CN108424442A - A kind of mutain of the coiled-coil domain albumen of people and its application - Google Patents
A kind of mutain of the coiled-coil domain albumen of people and its application Download PDFInfo
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Abstract
The present invention relates to a kind of mutain of the coiled-coil domain albumen of people and its applications, by by a large amount of cervical carcinomas, bladder cancer patients are as research case, genetic test is carried out to case and is analyzed, determine the mutain of the coiled-coil domain albumen of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the coiled-coil domain protein mutation albumen of the people, the gene diagnosis for cervical carcinoma and carcinoma of urinary bladder provides impulse, realizes the diagnosing and treating of relevant disease.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to a kind of mutation eggs of the coiled-coil domain albumen of people
Its application of bletilla.
Background technology
Coiled-coil domain albumen (coiled-coil domain-containingprotein) participation genetic transcription,
Apoptosis and cell cycle process, and the various biologicals behavior such as invasion and transfer for regulating and controlling malignant cell.
The coiled-coil domain albumen mutation of people can cause harmful effect to human health.To certain diseases,
During peripheral blood including cervical carcinoma, bladder cancer patients carries out gene sequencing, the coiled-coil domain of patient is found
Whether albumen is mutated, therefore, to the detection of the coiled-coil domain protein mutation of people to judging human body with correlation
Disease has certain impulse.
Invention content
Present invention aims at a kind of mutain of coiled-coil domain albumen of people of offer and its applications.
Technical solution of the present invention includes：
In a first aspect, providing a kind of coiled-coil domain protein mutation albumen of people, amino acid sequence such as SEQ ID
NO:Shown in 1.
Second aspect provides a kind of encoding gene of the mutain of the coiled-coil domain albumen of people, nucleotide
Sequence such as SEQ ID NO:Shown in 2.
The third aspect provides a kind of genetic chip, including：Solid phase carrier and fixed nucleotide probe on this carrier；
The nucleotide probe is according to the nucleotide sequence of the coiled-coil domain protein mutation albumen of people described above, the volume with people
The comparison result of the nucleotide sequence of bent helix domain protein normal albumen determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
It is anti-to provide a kind of monoclonal of the coiled-coil domain protein mutation albumen of specific recognition people for fourth aspect
Body, can be with SEQ ID NO:Amino acid sequencespecific shown in 1 combines.
5th aspect provides a kind of ELISA for detecting the antibody of the coiled-coil domain protein mutation albumen of people
Kit, the ELISA kit include：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection pair of said monoclonal antibody
Coiled-coil domain albumen, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and the termination of elephant
Liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
The present invention provides a kind of mutain of the coiled-coil domain albumen of people and its applications, by a large amount of uterine neck
Cancer, bladder cancer patients carry out genetic test to case and analyze, determine the coiled-coil domain egg of people as research case
White mutain, according to the coiled-coil domain protein mutation albumen of the people prepare genetic chip, monoclonal antibody and
ELISA kit, the gene diagnosis for cervical carcinoma, carcinoma of urinary bladder provide impulse, realize the diagnosing and treating of relevant disease.
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 Western blot testing result schematic diagrames that the embodiment of the present invention five provides；
Fig. 7 is the standard curve that the offer of the embodiment of the present invention six is protein content；
Fig. 8 is the Western blot testing result schematic diagrames that the embodiment of the present invention six provides.
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 the coiled-coil domain protein mutation albumen of people
Encoding gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the curling of people is determined according to the encoding gene
Helix domain protein mutation albumen, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, it according to the coiled-coil domain protein mutation albumen and its encoding gene of people, realizes as follows
The preparation of genetic chip.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the core of the coiled-coil domain protein mutation albumen of people
Nucleotide sequence determines with the comparison result of the nucleotide sequence of the coiled-coil domain protein normal albumen of people, and according to
The design principle of following probe, the nucleotide for designing the specificity of the coiled-coil domain protein mutation albumen for people are visited
Needle.
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 coiled-coiled structure of coiled-coil domain protein mutation the albumen corresponding amino acid sequence and people of people
The comparison result of the corresponding amino acid sequence of domain protein normal albumen please refers to Fig.1, wherein the Query sequences in Fig. 1 are people
The corresponding amino acid sequence of coiled-coil domain protein mutation albumen, Sbjct sequences are the coiled-coil domain albumen of people
The corresponding amino acid sequence of normal albumen, as can be seen from FIG. 1, the coiled-coil domain protein mutation albumen of people is relative to people's
Coiled-coil domain protein normal albumen, has amino acid sequence at one to be lacked, is inserted at one, and has several
Place is mutated.According to SEQ ID NO:The comparison result of nucleotide sequence and Fig. 1 shown in 2, in order to specificity
Identify whether the coiled-coil domain albumen of object to be detected is mutated, it, can be with then when choosing nucleotide probe
Nucleotide probe is designed according to any one of following several modes mode：
1. choosing the insertion nucleotides sequence column-generation nucleotide probe of insertion position；
2. before insertion position, and/or, after insertion position, several nucleotide sequences are respectively selected, by selection
Several consecutive nucleotides and the insertion nucleotide sequence of insertion position generate nucleotide probe jointly, wherein the nucleosides of generation
The sequencing of adjacent nucleotide and its sequence consensus in the corresponding nucleotide sequence of mutain in acid probe；
3. several nucleotide sequences are selected before insertion position, at the starting position of insertion position select it is several
A nucleotide sequence generates nucleotide probe；
4. several nucleotide sequences are selected after insertion position, at the end position of insertion position select it is several
A nucleotide sequence generates nucleotide probe；
5. before deletion sites with several nucleotide sequences (base sequence is constant) are respectively selected after deletion sites, it is raw
At nucleotide probe；
6. will include a nucleotide sequence of mutated site nucleotide 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, 2. designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, the nucleotide probe
For：ctcgaaggag caactga.
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether the coiled-coil domain albumen 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, ☆ is experimental group.
Wherein, the position of experimental group is the point sample position of nucleotide probe.
Point sample needed for negative internal reference Quality Control probe and positive control for point sample needed for blank control, negative control
Positive internal control Quality Control probe design it is as follows：
Blank control：The blank sampling liquid for being free from any genetic fragment refers to as the contamination monitoring in chip fabrication process
Mark.
Negative internal reference Quality Control probe：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 curling spiral shell of people
It revolves in domain protein mutain, selects sequence corresponding from nucleotide probe different, and can be with nucleotide probe base
Number it is identical, one section of nucleotide sequence that can also be different from the number of nucleotide probe base is visited as positive internal control Quality Control
Needle.Preferably, nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen.The present invention is real
It applies in example, the positive internal control probe sequence needed for genetic chip can be：agatctgcaa aagaaga.
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) take the 1.5mL EP pipes that DEPC is handled that detection object is added in pipe as detected sample processing tube
300 μ L of blood, add 700 μ L of Trizol, mix well, be placed at room temperature for 10min.
(3) chloroform of 140 μ L is added, covers tightly pipe lid, firmly shakes, be placed at room temperature for 3-5min, be placed in a centrifuge,
12000r/min, 4 DEG C of centrifugation 15min, carefully draws supernatant in centrifuge tube, the supernatant of absorption is transferred to clean centrifuge tube
In.
(4) isometric isopropanol is added in the centrifuge tube of supernatant in storing step (3), it is abundant gently overturns centrifuge tube
Mixing liquid is placed at room temperature for 10min, 12000r/min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(5) it is cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min centrifuges 15min, carefully sucks all supernatants, super
Dry 15min in net platform 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 |
| T7Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
Wherein, the addition X μ L of RNase-free Water are to subtract T7Promotor 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：
| 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 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
(2) 55 μ L 2 × GEx Hybridization Buffer are 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 LRNase 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.
| Cy3cRNA green fluorescences | 875ng |
| 10×Blocking Agent | 11μL |
| 25×Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ L 2 × GEx Hybridization Buffer are added.
(3) 100 μ L hybridization solutions is taken to be added drop-wise on chip ware, at the same be added dropwise respectively as shown in Figure 2 by chip layout blank,
On negative, positive, experimental group position.It covers chip and is sealed in hybridizing box, 60 DEG C 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 coiled-coil domain albumen of detection object is determined according to scanning result
Whether it is mutated.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, positive control is green
Color fluorescence shows that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence, then showing to detect
The coiled-coil domain albumen of object is mutated.In result shown in Fig. 4, negative control is colorless fluorescent, positive right
According to for green fluorescence, showing that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, then table
The coiled-coil domain albumen of bright detection object does not mutate.
Embodiment three, specific recognition people coiled-coil domain protein mutation albumen monoclonal antibody preparation
1, according to base sequence (such as SEQ ID NO of the coiled-coil domain protein mutation albumen of people:Shown in 2) 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)：atggcctcgg ctgagttgc
Downstream primer (R)：aataatggaa aactgtaca
2, the DNA of detection object is that template carries out PCR amplification
| 10×Buffer | 5uL |
| dNTP | 2uL |
| Ex Taq | 1uL |
| ddH2O | 5uL |
| Template DNA | 1uL |
| Primer (F) | 3uL |
| Primer (R) | 3uL |
| Total system | 20uL |
DNA to detect object carries out PCR amplification as template, obtains the coiled-coil domain protein mutation albumen base of people
Because of complete segment, and pMD19-TVector (Takara companies) is connected, is sequenced.Then it is prepared by special biotech firm
Antibody is a kind of humanization or Chimeric antibodies.Wherein, the monoclonal antibody prepared can be with SEQ ID NO:Ammonia shown in 1
Base acid sequence is specifically bound.The antibody of preparation is measured into content using ELISA method.
Example IV, coiled-coil domain protein mutation albumen for detecting people antibody ELISA kit
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, the coiled-coil domain albumen 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 benzidines；
Terminate liquid can be the sulfuric acid solution of 2mol/L.
Embodiment five
In embodiments of the present invention, using cervical cancer patient as detection object, and the uterine neck is detected using ELISA kit
Whether the coiled-coil domain albumen of cancer 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, the coiled-coil domain albumen of cervical cancer patient is diluted to various concentration gradient using sample diluting liquid, and
The coiled-coil domain albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and 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=0.99791, 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 volume of cervical cancer patient
The concentration of bent helix domain albumen.Utilize coiled coil knot in the ELISA method detection cervical cancer patient blood serum sample of foundation
The content of structure domain albumen.It is used in combination Western blot to be identified.Referring to FIG. 5, for the standard curve of light absorption value.Wherein, X-axis
For light absorption value, 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(PH8.8) | 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(PH6.8) | 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) it after the isometric mixing of Pierce ECLWestern Blotting Substrate kit A, B liquid, drips dropwise
It is added on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment Image J analyses.
Wherein, primary antibody is the coiled-coil domain protein mutation protein monoclonal antibody of the standby people of corporation, 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 52KD
There is apparent band, wherein the molecular weight of coiled-coil domain protein mutation albumen is about 52KD, shows that the cervical carcinoma is suffered from
The coiled-coil domain albumen of person is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, using bladder cancer patients as detection object, and the bladder is detected using ELISA kit
Whether the coiled-coil domain albumen of cancer 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, the coiled-coil domain albumen for detecting object is diluted to various concentration gradient using sample diluting liquid, and will
The coiled-coil domain albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and 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=0.99932, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire carcinoma of urinary bladder trouble in sample
The concentration of the coiled-coil domain albumen of person.It is detected in bladder cancer patients blood serum sample and is crimped using the ELISA method of foundation
The content of helix domain albumen.It is used in combination Western blot to be identified.Referring to FIG. 7, for the standard curve of light absorption value.Its
In, 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 52KD, wherein the molecular weight of coiled-coil domain protein mutation albumen is about 52KD, shows this
The coiled-coil domain albumen of bladder cancer patients 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>A kind of mutain of the coiled-coil domain albumen of people and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 218
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Ala Ser Ala Glu Leu Gln Gly Lys Tyr Gln Lys Leu Ala Gln Glu
1 5 10 15
Tyr Ser Lys Glu Gln Leu Lys Met Lys Asp Gln Ser Leu Arg Lys Leu
20 25 30
Gln Gln Glu Met Asp Ser Leu Thr Phe Arg Asn Leu Gln Leu Ala Lys
35 40 45
Arg Val Glu Leu Leu Gln Asp Glu Leu Ala Leu Ser Glu Pro Arg Gly
50 55 60
Lys Lys Asn Lys Lys Ser Gly Glu Ser Ser Ser Gln Leu Ser Gln Glu
65 70 75 80
Gln Lys Ser Val Phe Asp Glu Asp Leu Gln Lys Lys Ile Glu Glu Asn
85 90 95
Glu Arg Leu His Ile Gln Phe Phe Glu Ala Asp Glu Gln His Lys His
100 105 110
Val Glu Ala Glu Leu Arg Ser Arg Leu Ala Thr Leu Glu Thr Glu Ala
115 120 125
Ala Gln His Gln Ala Val Val Asp Gly Leu Thr Arg Lys Tyr Met Glu
130 135 140
Thr Ile Glu Lys Leu Gln Asn Asp Lys Ala Lys Leu Glu Val Lys Ser
145 150 155 160
Gln Thr Leu Glu Lys Glu Ala Lys Glu Cys Arg Leu Arg Thr Glu Glu
165 170 175
Cys Gln Leu Gln Leu Lys Thr Leu His Glu Asp Leu Ser Gly Arg Leu
180 185 190
Glu Glu Ser Leu Ser Ile Ile Asn Glu Lys Val Pro Phe Asn Asp Thr
195 200 205
Ser Arg Tyr Tyr Val Gln Phe Ser Ile Ile
210 215
<210> 2
<211> 654
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atggcctcgg ctgagttgca ggggaagtac cagaagctgg ctcaggagta ctcgaaggag 60
caactgaaaa tgaaggatca gtcattgaga aaactacaac aggaaatgga cagtttgaca 120
tttcgaaatc tgcagcttgc caagagggta gaactacttc aagatgaact agctctaagt 180
gaaccacgag gcaagaaaaa caagaaaagt ggagaatctt cttctcagtt gagtcaagag 240
cagaagagtg tctttgatga agatctgcaa aagaagatag aagagaatga acggttgcat 300
atacaatttt ttgaagctga tgagcagcac aagcatgtgg aagcagagct gaggagtcga 360
ctggccactc tggagacaga agcagcccag caccaagctg tggttgacgg tctcacccgg 420
aagtacatgg aaaccattga gaagctgcag aacgacaagg ctaaactaga agtgaaatct 480
cagactctag aaaaggaagc caaggaatgt cgacttcgaa cggaagaatg tcaattacag 540
ttaaagactc ttcatgaaga tttgtcaggt agattagagg aatccttatc aatcatcaat 600
gaaaaagtac cttttaatga tacaagtagg tattatgtac agttttccat tatt 654
<210> 3
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ctcgaaggag caactga 17
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggcctcgg ctgagttgc 19
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
aataatggaa aactgtaca 19
Claims (7)
1. the coiled-coil domain protein mutation albumen of people a kind of, which is characterized in that its amino acid sequence such as SEQ ID NO:1
It is shown.
2. a kind of encoding gene of the coiled-coil domain protein mutation albumen 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
The nucleotide sequence of the nucleotide probe coiled-coil domain protein mutation albumen of people according to claim 2, with people's
The comparison result of the nucleotide sequence of coiled-coil domain protein normal albumen 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 coiled-coil domain protein mutation albumen of specific recognition people, which is characterized in that its
It can be with SEQ ID NO:Amino acid sequencespecific shown in 1 combines.
6. a kind of ELISA kit for detecting the antibody of the coiled-coil domain protein mutation albumen 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 coiled-coil domain albumen, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and the end of object
Only liquid.
7. being used to detect the ELISA examinations of the antibody of the coiled-coil domain protein mutation albumen of people according to claim 6
Agent box, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810455838.8A CN108424442A (en) | 2018-05-14 | 2018-05-14 | A kind of mutain of the coiled-coil domain albumen of people and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810455838.8A CN108424442A (en) | 2018-05-14 | 2018-05-14 | A kind of mutain of the coiled-coil domain albumen of people and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108424442A true CN108424442A (en) | 2018-08-21 |
Family
ID=63162931
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810455838.8A Withdrawn CN108424442A (en) | 2018-05-14 | 2018-05-14 | A kind of mutain of the coiled-coil domain albumen of people and its application |
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| Country | Link |
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| CN (1) | CN108424442A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117233404A (en) * | 2023-11-13 | 2023-12-15 | 北京豪迈生物工程股份有限公司 | Kit for determining protein 2 content in field of human coiled coil structure |
-
2018
- 2018-05-14 CN CN201810455838.8A patent/CN108424442A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117233404A (en) * | 2023-11-13 | 2023-12-15 | 北京豪迈生物工程股份有限公司 | Kit for determining protein 2 content in field of human coiled coil structure |
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