 255
Ala Thr Leu Thr Gly Ala Gln Val Ser Ala Pro Trp Ile His Pro Leu
260 265 270
Ala Val Val Pro Gly Asn Pro Thr Pro Leu Leu Thr Ser Arg Trp Gly
275 280 285
Arg Gln Arg Val Gly Ala Ala Val Arg Lys Phe Ser
290 295 300
<210> 7
<211> 903
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 7
atggtctgcg agcagccgga ggtctttgct tccgcctgtg ccctggcccg ggccttcccg 60
ctgttcaccc accgctcagg tgcctctcgg cgcttggaga agaagacggt caccgtggag 120
tttttcctgg tgggacaaga caacgggccg gtggaggtgt ccacattgca gtgcttagcg 180
aatgccacag acggcgtgcg gctagcagcc cgcatcgtgg acacaccctg caatgagatg 240
aacaccgaca ccttcctcga ggagattaac aaagttggaa aggagctggg gatcatccca 300
accatcatcc gggatgagga actgaagacg agaggatttg gaggaatcta tggggttggc 360
aaagccgccc tgcatccccc agccctggcc gtcctcagcc acaccccaga tggagccacg 420
cagaccatcg cctgggtggg caaaggcatc gtctatgaca ctggaggcct cagcatcaaa 480
gggaagacta ccatgccggg gatgaagcga gactgcgggg gtgctgcggc cgtcctgggg 540
gccttcagag ccgcaatcaa gcagggtttc aaagacaacc tccacgctgt gttctgcttg 600
gctgagaact cggtggggcc caatgcgaca aggccagatg acatccacct gctgtactca 660
gggaagacgg tggaaatcaa caacacggat gccgagggca ggctggtgct ggcagatggc 720
gtgtcctatg cttgcaagga cctgggggcc gacatcatcc tggacatggc caccctgacc 780
ggggctcagg tgagtgctcc ctggatccac cccttagctg tagtcccagg gaaccccacc 840
ccactcttga cctcaaggtg gggaaggcag cgcgtggggg ctgccgtcag gaagttttca 900
taa 903
<210> 8
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
atggtctgcg agcagccgga ggtctttgct 30
<210> 9
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
tccgcctgtg ccctggcccg ggccttcccg 30
<210> 10
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ctgttcaccc accgctcagg tgcctctcgg 30
<210> 11
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
cgcttggaga agaagacggt caccgtggag 30
<210> 12
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
tttttcctgg tgggacaaga caacgggccg 30
<210> 13
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gtggaggtgt ccacattgca gtgcttagcg 30
<210> 14
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
aatgccacag acggcgtgcg gctagcagcc 30
<210> 15
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
cgcatcgtgg acacaccctg caatgagatg 30
<210> 16
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
aacaccgaca ccttcctcga ggagattaac 30
<210> 17
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
aaagttggaa aggagctggg gatcatccca 30
<210> 18
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
accatcatcc gggatgagga actgaagacg 30
<210> 19
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
agaggatttg gaggaatcta tggggttggc 30
<210> 20
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
aaagccgccc tgcatccccc agccctggcc 30
<210> 21
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
gtcctcagcc acaccccaga tggagccacg 30
<210> 22
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
cagaccatcg cctgggtggg caaagg 26
<210> 23
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
catcgtctat gacactggag 20
<210> 24
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
gcctcagcat caaagggaag 20
<210> 25
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
ttatgaaaac ttcctgacgg cagccccc 28
<210> 26
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
acgcgctgcc ttccccacct tgaggtca 28
<210> 27
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
agagtggggt ggggttccct gggactac 28
<210> 28
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
agctaagggg tggatccagg gagcactc 28
<210> 29
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
acctgagccc cggtcagggt ggccatgt 28
<210> 30
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
ccaggatgat gtcggccccc aggtcctt 28
<210> 31
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
gcaagcatag gacacgccat ctgccagc 28
<210> 32
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
accagcctgc cctcggcatc cgtgttgt 28
<210> 33
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
tgatttccac cgtcttccct gagtacag 28
<210> 34
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
caggtggatg tcatctggcc ttgtcgca 28
<210> 35
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
ttgggcccca ccgagttctc agccaagc 28
<210> 36
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
agaacacagc gtggaggttg tctttgaa 28
<210> 37
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
accctgcttg attgcggctc tgaaggcc 28
<210> 38
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
cccaggacgg ccgcagcacc cccgcagt 28
<210> 39
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
ctcgcttcat ccccggcatg gtagt 25
Claims (9)
1. a kind of mutain of the Aminopeptidases albumen -1 of people, which is characterized in that its amino acid sequence such as SEQ ID NO:1 institute
Show.
2. a kind of encoding gene of -1 mutain of Aminopeptidases albumen of people, which is characterized in that its nucleotide sequence such as SEQ
Shown in ID NO:2.
3. a kind of genetic chip characterized by comprising solid phase carrier and fixed nucleotide probe on this carrier；It is described
The nucleotide sequence of nucleotide probe -1 mutain of Aminopeptidases albumen of people according to claim 2, the ammonia peptide with people
The comparison result of the nucleotide sequence of the normal albumen of enzyme albumen -1 determines.
4. genetic chip according to claim 3, which is characterized in that
The nucleotide probe is base sequence shown in SEQ ID NO:3；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:4；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:5.
5. a kind of reagent, which is characterized in that contain the Aminopeptidases egg for detecting people described in claim 1 in the reagent
The primer pair of white -1 mutain.
6. reagent according to claim 5, which is characterized in that the primer pair include following any upstream primers with it is following
The combination of any downstream primer:
Upstream primer is the base sequence as shown in any in SEQ ID NO:8~NO:24；
Downstream primer is the base sequence as shown in any in SEQ ID NO:25~NO:39.
7. a kind of monoclonal antibody of -1 mutain of Aminopeptidases albumen of specific recognition people, which is characterized in that by such as weighing
Benefit require 5 or 6 described in reagent be prepared, can be in conjunction with amino acid sequencespecific shown in SEQ ID NO:1.
8. a kind of for detecting the ELISA kit of the antibody of -1 mutain of Aminopeptidases albumen of people, which is characterized in that institute
Stating ELISA kit includes: the ELISA ELISA Plate for being coated with monoclonal antibody described in claim 7, ELIAS secondary antibody, detection pair
- 1 albumen of Aminopeptidases albumen, sample diluting liquid, coating buffer, ELISA ELISA Plate cleaning solution, developing solution and the termination of elephant
Liquid.
9. according to claim 8 for detecting the ELISA kit of the antibody of -1 mutain of Aminopeptidases albumen of people,
It is characterized in that, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeled.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810959257.8A CN109251916A (en) | 2018-08-22 | 2018-08-22 | - 1 mutain of Aminopeptidases albumen of people a kind of and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810959257.8A CN109251916A (en) | 2018-08-22 | 2018-08-22 | - 1 mutain of Aminopeptidases albumen of people a kind of and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109251916A true CN109251916A (en) | 2019-01-22 |
Family
ID=65050266
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810959257.8A Pending CN109251916A (en) | 2018-08-22 | 2018-08-22 | - 1 mutain of Aminopeptidases albumen of people a kind of and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109251916A (en) |
-
2018
- 2018-08-22 CN CN201810959257.8A patent/CN109251916A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109251918A (en) | The uroporphyrinogen decarboxylase mutain of people a kind of and its application | |
| CN109251921A (en) | The uroporphyrinogen decarboxylase mutain of people a kind of and its application | |
| CN109182322A (en) | The Peptidyl prolyl cis-trans isomerases mutain of people a kind of and its application | |
| CN108949709A (en) | 1 mutain of Estradiol 17beta-dehydrogenase of people a kind of and its application | |
| CN108424898A (en) | 1 mutain of class aminopeptidase of people a kind of and its application | |
| CN109251906A (en) | I mutain of cytochrome C oxidase subunit base of people a kind of and its application | |
| CN109251910A (en) | It is assumed that ATP dependent form RNA helicase ROK1 mutain and application | |
| CN108866023A (en) | The serine/threonine protein kitase PINK1 mutain of people a kind of and its application | |
| CN108424902A (en) | The uroporphyrinogen decarboxylase mutain of people and its application | |
| CN109251916A (en) | - 1 mutain of Aminopeptidases albumen of people a kind of and its application | |
| CN108559741A (en) | The imidazolone propionase mutain of people a kind of and its application | |
| CN109207449A (en) | The tRNA transmethylase mutain of people a kind of and its application | |
| CN109251919A (en) | Uroporphyrinogen-III synzyme the mutain of people a kind of and its application | |
| CN109251915A (en) | 4 mutain of ubiquitin-specific peptase of people a kind of and its application | |
| CN109251900A (en) | The over-long chain fatty acid of people a kind of extends 1 mutain of enzyme and its application | |
| CN109182289A (en) | The mitochondrial inner membrane translocase Tim17B mutain of people a kind of and application | |
| CN109182290A (en) | The glutaminyl peptide cyclotransferase mutain of people a kind of and its application | |
| CN109182317A (en) | The acid ceramidase mutain of people a kind of and its application | |
| CN109022384A (en) | The mutation of tRNA (guanine-N (7))-transmethylase and application | |
| CN108949715A (en) | 1 mutain of glutathione s-transferase κ of people a kind of and its application | |
| CN109182288A (en) | The farnesyl pyrophosphate synthase mutain of people a kind of and its application | |
| CN109251238A (en) | The ADP- ribosylation factor interaction protein mutain of people a kind of and its application | |
| CN109206499A (en) | 5 mutain of HLA-B associated transcription factor of people a kind of and its application | |
| CN109337881A (en) | Istone lysine N- transmethylase KMT5B mutain and application | |
| CN109022391A (en) | Platelet-activating factor acetylhydrolase IB subunit γ mutain and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190122 |