CN108795888A - The over cure dioxygenase mutain of people a kind of and its application - Google Patents
The over cure dioxygenase mutain of people a kind of and its application Download PDFInfo
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- CN108795888A CN108795888A CN201810186518.7A CN201810186518A CN108795888A CN 108795888 A CN108795888 A CN 108795888A CN 201810186518 A CN201810186518 A CN 201810186518A CN 108795888 A CN108795888 A CN 108795888A
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Abstract
The present invention relates to the over cure dioxygenase mutain of people a kind of and its applications, by regarding a large amount of lung cancer and ethyl malonic acid encephalopathy patient as research case, genetic test is carried out to case and is analyzed, determine the mutain of the over cure dioxygenase of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the mutain of the over cure dioxygenase of the people, gene diagnosis for lung cancer and ethyl malonic acid encephalopathy provides impulse, and certain theoretical foundation is provided for the diagnosing and treating of relevant disease.
Description
Technical field
The present invention relates to the over cure dioxygenase mutain of a kind of genetic engineering field more particularly to a kind of people and its answer
With.
Background technology
Over cure dioxygenase plays key effect in mitochondrial matrix in hydrogen sulfide catabolic process；Once over cure
Object is transferred to close sulphur receptor, and over cure dioxygenase consumes the oxidation of molecular oxygen catalysis persulfide；It is protected by being metabolized hydrogen sulfide
Hold the metabolic balance in mitochondria.
The over cure dioxygenase mutation of people can cause to seriously affect to human health.To certain diseases, especially
During the peripheral blood of lung cancer and ethyl malonic acid encephalopathy patient carry out gene sequencing, the over cure dioxygenase of patient is found
It is mutated, therefore, has one to judging whether human body suffers from relevant disease to the detection of the over cure dioxygenase mutation of people
Fixed impulse.
Invention content
Present invention aims at the over cure dioxygenase mutain 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 over cure dioxygenase mutain of people, amino acid sequence such as SEQ ID NO:1 institute
Show.
Second aspect provides a kind of encoding gene of the over cure dioxygenase mutain of people, 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 the over cure dioxygenase mutain of the people, the over cure dioxygenase with people
The comparison result of the nucleotide sequence of normal albumen determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of monoclonal antibody of the over cure dioxygenase mutain of specific recognition people, compiles
The amino acid sequence of code is SEQ ID NO:Shown in 1.
5th aspect provides a kind of ELISA kit for detecting the antibody of the over cure dioxygenase mutain of people,
The ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, the over cure for detecting object of said monoclonal antibody
Dioxygenase albumen, 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 over cure dioxygenase mutation detecting people based on any of the above-described ELISA kit
The method of the antibody of albumen, 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, the over cure dioxygenase albumen for detecting object is diluted to various concentration gradient using the sample diluting liquid, and
The over cure dioxygenase albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, is incubated 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 the over cure dioxygenase mutain of people a kind of and its application, by a large amount of lung cancer and ethyl the third two
Sour encephalopathy patient carries out genetic test to case and analyzes, determine the mutation of the over cure dioxygenase of people as research case
Albumen prepares genetic chip, monoclonal antibody and ELISA kit according to the over cure dioxygenase mutain of the people, is lung
The gene diagnosis of cancer and ethyl malonic acid encephalopathy provides impulse, and certain theory is provided for the diagnosing and treating of relevant disease
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.
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 the coding of the over cure dioxygenase mutain of people
Gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the double oxygenations of the over cure of people are determined according to the encoding gene
Enzyme mutant albumen, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the over cure dioxygenase mutain and its encoding gene of people, gene core is realized as follows
The preparation of piece.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the nucleotides sequence of the over cure dioxygenase mutain of people
Row are determined with the comparison result of the nucleotide sequence of the normal albumen of over cure dioxygenase of people, and setting according to following probe
Principle is counted, the nucleotide probe of the specificity of the over cure dioxygenase mutain for people is designed.
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 over cure dioxygenase mutain of people and the normal egg of over cure dioxygenase of people
The comparison result of white corresponding amino acid sequence please refers to Fig.1, wherein the Query sequences in Fig. 1 are the over cure dioxygenases of people
The corresponding amino acid sequence of mutain, Sbjct sequences are the corresponding amino acid sequences of the normal albumen of over cure dioxygenase of people,
Sequence between Query sequences and Sbjct sequences is comparison result, as can be seen from FIG. 1, the over cure dioxygenase mutain of people
Relative to the normal albumen of over cure dioxygenase of people, several place's amino acid sequences are mutated, are lacked at one, according to
SEQ ID NO:The comparison result of nucleotide sequence and Fig. 1 shown in 2, in order to specific recognition object to be detected
Whether over cure dioxygenase is mutated, then when choosing nucleotide probe, it can be according to appointing in following several modes
A kind of mode designs nucleotide probe：
1. before deletion sites with several nucleotide sequences (base sequence is constant) are respectively selected after deletion sites, it is raw
At nucleotide probe；
2. will include a nucleotide sequence of mutated site nucleotide 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, 2. designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, the nucleotide probe
For：attacagctc gggctgct
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether the over cure dioxygenase 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, it is double in the over cure of people
In oxygenase mutain, select sequence corresponding from nucleotide probe different, and can be with the number of nucleotide probe base
Identical, 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.It is excellent
Selection of land chooses nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base.The embodiment of the present invention
In, the positive internal control probe sequence needed for genetic chip can be：gccagcgcgg cgggtctg.
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 at room temperature for 3-5min, be placed in a centrifuge,
12000r/min, 4 DEG C of centrifugation 15min, carefully draws 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
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, and 5 × First Strand B μ ffer are preheated at 65 DEG C
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.
| Cy3cRNA 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：
| DEPC-H2O | 700mL |
| 20×SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
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, determines that the over cure dioxygenase albumen of detection object is according to scanning result
It is no to be mutated.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, positive control is green
Fluorescence shows that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence, then showing detection pair
The over cure dioxygenase albumen of elephant is mutated.In result shown in Fig. 4, negative control is colorless fluorescent, and positive control is
Green fluorescence shows that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, then showing to examine
The over cure dioxygenase albumen for surveying object does not mutate.
Embodiment three, specific recognition people over cure dioxygenase mutain monoclonal antibody preparation
1, according to base sequence (such as SEQ ID NO of the over cure dioxygenase mutain of people:Shown in 2) design upstream draw
Object such as SEQ ID NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (P)：atggcggagg ctgtactga
Downstream primer (F)：ggcagtgggt gtctgcacc
2, the DNA of detection object is that template carries out PCR amplification
DNA to detect object carries out PCR amplification as template, and the over cure dioxygenase mutating protein gene for obtaining people is complete
Segment, and pMD19-TVector (Takara companies) is connected, it is sequenced.Then antibody is prepared by special biotech firm, is
A kind of humanization or Chimeric antibodies.The antibody of preparation is measured into content using ELISA method.
Example IV, over cure dioxygenase mutain 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, the over cure dioxygenase albumen for detecting object, sample diluting liquid, coating buffer solution, ELISA ELISA Plates are washed
Wash liquid, 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 the over cure dioxygenase 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, the over cure dioxygenase albumen of patients with lung cancer is diluted to various concentration gradient using sample diluting liquid, and will not
Over cure dioxygenase albumen with concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and 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=0.99814, 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 over cure of patients with lung cancer
The concentration of dioxygenase albumen.Utilize over cure dioxygenase albumen in the ELISA method detection Serum of Patients with Lung Cancer sample of foundation
Content.It is used in combination Westernblot to be identified.Referring to FIG. 5, for the standard curve of light absorption value.Wherein, X-axis is light absorption value,
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)
| ddH2O | 5.9mL |
| 30% acrylamide mixed liquor | 5.9mL |
| 1.5mol/LTris(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)
(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) after the isometric mixing of Pierce ECL Western Blotting Substrate kit A, B liquid, dropwise
It is added dropwise on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment Image J analyses.
Wherein, primary antibody is the over cure dioxygenase mutain monoclonal antibody of the standby people of corporation, and secondary antibody is horseradish mistake
The Goat anti-Human IgG of oxide enzyme label.
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 63KD
There is apparent band, wherein over cure dioxygenase mutant protein molecules amount is about 63KD, shows that the over cure of the patients with lung cancer is double
Oxygenation zymoprotein is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, it using ethyl malonic acid encephalopathy patient as detection object, and is examined using ELISA kit
Whether the over cure dioxygenase albumen for surveying ethyl malonic acid encephalopathy patient is mutated, and this method at least may include as follows
It is a kind of：
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, the over cure dioxygenase albumen for detecting object is diluted to various concentration gradient using sample diluting liquid, and will not
Over cure dioxygenase albumen with concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and 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=0.99754, therefore, this
It measures effective.Ethyl the third two can be acquired in sample by by the obtained wavelength of detection being the light absorption value substitution standard curve at the places 450nm
The concentration of the over cure dioxygenase albumen of sour encephalopathy patient.Ethyl malonic acid encephalopathy patient is detected using the ELISA method of foundation
The content of over cure dioxygenase albumen in blood serum sample.It is used in combination Westernblot to be identified.Referring to FIG. 7, for light absorption value
Standard 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. 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 63KD, wherein over cure dioxygenase mutant protein molecules amount is about 63KD, shows the ethyl the third two
The over cure dioxygenase albumen of sour encephalopathy patient 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>The over cure dioxygenase mutain of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 251
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Ala Glu Ala Val Leu Arg Val Ala Arg Arg Gln Leu Ser Gln Arg
1 5 10 15
Gly Gly Ser Gly Ala Pro Ile Leu Leu Arg Gln Met Phe Glu Pro Val
20 25 30
Ser Cys Thr Phe Thr Tyr Leu Leu Gly Asp Arg Glu Ser Arg Glu Ala
35 40 45
Val Leu Ile Asp Pro Val Leu Glu Thr Ala Pro Arg Asp Ala Gln Leu
50 55 60
Ile Lys Glu Leu Gly Leu Arg Leu Leu Tyr Ala Val Asn Thr His Cys
65 70 75 80
His Ala Asp His Ile Thr Ala Arg Ala Ala Pro Phe Pro Pro Pro Trp
85 90 95
Leu Pro Val Cys His Leu Pro Pro Trp Gly Pro Gly Leu Thr His Glu
100 105 110
Asp Gly Asp Ser Ile Arg Phe Gly Arg Phe Ala Leu Glu Thr Arg Ala
115 120 125
Ser Pro Gly His Thr Pro Gly Cys Val Thr Phe Val Leu Asn Asp His
130 135 140
Ser Met Ala Phe Thr Gly Asp Ala Leu Leu Ile Arg Gly Cys Gly Arg
145 150 155 160
Thr Asp Phe Gln Gln Gly Cys Ala Lys Thr Leu Tyr His Ser Val His
165 170 175
Glu Lys Ile Phe Thr Leu Pro Gly Asp Cys Leu Ile Tyr Pro Ala His
180 185 190
Asp Tyr His Gly Phe Thr Val Ser Thr Val Glu Glu Glu Arg Thr Leu
195 200 205
Asn Pro Arg Leu Thr Leu Ser Cys Glu Glu Phe Val Lys Ile Met Gly
210 215 220
Asn Leu Asn Leu Pro Lys Pro Gln Gln Ile Asp Phe Ala Val Pro Ala
225 230 235 240
Asn Met Arg Cys Gly Val Gln Thr Pro Thr Ala
245 250
<210> 2
<211> 759
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atggcggagg ctgtactgag ggtcgcccgg cggcagctga gccagcgcgg cgggtctgga 60
gcccccatcc tcctgcggca gatgttcgag cctgtgagct gcaccttcac gtacctgctg 120
ggtgacagag agtcccggga ggccgttctg atcgacccag tcctggaaac agcgcctcgg 180
gatgcccagc tgatcaagga gctggggctg cggctgctct atgctgtgaa tacccactgc 240
cacgcggacc acattacagc tcgggctgct ccgttccctc ctccctggct gccagtctgt 300
catctcccgc cttagtgggg cccaggctga cttacacatg aggatggaga ctccatccgc 360
ttcgggcgct tcgcgttgga gaccagggcc agccctggcc acaccccagg ctgtgtcacc 420
ttcgtcctga atgaccacag catggccttc actggagatg ccctgttgat ccgtgggtgt 480
gggcggacag acttccagca aggctgtgcc aagaccttgt accactcggt ccatgaaaag 540
atcttcacac ttccaggaga ctgtctgatc taccctgctc acgattacca tgggttcaca 600
gtgtccaccg tggaggagga gaggactctg aaccctcggc tcaccctcag ctgtgaggag 660
tttgtcaaaa tcatgggcaa cctgaacttg cctaaacctc agcagataga ctttgctgtt 720
ccagccaaca tgcgctgtgg ggtgcagaca cccactgcc 759
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
attacagctc gggctgct 18
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggcggagg ctgtactga 19
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ggcagtgggt gtctgcacc 19
Claims (9)
1. a kind of mutain of the over cure dioxygenase of people, which is characterized in that its amino acid sequence such as SEQ ID NO:1 institute
Show.
2. a kind of encoding gene of the over cure dioxygenase mutain of people, which is characterized in that its nucleotide sequence 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 normal with the over cure dioxygenase of people according to the nucleotide sequence of the over cure dioxygenase mutain of the people
The comparison result of the nucleotide sequence of albumen 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 the over cure dioxygenase mutain of specific recognition people, which is characterized in that it was encoded
Amino acid sequence is SEQ ID NO:Shown in 1.
6. a kind of ELISA kit for detecting the antibody of the over cure dioxygenase mutain of people, which is characterized in that described
ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection object of monoclonal antibody described in claim 5
Over cure dioxygenase albumen, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and terminate liquid.
7. it is used to detect the ELISA kit of the antibody of the over cure dioxygenase mutain of people according to claim 6,
It is characterized in that, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
8. a kind of antibody for the over cure dioxygenase mutain detecting people based on the ELISA kit of claim 6 or 7
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, the over cure dioxygenase albumen for detecting object is diluted to various concentration gradient using the sample diluting liquid, and will not
Over cure dioxygenase albumen with concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, 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. the method for the antibody of the over cure dioxygenase mutain of ELISA kit detection people according to claim 8,
It is characterized in that, further comprises：Blank control is tested.
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|---|---|---|---|
| CN201810186518.7A CN108795888A (en) | 2018-03-07 | 2018-03-07 | The over cure dioxygenase mutain of people a kind of and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810186518.7A CN108795888A (en) | 2018-03-07 | 2018-03-07 | The over cure dioxygenase mutain of people a kind of and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108795888A true CN108795888A (en) | 2018-11-13 |
Family
ID=64095054
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810186518.7A Pending CN108795888A (en) | 2018-03-07 | 2018-03-07 | The over cure dioxygenase mutain of people a kind of and its application |
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| Country | Link |
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2018
- 2018-03-07 CN CN201810186518.7A patent/CN108795888A/en active Pending
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