CN108424884A - The glutathione S-transferase κ 1b mutains of people a kind of and its application - Google Patents
The glutathione S-transferase κ 1b mutains of people a kind of and its application Download PDFInfo
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Abstract
The present invention relates to the glutathione S transferase κ 1b mutains of people a kind of and its applications, using a large amount of leukaemics as research case, genetic test is carried out to case and is analyzed, determine the mutain of the glutathione S transferase κ 1b of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the glutathione S transferase κ 1b mutains of the people, impulse is provided for the gene diagnosis of leukaemia, realizes the diagnosing and treating of leukaemia.
Description
Technical field
The present invention relates to the glutathione S-transferase κ 1b of a kind of genetic engineering field more particularly to a kind of people to be mutated egg
Its application of bletilla.
Background technology
Glutathione S-transferase (glutathione S-transferase, GST) is the pass of glutathione association reaction
Key enzyme is catalyzed the initial step of glutathione association reaction, is primarily present in cytosol.Glutathione S-transferase is in toxicology
On have certain importance.It can be catalyzed the association reaction of the glutathione and various electrophilic exogenous polyamines of nucleophilicity.
Many exogenous polyamines easily form certain bioactivity intermediate products in bioconversion first-phase reaction, they can be with cell
Covalent bond occurs for large biological molecule important component, is damaged to body.After glutathione is in connection, this can be prevented
Kind covalent bond, plays detoxication.It has been investigated that glutathione S-transferase and leukaemia important role, and paddy
The sweet peptide S- transferases κ 1b of Guang (1 isoform b of glutathione S-transferase kappa) are that glutathione S-turns
The homologous protein of enzyme is moved, therefore, to the detection of the glutathione S-transferase κ 1b mutation of people to judging human body whether with white blood
Disease has certain impulse.
Invention content
Present invention aims at the glutathione S-transferase κ 1b mutains of people of offer a kind of and its applications.
Technical solution of the present invention includes：
In a first aspect, providing a kind of glutathione S-transferase κ 1b mutains of people, amino acid sequence such as SEQ ID
NO:Shown in 1.
Second aspect provides a kind of encoding gene of the mutain of the glutathione S-transferase κ 1b of people, nucleotide
Sequence such as SEQ ID NO:Shown in 2.
The third aspect provides a kind of genetic chip, including：Solid phase carrier and fixed nucleotide probe on this carrier；
The nucleotide probe is according to the nucleotide sequence of the glutathione S-transferase κ 1b mutains of the people, the paddy Guang with people
The comparison result of the nucleotide sequence of the normal albumen of sweet peptide S- transferase κ 1b determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
It is anti-to provide a kind of monoclonal of the glutathione S-transferase κ 1b mutains of specific recognition people for fourth aspect
The amino acid sequence of body, coding is SEQ ID NO:Shown in 1.
5th aspect provides a kind of ELISA for detecting the antibody of the glutathione S-transferase κ 1b mutains of people
Kit, the ELISA kit include：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection pair of said monoclonal antibody
The glutathione S-transferase κ 1b albumen of elephant, 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 glutathione S-transferase κ detecting people based on any of the above-described ELISA kit
The method of the antibody of 1b mutains, including：
A, said monoclonal antibody is diluted using the coating buffer solution, and the monoclonal after dilution is resisted
Body is loaded into the hole of ELISA ELISA Plates, and is incubated at room temperature；
B, the glutathione S-transferase κ 1b albumen for detecting object is diluted to various concentration using the sample diluting liquid
Gradient, and the glutathione S-transferase κ 1b albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, room
Temperature is incubated；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature is protected from light incubation, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
Preferably, further comprise：Blank control is tested.
The present invention provides the glutathione S-transferase κ 1b mutains of people a kind of and its applications, by a large amount of leukaemia
Patient carries out genetic test to case and analyzes, determine the mutation of the glutathione S-transferase κ 1b of people as research case
Albumen prepares genetic chip, monoclonal antibody and ELISA reagents according to the glutathione S-transferase κ 1b mutains of the people
Box provides impulse for the gene diagnosis of leukaemia, realizes the diagnosing and treating of leukaemia.
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.
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 glutathione S-transferase κ 1b mutains of people
Encoding gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the paddy Guang of people is determined according to the encoding gene
Sweet peptide S- transferases κ 1b mutains, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, it according to the glutathione S-transferase κ 1b mutains and its encoding gene of people, realizes as follows
The preparation of genetic chip.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the core of the glutathione S-transferase κ 1b mutains of people
Nucleotide sequence determines with the comparison result of the nucleotide sequence of the normal albumen of glutathione S-transferase κ 1b of people, and according to
The design principle of following probe designs the nucleotide of the specificity of the glutathione S-transferase κ 1b mutains for people
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, which stablizes secondary structure pairing bases longs, is less than 4bp, can ensure in this way will not
Hybridization efficiency is influenced because of the secondary structure of nucleotide probe internal stability；
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 glutathione S-of glutathione S-transferase κ 1b the mutains corresponding amino acid sequence and people of people
The comparison result of the corresponding amino acid sequence of the normal albumen of transferase κ 1b please refers to Fig.1, wherein the Query sequences in Fig. 1 are
The corresponding amino acid sequence of glutathione S-transferase κ 1b mutains of people, Sbjct sequences are glutathione S-transfers of people
The corresponding amino acid sequence of the normal albumen of enzyme κ 1b, the sequence between Query sequences and Sbjct sequences is comparison result, according to figure
1 it is found that people glutathione S-transferase κ 1b normal albumen of the glutathione S-transferase κ 1b mutains relative to people,
It is mutated at several positions, is lacked at several positions, according to SEQ ID NO:Nucleotides sequence shown in 2
Whether the comparison result of row and Fig. 1, send out in order to the glutathione S-transferase κ 1b of specific recognition object to be detected
Mutation has been given birth to, then when choosing nucleotide probe, nucleotide can be designed according to any one of following several modes mode
Probe：
1. before deletion sites with respectively select several nucleotide sequences after deletion sites, generate 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, designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, which is：
ttcttccaga at。
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether the glutathione S-transferase κ 1b of detection people mutate
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 gluathione of people
In peptide S- transferase κ 1b mutains, select sequence corresponding from nucleotide probe different, and can be with nucleotide probe base
Number it is identical, one section of nucleotide sequence that can also be different from the number of nucleotide probe base is visited as positive internal control Quality Control
Needle.Preferably, nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen.The present invention is real
It applies in example, the positive internal control probe sequence needed for genetic chip can be：aaggactata ca.
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 to subtract T7 Promotor according to 11.5 μ L of total volume
The 5 μ L of addition of primer, then subtract the addition of total serum IgE and calculate and get.
(2) 10min, and ice bath 5min are kept the temperature at 65 DEG C, and 5 × First Strand B μ ffer are preheated at 65 DEG C
5min。
(3) following cDNA synthetic systems are configured：
| 5×First Strand Buffer | 4μL |
| 0.1M DTT | 2μL |
| 10mM dNTP mix | 1μL |
| MMLV RT | 1μL |
| RNase OUT | 0.5μL |
| Total volume | 8.5μL |
(4) above-mentioned 8.5 μ L are added after mixing after being denaturalized in the RNA of ice bath.
(5) pipette tips mixing is used to centrifuge later.
(6) 40 DEG C of reaction 2h.
3, aaUTP marks cRNA synthesis
The configuration of NTP：
It is spare to be distributed into 10 pipes, notes：40 DEG C of heat preservation 1min before 50%PEG (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcription mix；
(1) configuration Transcription mix
| RNase-free Water | 5.7μL |
| 4×Transcription Buffer | 20μL |
| NTP | 16μL |
| 0.1M DTT | 6μL |
| 50%PEG | 6.4μL |
| aa-UTP(25mM) | 4μL |
| Inorganic Pyrophosphatase | 0.6μL |
| T7RNA 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 LRNase free water are added, 350 μ L Buffer RLT are added and mix well.
(2) 250 μ L absolute ethyl alcohols are added, Tip mix well.
(3) total 700 solution of the μ L containing total serum IgE are transferred in the RNeasy pillars being sleeved in 2mL centrifuge tubes >=8000g from
Heart 15-30s, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=8000g centrifuge washings 15-30s discards filter
It crosses liquid and discards the casing of filtered solution and 2mL by RNeasy in >=8000g centrifuge washings 2min with 500 μ L Buffer RPE again
Mini pillars are transferred in a new 1.5mL Eppendorf pipes.
(5) water for drawing 30 μ L RNase free stands 1min, >=8000g centrifugation elutions 1min.
(6) it is primary to repeat step (5).
5, cRNA concentration mensurations
With spectrophotometric analysis cRNA concentration.It needs to measure the light absorption value at 260nm and 280nm to determine the dense of sample
Degree and purity, A260/A280 should be purer cRNA (ratio 1.9-2.1 also can) close to 2.0.
6, cRNA fluorescents mark；
(1) 4 μ g of above-mentioned cRNA are taken and are concentrated into 6.6 μ L.
(2) add 10 μ L DMSO mixings.
(3) add the sodium bicarbonate (NaHCO that the 0.3M pH of 3.4 μ L are 9.03) and mixing.
(4) above-mentioned 20 μ L cRNA mixtures are added in fluorescent dye Cy3 simultaneously mixing.25 DEG C of heat preservation 1h.
(5) plus 9 μ L 4M Hydroxylamine mixings after 25 DEG C heat preservation 15min.
7, fluorescent marker cRNA Sample Purification on Single
The process that specific steps are purified with cRNA in the step 4 of the present embodiment, this step are not repeating.
8, cRNA sample fragments and chip hybridization 4x44K microarrays
(1) according to the form below prepares fragmentation mixed liquor and then carries out fragmentation in 60 DEG C of warm bath 30min.
| 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 2 by chip layout blank,
On negative, positive, experimental group position.It covers chip and is sealed in hybridizing box, 60 DEG C roll hybridization 16h.
9, chip washs
Washing lotion 1 (1L) configures：
| DEPC-H2O | 700mL |
| 20×SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 2 (1L) configures：
| DEPC-H2O | 997mL |
| 20×SSPE | 3.0mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 3：Stabilization and Drying Solution
(1) it takes out chip and washs 1min in washing lotion 1；
(2) chip is put into washing lotion 2 again and washs 1min (37 DEG C)；
(3) chip is finally washed into 30s in washing lotion 3.
10, chip scanning
Chip is scanned in scanner, the glutathione S-transferase κ of detection object is determined according to scanning result
Whether 1b albumen is mutated.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence is positive right
According to for green fluorescence, showing that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence, then table
The glutathione S-transferase κ 1b albumen of bright detection object is mutated.In result shown in Fig. 4, negative control is colourless
Fluorescence, positive control are green fluorescence, show that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen
Fluorescence, then showing that the glutathione S-transferase κ 1b albumen for detecting object does not mutate.
Embodiment three, specific recognition people glutathione S-transferase κ 1b mutains monoclonal antibody preparation
1, according to base sequence (such as SEQ ID NO of the glutathione S-transferase κ 1b mutains of people:Shown in 2) it sets
Count sense primer such as SEQ ID NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (F)：atggggcccc tgccg
Downstream primer (R)：aagtctggca ttcacggctg g
2, the DNA of detection object is that template carries out PCR amplification
DNA to detect object carries out PCR amplification as template, obtains the glutathione S-transferase κ 1b mutains of people
The complete segment of gene, and pMD19-T Vector (Takara companies) are connected, it is sequenced.Then by special biotech firm's system
Standby antibody is a kind of humanization or Chimeric antibodies.The antibody of preparation is measured into content using ELISA method.
Example IV, glutathione S-transferase κ 1b mutains for detecting people antibody ELISA kit
In the present embodiment, the group of ELISA kit becomes：It is coated with the ELISA enzymes of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, the glutathione S-transferase κ 1b albumen for detecting object, sample diluting liquid, coating buffer solution, ELISA enzymes
Target cleaning solution, developing solution and terminate liquid.
Wherein, the parameter of above-mentioned each composition at least can be a kind of following parameter：
ELISA ELISA Plates can be the ELISA enzyme marks in 96 holes；
ELIAS secondary antibody can be the Goat anti-Human IgG of diluted HRP (horseradish peroxidase) labels；Wherein, dilution times
Number can be 8000 times.
It can be 1 × PBS, pH to be coated with buffer solution:7.4；
ELISA ELISA Plates cleaning solution can be 1 × PBS solution containing 0.05%Tween-20；
Developing solution can be 3,3 ', 5,5 '-tetramethyl benzidines；
Terminate liquid can be the sulfuric acid solution of 2mol/L.
Embodiment five
In embodiments of the present invention, using leukaemic as detection object, and the white blood is detected using ELISA kit
Whether the glutathione S-transferase κ 1b albumen 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, the glutathione S-transferase κ 1b albumen of leukaemic is diluted to various concentration ladder using sample diluting liquid
Degree, and the glutathione S-transferase κ 1b albumen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and room
Temperature is incubated 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, 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=0.99992, 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 paddy of leukaemic
The concentration of the sweet peptide S- transferases κ 1b albumen of Guang.Serum of leukaemia sample Glutathione is detected using the ELISA method of foundation
The content of peptide S- transferase κ 1b albumen.It is used in combination Western blot to be identified.Referring to FIG. 5, the standard for light absorption value is bent
Line.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot are identified
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(PH 8.8) | 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(PH 6.8) | 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 glutathione S-transferase κ 1b mutain monoclonal antibodies of the standby people of corporation, and secondary antibody is
The Goat anti-Human IgG of horseradish peroxidase-labeled.
J, Western blot Analysis of test results：It is shown according to Western blot experimental procedures as a result, such as Fig. 6 institutes
Show, wherein the Marker in Fig. 6 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 70KD
There is apparent band, wherein the molecular weight of glutathione S-transferase κ 1b mutains is about 70KD, shows the leukaemia
The glutathione S-transferase κ 1b albumen of 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 glutathione S-transferase κ 1b mutains of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 281
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Gly Pro Leu Pro Arg Thr Val Glu Leu Phe Tyr Asp Val Leu Ser
1 5 10 15
Pro Tyr Ser Trp Leu Gly Phe Glu Ile Leu Cys Arg Tyr Gln Asn Ile
20 25 30
Trp Asn Ile Asn Leu Gln Leu Arg Pro Ser Leu Ile Thr Gly Ile Met
35 40 45
Lys Asp Ser Gly Asn Lys Pro Pro Gly Leu Leu Pro Arg Lys Gly Leu
50 55 60
Tyr Met Ala Asn Asp Leu Lys Leu Leu Arg His His Leu Gln Ile Pro
65 70 75 80
Ile His Phe Pro Lys Asp Phe Leu Ser Val Met Leu Glu Lys Gly Ser
85 90 95
Leu Ser Ala Met Arg Phe Leu Thr Ala Val Asn Leu Glu His Pro Glu
100 105 110
Met Leu Glu Lys Ala Ser Arg Glu Leu Trp Met Arg Val Trp Ser Arg
115 120 125
Val Ser Val Gly Leu Trp Glu Ser Ser Gly Arg Thr Leu Asp Asp Phe
130 135 140
Leu Thr Phe Pro Arg His Val Phe Arg Val Met Ile Leu Pro Arg Pro
145 150 155 160
Gly Ile Tyr Cys Pro Pro Ser His Thr Pro Leu Pro Ala Pro Pro Ser
165 170 175
Cys Cys Leu Leu Phe Pro Ile Thr Val Ala His Val Asp Gly Gln Thr
180 185 190
His Met Leu Phe Gly Ser Asp Arg Met Glu Leu Leu Ala His Leu Leu
195 200 205
Gly Glu Lys Trp Met Gly Pro Ile Pro Pro Ala Val Asn Ala Arg Leu
210 215 220
Phe Gln Asn Glu Asp Ile Thr Glu Pro Gln Ser Ile Leu Ala Ala Ala
225 230 235 240
Glu Lys Ala Gly Met Ser Ala Glu Gln Ala Gln Gly Leu Leu Glu Lys
245 250 255
Ile Ala Thr Pro Lys Val Lys Asn Gln Leu Lys Glu Thr Thr Glu Ala
260 265 270
Ala Cys Arg Tyr Gly Ala Phe Gly Leu
275 280
<210> 2
<211> 843
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atggggcccc tgccgcgcac cgtggagctc ttctatgacg tgctgtcccc ctactcctgg 60
ctgggcttcg agatcctgtg ccggtatcag aatatctgga acatcaacct gcagttgcgg 120
cccagcctca taacagggat catgaaagac agtggaaaca agcctccagg tctgcttccc 180
cgcaaaggac tatacatggc aaatgactta aagctcctga gacaccatct ccagattccc 240
atccacttcc ccaaggattt cttgtctgtg atgcttgaaa aaggaagttt gtctgccatg 300
cgtttcctca ccgccgtgaa cttggagcat ccagagatgc tggagaaagc gtcccgggag 360
ctgtggatgc gcgtctggtc aagggtgagt gtggggctct gggaatcctc tgggaggacc 420
ttggatgact ttctgacctt ccccaggcac gttttcaggg tcatgatcct gccccgcccg 480
gggatctact gtcctcccag tcacacccct ctccccgcac cgccttcctg ctgtcttctc 540
ttcttccaga atgaagacat caccgagccg cagagcatcc tggcggctgc agagaaggct 600
ggtatgtctg cagaacaagc ccagggactt ctggaaaaga tcgcaacgcc aaaggtgaag 660
aaccagctca aggagaccac tgaggcagcc tgcagatacg gagcctttgg gctgcccatc 720
accgtggccc atgtggatgg ccaaacccac atgttatttg gctctgaccg gatggagctg 780
ctggcgcacc tgctgggaga gaagtggatg ggccctatac ctccagccgt gaatgccaga 840
ctt 843
<210> 3
<211> 12
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ttcttccaga at 12
<210> 4
<211> 15
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggggcccc tgccg 15
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
aagtctggca ttcacggctg g 21
Claims (9)
1. the glutathione S-transferase κ 1b mutains 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 the glutathione S-transferase κ 1b mutains of people, which is characterized in that its nucleotide sequence is such as
SEQ ID NO:Shown in 2.
3. a kind of genetic chip, which is characterized in that including：Solid phase carrier and fixed nucleotide probe on this carrier；It is described
Nucleotide probe is according to the nucleotide sequence of the glutathione S-transferase κ 1b mutains of the people, the glutathione with people
The comparison result of the nucleotide sequence of the normal albumen of S- transferase κ 1b 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 glutathione S-transferase κ 1b mutains of specific recognition people, which is characterized in that its
The amino acid sequence of coding is SEQ ID NO:Shown in 1.
6. a kind of ELISA kit for detecting the antibody of the glutathione S-transferase κ 1b mutains of people, feature exists
In the ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, inspection of monoclonal antibody described in claim 5
It surveys the glutathione S-transferase κ 1b albumen of object, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, develop the color
Liquid and terminate liquid.
7. being used to detect the ELISA examinations of the antibody of the glutathione S-transferase κ 1b mutains of people according to claim 6
Agent box, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
8. a kind of glutathione S-transferase κ 1b mutains detecting people based on the ELISA kit of claim 6 or 7
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, incubation at room temperature；
B, the glutathione S-transferase κ 1b albumen for detecting object is diluted to various concentration ladder using the sample diluting liquid
Degree, and the glutathione S-transferase κ 1b albumen of various concentration gradient 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, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature is protected from light incubation, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
9. ELISA kit detects the antibody of the glutathione S-transferase κ 1b mutains of people according to claim 8
Method, which is characterized in that further comprise：Blank control is tested.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810255181.0A CN108424884A (en) | 2018-03-27 | 2018-03-27 | The glutathione S-transferase κ 1b mutains of people a kind of and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108424884A true CN108424884A (en) | 2018-08-21 |
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ID=63159786
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|---|---|---|---|
| CN201810255181.0A Pending CN108424884A (en) | 2018-03-27 | 2018-03-27 | The glutathione S-transferase κ 1b mutains of people a kind of and its application |
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| Country | Link |
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| CN (1) | CN108424884A (en) |
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2018
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