CN108546693A - 1 mutain of metal tripolyphosphate esterase of people a kind of and its application - Google Patents
1 mutain of metal tripolyphosphate esterase of people a kind of and its application Download PDFInfo
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Abstract
The present invention relates to 1 mutain of metal tripolyphosphate esterase of people a kind of and its applications, by regarding a large amount of lung cancer, leukaemic as research case, genetic test is carried out to case and is analyzed, determine the mutain of the metal tripolyphosphate esterase 1 of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to 1 mutain of metal tripolyphosphate esterase of the people, gene diagnosis for lung cancer, leukaemia provides impulse, realizes the diagnosing and treating of relevant disease.
Description
Technical field
The present invention relates to 1 mutain of metal tripolyphosphate esterase of a kind of genetic engineering field more particularly to a kind of people and its
Using.
Background technology
Metal tripolyphosphate esterase 1 (metallophosphoesterase 1) participates in GPI anchorins from endoplasmic reticulum to Gao Er
The transfer of matrix composite.A side chain phosphoethanolamine on Man-2 by removing GPI intermediaries, participates in GPI anchorins
The lipid remodeling process of the committed step effectively transported-GPI anchorin maturations.
The mutation of metal tripolyphosphate esterase 1 of people can cause harmful effect to human health.To certain diseases, including lung
During cancer, the peripheral blood progress gene sequencing of leukaemic, it is prominent to find that the metal tripolyphosphate esterase 1 of patient has occurred
Become, therefore, there is certain guide to judging whether human body suffers from relevant disease to the detection that the metal tripolyphosphate esterase 1 of people is mutated
Effect.
Invention content
Present invention aims at 1 mutain of metal tripolyphosphate esterase 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 1 mutain of metal tripolyphosphate esterase of people, amino acid sequence such as SEQ ID NO:1
It is shown.
Second aspect provides a kind of encoding gene of the mutain of the metal tripolyphosphate esterase 1 of people, and nucleotide sequence is such as
SEQ IDNO: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 1 mutain of metal tripolyphosphate esterase of the people, the metal tripolyphosphate ester with people
The comparison result of the nucleotide sequence of 1 normal albumen of enzyme determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of monoclonal antibody of 1 mutain of metal tripolyphosphate esterase of specific recognition people, compiles
The amino acid sequence of code is SEQ ID NO:Shown in 1.
5th aspect provides a kind of ELISA reagents for detecting the antibody of 1 mutain of metal tripolyphosphate esterase of people
Box, the ELISA kit include：It is coated with the ELISA ELISA Plates of said monoclonal antibody, ELIAS secondary antibody, detects object
1 albumen of metal tripolyphosphate esterase, 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 mutation of metal tripolyphosphate esterase 1 detecting people based on any of the above-described ELISA kit
The method of the antibody of 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, 1 albumen of metal tripolyphosphate esterase for detecting object is diluted to various concentration gradient using the sample diluting liquid,
And 1 albumen of metal tripolyphosphate esterase 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.
Preferably, further comprise：Blank control is tested.
The present invention provides 1 mutain of metal tripolyphosphate esterase of people a kind of and its applications, and a large amount of lung cancer, leukaemia are suffered from
Person carries out genetic test to case and analyzes, determine the mutain of the metal tripolyphosphate esterase 1 of people, root as research case
Genetic chip, monoclonal antibody and ELISA kit are prepared according to 1 mutain of metal tripolyphosphate esterase of the people, is lung cancer, white blood
The gene diagnosis of disease provides impulse, realizes the diagnosing and treating of relevant disease.
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention and after coordinating attached drawing to be described in detail such as.
Description of the drawings
Fig. 1 is a kind of comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 2 is another comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 3 is a kind of genetic chip layout that the embodiment of the present invention one provides；
Fig. 4 is a kind of colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 5 is another colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 6 is the standard curve that the offer of the embodiment of the present invention five is protein content；
Fig. 7 is the Westernblot testing result schematic diagrames that the embodiment of the present invention five provides；
Fig. 8 is the standard curve that the offer of the embodiment of the present invention six is protein content；
Fig. 9 is the Westernblot testing result schematic diagrames that the embodiment of the present invention six provides.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment one, genetic chip
It is possible, firstly, to be a kind of " detection side of mutain according to application No. is " 201710429915.8 ", patent name
Detection method described in the Chinese invention patent of method and device " determines the coding of 1 mutain of metal tripolyphosphate esterase of people
Gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the metal tripolyphosphate ester of people is determined according to the encoding gene
1 mutain of enzyme, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to 1 mutain of metal tripolyphosphate esterase and its encoding gene of people, gene core is realized as follows
The preparation of piece.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the nucleotides sequence of 1 mutain of metal tripolyphosphate esterase of people
Row are determined with the comparison result of the nucleotide sequence of the 1 normal albumen of metal tripolyphosphate esterase of people, and setting according to following probe
Principle is counted, the nucleotide probe of the specificity of 1 mutain of metal tripolyphosphate esterase for people is designed.
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 corresponding amino acid sequence of 1 mutain of metal tripolyphosphate esterase of people and the metal tripolyphosphate esterase 1 of people are normal
The comparison result of the corresponding amino acid sequence of albumen please refers to Fig.1, wherein the Query sequences in Fig. 1 are the metal tripolyphosphate esters of people
The corresponding amino acid sequence of 1 mutain of enzyme, Sbjct sequences are the corresponding amino acid of 1 normal albumen of metal tripolyphosphate esterase of people
Sequence, as can be seen from FIG. 1, the 1 normal albumen of metal tripolyphosphate esterase of 1 mutain of metal tripolyphosphate esterase of people relative to people has
Amino acid sequence is mutated at one, is inserted at one.According to SEQ ID NO:Nucleotide sequence shown in 2, and
Whether the comparison result of Fig. 1, the metal tripolyphosphate esterase 1 in order to specific recognition object to be detected are mutated, then
When choosing nucleotide probe, nucleotide probe can be designed according to any one of following several modes mode：
1. choosing the insertion nucleotides sequence column-generation nucleotide probe of insertion position；
2. before insertion position, and/or, after insertion position, several nucleotide sequences are respectively selected, by selection
Several consecutive nucleotides and the insertion nucleotide sequence of insertion position generate nucleotide probe jointly, wherein the nucleosides of generation
The sequencing of adjacent nucleotide and its sequence consensus in the corresponding nucleotide sequence of mutain in acid probe；
3. several nucleotide sequences are selected before insertion position, at the starting position of insertion position select it is several
A nucleotide sequence generates nucleotide probe；
4. several nucleotide sequences are selected after insertion position, at the end position of insertion position select it is several
A nucleotide sequence generates nucleotide probe；
5. 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, 5. designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, the nucleotide probe
For：tcctccagag gaaa
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
When 1 mutain of metal tripolyphosphate esterase of people is compared in NCBI, the metal tripolyphosphate of chimpanzee in comparison
1 normal albumen of esterase, comparison result is referring to FIG. 2, Query sequences are 1 mutains pair of metal tripolyphosphate esterase of people in Fig. 2
The amino acid sequence answered, Sbjct sequences are the corresponding amino acid sequence of 1 normal albumen of metal tripolyphosphate esterase of chimpanzee, Query
Sequence between sequence and Sbjct sequences is comparison result.As can be seen from FIG. 2,1 mutain of metal tripolyphosphate esterase of people with it is black
The homology of the 1 normal albumen of metal tripolyphosphate esterase of orangutan is 93%, and according to 1 mutain of metal tripolyphosphate esterase of people and people
1 normal albumen of metal tripolyphosphate esterase homology be 93%, the homology with the 1 normal albumen of metal tripolyphosphate esterase of chimpanzee
It is identical with the homology of 1 normal albumen of metal tripolyphosphate esterase of people.Thus illustrate, detection object is sent out in metal tripolyphosphate esterase 1
There is atavism after raw mutation.
2, the preparation for the genetic chip whether the metal tripolyphosphate esterase 1 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 3.In figure 3, 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 metal phosphorus of people
In 1 mutain of acid esters enzyme, select sequence corresponding from nucleotide probe different, and can be with the number of nucleotide probe base
Identical, one section of nucleotide sequence that can also be different from the number of nucleotide probe base is as positive internal control Quality Control probe.It is excellent
Selection of land chooses nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base.The embodiment of the present invention
In, the positive internal control probe sequence needed for genetic chip can be：gtttggaaga caga.
It should be noted that in the deposition process of genetic chip, clicks and enters negative internal reference according to the layout of genetic chip and visit
Needle and positive internal control probe solution；The spotting buffer (10% aqueous trehalose) that reagent used in blank control is 1 times.
The application of embodiment two, genetic chip
1, sample treatment
(1) the blood 1-3mL of acquisition testing object.
(2) take the 1.5mL EP pipes that DEPC is handled that detection object is added in pipe as detected sample processing tube
300 μ L of blood, add 700 μ L of Trizol, mix well, be placed at room temperature for 10min.
(3) chloroform of 140 μ L is added, covers tightly pipe lid, firmly shakes, be placed at room temperature for 3-5min, be placed in a centrifuge,
12000r/min, 4 DEG C of centrifugation 15min, carefully draws supernatant in centrifuge tube, the supernatant of absorption is transferred to clean centrifuge tube
In.
(4) isometric isopropanol is added in the centrifuge tube of supernatant in storing step (3), it is abundant gently overturns centrifuge tube
Mixing liquid is placed at room temperature for 10min, 12000r/min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(5) it is cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min centrifuges 15min, carefully sucks all supernatants, super
Dry 15min in net platform is added 10 μ L DEPC and handles water dissolution.
(6) products therefrom is RNA can influence the labeling effciency and chip hybridization knot of probe if the purity of total serum IgE is not high
Fruit.So using QIAGENKit purifies total serum IgE.
2, the first chains of cDNA and the second chain one-step synthesis method
(1) 2 μ g RNA is taken to configure following reaction solution in the centrifuge tube of 1.5mL：
| Total serum IgE | The most 6.5 μ L of 2 μ g |
| T7Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
Wherein, the addition X μ L of RNase-free Water are subtracted according to 11.5 μ L of total volume
The 5 μ L of addition of T7Promotorprimer, then subtract the addition of total serum IgE and calculate and get.
(2) 10min, and ice bath 5min are kept the temperature at 65 DEG C, in advance by 5 × First Strand B μ ffer at 65 DEG C
Preheat 5min.
(3) following cDNA synthetic systems are configured：
| 5×First Strand Buffer | 4μL |
| 0.1M DTT | 2μL |
| 10mM dNTP mix | 1μL |
| MMLV RT | 1μL |
| RNase OUT | 0.5μL |
| Total volume | 8.5μL |
(4) above-mentioned 8.5 μ L are added after mixing after being denaturalized in the RNA of ice bath.
(5) pipette tips mixing is used to centrifuge later.
(6) 40 DEG C of reaction 2h.
3, aaUTP marks cRNA synthesis
The configuration of NTP：
| 100mM ATP | 250μL |
| 100mM GTP | 250μL |
| 100mM CTP | 250μL |
| 100mM UTP | 187.5μL |
| RNase free H2O | 62.5μL |
| Total volume | 1000μL |
It is spare to be distributed into 10 pipes, notes：40 DEG C of heat preservation 1min before 50%PEG (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcriptionmix；
(1) Transcriptionmix is 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) 55 μ L 2 × GEx Hybridization Buffer are added.
(3) 60 DEG C of hot lid in PCR instrument, 40 DEG C of reaction 2h.
4, cRNA is purified
QIAGEN RNeasy Mini kit purify cRNA, and specific method can be found in what QIAGEN companies provided with kit
Operation manual.
(1) 20 μ LRNase free water are added, 350 μ L BufferRLT 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 LBuffer 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 RNeasymini in >=8000g centrifuge washings 2min with 500 μ LBuffer RPE again
Pillar is 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) above-mentioned cRNA4 μ g 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 4x44Kmicroarrays
(1) according to the form below prepares fragmentation mixed liquor and then carries out fragmentation in 60 DEG C of warm bath 30min.
| Cy3cRNA green fluorescences | 875ng |
| 10×Blocking Agent | 11μL |
| 25×Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ L 2 × GEx Hybridization Buffer are added.
(3) 100 μ L hybridization solutions is taken to be added drop-wise on chip ware, at the same be added dropwise respectively as shown in Figure 3 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, 1 albumen of metal tripolyphosphate esterase of detection object is determined according to scanning result
Whether it is mutated.It please refers to Fig.4 and Fig. 5, in result shown in Fig. 4, negative control redgreen fluorescence, positive control is green
Color fluorescence shows that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence, then showing to detect
1 albumen of metal tripolyphosphate esterase of object is mutated.In result shown in fig. 5, negative control is colorless fluorescent, positive control
For green fluorescence, show that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, then showing
1 albumen of metal tripolyphosphate esterase of detection object does not mutate.
Embodiment three, specific recognition people 1 mutain of metal tripolyphosphate esterase monoclonal antibody preparation
1, according to base sequence (such as SEQ ID NO of 1 mutain of metal tripolyphosphate esterase of people:Shown in 2) design upstream
Primer such as SEQ ID NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (F)：atggcgatga tcgaattgg
Downstream primer (R)：acgctttccg agcaagttc
2, the DNA of detection object is that template carries out PCR amplification
DNA to detect object carries out PCR amplification as template, and 1 mutating protein gene of metal tripolyphosphate esterase for obtaining people is complete
Full wafer section, and pMD19-TVector (Takara companies) is connected, it is sequenced.Then antibody is prepared by special biotech firm,
It is a kind of humanization or Chimeric antibodies.The antibody of preparation is measured into content using ELISA method.
Example IV, 1 mutain of metal tripolyphosphate esterase 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, 1 albumen of metal tripolyphosphate esterase for detecting object, sample diluting liquid, coating buffer solution, ELISA ELISA Plates are washed
Wash liquid, 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 patients with lung cancer as detection object, and detects the lung cancer using ELISA kit and suffers from
Whether 1 albumen of metal tripolyphosphate esterase 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, 1 albumen of metal tripolyphosphate esterase of patients with lung cancer is diluted to various concentration gradient using sample diluting liquid, and will
1 albumen of metal tripolyphosphate esterase of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions are added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated 10-20min, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
H, standard curve is made：Using standard concentration as abscissa, the light absorption value that standard items measure is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measures effective when ＞ 0.99；Wherein it is possible to use ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc softwares2=0.99813, 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 metal of patients with lung cancer
The concentration of 1 albumen of phosphate.Utilize 1 egg of ELISA method detection Serum of Patients with Lung Cancer Gold Samples category phosphate of foundation
White content.It is used in combination Western blot to be identified.Referring to FIG. 6, for the standard curve of light absorption value.Wherein, X-axis is extinction
Value, Y-axis are corresponding concentration.
I, Westernblot is identified
1, glue
(1) it cleans glass plate and dries；
(2) reference molecule cloning process prepares SDS-PAGE glue：
A. lower layer's separation gel prepares system (Total Volum：15mL)
| ddH2O | 5.9mL |
| 30% acrylamide mixed liquor | 5.9mL |
| 1.5mol/L Tris(PH8.8) | 3.8mL |
| 10%SDS | 0.15mL |
| 10% ammonium persulfate | 0.15mL |
| TEMED | 0.006mL |
B. upper layer spacer gel concentration preparation system (Total Volum：8mL)
(3) lower layer's separation gel of respective volume is added in each offset plate, adds the sterile ddH of 1mL2O flattens separation
Glue blots remaining moisture in plastic plate after gelling to be separated is solid with filter paper, and upper layer is then added and concentrates glue, after being inserted into comb
Wait for upper layer concentration gelling solid.
2, albumen loading electrophoresis：
(1) comb is pulled up, duct label is carried out, 5 × SDS electrophoretic buffers is added, then to being added in protein sample
SDS-PAGE albumen sample-loading buffer (5 ×), boiling water bath heat 3-5min, loading；
(2) electrophoresis first is carried out with 80V voltages, after waiting for that band ran concentration glue, uses 100V voltages instead and run about 100min.
3, transferring film and incubation：
(1) first pvdf membrane is activated in methyl alcohol about 30s.Respectively by gel, foam-rubber cushion and pvdf membrane in advance in electrotransfer
30min is balanced in buffer solution.It is clipped by the sequence of filter paper-glue-film-filter paper, transferring film device is put into the principle of black flour according to black flour
In, refrigerator (ice bag) is added, about 120min is run with the electric current of 150mA in ice；
(2) it takes the film out after the completion of, is put into 5% skim milk at once, gently room temperature closes 1h on shaking table；
(3) PBST cleans closed film, cleans 3 times, each 10min；
(4) primary antibody for being diluted to corresponding multiple is added, 4 DEG C of incubator overnights are incubated；
(5) primary antibody is recycled, PBST cleans film 3 times, each 10min；
(6) secondary antibody is (with 3% milk with 1:5000-1:10000 dilutions) incubation at room temperature film 2h；
(7) film is cleaned 3 times with PBST, each 10min；
(8) it after the isometric mixing of Pierce ECLWestern Blotting Substrate kit A, B liquid, drips dropwise
It is added on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment Image J analyses.
Wherein, primary antibody is the 1 mutain monoclonal antibody of metal tripolyphosphate esterase of the standby people of corporation, and secondary antibody is horseradish mistake
The Goat anti-Human IgG of oxide enzyme label.
J, Western blot Analysis of test results：It is shown according to Western blot experimental procedures as a result, such as Fig. 7 institutes
Show, wherein the Marker in Fig. 7 is used to characterize the size of labelled protein.It is only attached in experimental group 100KD compared with blank control
Closely there is apparent band, wherein the molecular weight of 1 mutain of metal tripolyphosphate esterase is about 100KD, shows the patients with lung cancer
1 albumen of metal tripolyphosphate esterase is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, using leukaemic as detection object, and the white blood is detected using ELISA kit
Whether 1 albumen of metal tripolyphosphate esterase of patient is mutated, and this method at least may include following one kind：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h；
B, 1 albumen of metal tripolyphosphate esterase for detecting object is diluted to various concentration gradient using sample diluting liquid, and will
1 albumen of metal tripolyphosphate esterase of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions are added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated 10-20min, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
H, standard curve is made：Using standard concentration as abscissa, the light absorption value that standard items measure is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measures effective when ＞ 0.99；Wherein it is possible to use ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc softwares2=0.99837, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire leukaemia trouble in sample
The concentration of 1 albumen of metal tripolyphosphate esterase of person.Serum of leukaemia Gold Samples category phosphorus is detected using the ELISA method of foundation
The content of 1 albumen of acid esters enzyme.It is used in combination Westernblot to be identified.Referring to FIG. 8, for the standard curve of light absorption value.Wherein, X
Axis is light absorption value, and Y-axis is corresponding concentration.
I, Westernblot is identified
J, Westernblot 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. 9, in Fig. 9
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 100KD, wherein the molecular weight of 1 mutain of metal tripolyphosphate esterase is about 100KD, shows that this is white
1 albumen of metal tripolyphosphate esterase of blood patient is implicitly present in mutation.
The above is only a preferred embodiment of the present invention, it is not intended to restrict the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Sequence table
<110>Tianjin lakeside Pan Gu's genetic science Development Co., Ltd
<120>1 mutain of metal tripolyphosphate esterase of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 403
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Ala Met Ile Glu Leu Gly Phe Gly Arg Gln Asn Phe His Pro Leu
1 5 10 15
Lys Arg Lys Ser Ser Leu Leu Leu Lys Leu Ile Ala Val Val Phe Ala
20 25 30
Val Leu Leu Phe Cys Glu Phe Leu Ile Tyr Tyr Leu Ala Ile Phe Gln
35 40 45
Cys Asn Trp Pro Glu Val Lys Thr Thr Ala Ser Asp Gly Glu Gln Thr
50 55 60
Thr Arg Glu Pro Val Leu Lys Ala Met Phe Leu Ala Asp Thr His Leu
65 70 75 80
Leu Gly Glu Phe Leu Gly His Trp Leu Asp Lys Leu Arg Arg Glu Trp
85 90 95
Gln Met Glu Arg Ala Phe Gln Thr Ala Leu Trp Leu Leu Gln Pro Glu
100 105 110
Val Val Phe Ile Leu Gly Asp Ile Phe Asp Glu Gly Lys Trp Ser Thr
115 120 125
Pro Glu Ala Trp Ala Asp Asp Val Glu Arg Phe Gln Lys Met Phe Arg
130 135 140
His Pro Ser His Val Gln Leu Lys Val Val Ala Gly Asn His Asp Ile
145 150 155 160
Gly Phe His Tyr Glu Met Asn Thr Tyr Lys Val Glu Arg Phe Glu Lys
165 170 175
Val Phe Ser Ser Glu Arg Leu Phe Ser Trp Lys Gly Ile Asn Phe Val
180 185 190
Met Val Asn Ser Val Ala Leu Asn Gly Asp Gly Cys Gly Ile Cys Ser
195 200 205
Glu Thr Glu Ala Glu Leu Ile Glu Val Ser His Arg Leu Asn Cys Ser
210 215 220
Arg Glu His Tyr Pro Leu Tyr Arg Arg Ser Asp Ala Asn Cys Ser Gly
225 230 235 240
Glu Asp Ala Ala Pro Pro Glu Glu Arg Asp Ile Pro Phe Lys Glu Asn
245 250 255
Tyr Asp Val Leu Ser Arg Glu Ala Ser Gln Lys Glu Arg Ser Asp Gly
260 265 270
Gln Arg Pro Ser Ala Glu Arg Glu Pro Gly Gly Ala Asn Ser Leu Leu
275 280 285
Cys Cys Leu Leu Gly His Cys Gln Leu Leu Trp Trp Leu Gln Pro Arg
290 295 300
Leu Val Leu Ser Gly His Thr His Ser Ala Cys Glu Val His His Gly
305 310 315 320
Gly Arg Val Pro Glu Leu Ser Val Pro Ser Phe Ser Trp Arg Asn Arg
325 330 335
Asn Asn Pro Ser Phe Ile Met Gly Ser Ile Thr Pro Thr Asp Tyr Thr
340 345 350
Leu Ser Lys Cys Tyr Leu Pro Arg Glu Asp Val Val Leu Ile Ile Tyr
355 360 365
Cys Gly Val Val Gly Phe Leu Val Val Leu Thr Leu Thr His Phe Gly
370 375 380
Leu Leu Ala Ser Pro Phe Leu Ser Gly Leu Asn Leu Leu Gly Lys Arg
385 390 395 400
Lys Thr Arg
<210> 2
<211> 1218
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atggcgatga tcgaattggg gtttggaaga cagaattttc atccattaaa gaggaagagt 60
tcattgctgt tgaaactcat agctgttgtc tttgctgtgc ttctattttg tgaattttta 120
atctattact tagcgatctt tcagtgtaat tggcctgaag tgaaaaccac agcctctgat 180
ggtgaacaga ccacacgtga gcctgtgctc aaagccatgt ttttggctga cacccatttg 240
cttggggaat tcctaggcca ctggctggac aaattacgaa gggaatggca gatggagaga 300
gcgttccaga cagctctgtg gttgctgcag ccggaagtcg tcttcatcct gggggatatc 360
tttgatgaag ggaagtggag cacccctgag gcctgggcgg atgatgtgga gcggtttcag 420
aaaatgttca gacacccaag tcatgtacag ctgaaggtag ttgctggaaa ccatgacatt 480
ggcttccatt atgagatgaa cacatacaaa gtagaacgct ttgagaaagt gttcagctct 540
gaaagactgt tttcttggaa aggcattaac tttgtgatgg tcaacagcgt ggcgctgaac 600
ggggatggct gtggcatctg ctctgaaaca gaagcagagc tcattgaagt ttctcacaga 660
ctgaactgct cccgagagca ttatcctctg tatcggagaa gtgatgctaa ctgttctggg 720
gaagacgctg ctcctccaga ggaaagggac atcccattta aggagaacta tgacgtgctt 780
tcacgggagg catcacaaaa ggaaagaagc gatggccagc ggccgtctgc tgagcggtga 840
gagcctggtt gaggatgagc taacagcctg ctttgctgtc ttctcggcca ctgtcagctg 900
ctgtggtggc tccagccgcg cctggttctc agtggccaca cgcacagcgc ctgcgaggtg 960
caccacgggg gccgagtccc cgagctcagc gtcccatctt tcagttggag gaacagaaac 1020
aaccccagtt tcatcatggg tagcatcacg cccacagact acaccctctc caagtgctac 1080
ctcccacgtg aggatgtggt tttgatcatc tactgtggag tggtgggctt ccttgtggtc 1140
ctcacactca ctcactttgg gcttctagcc tcaccttttc tttctggttt gaacttgctc 1200
ggaaagcgta agacaaga 1218
<210> 3
<211> 14
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tcctccagag gaaa 14
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggcgatga tcgaattgg 19
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
acgctttccg agcaagttc 19
Claims (9)
1. 1 mutain of metal tripolyphosphate esterase of people a kind of, which is characterized in that its amino acid sequence such as SEQ ID NO:Shown in 1.
2. a kind of encoding gene of 1 mutain of metal tripolyphosphate esterase 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 1 mutain of metal tripolyphosphate esterase of the people, just with the metal tripolyphosphate esterase 1 of people
The comparison result of the nucleotide sequence of normal albumen determines.
4. genetic chip according to claim 3, which is characterized in that the nucleotide probe is SEQ ID NO:Shown in 3
Base sequence.
5. a kind of monoclonal antibody of 1 mutain of metal tripolyphosphate esterase of specific recognition people, which is characterized in that it was encoded
Amino acid sequence is SEQ ID NO:Shown in 1.
6. a kind of ELISA kit for detecting the antibody of 1 mutain of metal tripolyphosphate esterase of people, which is characterized in that institute
Stating ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection pair of monoclonal antibody described in claim 5
1 albumen of metal tripolyphosphate esterase, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and the terminate liquid of elephant.
7. it is used to detect the ELISA kit of the antibody of 1 mutain of metal tripolyphosphate esterase of people according to claim 6,
It is characterized in that, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
8. a kind of antibody for 1 mutain of metal tripolyphosphate esterase detecting people based on the ELISA kit of claim 6 or 7
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, incubation at room temperature；
B, 1 albumen of metal tripolyphosphate esterase for detecting object is diluted to various concentration gradient using the sample diluting liquid, and will
1 albumen of metal tripolyphosphate esterase of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, incubation 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. the method for the antibody of 1 mutain of metal tripolyphosphate esterase of ELISA kit detection people according to claim 8,
It is characterized in that, further comprising：Blank control is tested.
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| CN201810261488.1A CN108546693A (en) | 2018-03-28 | 2018-03-28 | 1 mutain of metal tripolyphosphate esterase of people a kind of and its application |
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|---|---|---|---|
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ID=63517347
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| CN201810261488.1A Pending CN108546693A (en) | 2018-03-28 | 2018-03-28 | 1 mutain of metal tripolyphosphate esterase of people a kind of and its application |
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| Country | Link |
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2018
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Application publication date: 20180918 |