CN109251903A - The sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] and its application - Google Patents
The sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] and its application Download PDFInfo
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Abstract
The present invention relates to a kind of sub- compound subunit 10 mutain of 1 β of the nadh dehydrogenase of people [coenzyme] and its applications, using several leukaemics as research case, genetic test is carried out to case and is analyzed, determine the mutain of the sub- compound subunit 10 of 1 β of nadh dehydrogenase [coenzyme] of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] of the people, impulse is provided for the gene diagnosis of leukaemia, to realize that the diagnosing and treating of leukaemia provides scientific basic.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to a kind of sub- compound Asias 1 β of nadh dehydrogenase of people [coenzyme]
10 mutain of base and its application.
Background technique
Reduced nicotinamide adenine dinucleotide (reduced form of nicotinamide edenine
Dinucleotide, NADH) dehydrogenase is located at mitochondrial inner membrane, and it is an important enzyme of tricarboxylic acid cycle, by 43 albumen
Composition, wherein having 7 is encoded by mitochondrial genomes, others are encoded by Matrix attachment region.Current research prompt, NADH are de-
Sub- compound subunit 10 (NADH dehydrogenase [ubiquinone] the 1beta subcomplex of 1 β of hydrogen enzyme [coenzyme]
Subunit 10) gene expression increase it is closely related with mitochondrial defects and oxidative stress, it is sub- to nadh dehydrogenase [coenzyme] 1 β
Effect of the compound subunit 10 in leukaemic is studied, to obtain the sub- compound subunit 10 of 1 β of nadh dehydrogenase [coenzyme]
To the positive effect of the treatment of leukaemia.
Summary of the invention
It is an object of that present invention to provide a kind of sub- compound subunit 10 mutain of the nadh dehydrogenase of people [coenzyme] 1 β and its
Using.
Technical solution of the present invention includes:
In a first aspect, a kind of sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] for providing people, amino acid
Sequence is as shown in SEQ ID NO:1.
Second aspect provides a kind of coding base of the mutain of the sub- compound subunit 10 of 1 β of nadh dehydrogenase [coenzyme] of people
Cause, nucleotide sequence is as shown in SEQ ID NO:2.
The third aspect provides a kind of genetic chip, comprising: solid phase carrier and fixed nucleotide probe on this carrier；
The nucleotide probe is according to the nucleotide of the sub- compound subunit 10 mutain of nadh dehydrogenase [coenzyme] 1 β of people described above
Sequence is determined with the comparison result of the nucleotide sequence of the sub- compound normal albumen of subunit 10 of 1 β of nadh dehydrogenase [coenzyme] of people.
Preferably, the nucleotide probe is base sequence shown in SEQ ID NO:3；
The nucleotide probe is base sequence shown in SEQ ID NO:4；
The nucleotide probe is base sequence shown in SEQ ID NO:5.
Fourth aspect provides a kind of reagent, contains the nadh dehydrogenase [coenzyme] 1 for detecting above-mentioned people in the reagent
The primer pair of the sub- compound subunit 10 mutain of β.
Preferably, the primer pair includes the combination of following any upstream primers Yu following any downstream primers:
Upstream primer is the base sequence as shown in any in SEQ ID NO:8~NO:20；
Downstream primer is the base sequence as shown in any in SEQ ID NO:21~NO:24.
5th aspect provides the sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] of specific recognition people a kind of
Monoclonal antibody, be prepared by above-mentioned reagent, can be with amino acid sequencespecific knot shown in SEQ ID NO:1
It closes.
6th aspect provides a kind of sub- compound subunit 10 mutain of nadh dehydrogenase [coenzyme] 1 β for detecting people
ELISA kit, the ELISA kit include: ELISA ELISA Plate, ELIAS secondary antibody, the sample for being coated with said monoclonal antibody
Product dilution, coating buffer, ELISA ELISA Plate cleaning solution, developing solution and terminate liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeled.
The present invention provides a kind of sub- compound subunit 10 mutain of 1 β of the nadh dehydrogenase of people [coenzyme] and its applications, will
Several leukaemics carry out genetic test to case and analyze, determine that the nadh dehydrogenase of people is [auxiliary as research case
Enzyme] the sub- compound subunit 10 of 1 β mutain, according to the sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] of the people
Genetic chip, monoclonal antibody and ELISA kit are prepared, provides impulse for the gene diagnosis of leukaemia, it is white to realize
The diagnosing and treating of blood disease provides scientific basic.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention and the accompanying drawings.
Detailed description of the invention
Fig. 1 is a kind of comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 2 is a kind of genetic chip layout that the embodiment of the present invention one provides；
Fig. 3 is a kind of colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 4 is another colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 5 is the standard curve for the protein content that the embodiment of the present invention five provides；
Fig. 6 is the Western blot testing result schematic diagram that the embodiment of the present invention five provides.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment one, genetic chip
It is possible, firstly, to be a kind of " detection side of mutain according to application No. is " 201710429915.8 ", patent name
Detection method described in the Chinese invention patent of method and device " determines the sub- compound Asia 1 β of nadh dehydrogenase [coenzyme] of people
The encoding gene of 10 mutain of base, nucleotide sequence is as shown in SEQ ID NO:2；Correspondingly, true according to the encoding gene
The sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] of people is made, amino acid sequence is as shown in SEQ ID NO:1.
Secondly, according to the sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] and its encoding gene of people, according to
As under type realizes the preparation of genetic chip.
1, the design of nucleotide probe
(1) design of nucleotide probe: nucleotide probe is according to the sub- compound subunit 10 of nadh dehydrogenase [coenzyme] 1 β of people
The nucleotide sequence of mutain, the nucleotide sequence with the sub- compound normal albumen of subunit 10 of 1 β of nadh dehydrogenase [coenzyme] of people
Comparison result determine that and according to the design principle of following probe, 1 β of nadh dehydrogenase [coenzyme] designed for people is sub-
The nucleotide probe of the specificity of compound subunit 10 mutain.
Wherein, the principle of nucleotide probe design is as follows:
1. nucleotide probe Tm value should be close to the average Tm value of whole gene group, 5 DEG C of fluctuation up and down；
2. the duplicate single base of nucleotide probe intramolecular is continuously no more than 4；
3. G+C content is 40%-60%, the specificity that non-specific hybridization guarantees hybridization is reduced；
4. it should be less than 4bp with the bases longs of probe molecule internal stability secondary structure pairing in nucleotide probe sequences,
Can guarantee that hybridization efficiency will not be influenced because of the secondary structure of nucleotide probe internal stability in this way；
5. the similitude through Homology search and other sequences is less than 40%；
6. being continuously no more than 20 bases with the homologous fragment of non-probe alternative sequence by contrast.
Wherein, the corresponding amino acid sequence of the sub- compound subunit 10 mutain of nadh dehydrogenase [coenzyme] 1 β of people and people
The comparison result of the corresponding amino acid sequence of the sub- compound normal albumen of subunit 10 of nadh dehydrogenase [coenzyme] 1 β referring to FIG. 1, its
In, the Query sequence in Fig. 1 is the corresponding amino acid sequence of the compound subunit 10 mutain in the Asia 1 β of nadh dehydrogenase [coenzyme] of people
Column, Sbjct sequence are the corresponding amino acid sequences of the compound normal albumen of subunit 10 in the Asia 1 β of nadh dehydrogenase [coenzyme] of people,
Sequence between Query sequence and Sbjct sequence is comparison result, and as can be seen from FIG. 1,1 β of nadh dehydrogenase [coenzyme] of people is sub-
Nadh dehydrogenase [coenzyme] 1 β sub- compound subunit 10 normal albumen of the compound subunit 10 mutain relative to people, in several positions
Place is lacked, according to nucleotide sequence shown in SEQ ID NO:2 and the comparison result of Fig. 1, in order to specificity
Identify whether the sub- compound subunit 10 of 1 β of nadh dehydrogenase [coenzyme] of object to be detected is mutated, then choosing nucleotide
When probe, nucleotide probe can be designed according to any mode in following several modes:
1. selecting several nucleotide sequences before deletion sites, and several nucleotide are selected after deletion sites
Sequence generates nucleotide probe.
In the present embodiment, it the nucleotide sequence according to shown in SEQ ID NO:2 and is set according to above-mentioned nucleotide probe
Principle is counted, at least can be designed that following several preferably nucleotide probes in the manner described above:
As shown in SEQ ID NO:3, the nucleotide probe are as follows: ttcacccagg；
As shown in SEQ ID NO:4, the nucleotide probe are as follows: agggggcctacagtt；
As shown in SEQ ID NO:5, the nucleotide probe are as follows: agttcacccagg.
(2) above-mentioned designed nucleotide probe the synthesis of nucleotide probe: is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether sub- compound subunit 10 of 1 β of nadh dehydrogenase [coenzyme] of detection people mutates
In order to guarantee the quality of test object sample, blank control, positive control and yin need to be also designed on genetic chip
Property control.Wherein, the layout of the genetic chip can be at least a kind of layout as shown in Figure 2.In Fig. 2, is blank pair
According to, zero is negative control,For positive control, ☆ 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: being one section does not have other genetic fragments of homology with detection gene, as hybridizing
The monitor control index of non-specific hybridization in journey, the nucleotide number that negative internal reference Quality Control probe includes can be with nucleotide probes
Base number is identical, can also be different.In the embodiment of the present invention, negative internal reference probe sequence needed for genetic chip can be with are as follows:
tgatgctgat aattgcat。
Positive internal control Quality Control probe: being one section of other genetic fragment for having homology with detection gene, de- in the NADH of people
In the sub- compound subunit 10 mutain of 1 β of hydrogen enzyme [coenzyme], select sequence corresponding from nucleotide probe different, and can be with core
The number of thuja acid probe base is identical, and one section of nucleotide sequence that can also be different from the number of nucleotide probe base is as sun
Property internal reference Quality Control probe.Preferably, the positive internal control Quality Control identical with the number of nucleotide probe base of nucleotide number is chosen
Probe.In the embodiment of the present invention, positive internal control probe sequence needed for genetic chip can be with are as follows: cgcaaagaac.
It should be noted that clicking and entering negative internal reference in the deposition process of genetic chip according to the layout of genetic chip and visiting
Needle and positive internal control probe solution；The spotting buffer (10% aqueous trehalose) that reagent used in blank control is 1 times.
The application of embodiment two, genetic chip
1, sample treatment
(1) the blood 1-3mL of acquisition testing object.
(2) the 1.5mL EP pipe for taking DEPC to handle, as detected sample processing tube, is added test object in pipe
300 μ L of blood, 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, it is careful to draw supernatant in centrifuge tube, the supernatant of absorption is transferred to clean centrifuge tube
In.
(4) isometric isopropanol is added in the centrifuge tube of supernatant in storing step (3), it is abundant gently overturns centrifuge tube
Liquid is mixed, 10min, 12000r/min, 4 DEG C of centrifugation 15min is placed at room temperature for, carefully sucks all supernatants.
(5) it is cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min is centrifuged 15min, all supernatants is carefully sucked, super
10 μ L DEPC processing water dissolution is added in dry 15min in net platform.
(6) products therefrom will affect the labeling effciency and chip hybridization knot of probe if the purity of total serum IgE is not high for RNA
Fruit.So using QIAGENKit purifies total serum IgE.
2, the first chain of cDNA and the second chain one-step synthesis method
(1) 2 μ g RNA is taken to configure following reaction solution in the centrifuge tube of 1.5mL:
| Total serum IgE | The most 6.5 μ L of 2 μ g |
| T7Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
Wherein, the additional amount X μ L of RNase-free Water is to subtract T7Promotor according to 11.5 μ L of total volume
The 5 μ L of additional amount of primer, then subtract the additional amount of total serum IgE and calculate and get.
(2) 10min, and ice bath 5min are kept the temperature at 65 DEG C, and 5 × First Strand B μ ffer is preheated at 65 DEG C
5min。
(3) following cDNA synthetic system is configured:
(4) above-mentioned 8.5 μ L is added after mixing after being denaturalized in the RNA of ice bath.
(5) it is centrifuged after being mixed with pipette tips.
(6) 40 DEG C of reaction 2h.
3, aaUTP marks cRNA synthesis
The configuration of NTP:
| 100mM ATP | 250μL |
| 100mM GTP | 250μL |
| 100mM CTP | 250μL |
| 100mM UTP | 187.5μL |
| RNase free H2O | 62.5μL |
| Total volume | 1000μL |
It is spare to be distributed into 10 pipes, note: 40 DEG C of heat preservation 1min before 50%PEG (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcription mix；
(1) Transcription mix is configured
| RNase-free Water | 5.7μL |
| 4×Transcription Buffer | 20μL |
| NTP | 16μL |
| 0.1M DTT | 6μL |
| 50%PEG | 6.4μL |
| aa-UTP(25mM) | 4μL |
| Inorganic Pyrophosphatase | 0.6μL |
| T7RNA Polymerase | 0.8μL |
| Total volume | 60μL |
(2) 60 μ L Transcription mix are added and mix.
(3) 60 DEG C of hot lid in PCR instrument, 40 DEG C of reaction 2h.
4, cRNA is purified
QIAGEN RNeasy Mini kit purifies cRNA, and specific method can be found in what QIAGEN company provided with kit
Operation manual.
(1) 20 μ LRNase free water are added, 350 μ L Buffer RLT are added and mix well.
(2) 250 μ L dehydrated alcohols are added, Tip mix well.
(3) total 700 solution of the μ L containing total serum IgE are transferred to cover in the RNeasy pillar in 2mL centrifuge tube >=8000g from
Heart 15-30s, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=8000g centrifuge washing 15-30s discards filter
It crosses liquid and discards the casing of filtered solution and 2mL for RNeasy in >=8000g centrifuge washing 2min with 500 μ L Buffer RPE again
Mini pillar is transferred in a new 1.5mL Eppendorf pipe.
(5) water for drawing 30 μ L RNase free stands 1min, >=8000g centrifugation elution 1min.
(6) it is primary that step (5) are repeated.
5, cRNA concentration mensuration
With spectrophotometric analysis cRNA concentration.Need to measure the light absorption value at 260nm and 280nm to determine the dense of sample
Degree and purity, A260/A280 should be purer cRNA (ratio 1.9-2.1 can also) close to 2.0.
6, cRNA fluorescent marks；
(1) it takes above-mentioned 4 μ g of cRNA and is concentrated into 6.6 μ L.
(2) plus 10 μ L DMSO are mixed.
(3) add the sodium bicarbonate (NaHCO that the 0.3M pH of 3.4 μ L is 9.03) and mix.
(4) above-mentioned 20 μ L cRNA mixture is added in fluorescent dye Cy3 and is mixed.25 DEG C of heat preservation 1h.
(5) 25 DEG C of heat preservation 15min after adding the 4M Hydroxylamine of 9 μ L to mix.
7, fluorescent marker cRNA Sample Purification on Single
The process that specific steps are purified with cRNA in the step 4 of the present embodiment, this step are not repeating.
8, cRNA sample fragment and 4 × 44K of chip hybridization microarrays
(1) according to the form below prepares fragmentation mixed liquor and then carries out fragmentation in 60 DEG C of warm bath 30min.
| Cy3cRNA green fluorescence | 875ng |
| 10×Blocking Agent | 11μL |
| 25×Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ L 2 × GEx Hybridization Buffer are added.
(3) 100 μ L hybridization solutions is taken to be added drop-wise on chip ware, at the same be added dropwise respectively as shown in Figure 2 by chip layout blank,
On negative, positive, experimental group position.It covers chip and is sealed in hybridizing box, 60 DEG C of rollings hybridize 16h.
9, chip washs
Washing lotion 1 (1L) configuration:
| DEPC-H2O | 700mL |
| 20×SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 2 (1L) configuration:
| DEPC-H2O | 997mL |
| 20×SSPE | 3.0mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 3:Stabilization and Drying Solution
(1) it takes out chip and washs 1min in washing lotion 1；
(2) chip is put into washing 1min (37 DEG C) in washing lotion 2 again；
(3) chip is finally washed into 30s in washing lotion 3.
10, chip scanning
Chip is scanned in scanner, 1 β of nadh dehydrogenase [coenzyme] of test object is determined according to scanning result
Whether sub- compound subunit 10 albumen is mutated.Please refer to Fig. 3 and Fig. 4, in result shown in Fig. 3, negative control redgreen
Fluorescence, positive control are green fluorescence, show that the sample quality of acquisition testing object is that there is no problem, and experimental group is green
Fluorescence, then showing that the sub- compound subunit 10 albumen of 1 β of nadh dehydrogenase [coenzyme] of test object is mutated.It is shown in Fig. 4
As a result in, negative control is colorless fluorescent, and positive control is green fluorescence, shows that the sample quality of acquisition testing object is that do not have
Problem, and experimental group redgreen fluorescence, then showing the sub- compound subunit 10 egg of 1 β of nadh dehydrogenase [coenzyme] of test object
Bai Wei mutates.
Embodiment three, specific recognition people the sub- compound subunit 10 mutain of nadh dehydrogenase [coenzyme] 1 β monoclonal
The preparation of antibody
1, the determination of reagent
Contain the sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] for detecting above-mentioned people in the reagent
Primer pair.
Wherein, according to the ORF of the sub- compound normal albumen of subunit 10 of the nadh dehydrogenase of people [coenzyme] 1 β (such as SEQ ID NO:
Shown in 6), it can determine the corresponding base sequence of ORF of the sub- compound normal albumen of subunit 10 of 1 β of nadh dehydrogenase [coenzyme] of people
It arranges (as shown in SEQ ID NO:7).
It is possible to further according to the base sequence of the sub- compound subunit 10 mutain of 1 β of the nadh dehydrogenase of people [coenzyme]
(as shown in SEQ ID NO:2), and 1 β of nadh dehydrogenase [coenzyme] the sub- compound normal albumen of subunit 10 of people according to people
The corresponding base sequence of ORF (as shown in SEQ ID NO:7), the primer pair designed include following any upstream primers with it is following
The combination of any downstream primer: upstream primer is the base sequence as shown in any in SEQ ID NO:8~NO:20；Draw in downstream
Object is the base sequence as shown in any in SEQ ID NO:21~NO:24:
As shown in SEQ ID NO:8: atgccggacagctgggacaaggatgtgtac
As shown in SEQ ID NO:9: cctgagcccccgcgccgcacgccggtgcag
As shown in SEQ ID NO:10: cccaatcccatcgtctacatgatgaaagcg
As shown in SEQ ID NO:11: ttcgacctcatcgtggaccgacccgtgacc
As shown in SEQ ID NO:12: ctcgtgagagaatttatagagcggcagcac
As shown in SEQ ID NO:13: gcaaagaacaggtattactactaccaccgg
As shown in SEQ ID NO:14: cagtaccgccgcgtgccagacatcactgag
As shown in SEQ ID NO:15: tgcaaggaggaggacatcatgtgcatgtat
As shown in SEQ ID NO:16: gaagccgaaatgcagtggaagagggactac
As shown in SEQ ID NO:17: aaagtcgaccaagaaattatcaacattatg
As shown in SEQ ID NO:18: caggatcggctcaaagcctgtcagcagagg
As shown in SEQ ID NO:19: gaaggacagaactaccagcagaactgtatc
As shown in SEQ ID NO:20: aaggaagtggagcagttcacccag
As shown in SEQ ID NO:21: tcaggaggtggcagcggcggcctcttttgc
As shown in SEQ ID NO:22: agcttttctctcttgcagcatcctctgcct
As shown in SEQ ID NO:23: ctgtttggccaggcacttcctggcagaact
As shown in SEQ ID NO:24: ggcacttcctggcagaactgtaggcccc
In the present embodiment, the sub- compound Asia 1 β of nadh dehydrogenase [coenzyme] in reagent containing one for detecting above-mentioned people
The primer pair of 10 mutain of base.
For the primer pair (primer (F) and primer (R)) of each combination producing, following PCR amplification steps are executed.
2, the DNA of test object is that template carries out PCR amplification
| 10×Buffer | 5uL |
| dNTP | 2uL |
| Ex Taq | 1uL |
| ddH2O | 5uL |
| Template DNA | 1uL |
| Primer (F) | 3uL |
| Primer (R) | 3uL |
| Total system | 20uL |
PCR amplification is carried out by template of the DNA of test object, obtains the sub- compound subunit of 1 β of nadh dehydrogenase [coenzyme] of people
The complete segment of 10 mutating protein genes, and pMD19-T Vector (Takara company) is connected, it is sequenced.Then by special
Biotech firm prepares antibody, is a kind of humanization or Chimeric antibodies.Wherein, the monoclonal antibody prepared can be with SEQ ID
Amino acid sequencespecific shown in NO:1 combines.The antibody of preparation is measured into content using ELISA method.
The ELISA examination of example IV, the sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] for detecting people
Agent box
In the present embodiment, the composition of ELISA kit are as follows: be coated with the ELISA enzyme of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, sample diluting liquid, coating buffer, ELISA ELISA Plate cleaning solution, developing solution and terminate liquid.
Wherein, the parameter of above-mentioned each composition can be at least a kind of following parameter:
ELISA ELISA Plate can be the ELISA enzyme mark in 96 holes；
ELIAS secondary antibody can be the Goat anti-Human IgG of diluted HRP (horseradish peroxidase) label；Wherein, dilution times
Number can be 8000 times.
Coating buffer can be 1 × PBS, pH:7.4；
ELISA ELISA Plate cleaning solution can be 1 × PBS solution containing 0.05%Tween-20；
Developing solution can be 3,3 ', 5,5 '-tetramethyl benzidines；
Terminate liquid can be the sulfuric acid solution of 2mol/L.
Embodiment five
Research method: choosing men and women leukaemic totally 20, as test object, and utilizes ELISA kit
Whether the sub- compound subunit 10 albumen of 1 β of nadh dehydrogenase [coenzyme] for detecting 20 patients is mutated.
By taking wherein a certain position patient as an example, the detection method to the patient includes:
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plate, it is incubated at room temperature 1-2h.
B, the sub- compound subunit 10 albumen of 1 β of nadh dehydrogenase [coenzyme] of patient is diluted to difference using sample diluting liquid
Concentration gradient, and the sub- compound subunit 10 albumen of 1 β of the nadh dehydrogenase of various concentration gradient [coenzyme] is loaded respectively to ELISA
In the hole of ELISA Plate, and it is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plate cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated for 10-20min, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
H, make standard curve: using standard concentration as abscissa, the light absorption value of standard items measurement is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measurement is effective when > 0.99；Wherein it is possible to use ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc software2=0.99979, therefore, this
Measurement is effective.The wavelength that will test, which substitutes into standard curve for the light absorption value at 450nm, can acquire the NADH dehydrogenation of patient
The concentration of the sub- compound subunit 10 albumen of 1 β of enzyme [coenzyme].It is de- using NADH in the ELISA method detection patient serum sample of foundation
The content of the sub- compound subunit 10 albumen of 1 β of hydrogen enzyme [coenzyme].And it is identified with Western blot.Referring to FIG. 5, being the trouble
Person corresponds to the standard curve of light absorption value.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot is identified
1, glue
(1) it cleans glass plate and dries；
(2) reference molecule cloning process prepares SDS-PAGE glue:
A. lower layer's separation gel prepares system (Total Volum:15mL)
| ddH2O | 5.9mL |
| 30% acrylamide mixed liquor | 5.9mL |
| 1.5mol/L Tris(PH 8.8) | 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 with filter paper after gelling to be separated is solid, upper layer is then added, glue is concentrated, after being inserted into comb
Wait upper layer concentration gelling solid.
2, albumen loading electrophoresis:
(1) comb is pulled up, duct label is carried out, 5 × SDS electrophoretic buffer is added, then to being added in protein sample
SDS-PAGE albumen sample-loading buffer (5 ×), boiling water bath heat 3-5min, loading；
(2) electrophoresis first is carried out with 80V voltage, after running concentration glue to band, uses 100V voltage instead and run about 100min.
3, transferring film and incubation:
(1) first pvdf membrane is activated in methyl alcohol about 30s.Respectively by gel, foam-rubber cushion and pvdf membrane in advance in electrotransfer
30min is balanced in buffer.It is clipped by filter paper-glue-film-filter paper sequence, is put into transferring film device according to principle of the black flour to black flour
In, it is added refrigerator (ice bag), about 120min is run with the electric current of 150mA in ice；
(2) it takes the film out, is put into 5% skim milk at once after the completion of, gently room temperature closes 1h on shaking table；
(3) PBST cleans closed film, cleans 3 times, each 10min；
(4) primary antibody for being diluted to corresponding multiple is added, 4 DEG C of shaking tables are incubated overnight；
(5) primary antibody is recycled, PBST is cleaned film 3 times, each 10min；
(6) secondary antibody (being diluted with 3% milk with 1:5000-1:10000) is incubated at room temperature film 2h；
(7) film 3 times are cleaned with PBST, each 10min；
(8) after Pierce ECL Western Blotting Substrate kit A, B liquid mixes in equal volume, dropwise
It is added dropwise on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment are analyzed with Image J.
Wherein, primary antibody is the sub- compound subunit 10 mutain monoclonal of 1 β of nadh dehydrogenase [coenzyme] of the standby people of corporation
Antibody, secondary antibody are the Goat anti-Human IgGs of horseradish peroxidase-labeled.
J, Western blot Analysis of test results: showing according to Western blot experimental procedure as a result, such as Fig. 6 institute
Show, wherein the Marker in Fig. 6 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 36KD
There is obvious band, wherein the molecular weight of the sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] is about 36KD, table
The sub- compound subunit 10 albumen of 1 β of nadh dehydrogenase [coenzyme] of the bright patient is implicitly present in mutation.
According to the studies above method, the sub- compound subunit 10 albumen of 1 β of nadh dehydrogenase [coenzyme] mutates in 20 patients
Probability be 39%, it is possible thereby to determine, the abrupt climatic change dialogue of the sub- compound subunit 10 albumen of 1 β of nadh dehydrogenase [coenzyme]
The gene diagnosis and treatment of blood disease have certain impulse.
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 sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] and its application
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 149
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 1
Met Pro Asp Ser Trp Asp Lys Asp Val Tyr Pro Glu Pro Pro Arg Arg
1 5 10 15
Thr Pro Val Gln Pro Asn Pro Ile Val Tyr Met Met Lys Ala Phe Asp
20 25 30
Leu Ile Val Asp Arg Pro Val Thr Leu Val Arg Glu Phe Ile Glu Arg
35 40 45
Gln His Ala Lys Asn Arg Tyr Tyr Tyr Tyr His Arg Gln Tyr Arg Arg
50 55 60
Val Pro Asp Ile Thr Glu Cys Lys Glu Glu Asp Ile Met Cys Met Tyr
65 70 75 80
Glu Ala Glu Met Gln Trp Lys Arg Asp Tyr Lys Val Asp Gln Glu Ile
85 90 95
Ile Asn Ile Met Gln Asp Arg Leu Lys Ala Cys Gln Gln Arg Glu Gly
100 105 110
Gln Asn Tyr Gln Gln Asn Cys Ile Lys Glu Val Glu Gln Phe Thr Gln
115 120 125
Gly Ala Tyr Ser Ser Ala Arg Lys Cys Leu Ala Lys Gln Arg Gln Arg
130 135 140
Met Leu Gln Glu Arg
145
<210> 2
<211> 447
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 2
atgccggaca gctgggacaa ggatgtgtac cctgagcccc cgcgccgcac gccggtgcag 60
cccaatccca tcgtctacat gatgaaagcg ttcgacctca tcgtggaccg acccgtgacc 120
ctcgtgagag aatttataga gcggcagcac gcaaagaaca ggtattacta ctaccaccgg 180
cagtaccgcc gcgtgccaga catcactgag tgcaaggagg aggacatcat gtgcatgtat 240
gaagccgaaa tgcagtggaa gagggactac aaagtcgacc aagaaattat caacattatg 300
caggatcggc tcaaagcctg tcagcagagg gaaggacaga actaccagca gaactgtatc 360
aaggaagtgg agcagttcac ccagggggcc tacagttctg ccaggaagtg cctggccaaa 420
cagaggcaga ggatgctgca agagaga 447
<210> 3
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ttcacccagg 10
<210> 4
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
agggggccta cagtt 15
<210> 5
<211> 12
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
agttcaccca gg 12
<210> 6
<211> 172
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 6
Met Pro Asp Ser Trp Asp Lys Asp Val Tyr Pro Glu Pro Pro Arg Arg
1 5 10 15
Thr Pro Val Gln Pro Asn Pro Ile Val Tyr Met Met Lys Ala Phe Asp
20 25 30
Leu Ile Val Asp Arg Pro Val Thr Leu Val Arg Glu Phe Ile Glu Arg
35 40 45
Gln His Ala Lys Asn Arg Tyr Tyr Tyr Tyr His Arg Gln Tyr Arg Arg
50 55 60
Val Pro Asp Ile Thr Glu Cys Lys Glu Glu Asp Ile Met Cys Met Tyr
65 70 75 80
Glu Ala Glu Met Gln Trp Lys Arg Asp Tyr Lys Val Asp Gln Glu Ile
85 90 95
Ile Asn Ile Met Gln Asp Arg Leu Lys Ala Cys Gln Gln Arg Glu Gly
100 105 110
Gln Asn Tyr Gln Gln Asn Cys Ile Lys Glu Val Glu Gln Phe Thr Gln
115 120 125
Val Ala Lys Ala Tyr Gln Asp Arg Tyr Gln Asp Leu Gly Ala Tyr Ser
130 135 140
Ser Ala Arg Lys Cys Leu Ala Lys Gln Arg Gln Arg Met Leu Gln Glu
145 150 155 160
Arg Lys Ala Ala Lys Glu Ala Ala Ala Ala Thr Ser
165 170
<210> 7
<211> 519
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 7
atgccggaca gctgggacaa ggatgtgtac cctgagcccc cgcgccgcac gccggtgcag 60
cccaatccca tcgtctacat gatgaaagcg ttcgacctca tcgtggaccg acccgtgacc 120
ctcgtgagag aatttataga gcggcagcac gcaaagaaca ggtattacta ctaccaccgg 180
cagtaccgcc gcgtgccaga catcactgag tgcaaggagg aggacatcat gtgcatgtat 240
gaagccgaaa tgcagtggaa gagggactac aaagtcgacc aagaaattat caacattatg 300
caggatcggc tcaaagcctg tcagcagagg gaaggacaga actaccagca gaactgtatc 360
aaggaagtgg agcagttcac ccaggtggcc aaggcctacc aggaccgcta tcaggacctg 420
ggggcctaca gttctgccag gaagtgcctg gccaaacaga ggcagaggat gctgcaagag 480
agaaaagctg caaaagaggc cgccgctgcc acctcctga 519
<210> 8
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
atgccggaca gctgggacaa ggatgtgtac 30
<210> 9
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cctgagcccc cgcgccgcac gccggtgcag 30
<210> 10
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
cccaatccca tcgtctacat gatgaaagcg 30
<210> 11
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ttcgacctca tcgtggaccg acccgtgacc 30
<210> 12
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
ctcgtgagag aatttataga gcggcagcac 30
<210> 13
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gcaaagaaca ggtattacta ctaccaccgg 30
<210> 14
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
cagtaccgcc gcgtgccaga catcactgag 30
<210> 15
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
tgcaaggagg aggacatcat gtgcatgtat 30
<210> 16
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
gaagccgaaa tgcagtggaa gagggactac 30
<210> 17
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
aaagtcgacc aagaaattat caacattatg 30
<210> 18
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
caggatcggc tcaaagcctg tcagcagagg 30
<210> 19
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gaaggacaga actaccagca gaactgtatc 30
<210> 20
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
aaggaagtgg agcagttcac ccag 24
<210> 21
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
tcaggaggtg gcagcggcgg cctcttttgc 30
<210> 22
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
agcttttctc tcttgcagca tcctctgcct 30
<210> 23
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
ctgtttggcc aggcacttcc tggcagaact 30
<210> 24
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
ggcacttcct ggcagaactg taggcccc 28
Claims (9)
1. a kind of sub- compound subunit 10 mutain of 1 β of the nadh dehydrogenase of people [coenzyme], which is characterized in that its amino acid sequence
As shown in SEQ ID NO:1.
2. a kind of encoding gene of the sub- compound subunit 10 mutain of 1 β of the nadh dehydrogenase of people [coenzyme], which is characterized in that its
Nucleotide sequence is as shown in SEQ ID NO:2.
3. a kind of genetic chip characterized by comprising solid phase carrier and fixed nucleotide probe on this carrier；It is described
The nucleotide of the nucleotide probe sub- compound subunit 10 mutain of nadh dehydrogenase [coenzyme] 1 β of people according to claim 2
Sequence is determined with the comparison result of the nucleotide sequence of the sub- compound normal albumen of subunit 10 of 1 β of nadh dehydrogenase [coenzyme] of people.
4. genetic chip according to claim 3, which is characterized in that
The nucleotide probe is base sequence shown in SEQ ID NO:3；
The nucleotide probe is base sequence shown in SEQ ID NO:4；
The nucleotide probe is base sequence shown in SEQ ID NO:5.
5. a kind of reagent, which is characterized in that contain the nadh dehydrogenase for detecting people described in claim 1 in the reagent
The primer pair of the sub- compound subunit 10 mutain of [coenzyme] 1 β.
6. reagent according to claim 5, which is characterized in that the primer pair include following any upstream primers with it is following
The combination of any downstream primer:
Upstream primer is the base sequence as shown in any in SEQ ID NO:8~NO:20；
Downstream primer is the base sequence as shown in any in SEQ ID NO:21~NO:24.
7. a kind of monoclonal antibody of the sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] of specific recognition people,
It is characterized in that, is prepared by reagent such as described in claim 5 or 6, it can be with amino acid sequence shown in SEQ ID NO:1
Specific binding.
8. a kind of ELISA kit of the sub- compound subunit 10 mutain of nadh dehydrogenase [coenzyme] 1 β for detecting people,
It is characterized in that, the ELISA kit includes: the ELISA ELISA Plate for being coated with monoclonal antibody described in claim 7, enzyme mark
Secondary antibody, sample diluting liquid, coating buffer, ELISA ELISA Plate cleaning solution, developing solution and terminate liquid.
9. the sub- compound subunit 10 mutain of 1 β of nadh dehydrogenase [coenzyme] for being used to detect people according to claim 8 is anti-
The ELISA kit of body, which is characterized in that the ELIAS secondary antibody is that the goat of diluted HRP horseradish peroxidase-labeled is anti-
Human IgG.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110396509A (en) * | 2019-07-24 | 2019-11-01 | 厦门大学 | Change the coenzyme activity of glucose dehydrogenase and the method and its application of Preference |
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2018
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110396509A (en) * | 2019-07-24 | 2019-11-01 | 厦门大学 | Change the coenzyme activity of glucose dehydrogenase and the method and its application of Preference |
| CN110396509B (en) * | 2019-07-24 | 2021-02-12 | 厦门大学 | Method for changing coenzyme activity and preference of glucose dehydrogenase and application thereof |
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