CN108048422A - A kind of GTP enzymes, 7 mutain of IMAP family members and its application - Google Patents
A kind of GTP enzymes, 7 mutain of IMAP family members and its application Download PDFInfo
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Abstract
The present invention relates to a kind of GTP enzymes, 7 mutain of IMAP family members and its application, by regarding a large amount of cancer patients as research case, genetic test is carried out to case and is analyzed, determines the GTP enzymes of people, the mutain of IMAP family members 7, according to the GTP enzymes of the people, 7 mutain of IMAP family members prepares genetic chip, monoclonal antibody and ELISA kit, is the impulse that the gene diagnosis of cancer provides, and realizes the early diagnosis, early discovery and associated treatment of cancer.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to a kind of GTP enzymes, 7 mutain of IMAP family members and
It is applied.
Background technology
GTP enzymes all play key player on signal transduction, protein biology synthesis, protein translocation and vesicle transport.
GTP enzymes, IMAP family members 7 have dimer protein activity and GTP enzymatic activitys.The GTP enzymes of people, IMAP family members 7 occur
Mutation can impact human health, the study found that some cancers, (cancer includes lung cancer, liver cancer, cancer of pancreas and white
Blood disease) patient peripheral blood carry out gene sequencing during, find patient GTP enzymes, IMAP family members 7 have occurred prominent
Become, therefore, to the GTP enzymes of people, the detection that IMAP family members 7 are mutated has centainly to judging whether human body suffers from cancer
Impulse.
The content of the invention
Present invention aims at provide a kind of GTP enzymes of people, 7 mutain of IMAP family members and its application.
Technical solution of the present invention includes：
In a first aspect, the GTP enzymes of people a kind of are provided, and 7 mutain of IMAP family members, amino acid sequence such as SEQ ID
NO:Shown in 1.
Second aspect provides the GTP enzymes of people a kind of, the encoding gene of the mutain of IMAP family members 7, 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 GTP enzymes of the people, the nucleotide sequence of 7 mutain of IMAP family members, the GTP with people
Enzyme, the comparison result of the nucleotide sequence of 7 normal albumen of IMAP family members determine.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect, provides a kind of GTP enzymes of specific recognition people, and the monoclonal of 7 mutain of IMAP family members resists
Body, the amino acid sequence of coding is SEQ ID NO:Shown in 1.
5th aspect provides a kind of GTP enzymes for being used to detect people, the ELISA of the antibody of 7 mutain of IMAP family members
Kit, the ELISA kit include：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection pair of said monoclonal antibody
The GTP enzymes of elephant, 7 albumen of IMAP family members, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and
Terminate liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
6th aspect, provides a kind of GTP enzymes based on any of the above-described ELISA kit detection people, IMAP families into
The method of the antibody of 7 mutains of member, 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 GTP enzymes of object will be detected using the sample diluting liquid, 7 albumen of IMAP family members is diluted to various concentration
Gradient, and by the GTP enzymes of various concentration gradient, 7 albumen of IMAP family members 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 GTP enzymes of people a kind of, a large amount of cancers are suffered from 7 mutain of IMAP family members and its application
Person carries out case genetic test and analyzes, determine the GTP enzymes of people as case is studied, IMAP family members' 7
Mutain, according to the GTP enzymes of the people, 7 mutain of IMAP family members prepares genetic chip, monoclonal antibody and ELISA
Kit 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 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 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 GTP enzymes of people, and IMAP family members 7 are mutated egg
White encoding gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the GTP of people is determined according to the encoding gene
Enzyme, 7 mutain of IMAP family members, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the GTP enzymes of people, 7 mutain of IMAP family members and its encoding gene are realized as follows
The preparation of genetic chip.
1st, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the GTP enzymes of people, the core of 7 mutain of IMAP family members
Nucleotide sequence, the GTP enzymes with people, the comparison result of the nucleotide sequence of 7 normal albumen of IMAP family members determine, and according to
The design principle of following probe designs the GTP enzymes for people, the nucleotide of the specificity of 7 mutain of IMAP family members
Probe.
Wherein, the principle of nucleotide probe design is as follows：
1. nucleotide probe Tm values should be close to the average Tm values of whole gene group, 5 DEG C of fluctuation up and down；
2. the single base that nucleotide probe intramolecular repeats continuously is no more than 4；
3. G+C contents are 40%-60%, the specificity that non-specific hybridization ensures hybridization is reduced；
4. 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 GTP enzymes of the GTP enzymes of people, the corresponding amino acid sequence of 7 mutain of IMAP family members and people, IMAP
The comparison result of the corresponding amino acid sequence of 7 normal albumen of family member please refers to Fig.1, wherein, the Query sequences in Fig. 1 are
The GTP enzymes of people, the corresponding amino acid sequence of 7 mutain of IMAP family members, Sbjct sequences are the GTP enzymes of people, IMAP family
The corresponding amino acid sequence of 7 normal albumen of family member, the sequence between Query sequences and Sbjct sequences are comparison result, according to
Fig. 1 understands that the GTP enzymes of people, 7 mutain of IMAP family members is compared with the GTP enzymes of people, 7 normal albumen of IMAP family members,
A part of amino acid sequence is lacked, according to SEQ ID NO:The comparison result of nucleotide sequence and Fig. 1 shown in 2, in order to
It is capable of the GTP enzymes of specific recognition object to be detected, whether IMAP family members 7 are mutated, then choosing nucleotide
During probe, can before deletion sites with respectively selecting several nucleotide sequences (base sequence is constant) after deletion sites,
Generate 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：
gacaccaagg aa
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2nd, the GTP enzymes of people, the preparation for the genetic chip whether IMAP family members 7 undergo mutation are detected
In order to ensure to detect the quality of subject sample, blank control, positive control and the moon need to be also designed on genetic chip
Property control.Wherein, the layout of the genetic chip at least can be a kind of layout as shown in Figure 2.In fig. 2, is blank pair
According to, zero is negative control,For positive control,For experimental group.
Wherein, the position of experimental group is the point sample position of nucleotide probe.
For point sample needed for the negative internal reference Quality Control probe and positive control of point sample needed for blank control, negative control
Positive internal control Quality Control probe design it is as follows：
Blank control：The blank sampling liquid for being free from any genetic fragment refers to as the contamination monitoring in chip fabrication process
Mark.
Negative internal reference Quality Control probe：Be one section does not have other genetic fragments of homology with detection gene, as hybridizing
The monitor control index of non-specific hybridization in journey, the nucleotide digit that negative internal reference Quality Control probe includes can be with nucleotide probes
Digit is identical.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：Be one section has other genetic fragments of homology with detection gene, in the GTP enzymes of people,
In 7 mutain of IMAP family members, select sequence corresponding from nucleotide probe different, and digit can be with nucleotide probe
Digit it is identical, one section of nucleotide sequence that can also be different from nucleotide probe digit is as positive internal control Quality Control probe.It is excellent
Selection of land chooses the nucleotide digit positive internal control Quality Control probe identical with nucleotide probe digit.In the embodiment of the present invention, gene
Positive internal control probe sequence needed for chip can be：ctccctaagg at.
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
| 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 |
| T7RNA Polymerase | 0.8μL |
| Total volume | 60μL |
(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 |
| 25X Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ L 2X GEx Hybridization Buffer are added in.
(3) 100 μ L hybridization solutions is taken to be added drop-wise on chip ware, at the same be added dropwise respectively as shown in Figure 2 by chip layout blank,
On negative, positive, experimental group position.It covers chip and is sealed in hybridizing box, 60 DEG C roll hybridization 16h.
9th, chip washs
(1 liter) configuration of washing lotion 1：
| DEPC-H2O | 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 is scanned in scanner, according to scanning result determine detection object GTP enzymes, IMAP families into
Whether 7 albumen of member are mutated.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence is positive
It compares as green fluorescence, showing the sample quality of acquisition testing object has no problem, and experimental group is green fluorescence, then
Show the GTP enzymes of detection object, 7 albumen of IMAP family members is mutated.In result shown in Fig. 4, negative control is nothing
Color fluorescence, positive control are green fluorescence, show the sample quality of acquisition testing object and have no problem, and experimental group is without green
Color fluorescence, then show to detect the GTP enzymes of object, 7 albumen of IMAP family members is not undergone mutation.
Embodiment three, the GTP enzymes of specific recognition people, the preparation of the monoclonal antibody of 7 mutain of IMAP family members
1st, according to the GTP enzymes of people, base sequence (such as SEQ ID NO of 7 mutain of IMAP family members: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)：atggctgaga gtgaggac
Anti-sense primer (F)：tagtttaatt tcttcatt
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 GTP enzymes of people, 7 mutain of IMAP family members
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 GTP enzymes for detecting people, the ELISA kits of the antibody of 7 mutain of IMAP family members
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 GTP enzymes for detecting object, 7 albumen of IMAP family members, 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 GTP enzymes of person, 7 albumen of IMAP family members are mutated, and this method can at least include following a kind of：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, 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, using sample diluting liquid by the GTP enzymes of patients with lung cancer, 7 albumen of IMAP family members is diluted to various concentration ladder
Degree, and by the GTP enzymes of various concentration gradient, 7 albumen of IMAP family members 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, 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 GTP enzymes of patients with lung cancer, 7 albumen of IMAP family members can be acquired by bringing the OD450 values that detection obtains into standard curve
Concentration.Using GTP enzymes in the ELISA method detection Serum of Patients with Lung Cancer sample of foundation, 7 albumen of IMAP family members contains
Amount.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
Corresponding concentration.
I, Western blot are identified
1st, glue
(1) clean glass plate and dry；
(2) reference molecule cloning process prepares SDS-PAGE glue：
A. lower floor's separation gel prepares system (Total Volum：15mL)
| ddH2O | 5.9mL |
| 30% acrylamide mixed liquor | 5.9mL |
| 1.5mol/L Tris(PH 8.8) | 3.8mL |
| 10%SDS | 0.15mL |
| 10% ammonium persulfate | 0.15mL |
| TEMED | 0.006mL |
B. upper strata spacer gel concentration preparation system (Total Volum：8mL)
(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 GTP enzymes of the standby people of company system, 7 mutain monoclonal antibody of IMAP family members, 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 53KD
There is apparent band, wherein, GTP enzymes, 7 mutant protein molecules amount of IMAP family members is about 53KD, shows the patients with lung cancer
GTP enzymes, 7 albumen of IMAP family members is 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 GTP enzymes of person, 7 albumen of IMAP family members are mutated, and this method can at least include following a kind of：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, 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 GTP enzymes of object will be detected using sample diluting liquid, 7 albumen of IMAP family members is diluted to various concentration ladder
Degree, and by the GTP enzymes of various concentration gradient, 7 albumen of IMAP family members 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, 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=0.9945, therefore, this measure
Effectively.The GTP enzymes of liver cancer patient in sample can be acquired by obtained OD450 values will be detected bringing standard curve into, IMAP families into
The concentration of 7 albumen of member.Utilize GTP enzymes in the ELISA method detection liver cancer patient blood serum sample of foundation, 7 egg of IMAP family members
White content.And it is identified with Western blot.Fig. 7 is refer to, is the standard curve of OD values.Wherein, X-axis is OD values,
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 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 53KD, wherein, GTP enzymes, 7 mutant protein molecules amount of IMAP family members is about 53KD, shows this
The GTP enzymes of liver cancer patient, 7 albumen of IMAP family members are 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 GTP enzymes of cancer patient, 7 albumen of IMAP family members are mutated, and this method can at least include following a kind of：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, 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, using sample diluting liquid by the GTP enzymes of Pancreas cancer patients, 7 albumen of IMAP family members is diluted to various concentration ladder
Degree, and by the GTP enzymes of various concentration gradient, 7 albumen of IMAP family members 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=0.9967, therefore, this measure
Effectively.The GTP enzymes of Pancreas cancer patients in sample, IMAP families can be acquired by bringing the OD450 values that detection obtains into standard curve
The concentration of 7 albumen of member.Using foundation ELISA method detection Pancreas cancer patients blood serum sample in GTP enzymes, IMAP families into
The content of 7 albumen of member.And it is identified with Western blot.Fig. 9 is refer to, is the standard curve of OD values.Wherein, X-axis is
OD values, 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 53KD, wherein, GTP enzymes, 7 mutant protein molecules amount of IMAP family members is about 53KD, shows this
The GTP enzymes of Pancreas cancer patients, 7 albumen of IMAP family members are implicitly present in mutation.
The above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Sequence table
<110>Tianjin lakeside Pan Gu's genetic science Development Co., Ltd
<120>A kind of GTP enzymes, 7 mutain of IMAP family members and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 220
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Ala Glu Ser Glu Asp Arg Ser Leu Arg Ile Val Leu Val Gly Lys
1 5 10 15
Thr Gly Ser Gly Lys Ser Ala Thr Ala Asn Thr Ile Leu Gly Glu Glu
20 25 30
Ile Phe Asp Ser Arg Ile Ala Ala Gln Ala Val Thr Lys Asn Cys Gln
35 40 45
Lys Ala Ser Arg Glu Trp Gln Gly Arg Asp Leu Leu Val Val Asp Thr
50 55 60
Lys Glu Ile Ser Arg Cys Ile Ile Ser Ser Cys Pro Gly Pro His Ala
65 70 75 80
Ile Val Leu Val Leu Leu Leu Gly Arg Tyr Thr Glu Glu Glu Gln Lys
85 90 95
Thr Val Ala Leu Ile Lys Ala Val Phe Gly Lys Ser Ala Met Lys His
100 105 110
Met Val Ile Leu Phe Thr Arg Lys Glu Glu Leu Glu Gly Gln Ser Phe
115 120 125
His Asp Phe Ile Ala Asp Ala Asp Val Gly Leu Lys Ser Ile Val Lys
130 135 140
Glu Cys Gly Asn Arg Cys Cys Ala Phe Ser Asn Ser Lys Lys Thr Ser
145 150 155 160
Lys Ala Glu Lys Glu Ser Gln Val Gln Glu Leu Val Glu Leu Ile Glu
165 170 175
Lys Met Val Gln Cys Asn Glu Gly Ala Tyr Phe Ser Asp Asp Ile Tyr
180 185 190
Lys Asp Thr Glu Glu Arg Leu Lys Gln Arg Glu Glu Val Leu Arg Lys
195 200 205
Ile Tyr Thr Asp Gln Leu Asn Glu Glu Ile Lys Leu
210 215 220
<210> 2
<211> 660
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atggctgaga gtgaggaccg ctccctaagg atcgttctgg tagggaaaac tggaagtggg 60
aaaagtgcaa cagcgaacac catccttgga gaggaaatct ttgattctag aattgctgcc 120
caagctgtta ccaagaactg tcaaaaagca tcccgggaat ggcaggggag agaccttctt 180
gttgtggaca ccaaggaaat cagccgctgc atcatctcct cctgcccagg gccccatgct 240
attgtcctag ttctgctgct gggccgctac acagaggagg agcagaaaac cgttgcattg 300
atcaaggctg tctttgggaa gtcagccatg aagcacatgg tcatcttgtt cactcgcaaa 360
gaagagttgg agggccagag cttccatgac ttcatagcag atgcggatgt gggcctaaaa 420
agcatcgtca aggagtgcgg gaaccgctgc tgtgccttta gcaacagcaa gaaaaccagt 480
aaggcagaga aggaaagtca agtgcaggag ttggtggagc tgatagagaa aatggtgcag 540
tgcaacgaag gggcttactt ttctgatgac atatacaagg acacagagga aaggctgaaa 600
caacgggaag aggttttgag gaaaatctac actgaccaat taaatgaaga aattaaacta 660
<210> 3
<211> 12
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gacaccaagg aa 12
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggctgaga gtgaggac 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
tagtttaatt tcttcatt 18
Claims (9)
1. the GTP enzymes of people a kind of, 7 mutain of IMAP family members, which is characterized in that its amino acid sequence such as SEQ ID NO:
Shown in 1.
2. the GTP enzymes of people a kind of, the encoding gene of 7 mutain of IMAP family members, 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 GTP enzymes of the people, the nucleotide sequence of 7 mutain of IMAP family members, the GTP enzymes with people,
The comparison result of the nucleotide sequence of 7 normal albumen of IMAP family members 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 GTP enzymes of specific recognition people, the monoclonal antibody of 7 mutain of IMAP family members, which is characterized in that its
The amino acid sequence of coding is SEQ ID NO:Shown in 1.
6. a kind of for detecting the GTP enzymes of people, the ELISA kit of the antibody of 7 mutain of IMAP family members, 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 the GTP enzymes of object, 7 albumen of IMAP family members, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, colour developing
Liquid and terminate liquid.
7. according to claim 6 for detecting the GTP enzymes of people, the ELISA of the antibody of 7 mutain of IMAP family members is tried
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 GTP enzymes based on the ELISA kit detection people of claim 6 or 7,7 mutain of IMAP family members
Antibody method, 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 GTP enzymes of object will be detected using the sample diluting liquid, 7 albumen of IMAP family members is diluted to various concentration ladder
Degree, and by the GTP enzymes of various concentration gradient, 7 albumen of IMAP family members is loaded respectively into the hole of ELISA ELISA Plates, and room
Temperature is incubated；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
D, the ELIAS secondary antibody is added in, and is incubated at room temperature；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
F, the developing solution is added in, room temperature is protected from light incubation, and adds in the terminate liquid and terminate reaction；
G, 450nm measures OD values in microplate reader.
9. the GTP enzymes of ELISA kit detection people according to claim 8, the antibody of 7 mutain of IMAP family members
Method, which is characterized in that further comprise：Blank control is tested.
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ID=62121444
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104812914A (en) * | 2012-09-21 | 2015-07-29 | 新加坡保健集团 | Methods of diagnosing liver cancer in a subject and a kit for diagnosing liver cancer |
-
2017
- 2017-11-30 CN CN201711233713.2A patent/CN108048422A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104812914A (en) * | 2012-09-21 | 2015-07-29 | 新加坡保健集团 | Methods of diagnosing liver cancer in a subject and a kit for diagnosing liver cancer |
Non-Patent Citations (1)
| Title |
|---|
| VENTER,J.C.等: ""GTPase, IMAP family member 7, isoform CRA_b, partial [Homo sapiens]"", 《GENBANK DATABASE》 * |
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