CN108410844A - 4 mutain of ubiquitin carboxy terminal hydrolase of people a kind of and its application - Google Patents
4 mutain of ubiquitin carboxy terminal hydrolase of people a kind of and its application Download PDFInfo
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Abstract
The present invention relates to 4 mutain of ubiquitin carboxy terminal hydrolase of people a kind of and its applications, by regarding a large amount of cancer patients as research case, genetic test is carried out to case and is analyzed, determine 4 mutain of ubiquitin carboxy terminal hydrolase of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to 4 mutain of ubiquitin carboxy terminal hydrolase of the people, impulse is provided for the gene diagnosis of cancer, and to realize that the diagnosing and treating of cancer is provided fundamental basis.
Description
Technical field
The present invention relates to the ubiquitin carboxy terminal hydrolases 4 of a kind of genetic engineering field more particularly to a kind of people to be mutated egg
Its application of bletilla.
Background technology
Ubiquitin-proteasome system mediates various kinds of cell activity by the ubiquitination and deubiquitination for regulating and controlling key protein.
Ubiquitin can be removed as deubiquitinating enzymes family member in ubiquitin carboxy terminal hydrolase unconventionality expression in kinds of tumors tissue
Change the ubiquitin molecule on albumen, the ubiquitination level of key protein, participates in the generation of kinds of tumors in occurring to mediate tumor
Evolution.
The ubiquitin carboxy terminal hydrolase 4 of people and the occurrence and development of cancer are closely related.To certain lung cancer, liver cancer and
During the peripheral blood of patient with breast cancer carries out gene sequencing, find that the ubiquitin carboxy terminal hydrolase 4 of patient has occurred
Therefore mutation has one to the detection that the ubiquitin carboxy terminal hydrolase 4 of people is mutated to judging whether human body suffers from associated cancer
Fixed impulse.
Invention content
Present invention aims at 4 mutain of ubiquitin carboxy terminal hydrolase 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 4 mutain of ubiquitin carboxy terminal hydrolase of people, amino acid sequence such as SEQ ID
NO:Shown in 1.
Second aspect provides a kind of encoding gene of 4 mutain of ubiquitin carboxy terminal hydrolase of people, nucleotides sequence
Row 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 4 mutain of ubiquitin carboxy terminal hydrolase of the people, the ubiquitin with people
The comparison result of the nucleotide sequence of 4 normal albumen of carboxyl-terminal hydrolase determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
It is anti-to provide a kind of monoclonal of 4 mutain of ubiquitin carboxy terminal hydrolase of specific recognition people for fourth aspect
The amino acid sequence of body, coding is SEQ ID NO:Shown in 1.
5th aspect provides a kind of ELISA for detecting the antibody of 4 mutain of ubiquitin carboxy terminal hydrolase of people
Kit, the ELISA kit include：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection pair of said monoclonal antibody
4 albumen of ubiquitin carboxy terminal hydrolase of elephant, 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 ubiquitin carboxy terminal hydrolysis detecting people based on any of the above-described ELISA kit
The method of the antibody of 4 mutain of enzyme, 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, 4 albumen of ubiquitin carboxy terminal hydrolase for detecting object is diluted to various concentration using the sample diluting liquid
Gradient, and 4 albumen of ubiquitin carboxy terminal hydrolase of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, room
Temperature is incubated；
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 4 mutain of ubiquitin carboxy terminal hydrolase of people a kind of and its applications, and a large amount of cancers are suffered from
Person carries out genetic test to case and analyzes, determine the mutation egg of the ubiquitin carboxy terminal hydrolase 4 of people as research case
In vain, genetic chip, monoclonal antibody and ELISA kit are prepared according to 4 mutain of ubiquitin carboxy terminal hydrolase of the people,
Impulse is provided for the gene diagnosis of associated cancer, and to realize that the diagnosing and treating of cancer is provided fundamental basis.
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 Westernblot 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 Westernblot 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 Westernblot 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 4 mutain of ubiquitin carboxy terminal hydrolase of people
Encoding gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the ubiquitin of people is determined according to the encoding gene
4 mutain of carboxyl-terminal hydrolase, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, it according to 4 mutain of ubiquitin carboxy terminal hydrolase and its encoding gene of people, realizes as follows
The preparation of genetic chip.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the core of 4 mutain of ubiquitin carboxy terminal hydrolase of people
Nucleotide sequence determines with the comparison result of the nucleotide sequence of the 4 normal albumen of ubiquitin carboxy terminal hydrolase of people, and according to
The design principle of following probe designs the nucleotide of the specificity of 4 mutain of ubiquitin carboxy terminal hydrolase for people
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. it should be less than 4bp with the bases longs of probe molecule internal stability secondary structure pairing in nucleotide probe sequences,
Can ensure 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 4 mutain of ubiquitin carboxy terminal hydrolase of people and the Ubiquitin carboxy of people end
The comparison result of the corresponding amino acid sequence of 4 normal albumen of end hydrolase please refers to Fig.1, wherein the Query sequences in Fig. 1 are
The corresponding amino acid sequence of 4 mutain of ubiquitin carboxy terminal hydrolase of people, Sbjct sequences are the ubiquitin carboxy terminal water of people
The corresponding amino acid sequence of 4 normal albumen of enzyme is solved, the sequence between Query sequences and Sbjct sequences is comparison result, according to figure
1 it is found that people ubiquitin carboxy terminal hydrolase 4 normal albumen of 4 mutain of ubiquitin carboxy terminal hydrolase relative to people, one
Place's amino acid sequence is mutated, and is lacked at one, according to SEQ ID NO:Nucleotide sequence and Fig. 1 shown in 2
Comparison result, whether the ubiquitin carboxy terminal hydrolase 4 in order to specific recognition object to be detected be mutated, that
When choosing nucleotide probe, nucleotide probe can be designed according to any one of following several modes mode：
1. will include a nucleotide sequence of mutated site nucleotide as nucleotide probe.
2. respectively selecting several nucleotide sequence (sequences of base sequence before deletion sites and after deletion sites
It is constant), generate 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, 2. designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, the nucleotide probe
For：caaagtaggt ag
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether the ubiquitin carboxy terminal hydrolase 4 of detection people mutates
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, ☆ is experimental group.
Wherein, the position of experimental group is the point sample position of nucleotide probe.
Point sample needed for negative internal reference Quality Control probe and positive control for point sample needed for blank control, negative control
Positive internal control Quality Control probe design it is as follows：
Blank control：The blank sampling liquid for being free from any genetic fragment refers to as the contamination monitoring in chip fabrication process
Mark.
Negative internal reference Quality Control probe：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, can also 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 ubiquitin carboxylic of people
In 4 mutain of base terminal hydrolase, select sequence corresponding from nucleotide probe different, and can be with nucleotide probe base
Number it is identical, one section of nucleotide sequence that can also be different from the number of nucleotide probe base is visited as positive internal control Quality Control
Needle.Preferably, nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen.The present invention is real
It applies in example, the positive internal control probe sequence needed for genetic chip can be：agatgtggtc tg.
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 QIAGENKit purifies 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 the centrifuge tube of 1.5mL：
| 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 Transcriptionmix；
(1) Transcriptionmix is configured
| RNase-freeWater | 5.7μL |
| 4×TranscriptionBuffer | 20μL |
| NTP | 16μL |
| 0.1MDTT | 6μL |
| 50%PEG | 6.4μL |
| aa-UTP(25mM) | 4μL |
| InorganicPyrophosphatase | 0.6μL |
| T7RNAPolymerase | 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 LBuffer 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 RNeasymini in >=8000g centrifuge washings 2min with 500 μ LBuffer RPE again
Pillar is 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) above-mentioned cRNA4 μ g 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×BlockingAgent | 11μL |
| 25×FragmentationBuffer | 2.2μL |
| Nuclease-freewater | 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
Washing lotion 1 (1L) configures：
Washing lotion 2 (1L) configures：
| 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 ubiquitin carboxy terminal hydrolase of detection object is determined according to scanning result
Whether 4 albumen are mutated.It please refers 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
4 albumen of ubiquitin carboxy terminal hydrolase of bright detection 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 4 albumen of ubiquitin carboxy terminal hydrolase for detecting object does not mutate.
Embodiment three, specific recognition people 4 mutain of ubiquitin carboxy terminal hydrolase monoclonal antibody preparation
1, according to base sequence (such as SEQ ID NO of 4 mutain of ubiquitin carboxy terminal hydrolase of people:Shown in 2) it sets
Count sense primer such as SEQ ID NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (P)：atggcggaag gtggaggctg
Downstream primer (F)：atacccaacc catatatata
2, the DNA of detection object is that template carries out PCR amplification
DNA to detect object carries out PCR amplification as template, obtains 4 mutain of ubiquitin carboxy terminal hydrolase of people
The complete segment of gene, and pMD19-T Vector (Takara companies) are connected, it is sequenced.Then by special biotech firm's system
Standby antibody is a kind of humanization or Chimeric antibodies.The antibody of preparation is measured into content using ELISA method.
Example IV, 4 mutain of ubiquitin carboxy terminal hydrolase for detecting people antibody ELISA kit
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, 4 albumen of ubiquitin carboxy terminal hydrolase for detecting object, sample diluting liquid, coating buffer solution, ELISA enzymes
Target 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 4 albumen of ubiquitin carboxy terminal hydrolase 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, 4 albumen of ubiquitin carboxy terminal hydrolase of patients with lung cancer is diluted to various concentration ladder using sample diluting liquid
Degree, and 4 albumen of ubiquitin carboxy terminal hydrolase of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and room
Temperature is incubated 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=0.99847, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire the ubiquitin of patients with lung cancer
The concentration of 4 albumen of carboxyl-terminal hydrolase.Utilize Ubiquitin carboxy end in the ELISA method detection Serum of Patients with Lung Cancer sample of foundation
The content of 4 albumen of end hydrolase.It is used in combination Westernblot to be identified.Referring to FIG. 5, for the standard curve of light absorption value.Its
In, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Westernblot 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)
B. upper layer spacer gel concentration preparation system (Total Volum：8mL)
| ddH2O | 5.5mL |
| 30% acrylamide mixed liquor | 1.3mL |
| 1.0mol/LTris(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；
(8) it after the isometric mixing of Pierce ECLWestern Blotting Substrate kit A, B liquid, drips dropwise
It is added on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment Image J analyses.
Wherein, primary antibody is the 4 mutain monoclonal antibody of ubiquitin carboxy terminal hydrolase 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：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 89KD
There is apparent band, wherein 4 mutant protein molecules amount of ubiquitin carboxy terminal hydrolase is about 89KD, shows the patients with lung cancer
4 albumen of ubiquitin carboxy terminal hydrolase be 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 4 albumen of ubiquitin carboxy terminal hydrolase 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, 4 albumen of ubiquitin carboxy terminal hydrolase for detecting object is diluted to various concentration ladder using sample diluting liquid
Degree, and 4 albumen of ubiquitin carboxy terminal hydrolase of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and room
Temperature is incubated 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=0.99950, 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
4 albumen of ubiquitin carboxy terminal hydrolase concentration.Utilize ubiquitin in the ELISA method detection liver cancer patient blood serum sample of foundation
The content of 4 albumen of carboxyl-terminal hydrolase.It is used in combination Westernblot to be identified.Referring to FIG. 7, the standard for light absorption value is bent
Line.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Westernblot is identified
J, Westernblot 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 89KD, wherein 4 mutant protein molecules amount of ubiquitin carboxy terminal hydrolase is about 89KD, shows this
4 albumen of ubiquitin carboxy terminal hydrolase of liver cancer patient is implicitly present in mutation.
Embodiment seven
In embodiments of the present invention, using patient with breast cancer as detection object, and the mammary gland is detected using ELISA kit
Whether 4 albumen of ubiquitin carboxy terminal hydrolase 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, 4 albumen of ubiquitin carboxy terminal hydrolase of patient with breast cancer is diluted to various concentration ladder using sample diluting liquid
Degree, and 4 albumen of ubiquitin carboxy terminal hydrolase of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and room
Temperature is incubated 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=0.99702, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire breast cancer trouble in sample
The concentration of 4 albumen of ubiquitin carboxy terminal hydrolase of person.Using in the ELISA method detection blood serum of patients with human breast carcinoma sample of foundation
The content of 4 albumen of ubiquitin carboxy terminal hydrolase.It is used in combination Westernblot to be identified.Referring to FIG. 9, for the mark of light absorption value
Directrix curve.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Westernblot is identified
J, Westernblot 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 89KD, wherein 4 mutant protein molecules amount of ubiquitin carboxy terminal hydrolase is about 89KD, shows this
4 albumen of ubiquitin carboxy terminal hydrolase of patient with breast cancer 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>4 mutain of ubiquitin carboxy terminal hydrolase of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 340
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Ala Glu Gly Gly Gly Cys Arg Glu Arg Pro Asp Ala Glu Thr Gln
1 5 10 15
Lys Ser Glu Leu Gly Pro Leu Met Arg Thr Thr Leu Gln Arg Gly Ala
20 25 30
Gln Trp Tyr Leu Ile Asp Ser Arg Trp Phe Lys Gln Trp Lys Lys Tyr
35 40 45
Val Gly Phe Asp Ser Trp Asp Met Tyr Asn Val Gly Glu His Asn Leu
50 55 60
Phe Pro Gly Pro Ile Asp Asn Ser Gly Leu Phe Ser Asp Pro Glu Ser
65 70 75 80
Gln Thr Leu Lys Glu His Leu Ile Asp Glu Leu Asp Tyr Val Leu Val
85 90 95
Pro Thr Glu Ala Trp Asn Lys Leu Leu Asn Trp Tyr Gly Cys Val Glu
100 105 110
Gly Gln Gln Pro Ile Val Arg Lys Val Val Glu His Gly Leu Phe Val
115 120 125
Lys His Cys Lys Val Glu Val Tyr Leu Leu Glu Leu Lys Leu Cys Glu
130 135 140
Asn Ser Asp Pro Thr Asn Val Leu Ser Cys His Phe Ser Lys Ala Asp
145 150 155 160
Thr Ile Ala Thr Ile Glu Lys Glu Met Arg Lys Leu Phe Asn Ile Pro
165 170 175
Ala Glu Arg Glu Thr Arg Leu Trp Asn Lys Tyr Met Ser Asn Thr Tyr
180 185 190
Glu Gln Leu Ser Lys Leu Asp Asn Thr Val Gln Asp Ala Gly Leu Tyr
195 200 205
Gln Gly Gln Val Leu Val Ile Glu Pro Gln Asn Glu Asp Gly Thr Trp
210 215 220
Pro Arg Gln Thr Leu Gln Ser Asn Gly Ser Gly Phe Ser Ala Ser Tyr
225 230 235 240
Asn Cys Gln Glu Pro Pro Ser Ser His Ile Gln Pro Gly Leu Cys Gly
245 250 255
Leu Gly Asn Leu Gly Asn Thr Cys Phe Met Asn Ser Ala Leu Gln Cys
260 265 270
Leu Ser Asn Thr Ala Pro Leu Thr Asp Tyr Phe Leu Lys Asp Glu Tyr
275 280 285
Glu Ala Glu Ile Asn Arg Asp Asn Pro Leu Gly Met Lys Gly Glu Ile
290 295 300
Ala Glu Ala Tyr Ala Glu Leu Ile Lys Gln Met Trp Ser Gly Arg Asp
305 310 315 320
Ala His Val Ala Pro Arg Met Phe Lys Val Gly Arg His Ile Tyr Ile
325 330 335
Trp Val Gly Tyr
340
<210> 2
<211> 1020
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atggcggaag gtggaggctg ccgtgagcga ccggatgcgg agactcagaa gtccgagctt 60
ggacccttaa tgaggaccac actccaacgc ggggcgcagt ggtatcttat tgacagccgg 120
tggttcaagc agtggaagaa gtatgtgggc tttgacagct gggacatgta caatgtgggt 180
gaacataacc tatttcctgg cccaatagac aactctgggc tattttcaga tcctgagagt 240
cagaccttga aagaacactt aattgatgaa ttggactatg tattggtccc taccgaggcg 300
tggaataaac tactaaactg gtacggctgt gtagaaggcc agcaacccat cgtcagaaaa 360
gttgtggagc atggcctgtt tgtcaagcac tgcaaagtcg aggtgtattt gctggaactg 420
aagctctgtg agaacagtga ccccaccaat gtgctgagtt gccatttcag caaggcagac 480
accattgcaa ccatcgagaa agagatgcgg aagctattca acatccctgc ggagcgtgaa 540
acacggctct ggaacaaata catgagcaac acctacgagc agttgagcaa gctagacaac 600
actgtccagg atgctgggct ataccagggt caggtgctag taattgagcc tcaaaatgaa 660
gatggcacat ggcccaggca gaccttgcag tcaaatggat ctggcttttc tgcttcgtat 720
aattgtcagg agccaccatc ctctcatata caacctgggc tctgtggact tggaaacctg 780
ggaaacacct gcttcatgaa ctccgctttg cagtgtttga gcaacactgc accactgact 840
gactactttc tcaaagatga gtatgaagcc gaaatcaaca gagacaaccc tctggggatg 900
aaaggggaaa ttgcagaagc ctatgctgaa ctcattaagc agatgtggtc tggaagggac 960
gcccatgtgg cacctcgcat gttcaaagta ggtagacata tatatatatg ggttgggtat 1020
<210> 3
<211> 12
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
caaagtaggt ag 12
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggcggaag gtggaggctg 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
atacccaacc catatatata 20
Claims (9)
1. 4 mutain of ubiquitin carboxy terminal hydrolase 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 4 mutain of ubiquitin carboxy terminal hydrolase of people, which is characterized in that its nucleotide sequence is 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 is according to the nucleotide sequence of 4 mutain of ubiquitin carboxy terminal hydrolase of the people, the Ubiquitin carboxy with people
The comparison result of the nucleotide sequence of 4 normal albumen of terminal hydrolase 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 4 mutain of ubiquitin carboxy terminal hydrolase of specific recognition people, which is characterized in that its
The amino acid sequence of coding is SEQ ID NO:Shown in 1.
6. a kind of ELISA kit for detecting the antibody of 4 mutain of ubiquitin carboxy terminal hydrolase of people, feature exists
In the ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, inspection of monoclonal antibody described in claim 5
It surveys 4 albumen of ubiquitin carboxy terminal hydrolase of object, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, develop the color
Liquid and terminate liquid.
7. being used to detect the ELISA examinations of the antibody of 4 mutain of ubiquitin carboxy terminal hydrolase of people according to claim 6
Agent box, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
8. a kind of 4 mutain of ubiquitin carboxy terminal hydrolase detecting people based on the ELISA kit of claim 6 or 7
Antibody method, 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, 4 albumen of ubiquitin carboxy terminal hydrolase for detecting object is diluted to various concentration ladder using the sample diluting liquid
Degree, and 4 albumen of ubiquitin carboxy terminal hydrolase of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, room temperature
It is incubated；
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. ELISA kit detects the antibody of 4 mutain of ubiquitin carboxy terminal hydrolase of people according to claim 8
Method, which is characterized in that further comprise：Blank control is tested.
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|---|---|---|---|
| CN201810178329.5A CN108410844A (en) | 2018-03-05 | 2018-03-05 | 4 mutain of ubiquitin carboxy terminal hydrolase of people a kind of and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810178329.5A CN108410844A (en) | 2018-03-05 | 2018-03-05 | 4 mutain of ubiquitin carboxy terminal hydrolase of people a kind of and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108410844A true CN108410844A (en) | 2018-08-17 |
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ID=63130027
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|---|---|---|---|
| CN201810178329.5A Pending CN108410844A (en) | 2018-03-05 | 2018-03-05 | 4 mutain of ubiquitin carboxy terminal hydrolase of people a kind of and its application |
Country Status (1)
| Country | Link |
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2018
- 2018-03-05 CN CN201810178329.5A patent/CN108410844A/en active Pending
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Application publication date: 20180817 |