CN108084254A - The antioncogene WWOX δ 6-8 mutains of people a kind of and its application - Google Patents
The antioncogene WWOX δ 6-8 mutains of people a kind of and its application Download PDFInfo
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Abstract
The present invention relates to 68 mutains of antioncogene WWOX δ of people a kind of and its applications, by regarding a large amount of cancer patients as research case, genetic test is carried out to case and is analyzed, determine the mutain of the antioncogene WWOX δ 68 of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to 68 mutains of antioncogene WWOX δ of the people, for the impulse that the gene diagnosis of cancer provides, the early diagnosis, early discovery and associated treatment of cancer are realized.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to the antioncogene WWOX δ 6-8 mutains of people a kind of
And its application.
Background technology
WWOX is one of generally acknowledged tumor suppressor gene, with tumor necrosis factor, p53 albumen and many other signals pathway proteins
Interaction plays regulating and controlling effect to cell.It is played in the occurrence and development of antioncogene WWOX δ 6-8 and cancer more crucial
Effect.The antioncogene WWOX δ 6-8 of people, which undergo mutation, to impact human health, the study found that some cancers
During the peripheral blood of (cancer includes lung cancer, liver cancer, cancer of pancreas and leukaemia) patient carries out gene sequencing, find
The antioncogene WWOX δ 6-8 of patient are mutated, therefore, the detection pair being mutated to the antioncogene WWOX δ 6-8 of people
Judge whether human body suffers from cancer and have certain impulse.
The content of the invention
Present invention aims at the antioncogene WWOX δ 6-8 mutains and its application for providing a kind of people.
Technical solution of the present invention includes：
In a first aspect, provide the antioncogene WWOX δ 6-8 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 mutain of the antioncogene WWOX δ 6-8 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 antioncogene WWOX δ 6-8 mutains of the people, the anticancer base with people
Because the comparison result of the nucleotide sequence of the normal albumen of WWOX δ 6-8 determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect, the monoclonal for providing the antioncogene WWOX δ 6-8 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 antioncogene WWOX δ 6-8 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
Antioncogene WWOX δ 6-8 albumen, 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.
6th aspect provides a kind of antioncogene WWOX δ based on any of the above-described ELISA kit detection people
The method of the antibody of 6-8 mutains, 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 antioncogene WWOX δ 6-8 albumen for detecting object is diluted to various concentration using the sample diluting liquid
Gradient, and the antioncogene WWOX δ 6-8 albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and
Incubation at room temperature；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
D, the ELIAS secondary antibody is added in, and is incubated at room temperature；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
F, the developing solution is added in, room temperature is protected from light incubation, and adds in the terminate liquid and terminate reaction；
G, 450nm measures OD values in microplate reader.
Preferably, further comprise：Blank control is tested.
The present invention provides the antioncogene WWOX δ 6-8 mutains of people a kind of and its application, by a large amount of cancer patients
As research case, genetic test is carried out to case and is analyzed, determines the mutation of the antioncogene WWOX δ 6-8 of people
Albumen prepares genetic chip, monoclonal antibody and ELISA reagents according to the antioncogene WWOX δ 6-8 mutains of the people
Box is the impulse that the gene diagnosis of cancer provides, and realizes the early diagnosis, early discovery and associated treatment of cancer.
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 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；
Fig. 9 is the standard curve that the offer of the embodiment of the present invention seven is protein content；
Figure 10 is the Westernblot testing result schematic diagrames that the embodiment of the present invention seven 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 antioncogene WWOX δ 6-8 mutains of people
Encoding gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the anticancer of people is determined according to the encoding gene
Gene WWOX δ 6-8 mutains, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the antioncogene WWOX δ 6-8 mutains 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 antioncogene WWOX δ 6-8 mutains of people
Nucleotide sequence determines with the comparison result of the nucleotide sequence of the normal albumen of antioncogene WWOX δ 6-8 of people, and according to
The design principle of following probe, the nucleotide for designing the specificity of the antioncogene WWOX δ 6-8 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 antioncogene WWOX δ 6-8 mutains of people and the antioncogene WWOX of people
The comparison result of the corresponding amino acid sequence of the normal albumen of δ 6-8 please refers to Fig.1, wherein, the Query sequences in Fig. 1 are people
The corresponding amino acid sequence of antioncogene WWOX δ 6-8 mutains, Sbjct sequences are the antioncogene WWOX δ 6-8 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 antioncogene WWOX δ 6-8 mutains of people have compared with the normal albumen of antioncogene WWOX δ 6-8 of people at two
Amino acid sequence is mutated, according to SEQ ID NO:The comparison result of nucleotide sequence and Fig. 1 shown in 2, in order to
Whether the antioncogene WWOX δ 6-8 of enough specific recognitions object to be detected are mutated, then are choosing nucleotide probe
When, it can 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, designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, which is：
tgttgaggtt cgaaaccgcc aa
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2nd, the preparation for the genetic chip whether the antioncogene WWOX δ 6-8 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, ☆ is 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.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 anticancer base of people
Because in WWOX δ 6-8 mutains, selecting sequence corresponding from nucleotide probe different, and the position of digit and nucleotide probe
Number is identical, and one section of nucleotide sequence that can also be different from the digit of nucleotide probe is as positive internal control Quality Control probe.It is preferred that
The nucleotide digit positive internal control Quality Control probe identical with nucleotide probe digit is chosen on ground.In the embodiment of the present invention, gene core
Positive internal control probe sequence needed for piece can be： ggggctggac gacacggaca gt.
It should be noted that in the deposition process of genetic chip, click and enter negative internal reference according to the layout of genetic chip and visit
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 10 Min.
(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：
| Total serum IgE | The most 6.5 μ L of 2 μ g |
| T7Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
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 4 μ g of cRNA 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 |
| 25XFragmentation 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 | 700mL |
| 20*SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
(1 liter) configuration of washing lotion 2：
| DEPC-H2O | 997mL |
| 20*SSPE | 3.0mL |
| 20%N-Lauroylsarcosine | 0.25mL |
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 antioncogene WWOX δ 6- of detection object are determined according to scanning result
Whether 8 albumen are mutated.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence is positive right
According to for green fluorescence, showing the sample quality of acquisition testing object has no problem, and experimental group is green fluorescence, then table
The antioncogene WWOX δ 6-8 albumen of bright detection object is 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 antioncogene WWOX δ 6-8 albumen for showing to detect object is not undergone mutation.
Embodiment three, specific recognition people antioncogene WWOX δ 6-8 mutains monoclonal antibody preparation
1st, according to base sequence (such as SEQ ID NO of the antioncogene WWOX δ 6-8 mutains of people:Shown in 2) it sets
Count 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)：atggcagcgc tgcgctacgc
Anti-sense primer (F)：ttggcggttt cgaacctcaa
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 antioncogene WWOX δ 6-8 mutains of people
The complete segment of gene, and pMD19-T Vector (Takara companies) are connected, it is sequenced.Then by special biotech firm's system
Standby 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 antioncogene WWOX δ 6-8 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 antioncogene WWOX δ 6-8 albumen 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 patients with lung cancer as detection object, and detect the lung cancer using ELISA kit and suffer from
Whether the antioncogene WWOX δ 6-8 albumen of person is 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, 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 antioncogene WWOX δ 6-8 albumen of patients with lung cancer is diluted to various concentration gradient using sample diluting liquid,
And the antioncogene WWOX δ 6-8 albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and room temperature is incubated
Educate 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；It is wherein it is possible to soft using ELISA Calc
Part draws the standard curve, can know regression coefficient R according to the ELISA Calc softwares2=0.9958, therefore, this measure
Effectively.The antioncogene WWOX δ 6-8 albumen of patients with lung cancer can be acquired by bringing the OD450 values that detection obtains into standard curve
Concentration.Utilize the content of antioncogene WWOX δ 6-8 albumen in the ELISA method detection Serum of Patients with Lung Cancer sample of foundation.
And it is identified with Western blot.Fig. 5 is refer to, is the standard curve of OD values.Wherein, X-axis is OD values, and Y-axis is correspondence
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)
(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 antioncogene WWOX δ 6-8 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 19KD
There is apparent band, wherein, the molecular weight of antioncogene WWOX δ 6-8 mutains is about 19KD, shows the patients with lung cancer
Antioncogene WWOX δ 6-8 albumen be implicitly present in mutation.
Embodiment six
In embodiments of the present invention, using liver cancer patient as detection object, and detect the liver cancer using ELISA kit and suffer from
Whether the antioncogene WWOX δ 6-8 albumen of person is 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, 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 antioncogene WWOX δ 6-8 albumen for detecting object is diluted to various concentration gradient using sample diluting liquid,
And the antioncogene WWOX δ 6-8 albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and room temperature is incubated
Educate 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；It is wherein it is possible to soft using ELISA Calc
Part draws the standard curve, can know regression coefficient R according to the ELISA Calc softwares2=0.9971, therefore, this measure
Effectively.The antioncogene WWOX δ 6- of liver cancer patient in sample can be acquired by bringing the OD450 values that detection obtains into standard curve
The concentration of 8 albumen.Utilize antioncogene WWOX δ 6-8 albumen in the ELISA methods detection liver cancer patient blood serum sample of foundation
Content.And it is identified with Western blot.Fig. 7 is refer to, is the standard curve of OD values.Wherein, X-axis be OD values, Y-axis
For corresponding concentration.
I, Western blot are identified
J, Western blot Analysis of test results
Wherein, step i and step j are identical in embodiment five, and details are not described herein, refer 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 19KD, wherein, the molecular weight of antioncogene WWOX δ 6-8 mutains is about 19KD, shows this
The antioncogene WWOX δ 6-8 albumen of liver cancer patient is implicitly present in mutation.
Embodiment seven
In embodiments of the present invention, using Pancreas cancer patients as detection object, and the pancreas is detected using ELISA kit
Whether the antioncogene WWOX δ 6-8 albumen of cancer patient is 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, 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 antioncogene WWOX δ 6-8 albumen of Pancreas cancer patients is diluted to various concentration ladder using sample diluting liquid
Degree, and the antioncogene WWOX δ 6-8 albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and room
Temperature 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, 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；It is wherein it is possible to soft using ELISA Calc
Part draws the standard curve, can know regression coefficient R according to the ELISA Calc softwares2=0.9951, therefore, this measure
Effectively.The antioncogene WWOX δ of Pancreas cancer patients in sample can be acquired by bringing the OD450 values that detection obtains into standard curve
The concentration of 6-8 albumen.Utilize antioncogene WWOX δ 6-8 in the ELISA method detection Pancreas cancer patients blood serum sample of foundation
The content of albumen.And it is identified with Western blot.Fig. 9 is refer to, is the standard curve of OD values.Wherein, X-axis OD
Value, Y-axis are corresponding concentration.
I, Western blot are identified
J, Western blot Analysis of test results
Wherein, step i and step j are identical in embodiment five, and details are not described herein, please refers to Fig.1 in 0, Figure 10
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 19KD, wherein, the molecular weight of antioncogene WWOX δ 6-8 mutains is about 19KD, shows this
The antioncogene WWOX δ 6-8 albumen of Pancreas cancer patients is 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>The antioncogene WWOX δ 6-8 mutains of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 82
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Ala Ala Leu Arg Tyr Ala Gly Leu Asp Asp Thr Asp Ser Glu Asp
1 5 10 15
Glu Leu Pro Pro Gly Trp Glu Glu Arg Thr Thr Lys Asp Gly Trp Val
20 25 30
Tyr Tyr Ala Asn His Thr Glu Glu Lys Thr Gln Trp Glu His Pro Lys
35 40 45
Thr Gly Lys Arg Lys Arg Val Ala Gly Asp Leu Pro Tyr Gly Trp Glu
50 55 60
Gln Glu Thr Asp Glu Asn Gly Gln Val Phe Phe Val Glu Val Arg Asn
65 70 75 80
Arg Gln
<210> 2
<211> 246
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atggcagcgc tgcgctacgc ggggctggac gacacggaca gtgaggacga gctgcctccg 60
ggctgggagg agagaaccac caaggacggc tgggtttact acgccaatca caccgaggag 120
aagactcagt gggaacatcc aaaaactgga aaaagaaaac gagtggcagg agatttgcca 180
tacggatggg aacaagaaac tgatgagaac ggacaagtgt tttttgttga ggttcgaaac 240
cgccaa 246
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tgttgaggtt cgaaaccgcc aa 22
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggcagcgc tgcgctacgc 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ttggcggttt cgaacctcaa 20
Claims (9)
- A kind of 1. antioncogene WWOX δ 6-8 mutains of people, which is characterized in that its amino acid sequence such as SEQ ID NO:1 institute Show.
- A kind of 2. encoding gene of the antioncogene WWOX δ 6-8 mutains of people, which is characterized in that its nucleotide sequence such as SEQ ID NO:Shown in 2.
- 3. a kind of genetic chip, which is characterized in that including：Solid phase carrier and fixed nucleotide probe on this carrier；It is described Nucleotide probe is according to the nucleotide sequence of the antioncogene WWOX δ 6-8 mutains of the people, the antioncogene WWOX with people The comparison result of the nucleotide sequence of the normal albumen of δ 6-8 determines.
- 4. genetic chip according to claim 3, which is characterized in that the nucleotide probe is SEQ ID NO:Shown in 3 Base sequence.
- 5. the monoclonal antibody of the antioncogene WWOX δ 6-8 mutains of a kind of specific recognition people, which is characterized in that it is compiled The amino acid sequence of code is SEQ ID NO:Shown in 1.
- 6. a kind of ELISA kit of the antibody for the antioncogene WWOX δ 6-8 mutains for being used to detect people, which is characterized in that The ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection of monoclonal antibody described in claim 5 Antioncogene WWOX δ 6-8 albumen, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and the end of object Only liquid.
- 7. it is used to detect the ELISA reagents of the antibody of the antioncogene WWOX δ 6-8 mutains of people according to claim 6 Box, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
- 8. a kind of antioncogene WWOX δ 6-8 mutains based on the ELISA kit detection people of claim 6 or 7 is anti- The method of body, which is characterized in that including：A, monoclonal antibody described in claim 5 is diluted 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 antioncogene WWOX δ 6-8 albumen for detecting object is diluted to various concentration gradient using the sample diluting liquid, and The antioncogene WWOX δ 6-8 albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and is incubated at room temperature；C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；D, the ELIAS secondary antibody is added in, and is incubated at room temperature；E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；F, the developing solution is added in, room temperature is protected from light incubation, and adds in the terminate liquid and terminate reaction；G, 450nm measures OD values in microplate reader.
- 9. the side of the antibody of the antioncogene WWOX δ 6-8 mutains of ELISA kit detection people according to claim 8 Method, which is characterized in that further comprise：Blank control is tested.
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Citations (4)
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|---|---|---|---|---|
| CN103517991A (en) * | 2010-10-27 | 2014-01-15 | 昆特拜克股份公司 | Capture of target DNA and RNA by probes comprising intercalator molecules |
| CN105324491A (en) * | 2013-03-15 | 2016-02-10 | 生物医学研究机构基金会 | Method for the prognosis and treatment of cancer metastasis |
| CN106337058A (en) * | 2016-03-10 | 2017-01-18 | 福建省立医院 | CRYL1-IFT88 fusion gene, and application thereof in diagnosis and treatment of primary hepatocellular carcinoma |
| CN106885908A (en) * | 2015-12-23 | 2017-06-23 | 中国人民解放军第二军医大学 | The detection kit of blood-serum P SMD4 albumen and its detection method and application |
-
2017
- 2017-11-30 CN CN201711232712.6A patent/CN108084254A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103517991A (en) * | 2010-10-27 | 2014-01-15 | 昆特拜克股份公司 | Capture of target DNA and RNA by probes comprising intercalator molecules |
| CN105324491A (en) * | 2013-03-15 | 2016-02-10 | 生物医学研究机构基金会 | Method for the prognosis and treatment of cancer metastasis |
| CN106885908A (en) * | 2015-12-23 | 2017-06-23 | 中国人民解放军第二军医大学 | The detection kit of blood-serum P SMD4 albumen and its detection method and application |
| CN106337058A (en) * | 2016-03-10 | 2017-01-18 | 福建省立医院 | CRYL1-IFT88 fusion gene, and application thereof in diagnosis and treatment of primary hepatocellular carcinoma |
Non-Patent Citations (2)
| Title |
|---|
| ANDRZEJ K. BEDNAREK等: "WWOX, the FRA16D Gene, Behaves as a Suppressor of Tumor Growth", 《CANCER RESEARCH》 * |
| BEDNAREK,A.K.等: "WWOXdelta6-8 [Homo sapiens]", 《GENBANK DATABASE》 * |
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