CN108148819A - 1 mutain of nadh dehydrogenase subunit of people a kind of and its application - Google Patents
1 mutain of nadh dehydrogenase subunit of people a kind of and its application Download PDFInfo
- Publication number
- CN108148819A CN108148819A CN201711221106.4A CN201711221106A CN108148819A CN 108148819 A CN108148819 A CN 108148819A CN 201711221106 A CN201711221106 A CN 201711221106A CN 108148819 A CN108148819 A CN 108148819A
- Authority
- CN
- China
- Prior art keywords
- nadh dehydrogenase
- people
- elisa
- dehydrogenase subunit
- mutain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
- C12Y106/99—Oxidoreductases acting on NADH or NADPH (1.6) with other acceptors (1.6.99)
- C12Y106/99003—NADH dehydrogenase (1.6.99.3)
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90209—Oxidoreductases (1.) acting on NADH or NADPH (1.6), e.g. those with a heme protein as acceptor (1.6.2) (general), Cytochrome-b5 reductase (1.6.2.2) or NADPH-cytochrome P450 reductase (1.6.2.4)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention relates to 1 mutain of nadh dehydrogenase subunit of people a kind of and its applications, using a large amount of leukaemics as research case, genetic test is carried out to case and is analyzed, determine the mutain of the nadh dehydrogenase subunit 1 of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to 1 mutain of nadh dehydrogenase subunit of the people, the impulse that gene diagnosis for human body leukaemia provides realizes the early diagnosis, early discovery and associated treatment of leukaemia.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to a kind of people 1 mutain of nadh dehydrogenase subunit and
It is applied.
Background technology
Dehydrogenase is again for nicotinamide adenine dinucleotide (Nicotinamide adenine dinucleotide, NADH)
Referred to as nadh dehydrogenase compound, NADH:Ubiquinone reductase or complex I, be it is a kind of be located at mitochondrial inner membrane be catalyzed electronics from
NADH passes to the enzyme of ubiquinone.This enzyme is oxidative phosphorylation in mitochondria " entrance enzyme ".DADH enzymes and leukaemia have
Close relationship, therefore, to the detection that the nadh dehydrogenase subunit 1 of people is mutated, to whether having with leukaemia for human body judged
Certain impulse.
Invention content
Present invention aims at 1 mutains of nadh dehydrogenase subunit and its application for providing a kind of people.
Technical solution of the present invention includes：
In a first aspect, 1 mutain of nadh dehydrogenase subunit 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 1 mutain of nadh dehydrogenase subunit of people, and nucleotide sequence is 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 1 mutain of nadh dehydrogenase subunit of the people, the NADH dehydrogenations with people
The comparison result of the nucleotide sequence of 1 normal albumen of enzyme subunit determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of monoclonal antibody of 1 mutain of nadh dehydrogenase subunit of specific recognition people,
The amino acid sequence of coding is SEQ ID NO:Shown in 1.
5th aspect provides a kind of ELISA reagents of the antibody for 1 mutain of nadh dehydrogenase subunit for being used to detect people
Box, the ELISA kit include：It is coated with the ELISA ELISA Plates of said monoclonal antibody, ELIAS secondary antibody, detects object
1 albumen of nadh dehydrogenase subunit, 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.
It is prominent to provide a kind of nadh dehydrogenase subunit 1 based on any of the above-described ELISA kit detection people for 6th aspect
Become the method for the antibody of albumen, 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, 1 albumen of nadh dehydrogenase subunit for detecting object is diluted to various concentration ladder using the sample diluting liquid
Degree, and 1 albumen of nadh dehydrogenase subunit of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and room temperature is incubated
It educates；
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, adds in the terminate liquid and terminates reaction；
G, 450nm measures OD values in microplate reader.
Preferably, further comprise：Blank control is tested.
The present invention provides 1 mutain of nadh dehydrogenase subunit of people a kind of and its application, by a large amount of leukaemics
As research case, genetic test is carried out to case and is analyzed, determines the mutation egg of the nadh dehydrogenase subunit 1 of people
In vain, genetic chip, monoclonal antibody and ELISA kit are prepared according to 1 mutain of nadh dehydrogenase subunit of the people, is people
The impulse that the gene diagnosis of body leukaemia provides realizes the early diagnosis, early discovery and associated treatment of leukaemia.
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.
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 volume of 1 mutain of nadh dehydrogenase subunit of people
Code gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the NADH dehydrogenations of people are determined according to the encoding gene
1 mutain of enzyme subunit, amino acid sequence such as SEQ IDNO:Shown in 1.
Secondly, according to 1 mutain of nadh dehydrogenase subunit and its encoding gene of people, gene is realized as follows
The preparation of chip.
1st, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the nucleotide of 1 mutain of nadh dehydrogenase subunit of people
Sequence determines, and with the comparison result of the nucleotide sequence of the 1 normal albumen of nadh dehydrogenase subunit of people according to following probe
Design principle, design for people 1 mutain of nadh dehydrogenase subunit specificity nucleotide probe.
Wherein, the principle of nucleotide probe design is as follows：
1. nucleotide probe Tm values should be close to the average Tm values of whole gene group, 5 DEG C of fluctuation up and down；
2. the single base that nucleotide probe intramolecular repeats continuously is no more than 4；
3. G+C contents are 40%-60%, the specificity that non-specific hybridization ensures hybridization is reduced；
4. 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 1 mutain of nadh dehydrogenase subunit of people and the nadh dehydrogenase subunit 1 of people
The comparison result of the corresponding amino acid sequence of normal albumen please refers to Fig.1, wherein, the Query sequences in Fig. 1 are that the NADH of people takes off
The corresponding amino acid sequence of 1 mutain of hydrogen enzyme subunit, Sbjct sequences are that the 1 normal albumen of nadh dehydrogenase subunit of people corresponds to
Amino acid sequence, sequence between Query sequences and Sbjct sequences is comparison result, and as can be seen from FIG. 1, the NADH of people takes off
1 mutain of hydrogen enzyme subunit is mutated at some positions relative to the 1 normal albumen of nadh dehydrogenase subunit of people,
It is lacked at some positions, is inserted at some positions.According to SEQ ID NO:Nucleotide sequence shown in 2,
And the comparison result of Fig. 1, in order to specific recognition object to be detected nadh dehydrogenase subunit 1 whether have occurred it is prominent
Become, then when choosing nucleotide probe, nucleotide probe can be designed according to any one of following several ways mode：
(1) whole nucleotides sequence column-generation nucleotide probe of insertion position is chosen；
(2) before insertion position and/or, after insertion position, respectively select several nucleotide sequences, the selection
Several sequences (sequence of base sequence position is constant) be inserted into whole nucleotide sequence generate nucleotide probe jointly；
(3) several nucleotide sequences are selected before insertion position, if with being selected at the starting position of insertion position
Dry nucleotide sequence, generates nucleotide probe；
(4) several nucleotide sequences are selected after insertion position, if with being selected at the end position of insertion position
Dry nucleotide sequence, generates nucleotide probe；
(5) several nucleotide sequences are selected before deletion sites, with selecting several nucleosides after deletion sites
Acid sequence generates nucleotide probe；
(6) nucleotide sequence of mutated site nucleotide will be included 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, in the manner described above (6) design one kind preferably nucleotide probe such as SEQ ID NO:Shown in 3, the nucleotide probe
For：atacccatgg cc
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2nd, the preparation of genetic chip whether the nadh dehydrogenase subunit 1 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, is taken off in the NADH of people
In 1 mutain of hydrogen enzyme subunit, select sequence corresponding from nucleotide probe different, the nucleosides that positive internal control Quality Control probe includes
Sour digit and the digit of nucleotide probe may be the same or different.Preferably, nucleotide digit and nucleotide probe are chosen
The identical positive internal control Quality Control probe of digit.In the embodiment of the present invention, the positive internal control probe sequence needed for genetic chip can
Think：actcctcatt gt.
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, 12000r/
Min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(4) isometric isopropanol is added in, the abundant mixing liquid of centrifuge tube is gently overturned, is placed at room temperature for 10min, 12000r/
Min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(5) one time, 7500r/min, 4 DEG C centrifugation 15min is washed with 1mL75% ethyl alcohol, all supernatants is carefully sucked, ultra-clean
Dry 15min in platform adds 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 QIAGENKit purifies 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 |
| T7Promotor 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：
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
| 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 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：
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 in scanner is scanned, 1 egg of nadh dehydrogenase subunit of detection object is determined according to scanning result
Whether it is mutated in vain.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, positive control is
Green fluorescence, the sample quality for showing acquisition testing object are that there is no problem, and experimental group is green fluorescence, then show to examine
1 albumen of nadh dehydrogenase subunit for surveying object is mutated.In result shown in Fig. 4, negative control is colorless fluorescent, positive
It compares as green fluorescence, the sample quality for showing acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, then
1 albumen of nadh dehydrogenase subunit for showing to detect object does not mutate.
Embodiment three, specific recognition people 1 mutain of nadh dehydrogenase subunit monoclonal antibody preparation
1st, according to base sequence (such as SEQ ID NO of 1 mutain of nadh dehydrogenase subunit of people:Shown in 2) in design
Swim primer such as SEQ ID NO:Shown in 4 and, downstream primer such as SEQ ID NO:Object shown in 5：
Sense primer (P)：atacccatgg ccaac
Downstream primer (F)：gtttgatgct caccct
2nd, the DNA of detection object carries out PCR amplification for template
PCR amplification is carried out as template using the DNA for detecting object, obtains 1 mutating protein gene of nadh dehydrogenase subunit of people
Complete segment, and pMD19-T Vector (Takara companies) are connected, it is sequenced.Then it is prepared by special biotech firm anti-
Body 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 1 mutain of nadh dehydrogenase subunit 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, 1 albumen of nadh dehydrogenase subunit for detecting 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, using leukaemic as detection object, and the white blood is detected using ELISA kit
Whether 1 albumen of nadh dehydrogenase subunit of patient is mutated, and this method can at least include following a kind of：
A, monoclonal antibody prepared by embodiment three is diluted, such as is diluted to using the coating buffer solution
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, 1 albumen of nadh dehydrogenase subunit of leukaemic is diluted to various concentration gradient using sample diluting liquid,
And 1 albumen of nadh dehydrogenase subunit of various 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, 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=0.99924, therefore, this measures effective.It will detect
To OD450 values bring the concentration that standard curve can acquire 1 albumen of nadh dehydrogenase subunit of leukaemic into.Utilize foundation
ELISA method detection serum of leukaemia sample in 1 albumen of nadh dehydrogenase subunit content.And with Western blot
It is identified.Fig. 5 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
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 1 mutain monoclonal antibody of nadh dehydrogenase subunit of the standby people of corporation, and secondary antibody is horseradish
The Goat anti-Human IgG of peroxidase labelling.
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 32KD
There is apparent band, wherein, the molecular weight of 1 mutain of nadh dehydrogenase subunit is about 32KD, shows the leukaemic
1 albumen of nadh dehydrogenase subunit 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>1 mutain of nadh dehydrogenase subunit of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 117
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Ile Pro Met Ala Asn Leu Leu Leu Leu Ile Val Pro Ile Leu Ile Ala
1 5 10 15
Met Ala Phe Leu Met Leu Thr Glu Arg Lys Ile Leu Gly Tyr Ile Gln
20 25 30
Leu Arg Lys Gly Pro Thr Leu Ala Leu Arg Ala Thr Thr Thr Leu Arg
35 40 45
Arg His Lys Thr Leu His Gln Arg Ala Leu Lys Pro Ala Thr Ser Thr
50 55 60
Ile Thr Leu Tyr Ile Thr Ala Pro Thr Leu Ala Leu Thr Ile Ala Leu
65 70 75 80
Leu Leu Thr Pro Ser His Thr Gln Pro Leu Val Asn Leu Asn Leu Gly
85 90 95
Leu Leu Phe Ile Leu Ala Thr Ser Ser Leu Ala Val Tyr Ser Ile Leu
100 105 110
Ser Gly Ala Ser Asn
115
<210> 2
<211> 366
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atacccatgg ccaacctcct actcctcatt gtacccattc taatcgcaat ggcattccta 60
atgcttaccg aacgaaaaat tctaggctat atacaactac gcaaaggccc aacgttgtag 120
gccctacggg ctactacaac ccttcgctga cgccataaaa ctcttcacca aagagcccta 180
aaacccgcca catctaccat caccctctac atcaccgccc cgaccttagc tctcaccatc 240
gctcttctac tatgaacccc ctcccatacc caacccctgg tcaacctcaa cctaggcctc 300
ctatttattc tagccacctc tagcctagcc gtttactcaa tcctctgatc agggtgagca 360
tcaaac 366
<210> 3
<211> 12
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
atacccatgg cc 12
<210> 4
<211> 15
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atacccatgg ccaac 15
<210> 5
<211> 16
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gtttgatgct caccct 16
Claims (9)
- A kind of 1. 1 mutain of nadh dehydrogenase subunit of people, which is characterized in that its amino acid sequence such as SEQ ID NO:1 institute Show.
- A kind of 2. encoding gene of 1 mutain of nadh dehydrogenase subunit 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 according to the nucleotide sequence of 1 mutain of nadh dehydrogenase subunit of the people, the nadh dehydrogenase Asia with people The comparison result of the nucleotide sequence of 1 normal albumen of base 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. the monoclonal antibody of 1 mutain of nadh dehydrogenase subunit of a kind of specific recognition people, which is characterized in that it is encoded Amino acid sequence be SEQ ID NO:Shown in 1.
- 6. a kind of ELISA kit of the antibody for 1 mutain of nadh dehydrogenase subunit 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, detection of monoclonal antibody described in claim 5 1 albumen of nadh dehydrogenase subunit, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and the end of object Only liquid.
- 7. it is used to detect the ELISA reagents of the antibody of 1 mutain of nadh dehydrogenase subunit of people according to claim 6 Box, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
- 8. a kind of 1 mutain of nadh dehydrogenase subunit based on the ELISA kit detection people of claim 6 or 7 is anti- The method of body, which is characterized in that including：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, 1 albumen of nadh dehydrogenase subunit for detecting object is diluted to various concentration gradient using the sample diluting liquid, and 1 albumen of nadh dehydrogenase subunit of various 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, adds in the terminate liquid and terminates reaction；G, 450nm measures OD values in microplate reader.
- 9. the side of the antibody of 1 mutain of nadh dehydrogenase subunit of ELISA kit detection people according to claim 8 Method, which is characterized in that further comprise：Blank control is tested.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711221106.4A CN108148819A (en) | 2017-11-29 | 2017-11-29 | 1 mutain of nadh dehydrogenase subunit of people a kind of and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711221106.4A CN108148819A (en) | 2017-11-29 | 2017-11-29 | 1 mutain of nadh dehydrogenase subunit of people a kind of and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108148819A true CN108148819A (en) | 2018-06-12 |
Family
ID=62469030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711221106.4A Pending CN108148819A (en) | 2017-11-29 | 2017-11-29 | 1 mutain of nadh dehydrogenase subunit of people a kind of and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108148819A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1352066A (en) * | 2000-11-02 | 2002-06-05 | 上海博德基因开发有限公司 | New polypeptide-human respiratory china NADH dehydrogenase subunit 8.91 and polynucleotide for encoding such polypeptide |
| 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 |
-
2017
- 2017-11-29 CN CN201711221106.4A patent/CN108148819A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1352066A (en) * | 2000-11-02 | 2002-06-05 | 上海博德基因开发有限公司 | New polypeptide-human respiratory china NADH dehydrogenase subunit 8.91 and polynucleotide for encoding such polypeptide |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 罗烈伟 等: ""白血病线粒体DNA突变的研究"", 《广东药学院学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108676785A (en) | A kind of ATP dependent forms RNA helicase DHX3 mutains and application | |
| CN108559741A (en) | The imidazolone propionase mutain of people a kind of and its application | |
| CN108192880A (en) | A kind of δ of people-aminolevulinic acid synzyme mutain and its application | |
| CN109251910A (en) | It is assumed that ATP dependent form RNA helicase ROK1 mutain and application | |
| CN108424447A (en) | 1 mutain of cytochrome b-c1 complex subunits of people a kind of and application | |
| CN110066772A (en) | 2 '-the 5 '-oligoadenylate synthetase mutains of people a kind of and its application | |
| CN108424888A (en) | 1 mutain of serine palmitoyltransferase of people a kind of and its application | |
| CN108546689A (en) | A kind of 1 mutain of vitamin K epoxy reductase complex subunit and application | |
| CN107828748B (en) | The NDUFA13 protein mutations albumen of people a kind of and its application | |
| CN108148819A (en) | 1 mutain of nadh dehydrogenase subunit of people a kind of and its application | |
| CN108152497A (en) | The atp synthase mutain of people a kind of and its application | |
| CN108059664A (en) | The ornithine decarboxylase antizyme mutain of people a kind of and its application | |
| CN108410828A (en) | 1 mutain of dehydrogenase SDR family members of people a kind of and its application | |
| CN108103035A (en) | Cytochrome b-c1 complexs the mutain of people a kind of and its application | |
| CN108085305A (en) | The 5-ALA mutant synthase albumen of people a kind of and its application | |
| CN108048422A (en) | A kind of GTP enzymes, 7 mutain of IMAP family members and its application | |
| CN108300708A (en) | A kind of compound subunit mutain of the cytochrome b-c1 of people and its application | |
| CN108359648A (en) | The nadh dehydrogenase mutain of people a kind of and its application | |
| CN108061805A (en) | The guanosine kinase b mutains of people a kind of and its application | |
| CN108977417A (en) | The Iduronate-2-sulfatase mutain of people a kind of and its application | |
| CN108037292A (en) | The phosphatidylserine receptor mutain of people a kind of and its application | |
| CN108084254A (en) | The antioncogene WWOX δ 6-8 mutains of people a kind of and its application | |
| CN109836485A (en) | A kind of mutain of the gamma-secretase activator protein of people and its application | |
| CN108148820A (en) | The glutathione S transferase M1 c mutains of people a kind of and its application | |
| CN108101977A (en) | The ACP protein mutations albumen of people a kind of and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180612 |
|
| WD01 | Invention patent application deemed withdrawn after publication |