CN108342369A - The GDP-L- fucose synzyme mutains of people a kind of and its application - Google Patents
The GDP-L- fucose synzyme mutains of people a kind of and its application Download PDFInfo
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Abstract
The present invention relates to the GDP L fucose synzyme mutains of people a kind of and its applications, by regarding a large amount of alimentary tract cancer patients as research case, genetic test is carried out to case and is analyzed, determine the mutain of the GDP L fucose synzyme of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the mutain of the GDP L fucose synzyme of the people, impulse is provided for the gene diagnosis of alimentary tract cancer, and certain theoretical foundation is provided for the diagnosing and treating of cancer.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to the GDP-L- fucose synzyme mutains of people a kind of
And its application.
Background technology
GDP-L- fucoses synzyme (GDP-L-fucose synthase) is a kind of GADP (H)-binding protein, it
The reaction of two step epimerases and reductase, catalysis GDP-4- ketone -6- deoxidations-can be catalyzed in GDP-D- mannose metabolic processes
Mannose generates GDP- fucoses.The biological behaviour of tumour cell is all along with the weight of the glycoprotein of cell surface and glycolipid
Structure, and the reconstruct of glycoprotein and glycolipid is to be incorporated in the structure of polysaccharide above by changing and realize.Fucose is as more
The important component and cancer of sugar are close.
Therefore, the GDP-L- fucose synzyme mutation of people can cause to seriously affect to human health.To certain
Alimentary tract cancer finds the GDP- of patient during carrying out gene sequencing such as the peripheral blood of liver cancer, gastric cancer, patients with bowel cancer
L-fucose synzyme is mutated, and therefore, the detection of enzyme mutant is synthesized to the GDP-L- fucoses of people is to judging human body
It is no that there is certain impulse with alimentary tract cancer.
Invention content
Present invention aims at the GDP-L- fucose synzyme mutains of people of offer a kind of and its applications.
Technical solution of the present invention includes：
In a first aspect, providing a kind of GDP-L- fucose synzyme mutains of people, amino acid sequence such as SEQ ID
NO:Shown in 1.
Second aspect provides a kind of encoding gene of the GDP-L- fucose synzyme 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 GDP-L- fucose synzyme mutains of the people, the GDP-L- with people
The comparison result of the nucleotide sequence of the normal albumen of fucose synzyme 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 GDP-L- fucose synzyme mutains of specific recognition people for fourth aspect
The amino acid sequence of body, coding is SEQ ID NO:Shown in 1.
5th aspect provides a kind of ELISA for detecting the antibody of the GDP-L- fucose synzyme mutains of people
Kit, the ELISA kit include：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection pair of said monoclonal antibody
GDP-L- fucoses synthetase 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 GDP-L- fucoses synthesis detecting people based on any of the above-described ELISA kit
The method of the antibody of enzyme mutant albumen, 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, incubation at room temperature；
B, the GDP-L- fucose synthetase albumens for detecting object are diluted to various concentration using the sample diluting liquid
Gradient, and the GDP-L- fucoses synthetase albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, room temperature
It is incubated；
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；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature is protected from light incubation, 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.
Preferably, further comprise：Blank control is tested.
The present invention provides the GDP-L- fucose synzyme mutains of people a kind of and its applications, by a large amount of digestive system cancers
Disease patient carries out genetic test to case and analyzes as research case, determines the prominent of the GDP-L- fucose synzyme of people
Become albumen, genetic chip, monoclonal antibody and ELISA reagents are prepared according to the GDP-L- fucose synzyme mutains of the people
Box provides impulse for the gene diagnosis of alimentary tract cancer, and certain theoretical foundation is provided for the diagnosing and treating of cancer.
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.
Fig. 9 is the standard curve that the offer of the embodiment of the present invention seven is protein content；
Figure 10 is the Western blot testing result schematic diagrames that the embodiment of the present invention seven 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 GDP-L- fucose synzyme mutains of people
Encoding gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the GDP- of people is determined according to the encoding gene
L-fucose synzyme mutain, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, it according to the GDP-L- fucose synzyme mutains 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 GDP-L- fucose synzyme mutains of people
Nucleotide sequence determines with the comparison result of the nucleotide sequence of the normal albumen of GDP-L- fucose synzyme of people, and according to
The design principle of following probe, the nucleotide for designing the specificity of the GDP-L- fucose synzyme mutains 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 GDP-L- fucoses of GDP-L- fucose synzyme the mutains corresponding amino acid sequence and people of people
The comparison result of the corresponding amino acid sequence of the normal albumen of synzyme please refers to Fig.1, wherein the Query sequences in Fig. 1 are people
The corresponding amino acid sequence of GDP-L- fucose synzyme mutains, Sbjct sequences are the GDP-L- fucose synzyme 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 normal albumen of GDP-L- fucose synzyme of the GDP-L- fucose synzyme mutains of people relative to people has one
Amino acid sequence is divided to be mutated.According to SEQ ID NO:The comparison result of nucleotide sequence and Fig. 1 shown in 2, in order to
Whether the GDP-L- fucoses synzyme for capableing of specific recognition object to be detected is mutated, then being visited choosing nucleotide
Can will include a nucleotide sequence of mutated site nucleotide as nucleotide probe when needle.
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：
atctcacgga tacagcacag accccgcgcc ctgttgagaa
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether the GDP-L- fucoses synzyme 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 GDP-L- of people
In fucose synzyme mutain, select 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：catgcggatt ctagtgacag ggggctctgg
gctggtaggc。
It should be noted that in the deposition process of genetic chip, clicks and enters negative internal reference according to the layout of genetic chip and visit
Needle and positive internal control probe solution；The spotting buffer (10% aqueous trehalose) that reagent used in blank control is 1 times.
The application of embodiment two, genetic chip
1, sample treatment
(1) the blood 1-3mL of acquisition testing object.
(2) the 1.5mL EP pipes that DEPC is handled are taken, as detected sample processing tube, in detected sample processing tube
The middle 300 μ L of blood that detection object is added, add 700 μ L of Trizol, mix well, be placed at room temperature for 10min.
(3) chloroform of 140 μ L is added, covers tightly pipe lid, firmly shakes, be placed 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 being cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min, 4 DEG C of centrifugation 15min carefully suck all supernatants,
The dry 15min in super-clean bench is added 10 μ L DEPC and handles water dissolution.
(6) products therefrom is RNA can influence the labeling effciency and chip hybridization knot of probe if the purity of total serum IgE is not high
Fruit.So using QIAGENKit purifies total serum IgE.
2, the first chains of cDNA and the second chain one-step synthesis method
(1) 2 μ g RNA is taken to configure following reaction solution in the centrifuge tube of 1.5mL：
| Total serum IgE | The most 6.5 μ L of 2 μ g |
| T7 Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
Wherein, the addition X μ L of RNase-free Water are to subtract T7 Promotor according to 11.5 μ L of total volume
The 5 μ L of addition of primer, then subtract the addition of total serum IgE and calculate and get.
(2) 10min, and ice bath 5min are kept the temperature at 65 DEG C, and 5 × First Strand B μ ffer are preheated at 65 DEG C
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：
It is spare to be distributed into 10 pipes, notes：40 DEG C of heat preservation 1min before 50%PEG (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcription mix；
(1) configuration Transcription mix
| RNase-free Water | 5.7μL |
| 4×Transcription Buffer | 20μL |
| NTP | 16μL |
| 0.1M DTT | 6μL |
| 50%PEG | 6.4μL |
| aa-UTP(25mM) | 4μL |
| Inorganic Pyrophosphatase | 0.6μL |
| T7 RNA Polymerase | 0.8μL |
| Total volume | 60μL |
(2) 60 μ L Transcription mix and mixing is added.
(3) 60 DEG C of hot lid in PCR instrument, 40 DEG C of reaction 2h.
4, cRNA is purified
QIAGEN RNeasy Mini kit purify cRNA, and specific method can be found in what QIAGEN companies provided with kit
Operation manual.
(1) 20 μ L RNase free water are added, 350 μ L Buffer RLT are added and mix well.
(2) 250 μ L absolute ethyl alcohols are added, Tip mix well.
(3) total 700 solution of the μ L containing total serum IgE are transferred in the RNeasy pillars being sleeved in 2mL centrifuge tubes >=8000g from
Heart 15-30s, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=8000g centrifuge washings 15-30s discards filter
It crosses liquid and discards the casing of filtered solution and 2mL by RNeasy in >=8000g centrifuge washings 2min with 500 μ L Buffer RPE again
Mini pillars are transferred in a new 1.5mL Eppendorf pipes.
(5) water for drawing 30 μ L RNase free stands 1min, >=8000g centrifugation elutions 1min.
(6) it is primary to repeat step (5).
5, cRNA concentration mensurations
With spectrophotometric analysis cRNA concentration.It needs to measure the light absorption value at 260nm and 280nm to determine the dense of sample
Degree and purity, A260/A280 should be purer cRNA (ratio 1.9-2.1 also can) close to 2.0.
6, cRNA fluorescents mark；
(1) 4 μ g of above-mentioned cRNA are taken and are concentrated into 6.6 μ L.
(2) add 10 μ L DMSO mixings.
(3) add the sodium bicarbonate (NaHCO that the 0.3M pH of 3.4 μ L are 9.03) and mixing.
(4) above-mentioned 20 μ L cRNA mixtures are added in fluorescent dye Cy3 simultaneously mixing.25 DEG C of heat preservation 1h.
(5) plus 9 μ L 4M Hydroxylamine mixings after 25 DEG C heat preservation 15min.
7, fluorescent marker cRNA Sample Purification on Single
The process that specific steps are purified with cRNA in the step 4 of the present embodiment, this step are not repeating.
8, cRNA sample fragments and 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.
| Cy3 cRNA 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 GDP-L- fucose synzyme of detection object is 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, show that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence, then showing
The GDP-L- fucose synthetase albumens of detection object are mutated.In result shown in Fig. 4, negative control is colourless glimmering
Light, positive control are green fluorescence, show that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen is glimmering
Light, then showing that the GDP-L- fucose synthetase albumens for detecting object do not mutate.
Embodiment three, specific recognition people GDP-L- fucose synzyme mutains monoclonal antibody preparation
1, according to base sequence (such as SEQ ID NO of the GDP-L- fucose synzyme mutains 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)：atgggtgaac cccaggga
Downstream primer (R)：catggtctcatctatcgg
2, the DNA of detection object is that template carries out PCR amplification
DNA to detect object carries out PCR amplification as template, obtains the GDP-L- fucose synzyme mutain bases of people
Because of complete segment, and pMD19-T Vector (Takara companies) are connected, is sequenced.Then it is 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, GDP-L- fucose synzyme mutains 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 GDP-L- fucoses synthetase 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) labels；Wherein, dilution times
Number can be 8000 times.
It can be 1 × PBS, pH to be coated with buffer solution:7.4；
ELISA ELISA Plates cleaning solution can be 1 × PBS solution containing 0.05%Tween-20；
Developing solution can be 3,3 ', 5,5 '-tetramethyl benzidines；
Terminate liquid can be the sulfuric acid solution of 2mol/L.
Embodiment five
In embodiments of the present invention, it using liver cancer patient as detection object, and detects the liver cancer using ELISA kit and suffers from
Whether the GDP-L- fucoses synthetase albumen of person is mutated, and this method at least may include following one kind：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h.
B, the GDP-L- fucose synthetase albumens of liver cancer patient are diluted to various concentration gradient using sample diluting liquid,
And the GDP-L- fucoses synthetase 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, 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=0.99915, 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 GDP- of liver cancer patient
The concentration of L-fucose synthetase albumen.Utilize GDP-L- rock algaes in the ELISA method detection liver cancer patient blood serum sample of foundation
The content of sugared synthetase albumen.It is used in combination Western blot to be identified.Referring to FIG. 5, 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
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)
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) after the isometric mixing of Pierce ECL Western Blotting Substrate kit A, B liquid, dropwise
It is added dropwise on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment Image J analyses.
Wherein, primary antibody is the GDP-L- fucose synzyme mutain monoclonal antibodies 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 32KD
There is apparent band, wherein GDP-L- fucose synzyme mutant protein molecules amounts are about 32KD, show the liver cancer patient
GDP-L- fucose synthetase albumens are implicitly present in mutation.
Embodiment six
In embodiments of the present invention, it using patients with gastric cancer as detection object, and detects the gastric cancer using ELISA kit and suffers from
Whether the GDP-L- fucoses synthetase albumen of person is mutated, and this method at least may include following one kind：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h；
B, the GDP-L- fucose synthetase albumens for detecting object are diluted to various concentration gradient using sample diluting liquid,
And the GDP-L- fucoses synthetase 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, 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=0.99843, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire patients with gastric cancer in sample
GDP-L- fucose synthetase albumens concentration.Utilize GDP- in the ELISA method detection Serum Obtained From Advance Gastric Cancer sample of foundation
The content of L-fucose synthetase albumen.It is used in combination Western blot to be identified.Referring to FIG. 7, the standard for light absorption value is bent
Line.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot are identified
J, Western blot Analysis of test results
Wherein, step i and step j are identical as in embodiment five, and details are not described herein, referring to FIG. 8, in Fig. 8
Marker is used to characterize the size of labelled protein.The experimental display result of the present embodiment, compared with blank control, only in experimental group
Nearby there is apparent band in 32KD, wherein GDP-L- fucose synzyme mutant protein molecules amounts are about 32KD, show the stomach
The GDP-L- fucose synthetase albumens of cancer patient are implicitly present in mutation.
Embodiment seven
In embodiments of the present invention, it using patients with bowel cancer as detection object, and detects the intestinal cancer using ELISA kit and suffers from
Whether the GDP-L- fucoses synthetase albumen of person is mutated, and this method at least may include following one kind：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h；
B, the GDP-L- fucose synthetase albumens of patients with bowel cancer are diluted to various concentration gradient using sample diluting liquid,
And the GDP-L- fucoses synthetase 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, washs and dries；
D, the ELIAS secondary antibody is added, 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=0.99858, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire patients with bowel cancer in sample
GDP-L- fucose synthetase albumens concentration.Utilize GDP- in the ELISA method detection patients with bowel cancer blood serum sample of foundation
The content of L-fucose synthetase albumen.It is used in combination Western blot to be identified.Referring to FIG. 9, the standard for light absorption value is bent
Line.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot are identified
J, Western blot Analysis of test results
Wherein, step i and step j are identical as in embodiment five, and details are not described herein, referring to FIG. 10, in 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 32KD, wherein the molecular weight of GDP-L- fucose synzyme mutains is about 32KD, shows this
The GDP-L- fucose synthetase albumens of patients with bowel cancer are 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>The GDP-L- fucose synzyme mutains of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 130
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Gly Glu Pro Gln Gly Ser Met Arg Ile Leu Val Thr Gly Gly Ser
1 5 10 15
Gly Leu Val Gly Lys Ala Ile Gln Lys Val Val Ala Asp Gly Ala Gly
20 25 30
Leu Pro Gly Glu Asp Trp Val Phe Val Ser Ser Lys Asp Ala Asp Leu
35 40 45
Thr Asp Thr Ala Gln Thr Pro Arg Pro Val Glu Lys Val Gln Pro Thr
50 55 60
His Val Ile His Leu Ala Ala Met Val Gly Gly Leu Phe Arg Asn Ile
65 70 75 80
Lys Tyr Asn Leu Asp Phe Trp Arg Lys Asn Val His Met Asn Asp Asn
85 90 95
Val Leu His Ser Ala Phe Glu Val Gly Ala Arg Lys Val Val Ser Cys
100 105 110
Leu Ser Thr Cys Ile Phe Pro Asp Lys Thr Thr Tyr Pro Ile Asp Glu
115 120 125
Thr Met
130
<210> 2
<211> 390
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atgggtgaac cccagggatc catgcggatt ctagtgacag ggggctctgg gctggtaggc 60
aaagccatcc agaaggtggt agcagatgga gctggacttc ctggagagga ctgggtgttt 120
gtctcctcta aagacgccga tctcacggat acagcacaga ccccgcgccc tgttgagaag 180
gtccaaccca cacacgtcat ccatcttgct gcaatggtgg ggggcctgtt ccggaatatc 240
aaatacaatt tggacttctg gaggaaaaac gtgcacatga acgacaacgt cctgcactcg 300
gcctttgagg tgggcgcccg caaggtggtg tcctgcctgt ccacctgtat cttccctgac 360
aagacgacct acccgataga tgagaccatg 390
<210> 3
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
atctcacgga tacagcacag accccgcgcc ctgttgagaa 40
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atgggtgaac cccaggga 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
catggtctca tctatcgg 18
Claims (9)
1. a kind of mutain of the GDP-L- fucose synzyme of people, which is characterized in that its amino acid sequence such as SEQ ID
NO:Shown in 1.
2. a kind of encoding gene of the GDP-L- fucose synzyme mutains 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 is according to the nucleotide sequence of the GDP-L- fucose synzyme mutains of the people, the GDP-L- rock algaes with people
The comparison result of the nucleotide sequence of the sugared normal albumen of synzyme 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 GDP-L- fucose synzyme 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 ELISA kit for detecting the antibody of the GDP-L- fucose synzyme 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 GDP-L- fucoses synthetase albumen, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, the developing solution of object
And terminate liquid.
7. being used to detect the ELISA examinations of the antibody of the GDP-L- fucose synzyme mutains 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.
8. a kind of GDP-L- fucose synzyme mutains detecting people based on the ELISA kit 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, incubation at room temperature；
B, the GDP-L- fucose synthetase albumens for detecting object are diluted to various concentration gradient using the sample diluting liquid,
And the GDP-L- fucoses synthetase albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, it is incubated at room temperature；
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；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature is protected from light incubation, 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.
9. ELISA kit detects the antibody of the GDP-L- fucose synzyme mutains of people according to claim 8
Method, which is characterized in that further comprise：Blank control is tested.
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| CN201810255646.2A CN108342369A (en) | 2018-03-27 | 2018-03-27 | The GDP-L- fucose synzyme mutains of people a kind of and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810255646.2A CN108342369A (en) | 2018-03-27 | 2018-03-27 | The GDP-L- fucose synzyme mutains of people a kind of and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108342369A true CN108342369A (en) | 2018-07-31 |
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ID=62957585
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810255646.2A Pending CN108342369A (en) | 2018-03-27 | 2018-03-27 | The GDP-L- fucose synzyme mutains of people a kind of and its application |
Country Status (1)
| Country | Link |
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| CN (1) | CN108342369A (en) |
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2018
- 2018-03-27 CN CN201810255646.2A patent/CN108342369A/en active Pending
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Application publication date: 20180731 |