CN108588048A - The transaldolase mutain of people a kind of and its application - Google Patents
The transaldolase mutain of people a kind of and its application Download PDFInfo
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Abstract
The present invention relates to the transaldolase mutain of people a kind of and its applications, by regarding a large amount of transaldolase defect diseases and 5 phosphate isomerase enzyme deficiency disease patient of ribose as research case, genetic test is carried out to case and is analyzed, determine the mutain of the transaldolase of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the mutain of the transaldolase of the people, gene diagnosis for transaldolase defect disease and 5 phosphate isomerase enzyme deficiency disease of ribose provides impulse, and certain theoretical foundation is provided for the diagnosing and treating of relevant disease.
Description
Technical field
The present invention relates to the transaldolase mutains and its application of a kind of genetic engineering field more particularly to a kind of people.
Background technology
Transaldolase (transaldolase) is that a kind of dihydroxyacetone basal orientation 3- phosphoric acid in sedoheptulose 7-phosphate is sweet
It plays catalytic action on oily aldehyde during first carbon atom transfer, generates D- erythrose-4-phosphates and D-Fructose -6- phosphoric acid
Enzyme.
The transaldolase mutation of people can cause to seriously affect to human health.To certain diseases, especially turn aldehyde
During the peripheral blood of alcohol enzyme defect disease and ribose-5-phosphate isomerase deficiency disease patient carry out gene sequencing, find to suffer from
Whether the transaldolase of person is mutated, therefore, to the detection of the transaldolase of people mutation to judging human body with correlation disease
Disease has certain impulse.
Invention content
Present invention aims at the transaldolase mutain of people of offer a kind of and its applications.
Technical solution of the present invention includes：
In a first aspect, a kind of transaldolase mutain of people is provided, amino acid sequence such as SEQ ID NO:Shown in 1.
Second aspect provides a kind of encoding gene of the transaldolase mutain of people, nucleotide sequence such as SEQ ID
NO:Shown in 2.
The third aspect provides a kind of genetic chip, including：Solid phase carrier and fixed nucleotide probe on this carrier；
The nucleotide probe is normal with the transaldolase of people according to the nucleotide sequence of the transaldolase mutain of people described above
The comparison result of the nucleotide sequence of albumen determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of monoclonal antibody of the transaldolase mutain of specific recognition people, can be with SEQ
ID NO:Amino acid sequencespecific shown in 1 combines.
5th aspect provides a kind of ELISA kit for detecting the antibody of the transaldolase mutain of people, described
ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, the transaldolase for detecting object of said monoclonal antibody
Albumen, 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.
The present invention provides the transaldolase mutain of people a kind of and its applications, by a large amount of transaldolase defects disease and core
Sugar -5- phosphate isomerase enzyme deficiency disease patients carry out genetic test to case and analyze, determine that people's turns aldehyde as research case
The mutain of alcoholase prepares genetic chip, monoclonal antibody and ELISA reagents according to the transaldolase mutain of the people
Box, the gene diagnosis for transaldolase defect disease and ribose-5-phosphate isomerase deficiency disease provide impulse, are relevant disease
Diagnosing and treating certain theoretical foundation is provided.
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention and after coordinating attached drawing to be described in detail such as.
Description of the drawings
Fig. 1 is a kind of comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 2 is a kind of genetic chip layout that the embodiment of the present invention one provides；
Fig. 3 is a kind of colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 4 is another colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 5 is the standard curve that the offer of the embodiment of the present invention five is protein content；
Fig. 6 is the Western blot testing result schematic diagrames that the embodiment of the present invention five provides；
Fig. 7 is the standard curve that the offer of the embodiment of the present invention six is protein content；
Fig. 8 is the Western blot testing result schematic diagrames that the embodiment of the present invention six provides.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment one, genetic chip
It is possible, firstly, to be a kind of " detection side of mutain according to application No. is " 201710429915.8 ", patent name
Detection method described in the Chinese invention patent of method and device " determines the encoding gene of the transaldolase mutain of people,
Its nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, determine that the transaldolase of people is mutated egg according to the encoding gene
In vain, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the transaldolase mutain and its encoding gene of people, genetic chip is realized as follows
It prepares.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe according to the nucleotide sequence of the transaldolase mutain of people, with
The comparison result of the nucleotide sequence of the normal albumen of transaldolase of people determines, and according to the design principle of following probe, if
Count out the nucleotide probe of the specificity of the transaldolase mutain for people.
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 transaldolase mutain of people is corresponding with the normal albumen of the transaldolase of people
The comparison result of amino acid sequence please refers to Fig.1, wherein the Query sequences in Fig. 1 are that the transaldolase mutain of people corresponds to
Amino acid sequence, Sbjct sequences are the corresponding amino acid sequence of the normal albumen of transaldolase of people, Query sequences and Sbjct
Sequence between sequence is comparison result, as can be seen from FIG. 1, the transaldolase mutain of people relative to people transaldolase just
Normal albumen has several place's amino acid sequences that mutation and missing has occurred, according to SEQ ID NO:Nucleotide sequence shown in 2, with
And the comparison result of Fig. 1, whether the transaldolase in order to specific recognition object to be detected is mutated, then selecting
When taking nucleotide probe, nucleotide probe can be designed according to any one of following several modes mode：
1. before deletion sites with several nucleotide sequences (base sequence is constant) are respectively selected after deletion sites, it is raw
At nucleotide probe；
2. will include a nucleotide sequence of mutated site nucleotide as nucleotide probe.
In the present embodiment, according to SEQ ID NO:It nucleotide sequence shown in 2 and is set according to above-mentioned nucleotide probe
Principle is counted, 2. designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, the nucleotide probe
For：aaggccagtt cca.
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether transaldolase 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 transaldolase of people
In enzyme mutant albumen, select sequence corresponding from nucleotide probe different, and can be identical as the number of nucleotide probe base,
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.Preferably,
Choose nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base.In the embodiment of the present invention, base
Because the positive internal control probe sequence needed for chip can be：aatgcagaga atg.
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 15mL：
| 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：
(4) above-mentioned 8.5 μ L are added after mixing after being denaturalized in the RNA of ice bath.
(5) pipette tips mixing is used to centrifuge later.
(6) 40 DEG C of reaction 2h.
3, aaUTP marks cRNA synthesis
The configuration of NTP：
| 100mM ATP | 250μL |
| 100mM GTP | 250μL |
| 100mM CTP | 250μL |
| 100mM UTP | 187.5μL |
| RNase free H2O | 62.5μL |
| Total volume | 1000μL |
It is spare to be distributed into 10 pipes, notes：40 DEG C of heat preservation 1min before 50%PEG (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcription mix；
(1) configuration Transcription mix
| 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 4 × 44K of chip hybridization microarrays
(1) according to the form below prepares fragmentation mixed liquor and then carries out fragmentation in 60 DEG C of warm bath 30min.
(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, determines whether the transaldolase albumen of detection object is sent out according to scanning result
Mutation is given birth to.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, positive control is that green is glimmering
Light 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 object
Transaldolase albumen be mutated.In result shown in Fig. 4, negative control is colorless fluorescent, and positive control is that green is glimmering
Light shows that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, then showing to detect object
Transaldolase albumen do not mutate.
Embodiment three, specific recognition people transaldolase mutain monoclonal antibody preparation
1, according to base sequence (such as SEQ ID NO of the transaldolase mutain of people:Shown in 2) design sense primer is such as
SEQ ID NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (F)：atgtcgagct cacccgtg
Downstream primer (R)：ctttccattc tctgcatt
2, the DNA of detection object is that template carries out PCR amplification
| 10×Buffer | 5uL |
| dNTP | 2uL |
| ExTaq | 1uL |
| ddH2O | 5uL |
| Template DNA | 1uL |
| Primer (F) | 3uL |
| Primer (R) | 3uL |
| Total system | 20uL |
DNA to detect object carries out PCR amplification as template, obtains the transaldolase mutating protein gene complete slice of people
Section, and pMD19-T Vector (Takara companies) are connected, it is sequenced.Then antibody is prepared by special biotech firm, is
A kind of humanization or Chimeric antibodies.Wherein, the monoclonal antibody prepared can be with SEQ ID NO:Amino acid sequence shown in 1
Specific binding.The antibody of preparation is measured into content using ELISA method.
Example IV, transaldolase mutain 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, detect the transaldolase albumen of object, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution,
Developing solution and terminate liquid.
Wherein, the parameter of above-mentioned each composition at least can be a kind of following parameter：
ELISA ELISA Plates can be the ELISA enzyme marks in 96 holes；
ELIAS secondary antibody can be the Goat anti-Human IgG of diluted HRP (horseradish peroxidase) labels；Wherein, dilution times
Number can be 8000 times.
It can be 1 × PBS, pH to be coated with buffer solution:7.4；
ELISA ELISA Plates cleaning solution can be 1 × PBS solution containing 0.05% Tween-20；
Developing solution can be 3,3 ', 5,5 '-tetramethyl benzidines；
Terminate liquid can be the sulfuric acid solution of 2mol/L.
Embodiment five
In embodiments of the present invention, it using transaldolase defect patient as detection object, and is examined using ELISA kit
Whether the transaldolase albumen for surveying transaldolase defect patient is mutated, and this method at least may include as next
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 transaldolase albumen of transaldolase defect patient is diluted to various concentration gradient using sample diluting liquid,
And the transaldolase albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and it 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=0.99878, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire transaldolase defect disease
The concentration of the transaldolase albumen of patient.Using in ELISA method detection transaldolase defect patient's blood serum sample of foundation
The content of transaldolase 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 layers separation gel prepares system (Total Volum：15mL)
| ddH2O | 5.9mL |
| 30% acrylamide mixed liquor | 5.9mL |
| 1.5mol/L Tris(PH8.8) | 3.8mL |
| 10% SDS | 0.15mL |
| 10% ammonium persulfate | 0.15mL |
| TEMED | 0.006mL |
B. upper layer spacer gel concentration preparation system (Total Volum：8mL)
| ddH2O | 5.5mL |
| 30% acrylamide mixed liquor | 1.3mL |
| 1.0mol/L Tris(PH6.8) | 1.0mL |
| 10% SDS | 0.08mL |
| 10% ammonium persulfate | 0.08mL |
| TEMED | 0.008mL |
(3) lower layer's separation gel of respective volume is added in each offset plate, adds the sterile ddH of 1mL2O flattens separation
Glue blots remaining moisture in plastic plate after gelling to be separated is solid with filter paper, and upper layer is then added and concentrates glue, after being inserted into comb
Wait for upper layer concentration gelling solid.
2, albumen loading electrophoresis：
(1) comb is pulled up, duct label is carried out, 5 × SDS electrophoretic buffers is added, then to being added in protein sample
SDS-PAGE albumen sample-loading buffer (5 ×), boiling water bath heat 3-5min, loading；
(2) electrophoresis first is carried out with 80V voltages, after waiting for that band ran concentration glue, uses 100V voltages instead and run about 100min.
3, transferring film and incubation：
(1) first pvdf membrane is activated in methyl alcohol about 30s.Respectively by gel, foam-rubber cushion and pvdf membrane in advance in electrotransfer
30min is balanced in buffer solution.It is clipped by the sequence of filter paper-glue-film-filter paper, transferring film device is put into the principle of black flour according to black flour
In, refrigerator (ice bag) is added, about 120min is run with the electric current of 150mA in ice；
(2) it takes the film out after the completion of, is put into 5% skim milk at once, gently room temperature closes 1h on shaking table；
(3) PBST cleans closed film, cleans 3 times, each 10min；
(4) primary antibody for being diluted to corresponding multiple is added, 4 DEG C of incubator overnights are incubated；
(5) primary antibody is recycled, PBST cleans film 3 times, each 10min；
(6) secondary antibody is (with 3% milk with 1:5000-1:10000 dilutions) incubation at room temperature film 2h；
(7) film is cleaned 3 times with PBST, each 10min；
(8) 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 transaldolase mutain monoclonal antibody of the standby people of corporation, and secondary antibody is horseradish peroxidating
The Goat anti-Human IgG of object enzyme label.
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 75KD
There is apparent band, wherein transaldolase mutant protein molecules amount is about 75KD, shows transaldolase defect patient's
Transaldolase albumen is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, it using ribose-5-phosphate isomerase deficiency disease patient as detection object, and utilizes
Whether the transaldolase albumen that ELISA kit detects ribose-5-phosphate isomerase deficiency disease patient is mutated, the party
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 transaldolase albumen for detecting object is diluted to various concentration gradient using sample diluting liquid, and will be different dense
The transaldolase albumen of degree 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=0.99936, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire ribose -5- phosphorus in sample
The concentration of the transaldolase albumen of acid isomer enzyme deficiency disease patient.Ribose -5- phosphate isomerases are detected using the ELISA method of foundation
The content of transaldolase zymoprotein in enzyme deficiency disease patient serum sample.It is used in combination Western blot to be identified.Referring to FIG. 7,
For the standard curve of light absorption value.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 75KD, wherein transaldolase mutant protein molecules amount is about 75KD, shows the ribose -5- phosphoric acid
The transaldolase albumen of isomery enzyme deficiency disease 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>The transaldolase mutain of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 306
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Ser Ser Ser Pro Val Lys Arg Gln Arg Met Glu Ser Ala Leu Asp
1 5 10 15
Gln Leu Lys Ala Ser Ser Thr His Thr Val Gln Trp Trp Ala Gln Thr
20 25 30
Thr Arg Ala Gly Thr Ile Ala Tyr Ala Thr Gly Ser Gln Glu Asp Gln
35 40 45
Ile Lys Asn Ala Ile Asp Lys Leu Phe Val Leu Phe Gly Ala Glu Ile
50 55 60
Leu Lys Lys Ile Pro Gly Arg Val Ser Thr Glu Val Asp Ala Arg Leu
65 70 75 80
Ser Phe Asp Lys Asp Ala Met Val Ala Arg Ala Arg Arg Leu Ile Glu
85 90 95
Leu Tyr Lys Glu Ala Gly Ile Ser Lys Asp Arg Ile Leu Ile Lys Leu
100 105 110
Ser Ser Thr Trp Glu Gly Ile Gln Ala Gly Lys Glu Leu Glu Glu Gln
115 120 125
His Gly Ile His Cys Asn Met Thr Leu Leu Phe Ser Phe Ala Gln Ala
130 135 140
Val Ala Cys Ala Glu Ala Gly Val Thr Leu Ile Ser Pro Phe Val Gly
145 150 155 160
Arg Ile Leu Asp Trp His Val Ala Asn Thr Asp Lys Lys Ser Tyr Glu
165 170 175
Pro Leu Glu Asp Pro Gly Val Lys Ser Val Thr Lys Ile Tyr Asn Tyr
180 185 190
Tyr Lys Lys Phe Ser Tyr Lys Thr Ile Val Met Gly Ala Ser Phe Arg
195 200 205
Asn Thr Gly Glu Ile Lys Ala Leu Ala Gly Cys Asp Phe Leu Thr Ile
210 215 220
Ser Pro Lys Leu Leu Gly Glu Leu Leu Gln Asp Asn Ala Lys Leu Val
225 230 235 240
Pro Val Leu Ser Ala Lys Ala Ala Gln Ala Ser Asp Leu Glu Lys Ile
245 250 255
His Leu Asp Glu Lys Ser Phe Arg Trp Leu His Asn Glu Asp Gln Met
260 265 270
Ala Val Glu Lys Leu Ser Asp Gly Ile Arg Lys Phe Ala Ala Asp Ala
275 280 285
Val Lys Leu Glu Arg Met Leu Thr Glu Arg Met Phe Asn Ala Glu Asn
290 295 300
Gly Lys
305
<210> 2
<211> 918
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atgtcgagct cacccgtgaa gcgtcagagg atggagtccg cgctggacca gctcaaggcc 60
agttccaccc acacggtgca gtggtgggcc cagacgacca gggcggggac gattgcctat 120
gccaccgggt cacaagagga ccagattaaa aatgctattg ataaactttt tgtgttgttt 180
ggagcagaaa tactaaagaa gattccgggc cgagtatcca cagaagtaga cgcaaggctc 240
tcctttgata aagatgcgat ggtggccaga gccaggcggc tcatcgagct ctacaaggaa 300
gctgggatca gcaaggaccg aattcttata aagctgtcat caacctggga aggaattcag 360
gctggaaagg agctcgagga gcagcacggc atccactgca acatgacgtt actcttctcc 420
ttcgcccagg ctgtggcctg tgccgaggcg ggtgtgaccc tcatctcccc atttgttggg 480
cgcatccttg attggcatgt ggcaaacacc gacaagaaat cctatgagcc cctggaagac 540
cctggggtaa agagtgtcac taaaatctac aactactaca agaagtttag ctacaaaacc 600
attgtcatgg gcgcctcctt ccgcaacacg ggcgagatca aagcactggc cggctgtgac 660
ttcctcacca tctcacccaa gctcctggga gagctgctgc aggacaacgc caagctggtg 720
cctgtgctct cagccaaggc ggcccaagcc agtgacctgg aaaaaatcca cctggatgag 780
aagtctttcc gttggttgca caacgaggac cagatggctg tggagaagct ctctgacggg 840
atccgcaagt ttgccgctga tgcagtgaag ctggagcgga tgctgacaga acgaatgttc 900
aatgcagaga atggaaag 918
<210> 3
<211> 13
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
aaggccagtt cca 13
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atgtcgagct cacccgtg 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ctttccattc tctgcatt 18
Claims (7)
1. a kind of mutain of the transaldolase 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 transaldolase mutain of people, which is characterized in that its nucleotide sequence such as SEQ ID NO:2
It is shown.
3. a kind of genetic chip, which is characterized in that including：Solid phase carrier and fixed nucleotide probe on this carrier；It is described
The nucleotide sequence of the nucleotide probe transaldolase mutain of people according to claim 2, just with the transaldolase 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 the transaldolase mutain of specific recognition people, which is characterized in that it can be with SEQ ID
NO:Amino acid sequencespecific shown in 1 combines.
6. a kind of ELISA kit for detecting the antibody of the transaldolase mutain of people, which is characterized in that described
ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection object of monoclonal antibody described in claim 5
Transaldolase albumen, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and terminate liquid.
7. being used to detect the ELISA kit of the antibody of the transaldolase mutain of people, feature according to claim 6
It is, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810457011.0A CN108588048A (en) | 2018-05-14 | 2018-05-14 | The transaldolase mutain of people a kind of and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810457011.0A CN108588048A (en) | 2018-05-14 | 2018-05-14 | The transaldolase mutain of people a kind of and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108588048A true CN108588048A (en) | 2018-09-28 |
Family
ID=63637413
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810457011.0A Withdrawn CN108588048A (en) | 2018-05-14 | 2018-05-14 | The transaldolase mutain of people a kind of and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108588048A (en) |
-
2018
- 2018-05-14 CN CN201810457011.0A patent/CN108588048A/en not_active Withdrawn
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Application publication date: 20180928 |