CN109180796A - 2 mutain of RAP1 gtpase activating protein of people a kind of and application - Google Patents
2 mutain of RAP1 gtpase activating protein of people a kind of and application Download PDFInfo
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Abstract
The present invention relates to 2 mutain of RAP1 gtpase activating protein of people a kind of and applications, by using a large amount of cervical cancer patients as research case, genetic test is carried out to case and is analyzed, determine the mutain of the RAP1 gtpase activating protein 2 of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the mutain of the RAP1 gtpase activating protein 2 of the people, impulse is provided for the gene diagnosis of cervical carcinoma, and certain theoretical basis is provided for the diagnosing and treating of cancer.
Description
Technical field
The present invention relates to the RAP1 gtpase activating proteins 2 of a kind of genetic engineering field more particularly to a kind of people to be mutated egg
Bletilla application.
Background technique
(rap1 GTPase-activating protein 2, the RAP1 GAP2) gene of RAP1 gtpase activating protein 2 is compiled
Code gtpase activating protein, activates the small guanine-nucleotide-binding protein RAP1 in blood platelet.The albumen and synapsin sample egg
White 1 and Rab27 interaction, and adjust the dense granule that blood platelet is secreted in interior skin damage location.
The mutation of RAP1 gtpase activating protein 2 of people can cause to seriously affect to human health.To certain uterine neck
During the peripheral blood of cancer patient carries out gene sequencing, the RAP1 gtpase activating protein 2 of discovery patient is mutated,
Therefore, the detection being mutated to the RAP1 gtpase activating protein 2 of people is to judge whether human body suffers from related disease and have centainly
Impulse.
Summary of the invention
It is an object of that present invention to provide 2 mutain of RAP1 gtpase activating protein of people a kind of and applications.
Technical solution of the present invention includes:
In a first aspect, 2 mutain of RAP1 gtpase activating protein of people a kind of is provided, amino acid sequence such as SEQ ID
Shown in NO:1.
Second aspect provides a kind of encoding gene of 2 mutain of RAP1 gtpase activating protein of people, nucleotides sequence
Column are 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；
The nucleotide probe is according to the nucleotide sequence of 2 mutain of RAP1 gtpase activating protein of people described above, with people's
The comparison result of the nucleotide sequence of the normal albumen of RAP1 gtpase activating protein 2 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, contains the RAP1 GTP enzyme activated protein for detecting above-mentioned people in the reagent
The primer pair of white 2 mutain.
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:17；
Downstream primer is the base sequence as shown in any in SEQ ID NO:18~NO:40.
5th aspect, the monoclonal for providing 2 mutain of RAP1 gtpase activating protein of specific recognition people a kind of are anti-
Body is prepared by above-mentioned reagent, can be in conjunction with amino acid sequencespecific shown in SEQ ID NO:1.
6th aspect, provides a kind of for detecting the ELISA of the antibody of 2 mutain of RAP1 gtpase activating protein of people
Kit, the ELISA kit include: the ELISA ELISA Plate for being coated with said monoclonal antibody, ELIAS secondary antibody, detection pair
2 albumen of RAP1 gtpase activating protein of elephant, 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 2 mutain of RAP1 gtpase activating protein of people a kind of and applications, and a large amount of cervical carcinomas are suffered from
Person carries out genetic test to case and analyzes, determine the mutation egg of the RAP1 gtpase activating protein 2 of people as research case
It is white, genetic chip, monoclonal antibody and ELISA kit are prepared according to 2 mutain of RAP1 gtpase activating protein of the people,
Impulse is provided for the gene diagnosis of cervical carcinoma, provides certain theoretical basis for the diagnosing and treating of cancer.
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 2 mutain of RAP1 gtpase activating protein of people
Encoding gene, nucleotide sequence is as shown in SEQ ID NO:2；Correspondingly, the RAP1 of people is determined according to the encoding gene
2 mutain of gtpase activating protein, amino acid sequence is as shown in SEQ ID NO:1.
Secondly, being realized as follows according to 2 mutain of RAP1 gtpase activating protein and its encoding gene of people
The preparation of genetic chip.
1, the design of nucleotide probe
(1) design of nucleotide probe: nucleotide probe is according to the core of 2 mutain of RAP1 gtpase activating protein of people
Nucleotide sequence determines with the comparison result of the nucleotide sequence of the normal albumen of RAP1 gtpase activating protein 2 of people, and according to
The design principle of following probe designs the nucleotide of the specificity of 2 mutain of RAP1 gtpase activating protein for people
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 2 mutain of RAP1 gtpase activating protein of people and the RAP1 GTP enzyme of people
The comparison result of the corresponding amino acid sequence of the normal albumen of activator protein 2 is referring to FIG. 1, wherein, the Query sequence in Fig. 1 is
The corresponding amino acid sequence of 2 mutain of RAP1 gtpase activating protein of people, Sbjct sequence are the RAP1 GTP enzyme activitions of people
The corresponding amino acid sequence of the normal albumen of albumen 2, the sequence between Query sequence and Sbjct sequence is comparison result, according to figure
1 it is found that people RAP1 gtpase activating protein 2 normal albumen of 2 mutain of RAP1 gtpase activating protein relative to people, have
A part of amino acid sequence is lacked.According to nucleotide sequence shown in SEQ ID NO:2 and the comparison result of Fig. 1,
Whether the RAP1 gtpase activating protein 2 in order to specific recognition object to be detected is mutated, then choosing core
When thuja acid probe, several nucleotide sequence (base sequences can be respectively selected before deletion sites and after deletion sites
Sequence it is constant), generate 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: gtcatcctgc caca；
The nucleotide probe as shown in SEQ ID NO:4 are as follows: atcgacgagg tcatcct；
The nucleotide probe as shown in SEQ ID NO:5 are as follows: aggtcatcct.
(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 RAP1 gtpase activating protein 2 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,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: 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 RAP1 of people
In 2 mutain of gtpase activating protein, select sequence corresponding from nucleotide probe different, and can be with nucleotide probe alkali
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 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
In embodiment, positive internal control probe sequence needed for genetic chip can be with are as follows: caagaccatg.
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:
| 5×First Strand Buffer | 4μL |
| 0.1M DTT | 2μL |
| 10mM dNTP mix | 1μL |
| MMLV RT | 1μL |
| RNase OUT | 0.5μL |
| Total volume | 8.5μL |
(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, the RAP1 gtpase activating protein of test object is determined according to scanning result
Whether 2 albumen are mutated.Please refer to Fig. 3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence is 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 is green fluorescence, then table
2 albumen of RAP1 gtpase activating protein of bright test object is mutated.In result shown in Fig. 4, negative control is colourless
Fluorescence, positive control are green fluorescence, show that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen
Fluorescence, then showing that 2 albumen of RAP1 gtpase activating protein of test object does not mutate.
Embodiment three, specific recognition people 2 mutain of RAP1 gtpase activating protein monoclonal antibody preparation
1, the determination of reagent
Contain the primer pair for detecting 2 mutain of RAP1 gtpase activating protein of above-mentioned people in the reagent.
It wherein, can be with according to the ORF of the normal albumen of the RAP1 gtpase activating protein of people 2 (as shown in SEQ ID NO:6)
Determine the corresponding base sequence of ORF of the normal albumen of RAP1 gtpase activating protein 2 of people (as shown in SEQ ID NO:7).
It is possible to further according to base sequence (such as SEQ ID of 2 mutain of RAP1 gtpase activating protein of people
Shown in NO:2), and (such as according to the corresponding base sequence of ORF of the normal albumen of RAP1 gtpase activating protein 2 of the people of people
Shown in SEQ ID NO:7), design following upstream primer and downstream primer:
Upstream primer (F):
As shown in SEQ ID NO:8: atgtttggcc ggaagcgcag tgtctccttt
As shown in SEQ ID NO:9: gggggcttcg gatggatcga caagaccatg
As shown in SEQ ID NO:10: ctggcaagtc tgaaggtcaa gaagcaggag
As shown in SEQ ID NO:11: ctggccaaca gctcggatgc gaccctccca
As shown in SEQ ID NO:12: gaccggccgc tctcccctcc tctcacggca
As shown in SEQ ID NO:13: cctcccacca tgaagtcgtc ggagttcttt
As shown in SEQ ID NO:14: gagatgctgg agaaaatgca ggggatcaag
As shown in SEQ ID NO:15: cttgaagagc agaagccggg accccagaag
As shown in SEQ ID NO:16: aacaaggacg actatatccc
As shown in SEQ ID NO:17: ataccccagc atcgacgagg tt
Downstream primer (R):
As shown in SEQ ID NO:18: ttagtgaccc gcaccagagc tggcatggct
As shown in SEQ ID NO:19: gagcttgtca aagcggaatt tcaggttcga
As shown in SEQ ID NO:20: tctcggggag tttctgcttt tggcatctgt
As shown in SEQ ID NO:21: gggactccgg ctcatgatga tcggggtggc
As shown in SEQ ID NO:22: agctgccccc aggctggggc tctcgctgct
As shown in SEQ ID NO:23: cggggacggg ctgtagacaa acacctcctg
As shown in SEQ ID NO:24: cttgaagggt gaggtcgtgg acggctggct
As shown in SEQ ID NO:25: gcccccactg tcgccctcct ccatggcctc
As shown in SEQ ID NO:26: gccctcccct gcagtgctgc tgacgctgct
As shown in SEQ ID NO:27: ggtgctggag gaggagcggg agatggcacg
As shown in SEQ ID NO:28: gccgttttcc ttcaacttca tgaagggctt
As shown in SEQ ID NO:29: ctccttgttg gggcagattt ccggagagct
As shown in SEQ ID NO:30: gggattggat gaggtctcag actttatctc
As shown in SEQ ID NO:31: ctgagaggag tgtccaccat ctggggtctt
As shown in SEQ ID NO:32: gggtgtgctg gatgtgctgt cacatcgtgc
As shown in SEQ ID NO:33: ccggctgtcg ccctggcctt ctgagcccgt
As shown in SEQ ID NO:34: gtgcaggcgg gggaagagcc ccgagcgtcg
As shown in SEQ ID NO:35: cttgatggga ctccgtgact ggttcttcac
As shown in SEQ ID NO:36: cgttgccgcc accactggag gcgagaaggt
As shown in SEQ ID NO:37: ggtcttggtg acctccatgc tgttgtggga
As shown in SEQ ID NO:38: gatgcccccg ctgaggctgc cagggatgcc
As shown in SEQ ID NO:39: cccactgtgc gacttcttct ggccgcccac
As shown in SEQ ID NO:40: catggtctcc atggagtggc tgcgtacgcg
In the present embodiment, what is contained in reagent is used to detect 2 mutain of RAP1 gtpase activating protein of above-mentioned people
Primer pair include: any of the above-described upstream primer Yu any of the above-described downstream primer combination.
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, obtains 2 mutain of RAP1 gtpase activating protein of people
The complete segment of gene, and pMD19-T Vector (Takara company) is connected, it is sequenced.Then by special biotech firm's system
Standby antibody is a kind of humanization or Chimeric antibodies.Wherein, the monoclonal antibody prepared can with shown in SEQ ID NO:1
Amino acid sequencespecific combines.The antibody of preparation is measured into content using ELISA method.
Example IV, 2 mutain of RAP1 gtpase activating protein 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, 2 albumen of RAP1 gtpase activating protein of test object, sample diluting liquid, coating buffer, ELISA enzyme
Target 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 cervical cancer patient as test object, and the uterine neck is detected using ELISA kit
Whether 2 albumen of RAP1 gtpase activating protein 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, 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, 2 albumen of RAP1 gtpase activating protein of cervical cancer patient is diluted to various concentration ladder using sample diluting liquid
Degree, and 2 albumen of RAP1 gtpase activating protein of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plate, and room
Temperature is incubated for 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 ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc software2=0.99895, therefore, this
Measurement is effective.The wavelength that will test is that the light absorption value substitution standard curve at 450nm can acquire cervical cancer patient
The concentration of 2 albumen of RAP1 gtpase activating protein.Utilize RAP1 in the ELISA method detection cervical cancer patient blood serum sample of foundation
The content of 2 albumen of gtpase activating protein.And it is identified with Western blot.Referring to FIG. 5, the standard for light absorption value is bent
Line.Wherein, X-axis is light absorption value, and Y-axis is 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(PH8.8) | 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(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 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 the 2 mutain monoclonal antibody of RAP1 gtpase activating protein of the standby people of corporation, and secondary antibody is
The Goat anti-Human IgG of horseradish peroxidase-labeled.
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 69KD
There is obvious band, wherein 2 mutant protein molecules amount of RAP1 gtpase activating protein is about 69KD, shows that the cervical carcinoma is suffered from
2 albumen of RAP1 gtpase activating protein of person 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>2 mutain of RAP1 gtpase activating protein of people a kind of and application
<160> 40
<170> SIPOSequenceListing 1.0
<210> 1
<211> 285
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 1
Met Phe Gly Arg Lys Arg Ser Val Ser Phe Gly Gly Phe Gly Trp Ile
1 5 10 15
Asp Lys Thr Met Leu Ala Ser Leu Lys Val Lys Lys Gln Glu Leu Ala
20 25 30
Asn Ser Ser Asp Ala Thr Leu Pro Asp Arg Pro Leu Ser Pro Pro Leu
35 40 45
Thr Ala Pro Pro Thr Met Lys Ser Ser Glu Phe Phe Glu Met Leu Glu
50 55 60
Lys Met Gln Gly Ile Lys Leu Glu Glu Gln Lys Pro Gly Pro Gln Lys
65 70 75 80
Asn Lys Asp Asp Tyr Ile Pro Tyr Pro Ser Ile Asp Glu Val Ile Leu
85 90 95
Pro Gln Phe Gly Gly Tyr Trp Ile Glu Asp Pro Glu Asn Val Gly Thr
100 105 110
Pro Thr Ser Leu Gly Ser Ser Ile Cys Glu Glu Glu Glu Glu Asp Asn
115 120 125
Leu Ser Pro Asn Thr Phe Gly Tyr Lys Leu Glu Cys Lys Gly Glu Ala
130 135 140
Arg Ala Tyr Arg Arg His Phe Leu Gly Lys Asp His Leu Asn Phe Tyr
145 150 155 160
Cys Thr Gly Ser Ser Leu Gly Asn Leu Ile Leu Ser Val Lys Cys Glu
165 170 175
Glu Ala Glu Gly Ile Glu Tyr Leu Arg Val Ile Leu Arg Ser Lys Leu
180 185 190
Lys Thr Val His Glu Arg Ile Pro Leu Ala Gly Leu Ser Lys Leu Pro
195 200 205
Ser Val Pro Gln Ile Ala Lys Ala Phe Cys Asp Asp Ala Val Gly Leu
210 215 220
Arg Phe Asn Pro Val Leu Tyr Pro Lys Ala Ser Gln Met Ile Val Ser
225 230 235 240
Tyr Asp Glu His Glu Val Asn Asn Thr Phe Lys Phe Gly Val Ile Tyr
245 250 255
Gln Lys Ala Arg Gln Thr Leu Glu Glu Glu Leu Phe Gly Asn Asn Glu
260 265 270
Glu Ser Pro Ala Phe Lys Glu Phe Leu Asp Leu Leu Gly
275 280 285
<210> 2
<211> 855
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 2
atgtttggcc ggaagcgcag tgtctccttt gggggcttcg gatggatcga caagaccatg 60
ctggcaagtc tgaaggtcaa gaagcaggag ctggccaaca gctcggatgc gaccctccca 120
gaccggccgc tctcccctcc tctcacggca cctcccacca tgaagtcgtc ggagttcttt 180
gagatgctgg agaaaatgca ggggatcaag cttgaagagc agaagccggg accccagaag 240
aacaaggacg actatatccc ataccccagc atcgacgagg tcatcctgcc acagtttggg 300
ggctattgga tcgaggaccc ggagaacgtg ggcaccccaa catcgctggg gagcagcatc 360
tgtgaggagg aggaagagga caacctcagc cccaacacat ttggctacaa gctcgagtgc 420
aagggtgaag ccagggccta ccggaggcac ttcctgggga aggatcatct aaacttttac 480
tgtaccggca gcagcctggg gaacttgatc ctgtccgtca agtgcgagga agcagagggg 540
atcgagtacc tccgggtcat cctcaggtcc aaactgaaga cggtacatga gcggatcccc 600
ttggctggac tgagcaagct tcccagtgtc cctcagattg caaaggcttt ctgtgatgat 660
gcagtgggac tgagattcaa tcctgtcctg taccccaagg cctcccaaat gattgtgtcc 720
tatgatgagc atgaagtcaa caacacattc aaattcggag tcatttatca aaaagccagg 780
cagaccctgg aggaggagct atttgggaac aatgaggaga gcccagcttt taaggagttc 840
ttggacctgc tgggg 855
<210> 3
<211> 14
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtcatcctgc caca 14
<210> 4
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
atcgacgagg tcatcct 17
<210> 5
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
aggtcatcct 10
<210> 6
<211> 730
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 6
Met Phe Gly Arg Lys Arg Ser Val Ser Phe Gly Gly Phe Gly Trp Ile
1 5 10 15
Asp Lys Thr Met Leu Ala Ser Leu Lys Val Lys Lys Gln Glu Leu Ala
20 25 30
Asn Ser Ser Asp Ala Thr Leu Pro Asp Arg Pro Leu Ser Pro Pro Leu
35 40 45
Thr Ala Pro Pro Thr Met Lys Ser Ser Glu Phe Phe Glu Met Leu Glu
50 55 60
Lys Met Gln Gly Ile Lys Leu Glu Glu Gln Lys Pro Gly Pro Gln Lys
65 70 75 80
Asn Lys Asp Asp Tyr Ile Pro Tyr Pro Ser Ile Asp Glu Val Val Glu
85 90 95
Lys Gly Gly Pro Tyr Pro Gln Val Ile Leu Pro Gln Phe Gly Gly Tyr
100 105 110
Trp Ile Glu Asp Pro Glu Asn Val Gly Thr Pro Thr Ser Leu Gly Ser
115 120 125
Ser Ile Cys Glu Glu Glu Glu Glu Asp Asn Leu Ser Pro Asn Thr Phe
130 135 140
Gly Tyr Lys Leu Glu Cys Lys Gly Glu Ala Arg Ala Tyr Arg Arg His
145 150 155 160
Phe Leu Gly Lys Asp His Leu Asn Phe Tyr Cys Thr Gly Ser Ser Leu
165 170 175
Gly Asn Leu Ile Leu Ser Val Lys Cys Glu Glu Ala Glu Gly Ile Glu
180 185 190
Tyr Leu Arg Val Ile Leu Arg Ser Lys Leu Lys Thr Val His Glu Arg
195 200 205
Ile Pro Leu Ala Gly Leu Ser Lys Leu Pro Ser Val Pro Gln Ile Ala
210 215 220
Lys Ala Phe Cys Asp Asp Ala Val Gly Leu Arg Phe Asn Pro Val Leu
225 230 235 240
Tyr Pro Lys Ala Ser Gln Met Ile Val Ser Tyr Asp Glu His Glu Val
245 250 255
Asn Asn Thr Phe Lys Phe Gly Val Ile Tyr Gln Lys Ala Arg Gln Thr
260 265 270
Leu Glu Glu Glu Leu Phe Gly Asn Asn Glu Glu Ser Pro Ala Phe Lys
275 280 285
Glu Phe Leu Asp Leu Leu Gly Asp Thr Ile Thr Leu Gln Asp Phe Lys
290 295 300
Gly Phe Arg Gly Gly Leu Asp Val Thr His Gly Gln Thr Gly Val Glu
305 310 315 320
Ser Val Tyr Thr Thr Phe Arg Asp Arg Glu Ile Met Phe His Val Ser
325 330 335
Thr Lys Leu Pro Phe Thr Asp Gly Asp Ala Gln Gln Leu Gln Arg Lys
340 345 350
Arg His Ile Gly Asn Asp Ile Val Ala Ile Ile Phe Gln Glu Glu Asn
355 360 365
Thr Pro Phe Val Pro Asp Met Ile Ala Ser Asn Phe Leu His Ala Tyr
370 375 380
Ile Val Val Gln Val Glu Thr Pro Gly Thr Glu Thr Pro Ser Tyr Lys
385 390 395 400
Val Ser Val Thr Ala Arg Glu Asp Val Pro Thr Phe Gly Pro Pro Leu
405 410 415
Pro Ser Pro Pro Val Phe Gln Lys Gly Pro Glu Phe Arg Glu Phe Leu
420 425 430
Leu Thr Lys Leu Thr Asn Ala Glu Asn Ala Cys Cys Lys Ser Asp Lys
435 440 445
Phe Ala Lys Leu Glu Asp Arg Thr Arg Ala Ala Leu Leu Asp Asn Leu
450 455 460
His Asp Glu Leu His Ala His Thr Gln Ala Met Leu Gly Leu Gly Pro
465 470 475 480
Glu Glu Asp Lys Phe Glu Asn Gly Gly His Gly Gly Phe Leu Glu Ser
485 490 495
Phe Lys Arg Ala Ile Arg Val Arg Ser His Ser Met Glu Thr Met Val
500 505 510
Gly Gly Gln Lys Lys Ser His Ser Gly Gly Ile Pro Gly Ser Leu Ser
515 520 525
Gly Gly Ile Ser His Asn Ser Met Glu Val Thr Lys Thr Thr Phe Ser
530 535 540
Pro Pro Val Val Ala Ala Thr Val Lys Asn Gln Ser Arg Ser Pro Ile
545 550 555 560
Lys Arg Arg Ser Gly Leu Phe Pro Arg Leu His Thr Gly Ser Glu Gly
565 570 575
Gln Gly Asp Ser Arg Ala Arg Cys Asp Ser Thr Ser Ser Thr Pro Lys
580 585 590
Thr Pro Asp Gly Gly His Ser Ser Gln Glu Ile Lys Ser Glu Thr Ser
595 600 605
Ser Asn Pro Ser Ser Pro Glu Ile Cys Pro Asn Lys Glu Lys Pro Phe
610 615 620
Met Lys Leu Lys Glu Asn Gly Arg Ala Ile Ser Arg Ser Ser Ser Ser
625 630 635 640
Thr Ser Ser Val Ser Ser Thr Ala Gly Glu Gly Glu Ala Met Glu Glu
645 650 655
Gly Asp Ser Gly Gly Ser Gln Pro Ser Thr Thr Ser Pro Phe Lys Gln
660 665 670
Glu Val Phe Val Tyr Ser Pro Ser Pro Ser Ser Glu Ser Pro Ser Leu
675 680 685
Gly Ala Ala Ala Thr Pro Ile Ile Met Ser Arg Ser Pro Thr Asp Ala
690 695 700
Lys Ser Arg Asn Ser Pro Arg Ser Asn Leu Lys Phe Arg Phe Asp Lys
705 710 715 720
Leu Ser His Ala Ser Ser Gly Ala Gly His
725 730
<210> 7
<211> 2193
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 7
atgtttggcc ggaagcgcag tgtctccttt gggggcttcg gatggatcga caagaccatg 60
ctggcaagtc tgaaggtcaa gaagcaggag ctggccaaca gctcggatgc gaccctccca 120
gaccggccgc tctcccctcc tctcacggca cctcccacca tgaagtcgtc ggagttcttt 180
gagatgctgg agaaaatgca ggggatcaag cttgaagagc agaagccggg accccagaag 240
aacaaggacg actatatccc ataccccagc atcgacgagg ttgtggagaa gggaggcccg 300
taccctcagg tcatcctgcc acagtttggg ggctattgga tcgaggaccc ggagaacgtg 360
ggcaccccaa catcgctggg gagcagcatc tgtgaggagg aggaagagga caacctcagc 420
cccaacacat ttggctacaa gctcgagtgc aagggtgaag ccagggccta ccggaggcac 480
ttcctgggga aggatcatct aaacttttac tgtaccggca gcagcctggg gaacttgatc 540
ctgtccgtca agtgcgagga agcagagggg atcgagtacc tccgggtcat ccttaggtcc 600
aaactgaaga cggtacatga gcggatcccc ttggctggac tgagcaagct tcccagtgtc 660
cctcagattg caaaggcttt ctgtgatgat gcagtgggac tgagattcaa tcctgtcctg 720
taccccaagg cctcccaaat gattgtgtcc tatgatgagc atgaagtcaa caacacattc 780
aaattcggag tcatttatca aaaagccagg cagaccctgg aggaggagct atttgggaac 840
aatgaggaga gcccagcttt taaggagttc ttggacctgc tgggggacac gatcacactg 900
caggatttca aaggtttccg aggaggcctg gacgtgaccc acggacagac aggggtggaa 960
tcagtgtaca caacattccg ggacagggag atcatgtttc acgtttccac aaagctgcca 1020
tttaccgacg gagacgccca gcagctccag agaaagagac acattggaaa tgacatcgtg 1080
gccatcatct tccaagagga aaacacgccg tttgtcccag acatgatagc ctccaatttc 1140
ttacatgcct acatcgtcgt gcaggtcgag accccaggca cagagacccc atcctacaag 1200
gtctctgtca ctgcgcggga agatgtgccc acctttggtc cacctctgcc cagtcccccc 1260
gttttccaga agggcccgga attcagggag tttctgctca ccaagctcac caatgccgag 1320
aacgcctgct gcaagtcgga caagtttgca aagctggagg accggaccag ggctgccctc 1380
ctggacaacc ttcacgatga gctccacgcc cacacacagg ccatgctggg actgggccca 1440
gaggaggaca agtttgagaa tggaggccac ggggggttcc tggagtcttt taagagggcc 1500
atccgcgtac gcagccactc catggagacc atggtgggcg gccagaagaa gtcgcacagt 1560
gggggcatcc ctggcagcct cagcgggggc atctcccaca acagcatgga ggtcaccaag 1620
accaccttct cgcctccagt ggtggcggca acggtgaaga accagtcacg gagtcccatc 1680
aagcgacgct cggggctctt cccccgcctg cacacgggct cagaaggcca gggcgacagc 1740
cgggcacgat gtgacagcac atccagcaca cccaagaccc cagatggtgg acactcctct 1800
caggagataa agtctgagac ctcatccaat cccagctctc cggaaatctg ccccaacaag 1860
gagaagccct tcatgaagtt gaaggaaaac ggccgtgcca tctcccgctc ctcctccagc 1920
accagcagcg tcagcagcac tgcaggggag ggcgaggcca tggaggaggg cgacagtggg 1980
ggcagccagc cgtccacgac ctcacccttc aagcaggagg tgtttgtcta cagcccgtcc 2040
ccgagcagcg agagccccag cctgggggca gctgccaccc cgatcatcat gagccggagt 2100
cccacagatg ccaaaagcag aaactccccg agatcgaacc tgaaattccg ctttgacaag 2160
ctcagccatg ccagctctgg tgcgggtcac taa 2193
<210> 8
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
atgtttggcc ggaagcgcag tgtctccttt 30
<210> 9
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gggggcttcg gatggatcga caagaccatg 30
<210> 10
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ctggcaagtc tgaaggtcaa gaagcaggag 30
<210> 11
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ctggccaaca gctcggatgc gaccctccca 30
<210> 12
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gaccggccgc tctcccctcc tctcacggca 30
<210> 13
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
cctcccacca tgaagtcgtc ggagttcttt 30
<210> 14
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gagatgctgg agaaaatgca ggggatcaag 30
<210> 15
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
cttgaagagc agaagccggg accccagaag 30
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
aacaaggacg actatatccc 20
<210> 17
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ataccccagc atcgacgagg tt 22
<210> 18
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
ttagtgaccc gcaccagagc tggcatggct 30
<210> 19
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gagcttgtca aagcggaatt tcaggttcga 30
<210> 20
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
tctcggggag tttctgcttt tggcatctgt 30
<210> 21
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
gggactccgg ctcatgatga tcggggtggc 30
<210> 22
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
agctgccccc aggctggggc tctcgctgct 30
<210> 23
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
cggggacggg ctgtagacaa acacctcctg 30
<210> 24
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
cttgaagggt gaggtcgtgg acggctggct 30
<210> 25
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
gcccccactg tcgccctcct ccatggcctc 30
<210> 26
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
gccctcccct gcagtgctgc tgacgctgct 30
<210> 27
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
ggtgctggag gaggagcggg agatggcacg 30
<210> 28
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
gccgttttcc ttcaacttca tgaagggctt 30
<210> 29
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
ctccttgttg gggcagattt ccggagagct 30
<210> 30
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
gggattggat gaggtctcag actttatctc 30
<210> 31
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
ctgagaggag tgtccaccat ctggggtctt 30
<210> 32
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
gggtgtgctg gatgtgctgt cacatcgtgc 30
<210> 33
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
ccggctgtcg ccctggcctt ctgagcccgt 30
<210> 34
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
gtgcaggcgg gggaagagcc ccgagcgtcg 30
<210> 35
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
cttgatggga ctccgtgact ggttcttcac 30
<210> 36
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
cgttgccgcc accactggag gcgagaaggt 30
<210> 37
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
ggtcttggtg acctccatgc tgttgtggga 30
<210> 38
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
gatgcccccg ctgaggctgc cagggatgcc 30
<210> 39
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
cccactgtgc gacttcttct ggccgcccac 30
<210> 40
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
catggtctcc atggagtggc tgcgtacgcg 30
Claims (9)
1. a kind of mutain of the RAP1GTP ras GTPase activating protein ras-GTP 2 of people, which is characterized in that its amino acid sequence such as SEQ ID
Shown in NO:1.
2. a kind of encoding gene of 2 mutain of RAP1GTP ras GTPase activating protein ras-GTP of people, which is characterized in that its nucleotide sequence is such as
Shown in SEQ 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 2 mutain of RAP1GTP ras GTPase activating protein ras-GTP of people according to claim 2, with people's
The comparison result of the nucleotide sequence of the normal albumen of RAP1GTP ras GTPase activating protein ras-GTP 2 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 swash in the reagent containing the RAP1GTP enzyme for detecting people described in claim 1
The primer pair of 2 mutain of living protein.
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:17；
Downstream primer is the base sequence as shown in any in SEQ ID NO:18~NO:40.
7. a kind of monoclonal antibody of 2 mutain of RAP1GTP ras GTPase activating protein ras-GTP of specific recognition people, which is characterized in that by
It, can be in conjunction with amino acid sequencespecific shown in SEQ ID NO:1 if reagent described in claim 5 or 6 is prepared.
8. a kind of for detecting the ELISA kit of the antibody of 2 mutain of RAP1GTP ras GTPase activating protein ras-GTP of people, feature exists
In the ELISA kit includes: the ELISA ELISA Plate for being coated with monoclonal antibody described in claim 7, ELIAS secondary antibody, inspection
Survey 2 albumen of RAP1GTP ras GTPase activating protein ras-GTP, sample diluting liquid, coating buffer, ELISA ELISA Plate cleaning solution, developing solution of object
And terminate liquid.
9. according to claim 8 for detecting the ELISA examination of the antibody of 2 mutain of RAP1GTP ras GTPase activating protein ras-GTP of people
Agent box, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeled.
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|---|---|---|---|
| CN201810959347.7A CN109180796A (en) | 2018-08-22 | 2018-08-22 | 2 mutain of RAP1 gtpase activating protein of people a kind of and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810959347.7A CN109180796A (en) | 2018-08-22 | 2018-08-22 | 2 mutain of RAP1 gtpase activating protein of people a kind of and application |
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| Publication Number | Publication Date |
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ID=64918943
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|---|---|---|---|
| CN201810959347.7A Pending CN109180796A (en) | 2018-08-22 | 2018-08-22 | 2 mutain of RAP1 gtpase activating protein of people a kind of and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109180796A (en) |
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2018
- 2018-08-22 CN CN201810959347.7A patent/CN109180796A/en active Pending
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