CN108359003A - A kind of S phases kinase-associated protein 1A（p19A）Mutain and application - Google Patents
A kind of S phases kinase-associated protein 1A（p19A）Mutain and application Download PDFInfo
- Publication number
- CN108359003A CN108359003A CN201810042827.7A CN201810042827A CN108359003A CN 108359003 A CN108359003 A CN 108359003A CN 201810042827 A CN201810042827 A CN 201810042827A CN 108359003 A CN108359003 A CN 108359003A
- Authority
- CN
- China
- Prior art keywords
- elisa
- people
- phase kinase
- associated proteins
- 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
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- 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
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Wood Science & Technology (AREA)
- Toxicology (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of S phases kinase-associated protein 1A (p19A) mutains and applications, by regarding a large amount of cancer patients as research case, genetic test is carried out to case and is analyzed, determine the mutain of the S phase kinase-associated protein 1A (p19A) of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to S phase kinase-associated proteins 1A (p19A) mutain of the people, impulse is provided for the gene diagnosis of cancer, realizes the early diagnosis, early discovery and associated treatment of cancer.
Description
Technical field
The present invention relates to a kind of genetic engineering fields more particularly to a kind of S phases kinase-associated protein 1A (p19A) to be mutated egg
Bletilla application.
Background technology
S phases kinase-associated protein 1 is the little albumen matter of about 160 amino acid.As the albumen mediated by 26S proteasomes
Matter is degraded to the cores of SCF type E3 ubiquitin ligases, and SKP1 is in cell cycle progression, transcriptional control, signal transduction, and
Play the part of pivotal player in many other cell processes in eucaryote.
S phase kinase-associated protein 1A (p19A) mutations of people can impact human health, the study found that right
During the peripheral blood of certain cancer (cancer includes lung cancer, liver cancer, cancer of pancreas and leukaemia) patients carries out gene sequencing,
Find that the S phase kinase-associated protein 1A (p19A) of patient are mutated, therefore, to the S phase kinase-associated proteins 1A of people
(p19A) detection being mutated has certain impulse to judging whether human body suffers from cancer.
Invention content
Present invention aims at S phase kinase-associated protein 1A (p19A) mutains of people of offer a kind of and its applications.
Technical solution of the present invention includes：
In a first aspect, providing a kind of S phase kinase-associated proteins 1A (p19A) mutain of people, amino acid sequence is such as
SEQ ID NO:Shown in 1.
Second aspect provides a kind of encoding gene of the mutain of the S phase kinase-associated protein 1A (p19A) of people, core
Nucleotide sequence such as SEQ ID NO:Shown in 2.
The third aspect provides a kind of genetic chip, including：Solid phase carrier and fixed nucleotide probe on this carrier；
The nucleotide probe is according to the nucleotide sequence of S phase kinase-associated proteins 1A (p19A) mutain of the people, the S with people
The comparison result of the nucleotide sequence of the normal albumen of phase kinase-associated protein 1A (p19A) determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of Dan Ke of S phase kinase-associated proteins 1A (p19A) mutain of specific recognition people
The amino acid sequence of grand antibody, coding is SEQ ID NO:Shown in 1.
5th aspect provides a kind of for detecting the antibody of S phase kinase-associated proteins 1A (p19A) mutain of people
ELISA kit, the ELISA kit include：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, inspection of said monoclonal antibody
Survey S phase kinase-associated proteins 1A (p19A) albumen of object, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution,
Developing solution and terminate liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
6th aspect provides a kind of S phase kinase-associated proteins 1A detecting people based on any of the above-described ELISA kit
(p19A) method of the antibody of mutain, including：
A, said monoclonal antibody is diluted using the coating buffer solution, and the monoclonal after dilution is resisted
Body is loaded into the hole of ELISA ELISA Plates, incubation at room temperature；
B, S phase kinase-associated proteins 1A (p19A) albumen for detecting object is diluted to difference using the sample diluting liquid
Concentration gradient, and S phase kinase-associated proteins 1A (p19A) albumen of various concentration gradient is loaded respectively to ELISA ELISA Plates
Kong Zhong, incubation at room temperature；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature is protected from light incubation, 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.
Preferably, further comprise：Blank control is tested.
The present invention provides S phase kinase-associated protein 1A (p19A) mutains of people a kind of and its applications, by a large amount of cancers
Disease patient carries out genetic test to case and analyzes, determine the S phase kinase-associated protein 1A (p19A) of people as research case
Mutain, according to S phase kinase-associated proteins 1A (p19A) mutain of the people prepare genetic chip, monoclonal antibody and
ELISA kit provides impulse for the gene diagnosis of cancer, realizes the early diagnosis, early discovery and associated treatment of cancer.
Above description is only the general introduction of technical solution 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, below with presently preferred embodiments of the present invention and after coordinating attached drawing to be described in detail such as.
Description of the drawings
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 diagrames that the embodiment of the present invention five provides；
Fig. 7 is the standard curve that the offer of the embodiment of the present invention six is protein content；
Fig. 8 is the Western blot testing result schematic diagrames that the embodiment of the present invention six provides；
Fig. 9 is the standard curve that the offer of the embodiment of the present invention seven is protein content；
Figure 10 is the Western blot testing result schematic diagrames that the embodiment of the present invention seven provides.
Specific implementation mode
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 S phase kinase-associated protein 1A (p19A) mutation of people
The encoding gene of albumen, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the S of people is determined according to the encoding gene
Phase kinase-associated protein 1A (p19A) mutain, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to S phase kinase-associated protein 1A (p19A) mutains and its encoding gene of people, as follows
Realize the preparation of genetic chip.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to S phase kinase-associated proteins 1A (p19A) mutain of people
Nucleotide sequence, come with the comparison result of the nucleotide sequence of the normal albumen of the S phase kinase-associated protein 1A (p19A) of people true
It is fixed, and according to the design principle of following probe, design the spy of S phase kinase-associated proteins 1A (p19A) mutain for people
Anisotropic nucleotide probe.
Wherein, the principle of nucleotide probe design is as follows：
1. nucleotide probe Tm values should be close to the average Tm values of whole gene group, 5 DEG C of fluctuation up and down；
2. the single base that nucleotide probe intramolecular repeats continuously is no more than 4；
3. G+C contents are 40%-60%, the specificity that non-specific hybridization ensures hybridization is reduced；
4. nucleotide probe intramolecule, which stablizes secondary structure pairing bases longs, is less than 4bp, can ensure in this way will not
Hybridization efficiency is influenced because of the secondary structure of nucleotide probe internal stability；
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 S phase kinases of S phase kinase-associated proteins 1A (p19A) mutain corresponding amino acid sequence and people of people
The comparison result of the corresponding amino acid sequence of the normal albumen of associated protein 1 A (p19A) please refers to Fig.1, wherein the Query in Fig. 1
Sequence is the corresponding amino acid sequence of S phase kinase-associated proteins 1A (p19A) mutain of people, and Sbjct sequences are the S phases of people
The corresponding amino acid sequence of the normal albumen of kinase-associated protein 1A (p19A), the sequence between Query sequences and Sbjct sequences are
Comparison result, as can be seen from FIG. 1, S phase kinase-associated protein 1A (p19A) mutains of people are related relative to the S phases kinases of people
Albumen 1A (p19A) normal albumen, several place's amino acid sequences are mutated, according to SEQ ID NO:Nucleotides sequence shown in 2
Row and Fig. 1 comparison result, be in order to the S phase kinase-associated protein 1A (p19A) of specific recognition object to be detected
It is no to be mutated, then when choosing nucleotide probe, can by include mutated site nucleotide a nucleotides sequence
Row are used as nucleotide probe.
In the present embodiment, according to SEQ ID NO:It nucleotide sequence shown in 2 and is set according to above-mentioned nucleotide probe
Principle is counted, designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, which is：
cctcctccca aagtttatga
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether the S phase kinase-associated protein 1A (p19A) of detection people mutate
In order to ensure to detect the quality of subject sample, blank control, positive control and the moon need to be also designed on genetic chip
Property control.Wherein, the layout of the genetic chip at least can be a kind of layout as shown in Figure 2.In fig. 2, is blank pair
According to, zero is negative control,For positive control,For 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：Be 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, also can be different.In the embodiment of the present invention, the negative internal reference probe sequence needed for genetic chip can be：
tgatgctgat aattgcat。
Positive internal control Quality Control probe：It is one section of other genetic fragment for having homology with detection gene, in the S phase kinases of people
In associated protein 1 A (p19A) mutain, select sequence corresponding from nucleotide probe different, and can be with nucleotide probe
The number of base 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 matter
Control probe.Preferably, nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen.This hair
In bright embodiment, the positive internal control probe sequence needed for genetic chip can be：ttgaagttga tgtggaaatt.
It should be noted that in the deposition process of genetic chip, clicks and enters negative internal reference according to the layout of genetic chip and visit
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 pipes that DEPC is handled are taken, as detected sample processing tube, in detected sample processing tube
The middle 300 μ L of blood that detection object is added, add 700 μ L of Trizol, mix well, be placed at room temperature for 10min.
(3) chloroform of 140 μ L is added, covers tightly pipe lid, firmly shakes, be placed in a centrifuge, 12000r/min, 4 DEG C of centrifugations
15min, it is careful to draw supernatant in centrifuge tube, the supernatant of absorption is transferred in clean centrifuge tube.
(4) isometric isopropanol is added in the centrifuge tube of supernatant in storing step (3), it is abundant gently overturns centrifuge tube
Mixing liquid is placed at room temperature for 10min, 12000r/min, 4 DEG C of centrifugation 15min, 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,
The dry 15min in super-clean bench is added 10 μ L DEPC and handles water dissolution.
(6) products therefrom is RNA can influence the labeling effciency and chip hybridization knot of probe if the purity of total serum IgE is not high
Fruit.So using QIAGENPurify total serum IgE.
2, the first chains of cDNA and the second chain one-step synthesis method
(1) 2 μ g RNA is taken to configure following reaction solution in 1.5mL centrifuge tubes：
| Total serum IgE | The most 6.5 μ L of 2 μ g |
| T7Promotorprimer | 5μL |
| RNase-freeWater | XμL |
| Total volume | 11.5μL |
Wherein, the addition X μ L of RNase-free Water are subtracted according to 11.5 μ L of total volume
The 5 μ L of addition of T7Promotorprimer, then subtract the addition of total serum IgE and calculate and get.
(2) 10min, and ice bath 5min are kept the temperature at 65 DEG C, in advance by 5 × First Strand B μ ffer at 65 DEG C
Preheat 5min.
(3) following cDNA synthetic systems are configured：
| 5×FirstStrandBuffer | 4μL |
| 0.1MDTT | 2μL |
| 10mMdNTPmix | 1μL |
| MMLVRT | 1μL |
| RNaseOUT | 0.5μL |
| Total volume | 8.5μL |
(4) above-mentioned 8.5 μ L are added after mixing after being denaturalized in the RNA of ice bath.
(5) pipette tips mixing is used to centrifuge later.
(6) 40 DEG C of reaction 2h.
3, aaUTP marks cRNA synthesis
The configuration of NTP：
It is spare to be distributed into 10 pipes, notes：40 DEG C of heat preservation 1min before 50%PEG (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcription mix；
(1) configuration Transcription mix
| 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 |
| T7RNA Polymerase | 0.8μL |
| Total volume | 60μL |
(2) 60 μ LTranscription mix and mixing is added.
(3) 60 DEG C of hot lid in PCR instrument, 40 DEG C of reaction 2h.
4, cRNA is purified
QIAGEN RNeasy Mini kit purify cRNA, and specific method can be found in what QIAGEN companies 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 absolute ethyl alcohols are added, Tip mix well.
(3) total 700 solution of the μ L containing total serum IgE are transferred in the RNeasy pillars being sleeved in 2mL centrifuge tubes >=8000g from
Heart 15-30s, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=8000g centrifuge washings 15-30s discards filter
It crosses liquid and discards the casing of filtered solution and 2mL by RNeasy in >=8000g centrifuge washings 2min with 500 μ L Buffer RPE again
Mini pillars are transferred in a new 1.5mL Eppendorf pipes.
(5) water for drawing 30 μ L RNase free stands 1min, >=8000g centrifugation elutions 1min.
(6) it is primary to repeat step (5).
5, cRNA concentration mensurations
With spectrophotometric analysis cRNA concentration.It needs 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 also can) close to 2.0.
6, cRNA fluorescents mark；
(1) 4 μ g of above-mentioned cRNA are taken and are concentrated into 6.6 μ L.
(2) add 10 μ L DMSO mixings.
(3) add the sodium bicarbonate (NaHCO that the 0.3M pH of 3.4 μ L are 9.03) and mixing.
(4) above-mentioned 20 μ L cRNA mixtures are added in fluorescent dye Cy3 simultaneously mixing.25 DEG C of heat preservation 1h.
(5) plus 9 μ L 4M Hydroxylamine mixings after 25 DEG C heat preservation 15min.
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 fragments and chip hybridization 4x44Kmicroarrays
(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 fluorescences | 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 roll hybridization 16h.
9, chip washs
((1L) is configured washing lotion 1：
((1L) is configured washing lotion 2：
| 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 lotion 2 again and washs 1min (37 DEG C)；
(3) chip is finally washed into 30s in washing lotion 3.
10, chip scanning
Chip is scanned in scanner, the S phase kinase-associated proteins 1A of detection object is determined according to scanning result
(p19A) whether albumen is mutated.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, sun
Property control be green fluorescence, show that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence, that
Show that S phase kinase-associated proteins 1A (p19A) albumen for detecting object is mutated.It is negative right in result shown in Fig. 4
According to for colorless fluorescent, positive control is green fluorescence, shows that the sample quality of acquisition testing object is that there is no problem, and tests
Group redgreen fluorescence, then showing that S phase kinase-associated proteins 1A (p19A) albumen for detecting object does not mutate.
Embodiment three, specific recognition people S phase kinase-associated proteins 1A (p19A) mutain monoclonal antibody
It prepares
1, according to base sequence (such as SEQ ID NO of S phase kinase-associated proteins 1A (p19A) mutain of people:2 institutes
Show) design sense primer such as SEQ ID NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (P)：atgccttcaa ttaagttg
Downstream primer (F)：agtaaagtca ttttttaa
2, the DNA of detection object is that template carries out PCR amplification
DNA to detect object carries out PCR amplification as template, obtains S phase kinase-associated protein 1A (p19A) mutation of people
The complete segment of protein gene, and pMD19-T Vector (Takara companies) are connected, it is sequenced.Then public by special biology
Department prepares antibody, is a kind of humanization or Chimeric antibodies.The antibody of preparation is measured into content using ELISA method.
Example IV, S phase kinase-associated proteins 1A (p19A) mutain for detecting people antibody ELISA reagents
Box
In the present embodiment, the group of ELISA kit becomes：It is coated with the ELISA enzymes of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, detect S phase kinase-associated proteins 1A (p19A) albumen of object, sample diluting liquid, coating buffer solution,
ELISA ELISA Plates cleaning solution, developing solution and terminate liquid.
Wherein, the parameter of above-mentioned each composition at least can be a kind of following parameter：
ELISA ELISA Plates can be the ELISA enzyme marks in 96 holes；
ELIAS secondary antibody can be the Goat anti-Human IgG of diluted HRP (horseradish peroxidase) labels；Wherein, dilution times
Number can be 8000 times.
It can be 1 × PBS, pH to be coated with buffer solution:7.4；
ELISA ELISA Plates 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, it using patients with lung cancer as detection object, and detects the lung cancer using ELISA kit and suffers from
Whether S phase kinase-associated proteins 1A (p19A) albumen of person 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 solution, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h.
B, S phase kinase-associated proteins 1A (p19A) albumen of patients with lung cancer is diluted to various concentration using sample diluting liquid
Gradient, and S phase kinase-associated proteins 1A (p19A) albumen of various concentration gradient is loaded respectively to the hole of ELISA ELISA Plates
In, and it is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates 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 solutions are added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated 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, standard curve is made：Using standard concentration as abscissa, the light absorption value that standard items measure is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measures effective when ＞ 0.99；Wherein it is possible to use ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc softwares2=0.99729, therefore, this
It measures effective.The S phases that the light absorption value substitution standard curve that the obtained wavelength of detection is the places 450nm can be acquired to patients with lung cancer swash
The concentration of enzyme associated protein 1 A (p19A) albumen.Utilize S phase kinases in the ELISA method detection Serum of Patients with Lung Cancer sample of foundation
The content of associated protein 1 A (p19A) albumen.It is used in combination Western blot to be identified.Referring to FIG. 5, for the standard of light absorption value
Curve.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot are 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)
B. upper layer spacer gel concentration preparation system (Total Volum：8mL)
| ddH2O | 5.5mL |
| 30% acrylamide mixed liquor | 1.3mL |
| 1.0mol/L Tris(PH6.8) | 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 after gelling to be separated is solid with filter paper, and upper layer is then added and concentrates glue, after being inserted into comb
Wait for upper layer concentration gelling solid.
2, albumen loading electrophoresis：
(1) comb is pulled up, duct label is carried out, 5 × SDS electrophoretic buffers 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 voltages, after waiting for that band ran concentration glue, uses 100V voltages 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 solution.It is clipped by the sequence of filter paper-glue-film-filter paper, transferring film device is put into the principle of black flour according to black flour
In, refrigerator (ice bag) is added, about 120min is run with the electric current of 150mA in ice；
(2) it takes the film out after the completion of, is put into 5% skim milk at once, 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 incubator overnights are incubated；
(5) primary antibody is recycled, PBST cleans film 3 times, each 10min；
(6) secondary antibody is (with 3% milk with 1:5000-1:10000 dilutions) incubation at room temperature film 2h；
(7) film is cleaned 3 times with PBST, each 10min；
After the isometric mixing of Pierce ECLWestern Blotting Substrate kit A, B liquid, it is added dropwise dropwise
On pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment Image J analyses.
Wherein, primary antibody is S phase kinase-associated proteins 1A (p19A) mutain monoclonal antibody of the standby people of corporation, two
Anti- is the Goat anti-Human IgG of horseradish peroxidase-labeled.
J, Western blot Analysis of test results：It is shown according to Western blot experimental procedures as a result, such as Fig. 6 institutes
Show, wherein the Marker in Fig. 6 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 36KD
There is apparent band, wherein the molecular weight of S phase kinase-associated proteins 1A (p19A) mutain is about 36KD, shows the lung
S phase kinase-associated proteins 1A (p19A) albumen of cancer patient is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, it using liver cancer patient as detection object, and detects the liver cancer using ELISA kit and suffers from
Whether S phase kinase-associated proteins 1A (p19A) albumen of person 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 solution, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h；
B, S phase kinase-associated proteins 1A (p19A) albumen for detecting object is diluted to various concentration using sample diluting liquid
Gradient, and S phase kinase-associated proteins 1A (p19A) albumen of various concentration gradient is loaded respectively to the hole of ELISA ELISA Plates
In, and it is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates 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 solutions are added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated 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, standard curve is made：Using standard concentration as abscissa, the light absorption value that standard items measure is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measures effective when ＞ 0.99；Wherein it is possible to use ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc softwares2=0.99605, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire liver cancer patient in sample
S phase kinase-associated proteins 1A (p19A) albumen concentration.Using in the ELISA method detection liver cancer patient blood serum sample of foundation
The content of S phase kinase-associated proteins 1A (p19A) albumen.It is used in combination Western blot to be identified.Referring to FIG. 7, being light absorption value
Standard curve.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot are identified
J, Western blot Analysis of test results
Wherein, step i and step j are identical as in embodiment five, and details are not described herein, referring to FIG. 8, in Fig. 8
Marker is used to characterize the size of labelled protein.The experimental display result of the present embodiment, compared with blank control, only in experimental group
Nearby there is apparent band in 36KD, wherein and the molecular weight of S phase kinase-associated proteins 1A (p19A) mutain is about 36KD,
Show that S phase kinase-associated proteins 1A (p19A) albumen of the liver cancer patient is implicitly present in mutation.
Embodiment seven
In embodiments of the present invention, using Pancreas cancer patients as detection object, and the pancreas is detected using ELISA kit
Whether S phase kinase-associated proteins 1A (p19A) albumen of cancer 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 solution, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h；
B, S phase kinase-associated proteins 1A (p19A) albumen of Pancreas cancer patients is diluted to using sample diluting liquid different dense
Gradient is spent, and S phase kinase-associated proteins 1A (p19A) albumen of various concentration gradient is loaded respectively to the hole of ELISA ELISA Plates
In, and it is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions are added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated 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, standard curve is made, using standard concentration as abscissa, the light absorption value that standard items measure is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measures effective when ＞ 0.99；Wherein it is possible to use ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc softwares2=0.99717, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire cancer of pancreas trouble in sample
The concentration of S phase kinase-associated proteins 1A (p19A) albumen of person.Pancreas cancer patients serum sample is detected using the ELISA method of foundation
The content of S phase kinase-associated proteins 1A (p19A) albumen in product.It is used in combination Western blot to be identified.Referring to FIG. 9, to inhale
The standard curve of light value.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot are identified
J, Western blot Analysis of test results
Wherein, step i and step j are identical as in embodiment five, and details are not described herein, referring to FIG. 10, in Figure 10
Marker is used to characterize the size of labelled protein.The experimental display result of the present embodiment, compared with blank control, only in experimental group
Nearby there is apparent band in 36KD, wherein and the molecular weight of S phase kinase-associated proteins 1A (p19A) mutain is about 36KD,
Show that S phase kinase-associated proteins 1A (p19A) albumen of the Pancreas cancer patients 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>A kind of S phases kinase-associated protein 1A（p19A）Mutain and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 146
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Pro Ser Ile Lys Leu Gln Ser Ser Gly Gly Glu Ile Phe Glu Val
1 5 10 15
Asp Val Glu Ile Val Lys Gln Ser Val Thr Ile Lys Thr Met Leu Glu
20 25 30
Asp Leu Gly Met Asn Asp Glu Gly Asp His Asp Pro Val Pro Leu Pro
35 40 45
Asn Val Asn Ala Ala Ile Leu Lys Lys Val Ile Gln Trp Cys Thr His
50 55 60
His Glu Asp Asp Ser Pro Pro Pro Lys Val Tyr Glu Asn Lys Glu Lys
65 70 75 80
Arg Thr Asp Asp Ile Pro Val Trp Asp Gln Glu Phe Leu Lys Val Asp
85 90 95
Gln Gly Thr Leu Phe Glu Leu Ile Leu Ala Ala Asn Tyr Leu Asp Ile
100 105 110
Lys Gly Leu Leu Asp Val Thr Cys Lys Thr Val Ala Asn Met Val Asn
115 120 125
Arg Lys Thr Pro Glu Glu Ile His Lys Thr Phe Asn Leu Lys Asn Asp
130 135 140
Phe Thr
145
<210> 2
<211> 438
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atgccttcaa ttaagttgca gagttctggt ggagagatat ttgaagttga tgtggaaatt 60
gtcaaacaat ctgtgactat caagaccatg ttggaagatt tgggaatgaa tgatgaagga 120
gatcatgacc cagttcctct accaaatgtt aatgcagcaa tattaaaaaa ggtcattcag 180
tggtgcaccc accatgagga tgactcacct cctcccaaag tttatgaaaa caaagaaaag 240
cgaacagacg atatccctgt ttgggaccaa gaatttctga aagttgacca aggaacactt 300
tttgaactca ttctggctgc aaactactta gacatcaaag gtttgcttga tgttacatgc 360
aagactgttg ccaatatggt caacaggaaa actcctgagg agattcacaa gaccttcaat 420
ttaaaaaatg actttact 438
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
cctcctccca aagtttatga 20
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atgccttcaa ttaagttg 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
agtaaagtca ttttttaa 18
Claims (9)
1. S phase kinase-associated proteins 1A (p19A) mutain of people a kind of, which is characterized in that its amino acid sequence such as SEQ ID
NO:Shown in 1.
2. a kind of encoding gene of S phase kinase-associated proteins 1A (p19A) mutain of people, which is characterized in that its nucleotides sequence
Row such as SEQ ID NO:Shown in 2.
3. a kind of genetic chip, which is characterized in that including：Solid phase carrier and fixed nucleotide probe on this carrier；It is described
Nucleotide probe swashs according to the nucleotide sequence of S phase kinase-associated proteins 1A (p19A) mutain of the people with the S phases of people
The comparison result of the nucleotide sequence of the normal albumen of enzyme associated protein 1 A (p19A) determines.
4. genetic chip according to claim 3, which is characterized in that the nucleotide probe is SEQ ID NO:Shown in 3
Base sequence.
5. a kind of monoclonal antibody of S phase kinase-associated proteins 1A (p19A) mutain of specific recognition people, feature exist
In the amino acid sequence of coding is SEQ ID NO:Shown in 1.
6. a kind of ELISA kit for detecting the antibody of S phase kinase-associated proteins 1A (p19A) mutain of people, special
Sign is that the ELISA kit includes：It is coated with ELISA ELISA Plates, the enzyme mark two of monoclonal antibody described in claim 5
Anti-, detection object S phase kinase-associated proteins 1A (p19A) albumen, sample diluting liquid, coating buffer solution, ELISA ELISA Plates are washed
Wash liquid, developing solution and terminate liquid.
7. being used to detect the antibody of S phase kinase-associated proteins 1A (p19A) mutain of people according to claim 6
ELISA kit, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human of diluted HRP horseradish peroxidase-labeleds
IgG。
8. a kind of S phase kinase-associated protein 1A (p19A) mutation detecting people based on the ELISA kit of claim 6 or 7
The method of the antibody of albumen, which is characterized in that including：
A, monoclonal antibody described in claim 5 is diluted using the coating buffer solution, and by the list after dilution
Clonal antibody is loaded into the hole of ELISA ELISA Plates, incubation at room temperature；
B, S phase kinase-associated proteins 1A (p19A) albumen for detecting object is diluted to various concentration using the sample diluting liquid
Gradient, and S phase kinase-associated proteins 1A (p19A) albumen of various concentration gradient is loaded respectively to the hole of ELISA ELISA Plates
In, incubation at room temperature；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature is protected from light incubation, 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.
9. S phase kinase-associated proteins 1A (p19A) mutain of ELISA kit detection people is anti-according to claim 8
The method of body, which is characterized in that further comprise：Blank control is tested.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810042827.7A CN108359003A (en) | 2018-01-17 | 2018-01-17 | A kind of S phases kinase-associated protein 1A（p19A）Mutain and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810042827.7A CN108359003A (en) | 2018-01-17 | 2018-01-17 | A kind of S phases kinase-associated protein 1A（p19A）Mutain and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108359003A true CN108359003A (en) | 2018-08-03 |
Family
ID=63006585
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810042827.7A Pending CN108359003A (en) | 2018-01-17 | 2018-01-17 | A kind of S phases kinase-associated protein 1A（p19A）Mutain and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108359003A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103118665A (en) * | 2010-03-29 | 2013-05-22 | 阿布拉科斯生物科学有限公司 | Methods of treating cancer |
| CN105203760A (en) * | 2015-07-24 | 2015-12-30 | 中国人民解放军第二军医大学 | PSMD4 protein ELISA detection kit as well as detection method and application thereof |
| CN106434936A (en) * | 2016-10-14 | 2017-02-22 | 浙江大学 | Primer group used for detecting lung cancer and detection method thereof |
-
2018
- 2018-01-17 CN CN201810042827.7A patent/CN108359003A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103118665A (en) * | 2010-03-29 | 2013-05-22 | 阿布拉科斯生物科学有限公司 | Methods of treating cancer |
| CN105203760A (en) * | 2015-07-24 | 2015-12-30 | 中国人民解放军第二军医大学 | PSMD4 protein ELISA detection kit as well as detection method and application thereof |
| CN106434936A (en) * | 2016-10-14 | 2017-02-22 | 浙江大学 | Primer group used for detecting lung cancer and detection method thereof |
Non-Patent Citations (2)
| Title |
|---|
| LI LQ等: "S-phase kinase-associated protein 1 isoform a [Homo sapiens]", 《GENBANK DATABASE》 * |
| 赵君等: "肺癌中细胞S期激酶相关蛋白2的表达及其对预后的影响", 《中华肿瘤杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108676785A (en) | A kind of ATP dependent forms RNA helicase DHX3 mutains and application | |
| CN109022388A (en) | The ribonuclease P protein protomer P30 mutain of people a kind of and its application | |
| CN108424447A (en) | 1 mutain of cytochrome b-c1 complex subunits of people a kind of and application | |
| CN109837269A (en) | The peptidyl-prolyl cis-trans isomerase B mutain of people a kind of and its application | |
| CN108192880A (en) | A kind of δ of people-aminolevulinic acid synzyme mutain and its application | |
| CN108359003A (en) | A kind of S phases kinase-associated protein 1A（p19A）Mutain and application | |
| CN109852595A (en) | Mediate rna plymerase ii transcripton subunit 11 mutain and its application | |
| CN108424888A (en) | 1 mutain of serine palmitoyltransferase of people a kind of and its application | |
| CN108559741A (en) | The imidazolone propionase mutain of people a kind of and its application | |
| CN107937375B (en) | The arginase mutain of people a kind of and its application | |
| CN108424898A (en) | 1 mutain of class aminopeptidase of people a kind of and its application | |
| CN108359657A (en) | The mannosidase β samples mutain of people a kind of and its application | |
| CN108300712A (en) | Different 2 mutain of chorismic acid synthetase domain protein of people a kind of and its application | |
| CN108424443A (en) | A kind of protein-bonded mutain of the ADP ribosylation factor of people and application | |
| CN108359660A (en) | The histone deacetylase 1 mutain of people a kind of and its application | |
| CN108342369A (en) | The GDP-L- fucose synzyme mutains of people a kind of and its application | |
| CN108329388A (en) | The silence distribution type information regulatory protein mutain of people a kind of and its application | |
| CN108795896A (en) | 1 mutain of tRNA transmethylases of people a kind of and its application | |
| CN108611335A (en) | A kind of interferon-induced gtp binding protein Mx2 mutains and its application | |
| CN108486077A (en) | 2 mutain of glycerine triphosphoric acid acyltransferase of people a kind of and its application | |
| CN108424895A (en) | 1 mutain of maintenance mitochondrial genomes exonuclease of people a kind of and application | |
| CN108359002A (en) | A kind of mutain of the calmodulin of people and its application | |
| CN109306345A (en) | The Protein-tyrosine-phosphatase type IV1b mutain of people a kind of and application | |
| CN108101977A (en) | The ACP protein mutations albumen of people a kind of and its application | |
| CN108060153A (en) | A kind of non-4 mutain of enzyme adjustment subunit of 26S proteasomes and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180803 |