CN108103035A - Cytochrome b-c1 complexs the mutain of people a kind of and its application - Google Patents
Cytochrome b-c1 complexs the mutain of people a kind of and its application Download PDFInfo
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Abstract
The present invention relates to the cytochrome b c1 complexs mutain 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 cytochrome b c1 complexs of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the cytochrome b c1 complex mutains of the people, for the impulse that the gene diagnosis of human body leukaemia provides, the early diagnosis, early discovery and associated treatment of leukaemia are realized.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to the cytochrome b-c1 complex mutains of people a kind of
And its application.
Background technology
UQCRH (ubiquinol-cytochrome c reductase hinge albumen) is a kind of protein coding gene.Base related to this
Because GO annotations include the activity of ubiquinone cytochrome b-c1 complexs.One important collateral line of the gene is UQCRHL.Cell
Pigment b-c1 complexs contain cytochrome b (b562, b566), cytochrome c 1 and a kind of moveable iron-sulfur protein
(Rieske protein), Rieske iron-sulfur proteins are then connected for 1 iron atom and two His residues.All iron-sulfur proteins
The transfer of an electronics is participated in, iron atom therein or aoxidize or is reduced, at least 8 iron-sulfur proteins are joined in mitochondria
With electron transmission.According to the study found that the cytochrome b-c1 complexs have close relationship with leukaemia, therefore, to people
Cytochrome b-c1 complexs mutation detection, to judge human body whether suffer from leukaemia have certain impulse.
The content of the invention
Present invention aims at the cytochrome b-c1 complexs mutains and its application for providing a kind of people.
Technical solution of the present invention includes：
In a first aspect, provide the cytochrome b-c1 complex mutains of people a kind of, amino acid sequence such as SEQ ID
NO:Shown in 1.
Second aspect provides a kind of encoding gene of the cytochrome b-c1 complex mutains of people, nucleotides sequence
Row such as SEQ ID NO:Shown in 2.
The third aspect provides a kind of genetic chip, including：Solid phase carrier and fixed nucleotide probe on this carrier；
The nucleotide probe is according to the nucleotide sequence of the cytochrome b-c1 complex mutains of the people, the cell color with people
The comparison result of the nucleotide sequence of the plain normal albumen of b-c1 complexs determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect, the monoclonal for providing the cytochrome b-c1 complex mutains of specific recognition people a kind of resist
Body, the amino acid sequence of coding is SEQ ID NO:Shown in 1.
5th aspect provides a kind of ELISA of the antibody for the cytochrome b-c1 complex mutains for being used to detect people
Kit, the ELISA kit include：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection pair of said monoclonal antibody
Cytochrome b-c1 complex proteins, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and the end of elephant
Only liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
It is compound to provide a kind of cytochrome b-c1 based on any of the above-described ELISA kit detection people for 6th aspect
The method of the antibody of body mutain, including：
A, said monoclonal antibody is diluted using the coating buffer solution, and the monoclonal after dilution is resisted
Body is loaded into the hole of ELISA ELISA Plates, and is incubated at room temperature；
B, the cytochrome b-c1 complex proteins for detecting object are diluted to various concentration using the sample diluting liquid
Gradient, and the cytochrome b-c1 complex proteins of various concentration gradient are loaded respectively into the hole of ELISA ELISA Plates, and room
Temperature is incubated；
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 the cytochrome b-c1 complexs mutain of people a kind of and its applications, and a large amount of leukaemia are suffered from
Person to case genetic test and analyze as research case, determine people cytochrome b-c1 complexs it is prominent
Become albumen, genetic chip, monoclonal antibody and ELISA reagents are prepared according to the cytochrome b-c1 complex mutains of the people
Box is the impulse that the gene diagnosis of human body leukaemia provides, and 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 practiced according to the content of specification, below with presently preferred embodiments of the present invention and coordinate attached drawing be described in detail as after.
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. " 201710429915.8 ", patent name
Detection method described in the Chinese invention patent of method and device " determines the cytochrome b-c1 complex mutains of people
Encoding gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the cell of people is determined according to the encoding gene
Pigment b-c1 complex mutains, amino acid sequence such as SEQID NO:Shown in 1.
Secondly, according to the cytochrome b-c1 complexs mutain and its encoding gene of people, realize as follows
The preparation of genetic chip.
1st, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the core of the cytochrome b-c1 complex mutains of people
Nucleotide sequence determines with the comparison result of the nucleotide sequence of the normal albumen of cytochrome b-c1 complexs of people, and according to
The design principle of following probe, the nucleotide for designing the specificity of the cytochrome b-c1 complex mutains for people are visited
Pin.
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 so ensure 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 cytochrome b-c1 complex mutains of people and the cytochrome b-c1 of people
The comparison result of the corresponding amino acid sequence of the normal albumen of complex please refers to Fig.1, wherein, the Query sequences in Fig. 1 are people
The corresponding amino acid sequence of cytochrome b-c1 complex mutains, Sbjct sequences are the cytochrome b-c1 complexs of people
The corresponding amino acid sequence of normal albumen, the sequence between Query sequences and Sbjct sequences are comparison result, can according to Fig. 1
Know, the cytochrome b-c1 complexs mutain of people compared with people the normal albumen of cytochrome b-c1 complexs, at one
It is mutated at position, is lacked at a position.According to SEQ ID NO:Nucleotide sequence shown in 2 and
Whether the comparison result of Fig. 1 is mutated in order to the cytochrome b-c1 complexs of specific recognition object to be detected,
So when choosing nucleotide probe, nucleotide probe can be designed according to any one of following several ways mode：
(1) several nucleotide sequences are selected before deletion sites, with selecting several nucleosides after deletion sites
Acid sequence generates nucleotide probe；
(2) 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 (1) design one kind preferably nucleotide probe such as SEQ ID NO:Shown in 3, the nucleotide probe
For：gccaccccgg ag
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2nd, the preparation for the genetic chip whether cytochrome b-c1 complexs of detection people undergo mutation
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 not also be same.In the embodiment of the present invention, the negative internal reference probe sequence needed for genetic chip can be：
tgatgctgat aattgcatag。
Positive internal control Quality Control probe：It is one section of other genetic fragment for having homology with detection gene, in the cell color of people
In plain b-c1 complexs mutain, selection sequence corresponding from nucleotide probe is different, and positive internal control Quality Control probe includes
Nucleotide digit and the digit of nucleotide probe may be the same or different.Preferably, nucleotide digit and nucleotide are chosen
The identical positive internal control Quality Control probe of the digit of probe.In the embodiment of the present invention, the positive internal control probe sequence needed for genetic chip
Arranging to be：ctcgggcccg tt.
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
Pin 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) 2 μ g RNA is taken to configure following reaction solution in 1.5mL centrifuge tubes：
10 minutes ice baths of (2) 65 DEG C of heat preservations 5 minutes, in advance 5 × First Strand B μ ffer in 65 DEG C of 5 points of preheatings
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 denatured in the RNA of ice bath.
(5) centrifuged with after pipette tips mixing.
(6) 40 DEG C of reaction 2h.
3rd, aaUTP marks cRNA synthesis
The configuration of NTP：
| 100mM ATP | 250μL |
| 100mM GTP | 250μL |
| 100mM CTP | 250μL |
| 100mM UTP | 187.5μL |
| RNase free H2O | 62.5μL |
| Total volume | 1000μL |
It is spare to be distributed into 10 pipes, notes：40 DEG C of heat preservation 1min before 50%PEG uses.Simultaneously by following operative configuration
Transcription mix；
(1) Transcription mix are configured
(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) step (5) is repeated once.
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 cRNA4 μ g are taken and are concentrated into 6.6 μ L.
(2) 10 μ L DMSO mixings are added.
(3) 0.3M sodium acid carbonates (NaHCO3) pH9.0 and mixing of 3.4 μ L is added.
(4) above-mentioned 20 μ L cRNA mixtures are added in fluorescent dye Cy3 simultaneously mixing.25 DEG C of heat preservation 1h.
(5) plus 9 μ L 4M Hydroxylamine mixings after 25 DEG C heat preservation 15min.
7th, fluorescent marker cRNA Sample Purification on Single
The process that specific steps are purified with cRNA in the step 4 of the present embodiment, this step are not repeating.
8th, cRNA sample fragments and chip hybridization 4x44K microarrays
(1) according to the form below prepares fragmentation mixed liquor and then carries out fragmentation in 60 DEG C of warm bath 30min.
| Cy3cRNA green fluorescences | 875ng |
| 10XBlocking Agent | 11μL |
| 25X Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ L 2X GEx Hybridization Buffer are added in.
(3) 100 μ L hybridization solutions is taken to be added drop-wise on chip ware, at the same be added dropwise respectively as shown in Figure 2 by chip layout blank,
On negative, positive, experimental group position.It covers chip and is sealed in hybridizing box, 60 DEG C roll hybridization 16h.
9th, chip washs
(1 liter) configuration of washing lotion 1：
| DEPC-H2O | 700m L |
| 20*SSPE | 300m L |
| 20%N-Lauroylsarcosine | 0.25m L |
(1 liter) configuration of washing lotion 2：
| DEPC-H2O | 997m L |
| 20*SSPE | 3.0m L |
| 20%N-Lauroylsarcosine | 0.25m L |
Washing lotion 3：Stabilization and Drying Solution
(1) chip is taken out to wash 1 minute in washing lotion 1；
(2) chip is put into washing lotion 2 again and washs 1 minute (37 DEG C)；
(3) finally chip is washed 30 seconds in washing lotion 3.
10th, chip scanning
Chip in scanner is scanned, the cytochrome b-c1 complexs of detection object are determined according to scanning result
Whether albumen is mutated.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, positive control
For green fluorescence, showing the sample quality of acquisition testing object has no problem, and experimental group is green fluorescence, then shows
Cytochrome b-c1 the complex proteins of detection object are mutated.In result shown in Fig. 4, negative control is colourless glimmering
Light, positive control are green fluorescence, show the sample quality of acquisition testing object and have no problem, and experimental group redgreen is glimmering
Light, then the cytochrome b-c1 complex proteins for showing to detect object are not undergone mutation.
Embodiment three, specific recognition people cytochrome b-c1 complex mutains monoclonal antibody preparation
1st, according to base sequence (such as SEQ ID NO of the cytochrome b-c1 complex mutains of people:Shown in 2) design
Sense primer such as SEQ ID NO:Shown in 4 and, anti-sense primer such as SEQ ID NO:Shown in 5：
Sense primer (P)：atgttgtcgg tagca
Anti-sense primer (F)：tactccagtt accaaata
2nd, detect the DNA of object and carry out PCR amplification for template
| 10×Buffer | 5uL |
| dNTP | 2uL |
| Ex Taq | 1uL |
| ddH2O | 5uL |
| Template DNA | 1uL |
| Primer (P) | 3uL |
| Primer (F) | 3uL |
| Total system | 20uL |
PCR amplification is carried out as template using the DNA for detecting object, obtains the cytochrome b-c1 complex mutain bases of people
Because of complete segment, and pMD19-T Vector (Takara companies) are connected, be sequenced.Then prepared by special biotech firm
Antibody is a kind of humanization or Chimeric antibodies.The antibody of preparation is measured into content using ELISA method.
Example IV, the ELISA kit for detecting the antibody of the cytochrome b-c1 complex mutains 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, the cytochrome b-c1 complex proteins for detecting object, sample diluting liquid, coating buffer solution, ELISA enzymes
Target 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) marks；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 cytochrome b-c1 the complex proteins of patient are mutated, and this method can at least include following a kind of：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, such as be diluted to
2.0ug/ml, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h；
B, the cytochrome b-c1 complex proteins of leukaemic are diluted to various concentration ladder using sample diluting liquid
Degree, and the cytochrome b-c1 complex proteins of various concentration gradient are loaded respectively into the hole of ELISA ELISA Plates, and room temperature
It is incubated 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；According to ELISACalc Software on Drawing bids
Directrix curve can draw the R according to the ELISACalc softwares2=0.99949, therefore, this measures effective.Detection is obtained
OD450 values bring the concentration that standard curve can acquire the cytochrome b-c1 complex proteins of leukaemic into.Using building
The content of vertical ELISA method detection serum of leukaemia cells in sample pigment b-c1 complex proteins.And use Western
Blot is identified.Fig. 5 is refer 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) clean glass plate and dry；
(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 upper strata concentration gelling solid.
2nd, albumen loading electrophoresis：
(1) comb is pulled up, duct mark 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 order 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) take the film out after the completion of, be put at once in 5% skim milk, 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 cytochrome b-c1 complex mutain monoclonal antibodies of the standby people of company system, and secondary antibody 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.Compared with blank control, only near experimental group 25KD
There is apparent band, wherein, the molecular weight of cytochrome b-c1 complex mutains is about 25KD, shows that the leukaemia is suffered from
Cytochrome b-c1 the complex proteins of person are implicitly present in mutation.
The above is only the preferred embodiment of the present invention, is not intended to limit 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>Cytochrome b-c1 complexs the mutain of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 101
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Leu Ser Val Ala Ala Arg Ser Gly Pro Phe Ala Pro Val Leu Ser
1 5 10 15
Ala Thr Pro Glu Gln Pro Val Leu Asp Leu Lys Arg Pro Phe Leu Ser
20 25 30
Arg Glu Ser Leu Ser Gly Gln Ala Val Arg Arg Pro Leu Val Ala Ser
35 40 45
Val Gly Leu Asn Val Pro Ala Ser Val Cys Tyr Ser His Thr Asp Ile
50 55 60
Lys Val Pro Asp Phe Ser Glu Tyr Arg Arg Leu Glu Val Leu Asp Ser
65 70 75 80
Thr Lys Ser Ser Arg Glu Ser Ser Glu Ala Arg Lys Gly Phe Ser Tyr
85 90 95
Leu Val Thr Gly Val
100
<210> 2
<211> 303
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atgttgtcgg tagcagcccg ctcgggcccg ttcgcgcccg tcctgtcggc caccccggag 60
cagcctgtgt tggacctgaa gcggcccttc ctcagccggg agtcgctgag cggccaggcc 120
gtgcgccggc ctttggtcgc ctccgtgggc ctcaatgtcc ctgcttctgt ttgttattcc 180
cacacagaca tcaaggtgcc tgacttctct gaataccgcc gccttgaagt tttagatagt 240
acgaagtctt caagagaaag cagcgaggct aggaaaggtt tctcctattt ggtaactgga 300
gta 303
<210> 3
<211> 12
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gccaccccgg ag 12
<210> 4
<211> 15
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atgttgtcgg tagca 15
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
tactccagtt accaaata 18
Claims (9)
- A kind of 1. cytochrome b-c1 complex mutains of people, which is characterized in that its amino acid sequence such as SEQ ID NO:1 It is shown.
- 2. the encoding gene of the cytochrome b-c1 complex mutains of a kind of people, which is characterized in that its nucleotide sequence is 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 according to the nucleotide sequences of the cytochrome b-c1 complex mutains of the people, with the cytochrome b of people- The comparison result of the nucleotide sequence of the normal albumen of c1 complexs determines.
- 4. genetic chip according to claim 3, which is characterized in that the nucleotide probe is SEQ ID NO:Shown in 3 Base sequence.
- 5. a kind of monoclonal antibody of the cytochrome b-c1 complex mutains of specific recognition people, which is characterized in that its The amino acid sequence of coding is SEQ ID NO:Shown in 1.
- 6. a kind of for detecting the ELISA kit of the antibody of the cytochrome b-c1 complex mutains of people, feature exists In the ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, inspection of monoclonal antibody described in claim 5 Survey cytochrome b-c1 complex proteins, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, the developing solution of object And terminate liquid.
- 7. it is tried according to claim 6 for detecting the ELISA of the antibody of the cytochrome b-c1 complex mutains of people Agent 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 cytochrome b-c1 complex mutains based on the ELISA kit detection people of claim 6 or 7 The method of antibody, which is characterized in that including：A, monoclonal antibody described in claim 5 is diluted using the coating buffer solution, and by the list after dilution Clonal antibody is loaded into the hole of ELISA ELISA Plates, and is incubated at room temperature；B, the cytochrome b-c1 complex proteins for detecting object are diluted to various concentration gradient using the sample diluting liquid, And cytochrome b-c1 the complex proteins of various concentration gradient are 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.
- 9. ELISA kit detects the antibody of the cytochrome b-c1 complex mutains of people according to claim 8 Method, which is characterized in that further comprise：Blank control is tested.
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| Country | Link |
|---|---|
| CN (1) | CN108103035A (en) |
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2017
- 2017-11-29 CN CN201711220853.6A patent/CN108103035A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 柳萍: "B细胞急性淋巴细胞白血病预后新分子标记的研究进展", 《国际输血及血液学杂志》 * |
| 王鸿利: "白血病的分子标记检测和血友病的基因诊断", 《中国实用内科杂志》 * |
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