CN108152497A - The atp synthase mutain of people a kind of and its application - Google Patents
The atp synthase mutain of people a kind of and its application Download PDFInfo
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Abstract
The present invention relates to the atp synthase mutain 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 the mutain of the atp synthase of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the atp synthase mutain of the people, the impulse that gene diagnosis for cancer provides realizes the early diagnosis, early discovery and associated treatment of cancer.
Description
Technical field
The present invention relates to the atp synthase mutains and its application of a kind of genetic engineering field more particularly to a kind of people.
Background technology
Atp synthase is the key enzyme of bioenergetic metabolism.It participates in oxidative phosphorylation to react with photophosphorylation, in cross-film
ATP is catalyzed and synthesized under the promotion of proton dynamics.The atp synthase mutation of people can impact human health, research hair
It is existing, carry out gene sequencing in the peripheral blood to certain cancers (cancer includes lung cancer, liver cancer, cancer of pancreas and leukaemia) patient
In the process, find that the atp synthase of patient is mutated, therefore, the detection being mutated to the atp synthase of people is to judging human body
It is no that there is certain impulse with cancer.
Invention content
Present invention aims at the atp synthase mutains and its application for providing a kind of people.
Technical solution of the present invention includes：
In a first aspect, the atp synthase mutain of people a kind of is provided, amino acid sequence such as SEQ ID NO:Shown in 1.
Second aspect provides a kind of encoding gene of the mutain of the atp synthase 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 atp synthase mutain of the people, with the normal albumen of atp synthase of people
The comparison result of nucleotide sequence determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of monoclonal antibody of the atp synthase mutain of specific recognition people, the ammonia of coding
Base acid sequence is SEQ ID NO:Shown in 1.
5th aspect provides a kind of ELISA kit of the antibody for the atp synthase mutain for being used to detect people, described
ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, the atp synthase for detecting object of said monoclonal antibody
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 atp synthase mutain based on any of the above-described ELISA kit detection people
The method of antibody, including：
A, said monoclonal antibody is diluted, and the monoclonal after dilution is resisted using the coating buffer solution
Body is loaded into the hole of ELISA ELISA Plates, and is incubated at room temperature；
B, the atp synthase albumen for detecting object is diluted to various concentration gradient, and will not using the sample diluting liquid
Atp synthase albumen with concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and be incubated at room temperature；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
D, the ELIAS secondary antibody is added in, and is incubated at room temperature；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
F, the developing solution is added in, room temperature is protected from light incubation, and adds in the terminate liquid and terminate reaction；
G, 450nm measures OD values in microplate reader.
Preferably, further comprise：Blank control is tested.
The present invention provides the atp synthase mutain of people a kind of and its application, using a large amount of cancer patients as research disease
Example carries out case genetic test and analyzes, the mutain of the atp synthase of people determined, according to the atp synthase of the people
Mutain prepares genetic chip, monoclonal antibody and ELISA kit, the impulse that the gene diagnosis for cancer provides,
Realize the early diagnosis, early discovery and associated treatment of cancer.
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technological 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 attached drawing is coordinated to be described in detail such as.
Description of the drawings
Fig. 1 is a kind of comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 2 is a kind of genetic chip layout that the embodiment of the present invention one provides；
Fig. 3 is a kind of colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 4 is another colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 5 is the standard curve that the offer of the embodiment of the present invention five is protein content；
Fig. 6 is the Western blot testing result schematic diagrames that the embodiment of the present invention five provides；
Fig. 7 is the standard curve that the offer of the embodiment of the present invention six is protein content；
Fig. 8 is the Western blot testing result schematic diagrames that the embodiment of the present invention six provides；
Fig. 9 is the standard curve that the offer of the embodiment of the present invention seven is protein content；
Figure 10 is the Western blot testing result schematic diagrames that the embodiment of the present invention seven provides.
Specific 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 it is 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 encoding gene of the atp synthase mutain of people,
Its nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the atp synthase mutain of people is determined according to the encoding gene,
Its amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the atp synthase mutain and its encoding gene of people, the system of genetic chip is realized as follows
It is standby.
1st, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe according to the nucleotide sequence of the atp synthase mutain of people, with
The comparison result of the nucleotide sequence of the normal albumen of atp synthase of people determines, and according to the design principle of following probe, design
Go out the nucleotide probe of the specificity of the atp synthase mutain for people.
Wherein, the principle of nucleotide probe design is as follows：
1. nucleotide probe Tm values should be close to the average Tm values of whole gene group, 5 DEG C of fluctuation up and down；
2. the single base that nucleotide probe intramolecular repeats continuously is no more than 4；
3. G+C contents are 40%-60%, the specificity that non-specific hybridization ensures hybridization is reduced；
4. nucleotide probe intramolecule stablizes secondary structure pairing bases longs less than 4bp, can ensure in this way will not
Hybridization efficiency is influenced due to the secondary structure of nucleotide probe internal stability；
5. the similitude through Homology search and other sequences is less than 40%；
6. continuously it is no more than 20 bases with the homologous fragment of non-probe alternative sequence by contrast.
Wherein, the corresponding amino acid sequence of atp synthase mutain of people ammonia corresponding with the normal albumen of the atp synthase of people
The comparison result of base acid sequence please refers to Fig.1, wherein, the Query sequences in Fig. 1 are that the atp synthase mutain of people is corresponding
Amino acid sequence, Sbjct sequences are the corresponding amino acid sequence of the normal albumen of atp synthase of people, Query sequences and Sbjct sequences
Sequence between row is comparison result, as can be seen from FIG. 1, the atp synthase mutain of people relative to people the normal egg of atp synthase
In vain, a part of amino acid sequence has been lacked at a position.According to SEQ ID NO:Nucleotide sequence and Fig. 1 shown in 2
Comparison result, whether the atp synthase in order to specific recognition object to be detected be mutated, then is choosing nucleosides
During acid probe, nucleotide probe can be designed as follows：
Several nucleotide sequences are selected before deletion sites, with selecting several nucleotides sequences after deletion sites
Row 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
Count principle, design one kind preferably nucleotide probe such as SEQ ID NO:Shown in 3, which is：tggtgtaaca
gactttgctg
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2nd, the preparation of the genetic chip whether atp synthase 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,For experimental group.
Wherein, the position of experimental group is the point sample position of nucleotide probe.
For point sample needed for the negative internal reference Quality Control probe and positive control of 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 digit that negative internal reference Quality Control probe includes can be with nucleotide probes
Digit 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 aattgcatag。
Positive internal control Quality Control probe：It is one section of other genetic fragment for having homology with detection gene, in the atp synthase of people
In mutain, select corresponding from nucleotide probe sequence is different, and positive internal control Quality Control probe includes nucleotide digit and
The digit of nucleotide probe may be the same or different.Preferably, nucleotide digit and the digit phase of nucleotide probe are chosen
Same positive internal control Quality Control probe.In the embodiment of the present invention, the positive internal control probe sequence needed for genetic chip can be：
atgctgtccc gggtggtact。
It should be noted that in the deposition process of genetic chip, click and enter 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
1st, sample treatment
(1) the blood 1-3mL of acquisition testing object.
(2) the 1.5mLEP pipes that DEPC is handled are taken, to be detected sample processing tube, are added in sample processing tube is detected
The 300 μ L of blood of object are detected, add 700 μ L of Trizol, abundant mixing is placed at room temperature for 10Min.
(3) chloroform of 200 μ L is added in, centrifuge tube lid is covered tightly, firmly shakes centrifuge tube, be placed at room temperature for 3-5min, 12000
R/min, 4 DEG C of 15 min of centrifugation, carefully sucks all supernatants.
(4) isometric isopropanol is added in, gently overturns the abundant mixing liquid of centrifuge tube, is placed at room temperature for 10 min, 12000
R/min, 4 DEG C of 15 min of centrifugation, carefully sucks all supernatants.
(5) one time, 7500 r/min, 4 DEG C 15 min of centrifugation are washed with 1mL75% ethyl alcohol, all supernatants is carefully sucked, super
Dry 15 min in net platform add in 10 μ L DEPC processing water dissolutions.
(6) products therefrom is RNA, if the purity of total serum IgE is not high, can influence the labeling effciency of probe and chip hybridization knot
Fruit.So use QIAGENPurify total serum IgE.
2nd, the first chains of cDNA and the second chain one-step synthesis method
(1) take 2 μ g RNA that following reaction solution is configured in 1.5mL centrifuge tubes：
| 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 |
5 × First Strand B μ ffer, are preheated 5 points by 10 minutes ice baths of (2) 65 DEG C of heat preservations 5 minutes at 65 DEG C in advance
Clock.
(3) following cDNA synthetic systems are 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 mix are added in after being denaturalized in the RNA of ice bath.
(5) it is centrifuged with after pipette tips mixing.
(6) 40 DEG C of reaction 2h.
3rd, 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, notes：40 DEG C of heat preservation 1min before 50%PEG uses.Simultaneously by following operative configuration
Transcription mix；
(1) configuration Transcription mix
(2) 60 μ l Transcription mix and mixing are added in
(3) hot 60 DEG C of lid in PCR instrument, 40 DEG C of reaction 2h.
4th, 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 LRNase free water are added in, add in 350 μ L Buffer RLT and abundant mixing.
(2) 250 μ L absolute ethyl alcohols, Tip abundant mixing are added in.
(3) 700 solution of the μ L containing total serum IgE altogether are transferred in the RNeasy pillars being sleeved in 2mL centrifuge tubes >=8000g from
Heart 15-30sec, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=15-30sec of 8000g centrifuge washings abandons
Filtered solution is gone to discard the casing of filtered solution and 2mL in >=8000g centrifuge washings 2min with 500 μ L Buffer RPE again will
RNeasy mini pillars are transferred in a new 1.5mL Eppendorf pipes.
(5) water for drawing 30 μ L RNase free stands 1min, >=8000g centrifugation elutions 1min.
(6) it is primary to repeat step (5).
5th, cRNA concentration mensurations
With spectrophotometric analysis RNA concentration.Need 260 and 280nm measure absorbance come determine the concentration of sample and
Purity A260/A280 should be purer RNA (ratio 1.9-2.1 also can) close to 2.0.
6th, cRNA fluorescents mark；
(1) above-mentioned 4 μ g of cRNA are taken and are concentrated into 6.6 μ L.
(2) add 10 μ L DMSO mixings.
(3) add 0.3M sodium bicarbonates (NaHCO3) pH9.0 and mixing of 3.4 μ L.
(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.
7th, 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.
8th, cRNA sample fragments and chip hybridization 4x44K microarrays
(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 |
| 10XBlocking Agent | 11μL |
| 25X Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ L 2X GEx Hybridization Buffer are added in.
(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.
9th, chip washs
(1 liter) configuration of washing lotion 1：
| DEPC-H2O | 700m L |
| 20*SSPE | 300m L |
| 20%N-Lauroylsarcosine | 0.25m L |
(1 liter) configuration of washing lotion 2：
| DEPC-H2O | 997m L |
| 20*SSPE | 3.0m L |
| 20%N-Lauroylsarcosine | 0.25m L |
Washing lotion 3：Stabilization and Drying Solution
(1) chip is taken out to wash 1 minute in washing lotion 1；
(2) chip is put into washing lotion 2 again and washs 1 minute (37 DEG C)；
(3) finally chip is washed 30 seconds in washing lotion 3.
10th, chip scanning
Chip is scanned in scanner, whether the atp synthase albumen for determining detection object according to scanning result is sent out
Mutation is given birth to.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, positive control is glimmering for green
Light, the sample quality for showing acquisition testing object are that there is no problem, and experimental group is green fluorescence, then show to detect object
Atp synthase albumen be mutated.In result shown in Fig. 4, negative control is colorless fluorescent, and positive control is glimmering for green
Light, the sample quality for showing acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, then shows to detect object
Atp synthase albumen do not mutate.
Embodiment three, specific recognition people atp synthase mutain monoclonal antibody preparation
1st, according to base sequence (such as SEQ ID NO of the atp synthase mutain of people:Shown in 2) design sense primer is such as
SEQ ID NO:Shown in 4 and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (P)：atgctgtccc gggtggtact
Downstream primer (F)：ggcaagtt tttgctcatt
2nd, the DNA of detection object carries out PCR amplification for template
| 10×Buffer | 5uL |
| dNTP | 2uL |
| Ex Taq | 1uL |
| ddH2O | 5uL |
| Template DNA | 1uL |
| Primer (P) | 3uL |
| Primer (F) | 3uL |
| Total system | 20uL |
PCR amplification is carried out as template using the DNA for detecting object, obtains the complete segment of atp synthase mutating protein gene of people,
And pMD19-T Vector (Takara companies) are 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, the ELISA kit for detecting the antibody of the atp synthase mutain of people
In the present embodiment, the composition of ELISA kit is：It is coated with the ELISA enzymes of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, detect the atp synthase albumen of object, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution,
Developing solution and terminate liquid.
Wherein, the parameter of above-mentioned each composition at least can be a kind of following parameter：
ELISA ELISA Plates can be the ELISA enzyme marks in 96 holes；
ELIAS secondary antibody can be the Goat anti-Human IgG of diluted HRP (horseradish peroxidase) labels；Wherein, dilution times
Number can be 8000 times.
It can be 1 × PBS, pH to be coated with buffer solution:7.4；
ELISA ELISA Plates cleaning solution can be 1 × PBS solution containing 0.05%Tween-20；
Developing solution can be 3,3 ', 5,5 '-tetramethyl benzidine；
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 atp synthase albumen of person is mutated, and this method can at least include following 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 atp synthase albumen of patients with lung cancer is diluted to various concentration gradient, and will be different dense using sample diluting liquid
The atp synthase albumen of degree gradient is loaded respectively into the hole of ELISA ELISA Plates, and be incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
D, the ELIAS secondary antibody is added in, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions is added in, washs and dry；
F, the developing solution is added in, room temperature, which is protected from light, is incubated 10-20min, adds in the terminate liquid and terminates reaction；
G, 450nm measures OD values in microplate reader.
H, standard curve is made：Using standard concentration as abscissa, the OD values that standard items measure are ordinate, make standard
Curve；Calculate standard curve regression coefficient R2, work as R2This measures effective during ＞ 0.99；Gone out according to ELISA Calc Software on Drawing
Standard curve can obtain the R according to the ELISA Calc softwares2=0.99215, therefore, this measures effective.It will detect
To OD450 values bring the concentration that standard curve can acquire the atp synthase albumen of patients with lung cancer into.Utilize the ELISA side of foundation
The content of atp synthase albumen in method detection Serum of Patients with Lung Cancer sample.And it is identified with Western blot.Please refer to Fig. 5,
Standard curve for OD values.Wherein, X-axis is OD values, and Y-axis is corresponding concentration.
I, Western blot are identified
1st, glue
(1) it cleans glass plate and dries；
(2) reference molecule cloning process prepares SDS-PAGE glue：
A. lower floor's separation gel prepares system (Total Volum：15mL)
| ddH2O | 5.9mL |
| 30% acrylamide mixed liquor | 5.9mL |
| 1.5mol/L Tris(PH 8.8) | 3.8mL |
| 10%SDS | 0.15mL |
| 10% ammonium persulfate | 0.15mL |
| TEMED | 0.006mL |
B. upper strata spacer gel concentration preparation system (Total Volum：8mL)
| ddH2O | 5.5mL |
| 30% acrylamide mixed liquor | 1.3mL |
| 1.0mol/L Tris(PH 6.8) | 1.0mL |
| 10%SDS | 0.08mL |
| 10% ammonium persulfate | 0.08ml |
| TEMED | 0.008mL |
(3) lower floor's separation gel of respective volume is added in each offset plate, adds the sterile ddH of 1mL2O flattens separation
Glue, after gelling to be separated is solid, remaining moisture in plastic plate is blotted with filter paper, upper strata concentration glue is then added in, after being inserted into comb
Wait for upper strata concentration gelling solid.
2nd, albumen loading electrophoresis：
(1) comb is pulled up, duct label is carried out, adds in 5 × SDS electrophoretic buffers, then to being added in protein sample
SDS-PAGE albumen sample-loading buffer (5 ×), boiling water bath heating 3-5min, loading；
(2) electrophoresis first is carried out with 80V voltages, after treating that band ran concentration glue, uses 100V voltages instead and run about 100min.
3rd, 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 in, 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 close membrane 1h on shaking table；
(3) film that PBST cleanings have been closed, is cleaned 3 times, each 10min；
(4) primary antibody for being diluted to corresponding multiple is added in, 4 DEG C of incubator overnights are incubated；
(5) primary antibody is recycled, PBST cleans film three times, each 10min；
(6) secondary antibody is (with 3% milk with 1:5000-10000 dilutes) incubation at room temperature film 2h；
(7) film is cleaned three 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 are analyzed with Image J.
Wherein, primary antibody is the atp synthase mutain monoclonal antibody of the standby people of corporation, and secondary antibody is horseradish peroxidase
The Goat anti-Human IgG of 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.Wherein, the molecular size range of atp synthase mutain
For 24KD.Compared with blank control, only occur apparent band near experimental group 24KD, show the atp synthase of the patients with lung cancer
Albumen is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, it using liver cancer patient as detection object, and detects the liver cancer using ELISA kit and suffers from
Whether the atp synthase albumen of person is mutated, and this method can at least include following 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 atp synthase albumen for detecting object is diluted to various concentration gradient, and will be different dense using sample diluting liquid
The atp synthase albumen of degree gradient is loaded respectively into the hole of ELISA ELISA Plates, and be incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
D, the ELIAS secondary antibody is added in, and is incubated at room temperature 1-2hh；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions is added in, washs and dry；
F, the developing solution is added in, room temperature, which is protected from light, is incubated 10-20min, adds in the terminate liquid and terminates reaction；
G, 450nm measures OD values in microplate reader.
H, standard curve is made：Using standard concentration as abscissa, the OD values that standard items measure are ordinate, make standard
Curve；Calculate standard curve regression coefficient R2, work as R2This measures effective during ＞ 0.99；Gone out according to ELISA Calc Software on Drawing
Standard curve can obtain the R according to the ELISA Calc softwares2=0.99819, therefore, this measures effective.It will detect
To OD450 values bring the concentration that standard curve can acquire the atp synthase albumen of liver cancer patient in sample into.Utilize foundation
The content of atp synthase albumen in ELISA method detection liver cancer patient blood serum sample.And it is identified with Western blot.Please
It is the standard curve of OD values with reference to figure 7.Wherein, X-axis is OD values, and Y-axis is corresponding concentration.
I, Western blot are identified
J, Western blot Analysis of test results
Wherein, step i and step j are identical in embodiment five, and details are not described herein, please refer to Fig. 8, in Fig. 8
Marker is used to characterize the size of labelled protein.Wherein, the molecular size range of atp synthase mutain is 24KD.The present embodiment
Experimental display result, compared with blank control, only occur apparent band near experimental group 24KD, show the liver cancer patient
Atp synthase albumen is implicitly present in mutation.
Embodiment seven
In embodiments of the present invention, using Pancreas cancer patients as detection object, and the pancreas is detected using ELISA kit
Whether the atp synthase albumen of cancer patient is mutated, and this method can at least include following 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 atp synthase albumen of Pancreas cancer patients is diluted to various concentration gradient, and will be different using sample diluting liquid
The atp synthase albumen of concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and be incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
D, the ELIAS secondary antibody is added in, is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions is added in, washs and dry；
F, the developing solution is added in, room temperature, which is protected from light, is incubated 10-20min, adds in the terminate liquid and terminates reaction；
G, 450nm measures OD values in microplate reader.
H, standard curve is made, using standard concentration as abscissa, the OD values that standard items measure are ordinate, make standard
Curve；Calculate standard curve regression coefficient R2, work as R2This measures effective during ＞ 0.99；Gone out according to ELISA Calc Software on Drawing
Standard curve can obtain the R according to the ELISA Calc softwares2=0.99311, therefore, this measures effective.It will detect
To OD450 values bring the concentration that standard curve can acquire the atp synthase albumen of Pancreas cancer patients in sample into.Utilize foundation
The content of atp synthase albumen in ELISA method detection Pancreas cancer patients blood serum sample.And it is identified with Western blot.
Fig. 9 is please referred to, is the standard curve of OD values.Wherein, X-axis is OD values, and Y-axis is corresponding concentration.
I, Western blot are identified
J, Western blot Analysis of test results
Wherein, step i and step j are identical in embodiment five, and details are not described herein, please refers to Fig.1 in 0, Figure 10
Marker is used to characterize the size of labelled protein.Wherein, the molecular size range of atp synthase mutain is 24KD.The present embodiment
Experimental display result, compared with blank control, only occur apparent band near experimental group 24KD, show the Pancreas cancer patients
Atp synthase albumen be implicitly present in mutation.
The above is only the preferred embodiment of the present invention, 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 atp synthase mutain of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 86
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Leu Ser Arg Val Val Leu Ser Ala Ala Ala Thr Ala Ala Pro Ser
1 5 10 15
Leu Lys Asn Ala Ala Phe Leu Gly Pro Gly Val Leu Gln Ala Thr Arg
20 25 30
Thr Phe His Thr Gly Gln Pro His Leu Val Pro Val Pro Pro Leu Pro
35 40 45
Glu Tyr Gly Gly Lys Val Arg Tyr Gly Leu Ile Pro Glu Glu Phe Phe
50 55 60
Gln Phe Leu Tyr Pro Lys Thr Gly Val Thr Asp Phe Ala Asp Lys Leu
65 70 75 80
Asn Glu Gln Lys Leu Ala
85
<210> 2
<211> 258
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atgctgtccc gggtggtact ttccgccgcc gccacagcgg ccccctctct gaagaatgca 60
gccttcctag gtccaggggt attgcaggca acaaggacct ttcatacagg gcagccacac 120
cttgtccctg taccacctct tcctgaatac ggaggaaaag ttcgttatgg actgatccct 180
gaggaattct tccagtttct ttatcctaaa actggtgtaa cagactttgc tgataaactc 240
aatgagcaaa aacttgcc 258
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tggtgtaaca gactttgctg 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atgctgtccc gggtggtact 20
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ggcaagtttt tgctcatt 18
Claims (9)
- A kind of 1. atp synthase mutain of people, which is characterized in that its amino acid sequence such as SEQ ID NO:Shown in 1.
- A kind of 2. encoding gene of the atp synthase mutain of people, which is characterized in that its nucleotide sequence such as SEQ ID NO:2 It is shown.
- 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 the atp synthase mutain of the people, the nucleosides with the normal albumen of atp synthase of people The comparison result of acid sequence 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.
- A kind of 5. monoclonal antibody of the atp synthase mutain of specific recognition people, which is characterized in that its amino acid encoded Sequence is SEQ ID NO:Shown in 1.
- A kind of 6. ELISA kit of the antibody for the atp synthase mutain for being used to detect people, which is characterized in that the ELISA Kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, the ATP for detecting object of monoclonal antibody described in claim 5 Synthase protein, 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 atp synthase mutain of people, feature according to claim 6 It is, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
- 8. a kind of method of the antibody of the atp synthase mutain based on the ELISA kit detection people of claim 6 or 7, It is characterised in that it includes：A, monoclonal antibody described in claim 5 is diluted, and by the list after dilution using the coating buffer solution Clonal antibody is loaded into the hole of ELISA ELISA Plates, and is incubated at room temperature；B, the atp synthase albumen for detecting object is diluted to various concentration gradient, and will be different dense using the sample diluting liquid The atp synthase albumen of degree gradient is loaded respectively into the hole of ELISA ELISA Plates, and be incubated at room temperature；C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；D, the ELIAS secondary antibody is added in, and is incubated at room temperature；E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；F, the developing solution is added in, room temperature is protected from light incubation, and adds in the terminate liquid and terminate reaction；G, 450nm measures OD values in microplate reader.
- 9. the method for the antibody of the atp synthase mutain of ELISA kit detection people according to claim 8, feature It is, further comprises：Blank control is tested.
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| CN1363656A (en) * | 2001-01-05 | 2002-08-14 | 上海博德基因开发有限公司 | Polypeptide-ATP systhetase 11.55 and polynucleotide for coding it |
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| CN101137760A (en) * | 2005-03-18 | 2008-03-05 | 香港中文大学 | Method for the detection of chromosomal aneuploidies |
| CN101440397A (en) * | 2008-08-13 | 2009-05-27 | 中国人民解放军第三军医大学第二附属医院 | Gene chip for detecting differential expression of human mitochondria gene |
| CN105652016A (en) * | 2016-01-28 | 2016-06-08 | 深圳大学 | Autism detection marker and detection method thereof |
| CN107403074A (en) * | 2017-06-09 | 2017-11-28 | 天津市湖滨盘古基因科学发展有限公司 | A kind of detection method and device of mutain |
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2017
- 2017-11-29 CN CN201711220796.1A patent/CN108152497A/en active Pending
Patent Citations (6)
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| CN1363656A (en) * | 2001-01-05 | 2002-08-14 | 上海博德基因开发有限公司 | Polypeptide-ATP systhetase 11.55 and polynucleotide for coding it |
| CN101061235A (en) * | 2004-11-19 | 2007-10-24 | 大塚制药株式会社 | Method of diagnosing the risk of thermolabile phenotype diseases by using gene |
| CN101137760A (en) * | 2005-03-18 | 2008-03-05 | 香港中文大学 | Method for the detection of chromosomal aneuploidies |
| CN101440397A (en) * | 2008-08-13 | 2009-05-27 | 中国人民解放军第三军医大学第二附属医院 | Gene chip for detecting differential expression of human mitochondria gene |
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Application publication date: 20180612 |