CN108424447A - 1 mutain of cytochrome b-c1 complex subunits of people a kind of and application - Google Patents
1 mutain of cytochrome b-c1 complex subunits of people a kind of and application Download PDFInfo
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Abstract
The present invention relates to 1 mutain of cytochrome b c1 complex subunits of people a kind of and applications, by regarding a large amount of lung cancer and leukaemic as research case, genetic test is carried out to case and is analyzed, determine 1 mutain of cytochrome b c1 complex subunits of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to 1 mutain of cytochrome b c1 complex subunits of the people, gene diagnosis for lung cancer and leukaemia provides impulse, to realize that the diagnosing and treating of cancer provides theoretical foundation.
Description
Technical field
It is prominent that the present invention relates to the cytochrome b-c1 complex subunits 1 of a kind of genetic engineering field more particularly to a kind of people
Kink of preserved egg bletilla application.
Background technology
Cytochrome b-c1 compounds (cytochrome b-c1 complex), contain cytochrome b, cytochrome c 1
With a kind of moveable iron-sulfur protein (Rieske protein).Rieske iron-sulfur proteins are 1 iron atom and two His residues
It is connected.All iron-sulfur protein participates in the transfer of an electronics, iron atom therein or is aoxidized or is reduced, in mitochondria
At least 8 iron-sulfur proteins participate in electron transmission.
During the peripheral blood to certain lung cancer and leukaemic carries out gene sequencing, the cell of patient is found
Pigment b-c1 complex subunits 1 are mutated, therefore, the detection pair that the cytochrome b-c1 complex subunits 1 of people are mutated
Judge whether human body suffers from cancer and have certain impulse.
Invention content
Present invention aims at 1 mutain of cytochrome b-c1 complex subunits of people of offer a kind of and applications.
Technical solution of the present invention includes：
In a first aspect, providing a kind of 1 mutain of cytochrome b-c1 complex subunits of people, amino acid sequence is such as
SEQ ID NO:Shown in 1.
Second aspect provides a kind of encoding gene of 1 mutain of cytochrome b-c1 complex subunits of people, nucleosides
Acid 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 according to the nucleotide sequence of 1 mutain of cytochrome b-c1 complex subunits of people described above, with
The comparison result of the nucleotide sequence of the 1 normal albumen of cytochrome b-c1 complex subunits of people determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of Dan Ke of 1 mutain of cytochrome b-c1 complex subunits of specific recognition people
Grand antibody, can be with SEQ ID NO:Amino acid sequencespecific shown in 1 combines.
5th aspect provides a kind of for detecting the antibody of 1 mutain of cytochrome b-c1 complex subunits of people
ELISA kit, the ELISA kit include：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, inspection of said monoclonal antibody
Survey 1 albumen of cytochrome b-c1 complex subunits of object, 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.
The present invention provides 1 mutain of cytochrome b-c1 complex subunits of people a kind of and applications, by a large amount of lung cancer
With leukaemic as research case, genetic test is carried out to case and is analyzed, determines that the cytochrome b-c1 of people is compound
The mutain of object subunit 1 prepares genetic chip, Dan Ke according to 1 mutain of cytochrome b-c1 complex subunits of the people
Grand antibody and ELISA kit, the gene diagnosis for lung cancer and leukaemia provide impulse, to realize the diagnosis of cancer and controlling
It treats and theoretical foundation is provided.
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention and after coordinating attached drawing to be described in detail such as.
Description of the drawings
Fig. 1 is a kind of comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 2 is a kind of genetic chip layout that the embodiment of the present invention one provides；
Fig. 3 is a kind of colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 4 is another colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 5 is the standard curve that the offer of the embodiment of the present invention five is protein content；
Fig. 6 is the Westernblot testing result schematic diagrames that the embodiment of the present invention five provides；
Fig. 7 is the standard curve that the offer of the embodiment of the present invention six is protein content；
Fig. 8 is the Westernblot testing result schematic diagrames that the embodiment of the present invention six provides.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment one, genetic chip
It is possible, firstly, to be a kind of " detection side of mutain according to application No. is " 201710429915.8 ", patent name
Detection method described in the Chinese invention patent of method and device " determines that the cytochrome b-c1 complex subunits 1 of people are mutated
The encoding gene of albumen, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, determine people's according to the encoding gene
1 mutain of cytochrome b-c1 complex subunits, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to 1 mutain of cytochrome b-c1 complex subunits and its encoding gene of people, as follows
Realize the preparation of genetic chip.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to 1 mutain of cytochrome b-c1 complex subunits of people
Nucleotide sequence, come with the comparison result of the nucleotide sequence of the 1 normal albumen of cytochrome b-c1 complex subunits of people true
It is fixed, and according to the design principle of following probe, design the spy of 1 mutain of cytochrome b-c1 complex subunits for people
Anisotropic 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. it should be less than 4bp with the bases longs of probe molecule internal stability secondary structure pairing in nucleotide probe sequences,
Can ensure that hybridization efficiency will not be influenced because of the secondary structure of nucleotide probe internal stability in this way；
5. the similitude through Homology search and other sequences is less than 40%；
6. being continuously no more than 20 bases with the homologous fragment of non-probe alternative sequence by contrast.
Wherein, the cell color of 1 mutain of cytochrome b-c1 the complex subunits corresponding amino acid sequence and people of people
The comparison result of the corresponding amino acid sequence of 1 normal albumen of plain b-c1 complex subunits please refers to Fig.1, wherein in Fig. 1
Query sequences are the corresponding amino acid sequences of 1 mutain of cytochrome b-c1 complex subunits of people, and Sbjct sequences are people
The corresponding amino acid sequence of 1 normal albumen of cytochrome b-c1 complex subunits, between Query sequences and Sbjct sequences
Sequence is comparison result, as can be seen from FIG. 1, the cell of 1 mutain of cytochrome b-c1 complex subunits of people relative to people
1 normal albumen of pigment b-c1 complex subunits, amino acid sequence is lacked at one.According to SEQ ID NO:Core shown in 2
The comparison result of nucleotide sequence and Fig. 1, it is sub- in order to the cytochrome b-c1 compounds of specific recognition object to be detected
Whether base 1 is mutated, then when choosing nucleotide probe, it can be each before deletion sites and after deletion sites
Several nucleotide sequences (sequence of base sequence is constant) are selected, nucleotide probe is generated.
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, designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, which is：
tcggtgggag tgtggatt。
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether cytochrome b-c1 complex subunits 1 of detection people mutate
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.
Point sample needed for negative internal reference Quality Control probe and positive control for point sample needed for blank control, negative control
Positive internal control Quality Control probe design it is as follows：
Blank control：The blank sampling liquid for being free from any genetic fragment refers to as the contamination monitoring in chip fabrication process
Mark.
Negative internal reference Quality Control probe：Be one section does not have other genetic fragments of homology with detection gene, as hybridizing
The monitor control index of non-specific hybridization in journey, the nucleotide number that negative internal reference Quality Control probe includes can be with nucleotide probes
Base number is identical, can also be different.In the embodiment of the present invention, the negative internal reference probe sequence needed for genetic chip can be：
tgatgctgat aattgcat。
Positive internal control Quality Control probe：It is one section of other genetic fragment for having homology with detection gene, in the cell color of people
In plain 1 mutain of b-c1 complex subunits, select sequence corresponding from nucleotide probe different, and can be with nucleotide probe
The number of base is identical, and one section of nucleotide sequence that can also be different from the number of nucleotide probe base is as positive internal control matter
Control probe.Preferably, nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen.This hair
In bright embodiment, the positive internal control probe sequence needed for genetic chip can be：agcagtggct ggatatgg.
It should be noted that in the deposition process of genetic chip, clicks and enters negative internal reference according to the layout of genetic chip and visit
Needle and positive internal control probe solution；The spotting buffer (10% aqueous trehalose) that reagent used in blank control is 1 times.
The application of embodiment two, genetic chip
1, sample treatment
(1) the blood 1-3mL of acquisition testing object.
(2) the 1.5mL EP pipes that DEPC is handled are taken, as detected sample processing tube, in detected sample processing tube
The middle 300 μ L of blood that detection object is added, add 700 μ L of Trizol, mix well, be placed at room temperature for 10min.
(3) chloroform of 140 μ L is added, covers tightly pipe lid, firmly shakes, be placed in a centrifuge, 12000r/min, 4 DEG C of centrifugations
15min, it is careful to draw supernatant in centrifuge tube, the supernatant of absorption is transferred in clean centrifuge tube.
(4) isometric isopropanol is added in the centrifuge tube of supernatant in storing step (3), it is abundant gently overturns centrifuge tube
Mixing liquid is placed at room temperature for 10min, 12000r/min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(5) it being cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min, 4 DEG C of centrifugation 15min carefully suck all supernatants,
The dry 15min in super-clean bench is added 10 μ L DEPC and handles water dissolution.
(6) products therefrom is RNA can influence the labeling effciency and chip hybridization knot of probe if the purity of total serum IgE is not high
Fruit.So using QIAGENKit purifies total serum IgE.
2, the first chains of cDNA and the second chain one-step synthesis method
(1) 2 μ g RNA is taken to configure following reaction solution in the centrifuge tube of 1.5mL：
| Total serum IgE | The most 6.5 μ L of 2 μ g |
| T7 Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
Wherein, the addition X μ L of RNase-free Water are to subtract T7 Promotor according to 11.5 μ L of total volume
The 5 μ L of addition of primer, then subtract the addition of total serum IgE and calculate and get.
(2) 10min, and ice bath 5min are kept the temperature at 65 DEG C, in advance by 5 × First Strand B μ ffer at 65 DEG C
Preheat 5min.
(3) following cDNA synthetic systems are configured：
(4) above-mentioned 8.5 μ L are added after mixing after being denaturalized in the RNA of ice bath.
(5) pipette tips mixing is used to centrifuge later.
(6) 40 DEG C of reaction 2h.
3, aaUTP marks cRNA synthesis
The configuration of NTP：
| 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 (polyethylene glycol) use.Simultaneously by following behaviour
Make 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 |
| T7 RNA Polymerase | 0.8μL |
| Total volume | 60μL |
(2) 60 μ L Transcription mix and mixing is added.
(3) 60 DEG C of hot lid in PCR instrument, 40 DEG C of reaction 2h.
4, cRNA is purified
QIAGEN RNeasy Mini kit purify cRNA, and specific method can be found in what QIAGEN companies provided with kit
Operation manual.
(1) 20 μ L RNase free water are added, 350 μ L Buffer RLT are added and mix well.
(2) 250 μ L absolute ethyl alcohols are added, Tip mix well.
(3) total 700 solution of the μ L containing total serum IgE are transferred in the RNeasy pillars being sleeved in 2mL centrifuge tubes >=8000g from
Heart 15-30s, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=8000g centrifuge washings 15-30s discards filter
It crosses liquid and discards the casing of filtered solution and 2mL by RNeasy in >=8000g centrifuge washings 2min with 500 μ L Buffer RPE again
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).
5, cRNA concentration mensurations
With spectrophotometric analysis cRNA concentration.It needs to measure the light absorption value at 260nm and 280nm to determine the dense of sample
Degree and purity, A260/A280 should be purer cRNA (ratio 1.9-2.1 also can) close to 2.0.
6, cRNA fluorescents mark；
(1) 4 μ g of above-mentioned cRNA are taken and are concentrated into 6.6 μ L.
(2) add 10 μ L DMSO mixings.
(3) add the sodium bicarbonate (NaHCO that the 0.3M pH of 3.4 μ L are 9.03) and mixing.
(4) above-mentioned 20 μ L cRNA mixtures are added in fluorescent dye Cy3 simultaneously mixing.25 DEG C of heat preservation 1h.
(5) plus 9 μ L 4M Hydroxylamine mixings after 25 DEG C heat preservation 15min.
7, fluorescent marker cRNA Sample Purification on Single
The process that specific steps are purified with cRNA in the step 4 of the present embodiment, this step are not repeating.
8, cRNA sample fragments and 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.
(2) 55 μ L 2 × GEx Hybridization Buffer are added.
(3) 100 μ L hybridization solutions is taken to be added drop-wise on chip ware, at the same be added dropwise respectively as shown in Figure 2 by chip layout blank,
On negative, positive, experimental group position.It covers chip and is sealed in hybridizing box, 60 DEG C roll hybridization 16h.
9, chip washs
Washing lotion 1 (1L) configures：
| DEPC-H2O | 700mL |
| 20×SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 2 (1L) configures：
| DEPC-H2O | 997mL |
| 20×SSPE | 3.0mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 3：Stabilization and Drying Solution
(1) it takes out chip and washs 1min in washing lotion 1；
(2) chip is put into washing lotion 2 again and washs 1min (37 DEG C)；
(3) chip is finally washed into 30s in washing lotion 3.
10, chip scanning
Chip is scanned in scanner, the cytochrome b-c1 compounds of detection object are determined according to scanning result
Whether 1 albumen of subunit is mutated.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, sun
Property control be green fluorescence, show that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence, that
Show that 1 albumen of cytochrome b-c1 complex subunits for detecting object is mutated.It is negative right in result shown in Fig. 4
According to for colorless fluorescent, positive control is green fluorescence, shows that the sample quality of acquisition testing object is that there is no problem, and tests
Group redgreen fluorescence, then showing that 1 albumen of cytochrome b-c1 complex subunits for detecting object does not mutate.
Embodiment three, specific recognition people cytochrome b-c1 complex subunits 1 mutains monoclonal antibody
It prepares
1, according to base sequence (such as SEQ ID NO of 1 mutain of cytochrome b-c1 complex subunits of people:2 institutes
Show) design sense primer such as SEQ ID NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (F)：atggcggcgt ccgtggtct
Downstream primer (R)：gaagcgcagc cagaacatg
2, the DNA of detection object is that template carries out PCR amplification
| 10×Buffer | 5uL |
| dNTP | 2uL |
| ExTaq | 1uL |
| ddH2O | 5uL |
| Template DNA | 1uL |
| Primer (F) | 3uL |
| Primer (R) | 3uL |
| Total system | 20uL |
DNA to detect object carries out PCR amplification as template, and the cytochrome b-c1 complex subunits 1 for obtaining people are mutated
The complete segment of protein gene, and pMD19-T Vector (Takara companies) are connected, it is sequenced.Then public by special biology
Department prepares antibody, is a kind of humanization or Chimeric antibodies.Wherein, the monoclonal antibody prepared can be with SEQ ID NO:1 institute
The amino acid sequencespecific shown combines.The antibody of preparation is measured into content using ELISA method.
Example IV, 1 mutain of cytochrome b-c1 complex subunits for detecting people antibody ELISA reagents
Box
In the present embodiment, the group of ELISA kit becomes：It is coated with the ELISA enzymes of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, detect 1 albumen of cytochrome b-c1 complex subunits 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 benzidines；
Terminate liquid can be the sulfuric acid solution of 2mol/L.
Embodiment five
In embodiments of the present invention, it using patients with lung cancer as detection object, and detects the lung cancer using ELISA kit and suffers from
Whether 1 albumen of cytochrome b-c1 complex subunits of person is mutated, and this method at least may include following one kind：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h.
B, 1 albumen of cytochrome b-c1 complex subunits of patients with lung cancer is diluted to various concentration using sample diluting liquid
Gradient, and 1 albumen of cytochrome b-c1 complex subunits of various concentration gradient is loaded respectively to the hole of ELISA ELISA Plates
In, and it is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions are added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated 10-20min, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
H, standard curve is made：Using standard concentration as abscissa, the light absorption value that standard items measure is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measures effective when ＞ 0.99；Wherein it is possible to use ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc softwares2=0.99899, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire the cell of patients with lung cancer
The concentration of 1 albumen of pigment b-c1 complex subunits.Serum of Patients with Lung Cancer cells in sample color is detected using the ELISA method of foundation
The content of plain 1 albumen of b-c1 complex subunits.It is used in combination Western blot to be identified.Referring to FIG. 5, for the standard of light absorption value
Curve.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot are 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)
| 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 layer's separation gel of respective volume is added in each offset plate, adds the sterile ddH of 1mL2O flattens separation
Glue blots remaining moisture in plastic plate after gelling to be separated is solid with filter paper, and upper layer is then added and concentrates glue, after being inserted into comb
Wait for upper layer concentration gelling solid.
2, albumen loading electrophoresis：
(1) comb is pulled up, duct label is carried out, 5 × SDS electrophoretic buffers is added, then to being added in protein sample
SDS-PAGE albumen sample-loading buffer (5 ×), boiling water bath heat 3-5min, loading；
(2) electrophoresis first is carried out with 80V voltages, after waiting for that band ran concentration glue, uses 100V voltages instead and run about 100min.
3, transferring film and incubation：
(1) first pvdf membrane is activated in methyl alcohol about 30s.Respectively by gel, foam-rubber cushion and pvdf membrane in advance in electrotransfer
30min is balanced in buffer solution.It is clipped by the sequence of filter paper-glue-film-filter paper, transferring film device is put into the principle of black flour according to black flour
In, refrigerator (ice bag) is added, about 120min is run with the electric current of 150mA in ice；
(2) it takes the film out after the completion of, is put into 5% skim milk at once, gently room temperature closes 1h on shaking table；
(3) PBST cleans closed film, cleans 3 times, each 10min；
(4) primary antibody for being diluted to corresponding multiple is added, 4 DEG C of incubator overnights are incubated；
(5) primary antibody is recycled, PBST cleans film 3 times, each 10min；
(6) secondary antibody is (with 3% milk with 1:5000-1:10000 dilutions) incubation at room temperature film 2h；
(7) film is cleaned 3 times with PBST, each 10min；
(8) after the isometric mixing of Pierce ECL Western Blotting Substrate kit A, B liquid, dropwise
It is added dropwise on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment Image J analyses.
Wherein, primary antibody is the 1 mutain monoclonal antibody of cytochrome b-c1 complex subunits of the standby people of corporation, two
Anti- is the Goat anti-Human IgG of horseradish peroxidase-labeled.
J, Western blot Analysis of test results：It is shown according to Western blot experimental procedures as a result, such as Fig. 6 institutes
Show, wherein the Marker in Fig. 6 is used to characterize the size of labelled protein.It is only attached in experimental group 106KD compared with blank control
Closely there is apparent band, wherein 1 mutant protein molecules amount of cytochrome b-c1 complex subunits is about 106KD, shows the lung
1 albumen of cytochrome b-c1 complex subunits of cancer patient is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, using leukaemic as detection object, and the white blood is detected using ELISA kit
Whether 1 albumen of cytochrome b-c1 complex subunits of patient is mutated, and this method at least may include following one kind：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h；
B, 1 albumen of cytochrome b-c1 complex subunits for detecting object is diluted to various concentration using sample diluting liquid
Gradient, and 1 albumen of cytochrome b-c1 complex subunits of various concentration gradient is loaded respectively to the hole of ELISA ELISA Plates
In, and it is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions are added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated 10-20min, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
H, standard curve is made：Using standard concentration as abscissa, the light absorption value that standard items measure is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measures effective when ＞ 0.99；Wherein it is possible to use ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc softwares2=0.99684, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire leukaemia trouble in sample
The concentration of 1 albumen of cytochrome b-c1 complex subunits of person.Serum of leukaemia sample is detected using the ELISA method of foundation
The content of 1 albumen of cytochrome b-c1 complex subunits in product.It is used in combination Western blot to be identified.Referring to FIG. 7, to inhale
The standard curve of light value.Wherein, X-axis is light absorption value, 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 as in embodiment five, and details are not described herein, referring to FIG. 8, in Fig. 8
Marker is used to characterize the size of labelled protein.The experimental display result of the present embodiment, compared with blank control, only in experimental group
Nearby there is apparent band in 106KD, wherein and 1 mutant protein molecules amount of cytochrome b-c1 complex subunits is about 106KD,
Show that 1 albumen of cytochrome b-c1 complex subunits of the leukaemic is implicitly present in mutation.
The above is only a preferred embodiment of the present invention, it is not intended to restrict the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Sequence table
<110>Tianjin lakeside Pan Gu's genetic science Development Co., Ltd
<120>1 mutain of cytochrome b-c1 complex subunits of people a kind of and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 433
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Ala Ala Ser Val Val Cys Arg Ala Ala Thr Ala Gly Ala Gln Val
1 5 10 15
Leu Leu Arg Ala Arg Arg Ser Val Gly Val Trp Ile Asp Val Gly Ser
20 25 30
Arg Phe Glu Thr Glu Lys Asn Asn Gly Ala Gly Tyr Phe Leu Glu His
35 40 45
Leu Ala Phe Lys Gly Thr Lys Asn Arg Pro Gly Ser Ala Leu Glu Lys
50 55 60
Glu Val Glu Ser Met Gly Ala His Leu Asn Ala Tyr Ser Thr Arg Glu
65 70 75 80
His Thr Ala Tyr Tyr Ile Lys Ala Leu Ser Lys Asp Leu Pro Lys Ala
85 90 95
Val Glu Leu Leu Gly Asp Ile Val Gln Asn Cys Ser Leu Glu Asp Ser
100 105 110
Gln Ile Glu Lys Glu Arg Asp Val Ile Leu Arg Glu Met Gln Glu Asn
115 120 125
Asp Ala Ser Met Arg Asp Val Val Phe Asn Tyr Leu His Ala Thr Ala
130 135 140
Phe Gln Gly Thr Pro Leu Ala Gln Ala Val Glu Gly Pro Ser Glu Asn
145 150 155 160
Val Arg Lys Leu Ser Arg Ala Asp Leu Thr Glu Tyr Leu Ser Thr His
165 170 175
Tyr Lys Ala Pro Arg Met Val Leu Ala Ala Ala Gly Gly Val Glu His
180 185 190
Gln Gln Leu Leu Asp Leu Ala Gln Lys His Leu Gly Gly Ile Pro Trp
195 200 205
Thr Tyr Ala Glu Asp Ala Val Pro Thr Leu Thr Pro Cys Arg Phe Thr
210 215 220
Gly Ser Glu Ile Arg His Arg Asp Asp Ala Leu Pro Phe Ala His Val
225 230 235 240
Ala Ile Ala Val Glu Gly Pro Gly Trp Ala Ser Pro Asp Asn Val Ala
245 250 255
Leu Gln Val Ala Asn Ala Ile Ile Gly His Tyr Asp Cys Thr Tyr Gly
260 265 270
Gly Gly Val His Leu Ser Ser Pro Leu Ala Ser Gly Ala Val Ala Asn
275 280 285
Lys Leu Cys Gln Ser Phe Gln Thr Phe Ser Ile Cys Tyr Ala Glu Thr
290 295 300
Gly Leu Leu Gly Ala His Phe Val Cys Asp Arg Met Lys Ile Asp Asp
305 310 315 320
Met Met Phe Val Leu Gln Gly Gln Trp Met Arg Leu Cys Thr Ser Ala
325 330 335
Thr Glu Ser Glu Val Ala Arg Gly Lys Asn Ile Leu Arg Asn Ala Leu
340 345 350
Val Ser His Leu Asp Gly Thr Thr Pro Val Cys Glu Asp Ile Gly Arg
355 360 365
Ser Leu Leu Thr Tyr Gly Arg Arg Ile Pro Leu Ala Glu Trp Glu Ser
370 375 380
Arg Ile Ala Glu Val Asp Ala Ser Val Val Arg Glu Ile Cys Ser Lys
385 390 395 400
Tyr Ile Tyr Asp Gln Cys Pro Ala Val Ala Gly Tyr Gly Pro Ile Glu
405 410 415
Gln Leu Pro Asp Tyr Asn Arg Ile Arg Ser Gly Met Phe Trp Leu Arg
420 425 430
Phe
<210> 2
<211> 1299
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atggcggcgt ccgtggtctg tcgggccgct accgccgggg cacaagtgct attgcgcgcc 60
cgccgctcgg tgggagtgtg gattgatgtt ggcagccgtt ttgagactga gaagaataat 120
ggggcaggct actttttgga gcatctggct ttcaagggaa caaagaatcg gcctggcagt 180
gccctggaga aggaggtgga gagcatgggg gcccatctta atgcctacag cacccgggag 240
cacacagctt actacatcaa ggcgctgtcc aaggatctgc cgaaagctgt ggagctcctg 300
ggtgacattg tgcagaactg tagtctggaa gactcacaga ttgagaagga acgtgatgtg 360
atcctgcggg agatgcagga gaatgatgca tctatgcgag atgtggtctt taactacctg 420
catgccacag cattccaggg cacacctcta gcccaggctg tggaggggcc cagtgagaat 480
gtcaggaagc tgtctcgtgc agacttgacc gagtacctca gcacacatta caaggcccct 540
cgaatggtgc tggcagcagc tggaggagtg gagcaccagc aactgttaga cctcgcccag 600
aagcacctcg gtggcatccc atggacatat gcagaggacg ctgtgcccac tcttactcca 660
tgccgcttca ctggcagtga gatccgccac cgtgatgatg ctctaccttt tgcccacgtg 720
gccattgcag tagagggtcc tggctgggcc agcccggaca atgtggcctt gcaagtggcc 780
aatgccatca tcggccacta tgactgcact tatggtggtg gcgtgcacct gtccagccca 840
ctggcttcag gtgctgtggc caacaagcta tgccagagtt tccagacctt cagcatctgc 900
tatgcagaga cgggcttgct gggtgcacac tttgtctgtg accgaatgaa aatcgatgac 960
atgatgttcg tcctgcaagg gcagtggatg cgcctgtgta ccagtgccac ggagagtgag 1020
gtggcccggg gcaaaaacat cctcagaaat gccctggtat ctcatctaga tggcactact 1080
cctgtgtgtg aggacatcgg acgcagcctc ctgacctatg gccgccgcat ccccctggct 1140
gaatgggaaa gccggattgc ggaggtggat gccagtgtgg tacgtgagat ctgctccaag 1200
tacatctatg accagtgccc agcagtggct ggatatggcc ccattgagca gctcccagac 1260
tacaaccgga tccgtagcgg catgttctgg ctgcgcttc 1299
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tcggtgggag tgtggatt 18
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggcggcgt ccgtggtct 19
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gaagcgcagc cagaacatg 19
Claims (7)
1. 1 mutain of cytochrome b-c1 complex subunits of people a kind of, which is characterized in that its amino acid sequence such as SEQ ID
NO:Shown in 1.
2. a kind of encoding gene of 1 mutain of cytochrome b-c1 complex subunits of people, which is characterized in that its nucleotides sequence
Row 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
The nucleotide sequence of nucleotide probe 1 mutain of cytochrome b-c1 complex subunits of people according to claim 2, with
The comparison result of the nucleotide sequence of the 1 normal albumen of cytochrome b-c1 complex subunits of people determines.
4. genetic chip according to claim 3, which is characterized in that the nucleotide probe is SEQ ID NO:Shown in 3
Base sequence.
5. a kind of monoclonal antibody of 1 mutain of cytochrome b-c1 complex subunits of specific recognition people, feature exist
In can be with SEQ ID NO:Amino acid sequencespecific shown in 1 combines.
6. a kind of ELISA kit for detecting the antibody of 1 mutain of cytochrome b-c1 complex subunits of people, special
Sign is that the ELISA kit includes：It is coated with ELISA ELISA Plates, the enzyme mark two of monoclonal antibody described in claim 5
Anti-, detection object 1 albumen of cytochrome b-c1 complex subunits, sample diluting liquid, coating buffer solution, ELISA ELISA Plates are washed
Wash liquid, developing solution and terminate liquid.
7. being used to detect the antibody of 1 mutain of cytochrome b-c1 complex subunits of people according to claim 6
ELISA kit, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human of diluted HRP horseradish peroxidase-labeleds
IgG。
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Publications (1)
| Publication Number | Publication Date |
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ID=63163018
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112778415A (en) * | 2021-03-01 | 2021-05-11 | 大连工业大学 | Preparation method of apostichopus japonicus AjCYTB polyclonal antibody |
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2018
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112778415A (en) * | 2021-03-01 | 2021-05-11 | 大连工业大学 | Preparation method of apostichopus japonicus AjCYTB polyclonal antibody |
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