CN108866023A - The serine/threonine protein kitase PINK1 mutain of people a kind of and its application - Google Patents
The serine/threonine protein kitase PINK1 mutain of people a kind of and its application Download PDFInfo
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Abstract
The present invention relates to the serine/threonine protein kitase PINK1 mutain of people a kind of and its applications, using several leukaemia, Parkinsonian as research case, genetic test is carried out to case and is analyzed, determine the mutain of the serine/threonine protein kitase PINK1 of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the serine/threonine protein kitase PINK1 mutain of the people, gene diagnosis for leukaemia, Parkinson's disease provides impulse, to realize that leukaemia, the diagnosing and treating of Parkinson's disease provide scientific basic.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to the serine/threonine protein kitase PINK1 of people a kind of
Mutain and its application.
Background technique
PINK1 (PTEN induced putative kinase 1) is a kind of protein kinase, can be in the thin of entire body
It is expressed in born of the same parents, in heart, the expression that muscle and brain etc. consume energy in high organ is especially prominent.In the cell, it is predominantly located at
In the inner membrance of mitochondria.The function of PINK1 is understood completely not yet.By inference during cellular stress, such as there is exception in cell
When high energy demand, have the function of protecting intracellular mitochondria.
Recently, research finds that the mutation of PINK1 gene and leukaemia have certain association, and PINK1 gene is prominent
Change will lead to autosomal recessive inheritance early onset Parkinson's disease, therefore, to the PINK1 serine/threonine protein kitase of people
Whether the detection of PINK1 can suffer from leukaemia for human body, Parkinson's disease provides certain impulse.
Summary of the invention
It is an object of that present invention to provide the serine/threonine protein kitase PINK1 mutain of people a kind of and its applications.
Technical solution of the present invention includes：
In a first aspect, providing the serine/threonine protein kitase PINK1 mutain of people a kind of, amino acid sequence
Such as SEQ ID NO:Shown in 1.
Second aspect provides a kind of encoding gene of the mutain of the serine/threonine protein kitase PINK1 of people,
Its 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 according to the nucleotide sequence of the serine/threonine protein kitase PINK1 mutain of people described above,
It is determined with the comparison result of the nucleotide sequence of the normal albumen of serine/threonine protein kitase PINK1 of people.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3；
Or, the nucleotide probe is SEQ ID NO:Base sequence shown in 4；
Or, the nucleotide probe is SEQ ID NO:Base sequence shown in 5.
Fourth aspect provides a kind of list of the serine/threonine protein kitase PINK1 mutain of specific recognition people
Clonal antibody, can be with SEQ ID NO:Amino acid sequencespecific shown in 1 combines.
5th aspect, provides a kind of for detecting the antibody of the serine/threonine protein kitase PINK1 mutain of people
ELISA kit, the ELISA kit includes：Be coated with the ELISA ELISA Plate of said monoclonal antibody, ELIAS secondary antibody,
Serine/threonine protein kitase PINK1 albumen, sample diluting liquid, coating buffer, the ELISA ELISA Plate of test object are washed
Wash liquid, developing solution and terminate liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeled.
The present invention provides the serine/threonine protein kitase PINK1 mutain of people a kind of and its applications, if will be white
Blood disease, Parkinsonian carry out genetic test to case and analyze, determine serine/Soviet Union's ammonia of people as research case
The mutain of pka acid PINK1 prepares base according to the serine/threonine protein kitase PINK1 mutain of the people
Because of chip, monoclonal antibody and ELISA kit, the gene diagnosis for leukaemia, Parkinson's disease provides impulse, to realize
Leukaemia, the diagnosing and treating of Parkinson's disease provide scientific basic.
The above description is only an overview of the technical scheme 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, the following is a detailed description of the preferred embodiments of the present invention and the accompanying drawings.
Detailed description of the invention
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 another comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 4 is a kind of genetic chip layout that the embodiment of the present invention one provides；
Fig. 5 is a kind of colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 6 is another colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 7 is the standard curve for the protein content that the embodiment of the present invention five provides；
Fig. 8 is the Western blot testing result schematic diagram that the embodiment of the present invention five provides；
Fig. 9 is the standard curve for the protein content that the embodiment of the present invention six provides；
Figure 10 is the Western blot testing result schematic diagram that the embodiment of the present invention six provides.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment one, genetic chip
It is possible, firstly, to 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 serine/threonine protein kitase PINK1 of people
The encoding gene of mutain, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, it is determined according to the encoding gene
The serine/threonine protein kitase PINK1 mutain of people, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the serine/threonine protein kitase PINK1 mutain and its encoding gene of people, according to as follows
Mode realizes the preparation of genetic chip.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is mutated according to the serine/threonine protein kitase PINK1 of people
The nucleotide sequence of albumen, the comparison knot with the nucleotide sequence of the normal albumen of serine/threonine protein kitase PINK1 of people
Fruit determines, and according to the design principle of following probe, and the serine/threonine protein kitase PINK1 designed for people is prominent
The nucleotide probe of the white specificity of a kink of preserved egg.
Wherein, the principle of nucleotide probe design is as follows：
1. nucleotide probe Tm value should be close to the average Tm value of whole gene group, 5 DEG C of fluctuation up and down；
2. the duplicate single base of nucleotide probe intramolecular is continuously no more than 4；
3. G+C content is 40%-60%, the specificity that non-specific hybridization guarantees 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 guarantee 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 silk ammonia of serine/threonine protein kitase PINK1 the mutain corresponding amino acid sequence and people of people
Acid/normal the albumen of Serineprotein kinase PINK1 corresponding amino acid sequence comparison result is referring to FIG. 1, wherein, in Fig. 1
Query sequence be people the corresponding amino acid sequence of serine/threonine protein kitase PINK1 mutain, Sbjct sequence
It is the corresponding amino acid sequence of the normal albumen of serine/threonine protein kitase PINK1 of people, Query sequence and Sbjct sequence
Between sequence be comparison result, as can be seen from FIG. 1, the serine/threonine protein kitase PINK1 mutain of people relative to
The normal albumen of serine/threonine protein kitase PINK1 of people, is inserted into and is mutated, according to SEQ ID NO:Shown in 2
The comparison result of nucleotide sequence and Fig. 1, in order to the serine/threonine protein of specific recognition object to be detected
Whether kinases PINK1 is mutated, then when choosing nucleotide probe, it can be according to any one of following several modes
Mode designs nucleotide probe：
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, at least can be designed that following several preferably nucleotide probes in the manner described above：
Such as SEQ ID NO:Nucleotide probe shown in 3 is：ccaggtacca gt；
Such as SEQ ID NO:Nucleotide probe shown in 4 is：tgccttcccc tt；
Such as SEQ ID NO:Nucleotide probe shown in 5 is：agaggtccaa g.
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
When the serine/threonine protein kitase PINK1 mutain of people is compared in NCBI, Soviet Union's door in comparison
The normal albumen of serine/threonine protein kitase PINK1 of cured orangutan is answered, comparison result is referring to FIG. 2, Query sequence in Fig. 2
Column are the corresponding amino acid sequences of serine/threonine protein kitase PINK1 mutain of people, and Sbjct sequence is that Soviet Union's door is answered
The corresponding amino acid sequence of the normal albumen of serine/threonine protein kitase PINK1 of cured orangutan, Query sequence and Sbjct sequence
Sequence between column is comparison result, as can be seen from FIG. 2, the serine/threonine protein kitase PINK1 mutain of people and Soviet Union
The homology that door answers the normal albumen of serine/threonine protein kitase PINK1 of cured orangutan is 77%, and according to the silk ammonia of people
The normal albumen of acid/Serineprotein kinase PINK1 mutain and people serine/threonine protein kitase PINK1 it is homologous
Rate is 77%, and homology is identical, is thus illustrated, test object goes out after serine/threonine protein kitase PINK1 mutation
Existing atavism.
When the serine/threonine protein kitase PINK1 mutain of people is compared in NCBI, grivet in comparison
The normal albumen of serine/threonine protein kitase PINK1, comparison result is referring to FIG. 3, Query sequence is people in Fig. 3
The corresponding amino acid sequence of serine/threonine protein kitase PINK1 mutain, Sbjct sequence are serine/Soviet Unions of grivet
The corresponding amino acid sequence of the normal albumen of propylhomoserin protein kinase PINK1, sequence between Query sequence and Sbjct sequence be than
Pair as a result, as can be seen from FIG. 3, the serine/threonine protein kitase PINK1 mutain of people and serine/Soviet Union's ammonia of grivet
The homology of the normal albumen of pka acid PINK1 is 75%.
2, the preparation for the genetic chip whether the serine/threonine protein kitase PINK1 of detection people mutates
In order to guarantee the quality of test object sample, blank control, positive control and yin need to be also designed on genetic chip
Property control.Wherein, the layout of the genetic chip can be at least a kind of layout as shown in Figure 4.In Fig. 4, is blank pair
According to, zero is negative control,For positive control,For experimental group.
Wherein, the position of experimental group is the point sample position of nucleotide probe.
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：Being 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, negative internal reference probe sequence needed for genetic chip can be：
tgatgctgat aattgcat。
Positive internal control Quality Control probe：Being one section has other genetic fragments of homology with detection gene, people serine/
In Serineprotein kinase PINK1 mutain, select sequence corresponding from nucleotide probe different, and can visit with nucleotide
The number of needle base is identical, and 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, nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen.This
In inventive embodiments, positive internal control probe sequence needed for genetic chip can be：gttggacacg ag.
It should be noted that clicking and entering negative internal reference in the deposition process of genetic chip according to the layout of genetic chip and visiting
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 pipe for taking DEPC to handle, as detected sample processing tube, is added test object in pipe
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, it is careful to draw 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
Liquid is mixed, 10min, 12000r/min, 4 DEG C of centrifugation 15min is placed at room temperature for, carefully sucks all supernatants.
(5) it is cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min is centrifuged 15min, all supernatants is carefully sucked, super
10 μ L DEPC processing water dissolution is added in dry 15min in net platform.
(6) products therefrom will affect the labeling effciency and chip hybridization knot of probe if the purity of total serum IgE is not high for RNA
Fruit.So using QIAGENKit purifies total serum IgE.
2, the first chain 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 additional amount X μ L of RNase-free Water is to subtract T7Promotor according to 11.5 μ L of total volume
The 5 μ L of additional amount of primer, then subtract the additional amount 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 is preheated at 65 DEG C
5min。
(3) following cDNA synthetic system is 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 is added after mixing after being denaturalized in the RNA of ice bath.
(5) it is centrifuged after being mixed with pipette tips.
(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, infuses：40 DEG C of heat preservation 1min before 50%PEG (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcription mix；
(1) Transcription mix is configured
(2) 60 μ L Transcription mix are added and mix.
(3) 60 DEG C of hot lid in PCR instrument, 40 DEG C of reaction 2h.
4, cRNA is purified
QIAGEN RNeasy Mini kit purifies cRNA, and specific method can be found in what QIAGEN company 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 dehydrated alcohols are added, Tip mix well.
(3) total 700 solution of the μ L containing total serum IgE are transferred to cover in the RNeasy pillar in 2mL centrifuge tube >=8000g from
Heart 15-30s, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=8000g centrifuge washing 15-30s discards filter
It crosses liquid and discards the casing of filtered solution and 2mL for RNeasy in >=8000g centrifuge washing 2min with 500 μ L Buffer RPE again
Mini pillar is transferred in a new 1.5mL Eppendorf pipe.
(5) water for drawing 30 μ L RNase free stands 1min, >=8000g centrifugation elution 1min.
(6) it is primary that step (5) are repeated.
5, cRNA concentration mensuration
With spectrophotometric analysis cRNA concentration.Need 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 can also) close to 2.0.
6, cRNA fluorescent marks；
(1) it takes above-mentioned 4 μ g of cRNA and is concentrated into 6.6 μ L.
(2) plus 10 μ L DMSO are mixed.
(3) add the sodium bicarbonate (NaHCO that the 0.3M pH of 3.4 μ L is 9.03) and mix.
(4) above-mentioned 20 μ L cRNA mixture is added in fluorescent dye Cy3 and is mixed.25 DEG C of heat preservation 1h.
(5) 25 DEG C of heat preservation 15min after adding the 4M Hydroxylamine of 9 μ L to mix.
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 fragment 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.
| Cy3cRNA green fluorescence | 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 4 by chip layout blank,
On negative, positive, experimental group position.It covers chip and is sealed in hybridizing box, 60 DEG C of rollings hybridize 16h.
9, chip washs
Washing lotion 1 (1L) configuration：
| DEPC-H2O | 700mL |
| 20×SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 2 (1L) configuration：
| 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 1min (37 DEG C) in washing lotion 2 again；
(3) chip is finally washed into 30s in washing lotion 3.
10, chip scanning
Chip is scanned in scanner, the serine/threonine protein of test object is determined according to scanning result
Whether kinases PINK1 albumen is mutated.Please refer to Fig. 5 and Fig. 6, in result shown in fig. 5, negative control redgreen is glimmering
Light, positive control are green fluorescence, show that the sample quality of acquisition testing object is that there is no problem, and experimental group is that green is glimmering
Light, then showing that the serine/threonine protein kitase PINK1 albumen of test object is mutated.Result shown in fig. 6
In, negative control is colorless fluorescent, and positive control is green fluorescence, shows that the sample quality of acquisition testing object is that there is no problem
, and experimental group redgreen fluorescence, then showing that the serine/threonine protein kitase PINK1 albumen of test object does not occur
Mutation.
Embodiment three, specific recognition people serine/threonine protein kitase PINK1 mutain monoclonal antibody
Preparation
1, according to base sequence (such as SEQ ID NO of the serine/threonine protein kitase PINK1 mutain of people:2
It is shown) design upstream primer such as SEQ ID NO:Shown in 6, and, downstream primer such as SEQ ID NO:Shown in 7：
Upstream primer (F)：gagatccagg caattttta
Downstream primer (R)：gtcggatttc aggtctctg
2, the DNA of test object is that template carries out PCR amplification
| 10×Buffer | 5uL |
| dNTP | 2uL |
| Ex Taq | 1uL |
| ddH2O | 5uL |
| Template DNA | 1uL |
| Primer (F) | 3uL |
| Primer (R) | 3uL |
| Total system | 20uL |
PCR amplification is carried out by template of the DNA of test object, the serine/threonine protein kitase PINK1 for obtaining people is prominent
Become the complete segment of protein gene, and connect pMD19-T Vector (Takara company), is sequenced.Then by special biology
Corporation is a kind of humanization or Chimeric antibodies for antibody.Wherein, the monoclonal antibody prepared can be with SEQ ID NO:1
Shown in amino acid sequencespecific combine.The antibody of preparation is measured into content using ELISA method.
Example IV, serine/threonine protein kitase PINK1 mutain for detecting people antibody ELISA
Kit
In the present embodiment, the group of ELISA kit becomes：It is coated with the ELISA enzyme of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, the serine/threonine protein kitase PINK1 albumen of test object, sample diluting liquid, coating buffer,
ELISA ELISA Plate cleaning solution, developing solution and terminate liquid.
Wherein, the parameter of above-mentioned each composition can be at least a kind of following parameter：
ELISA ELISA Plate can be the ELISA enzyme mark in 96 holes；
ELIAS secondary antibody can be the Goat anti-Human IgG of diluted HRP (horseradish peroxidase) label；Wherein, dilution times
Number can be 8000 times.
Coating buffer can be 1 × PBS, pH:7.4；
ELISA ELISA Plate 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
Research method：Men and women leukaemic totally 22 are chosen, as test object, and utilizes ELISA kit
Whether the serine/threonine protein kitase PINK1 albumen for detecting 22 patients is mutated.
By taking wherein a certain position patient as an example, the detection method to the patient includes：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plate, it is incubated at room temperature 1-2h.
B, the serine/threonine protein kitase PINK1 albumen of patient is diluted to various concentration using sample diluting liquid
Gradient, and the serine/threonine protein kitase PINK1 albumen of various concentration gradient is loaded respectively to ELISA ELISA Plate
Kong Zhong, and it is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plate 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 solution is added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated for 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 of standard items measurement is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measurement is 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 software2=0.99930, therefore, this
Measurement is effective.The wavelength that will test, which substitutes into standard curve for the light absorption value at 450nm, can acquire serine/Soviet Union of patient
The concentration of propylhomoserin protein kinase PINK1 albumen.Utilize serine/Soviet Union's ammonia in the ELISA method detection patient serum sample of foundation
The content of pka acid PINK1 albumen.And it is identified with Western blot.Referring to FIG. 7, corresponding to extinction for the patient
The standard curve of value.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot 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(PH 8.8) | 3.8mL |
| 10%SDS | 0.15mL |
| 10% ammonium persulfate | 0.15mL |
| TEMED | 0.006mL |
B. preparation system (Total Volum is concentrated in upper layer spacer gel：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 with filter paper after gelling to be separated is solid, upper layer is then added, glue is concentrated, after being inserted into comb
Wait upper layer concentration gelling solid.
2, albumen loading electrophoresis：
(1) comb is pulled up, duct label is carried out, 5 × SDS electrophoretic buffer 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 voltage, after running concentration glue to band, uses 100V voltage 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.It is clipped by filter paper-glue-film-filter paper sequence, is put into transferring film device according to principle of the black flour to black flour
In, it is added refrigerator (ice bag), about 120min is run with the electric current of 150mA in ice；
(2) it takes the film out, is put into 5% skim milk at once after the completion of, 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 shaking tables are incubated overnight；
(5) primary antibody is recycled, PBST is cleaned 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 3 times are cleaned with PBST, each 10min；
(8) after Pierce ECL Western Blotting Substrate kit A, B liquid mixes in equal volume, dropwise
It is added dropwise on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment are analyzed with Image J.
Wherein, primary antibody is that the serine/threonine protein kitase PINK1 mutain monoclonal of the standby people of corporation is anti-
Body, secondary antibody are the Goat anti-Human IgGs of horseradish peroxidase-labeled.
J, Western blot Analysis of test results：It is shown according to Western blot experimental procedure as a result, such as Fig. 8 institute
Show, wherein the Marker in Fig. 8 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 60KD
There is obvious band, wherein the molecular weight of serine/threonine protein kitase PINK1 mutain is about 60KD, shows this
The serine/threonine protein kitase PINK1 albumen of patient is implicitly present in mutation.
According to the studies above method, serine/threonine protein kitase PINK1 albumen mutates general in 22 patients
Rate is 42%, it is possible thereby to determine, the base of the abrupt climatic change of serine/threonine protein kitase PINK1 albumen to leukaemia
Because diagnosis has certain impulse.
Embodiment six
Research method：Men and women Parkinsonian totally 42 are chosen, as test object, and utilizes ELISA reagent
Whether the serine/threonine protein kitase PINK1 albumen that box detects 42 patients is mutated.
By taking wherein a certain position patient as an example, the detection method to the patient includes：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plate, it is incubated at room temperature 1-2h；
B, difference is diluted to using the serine/threonine protein kitase PINK1 albumen that sample diluting liquid will test object
Concentration gradient, and the serine/threonine protein kitase PINK1 albumen of various concentration gradient is loaded respectively to ELISA enzyme mark
In the hole of plate, and it is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plate 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 solution is added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated for 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 of standard items measurement is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measurement is 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 software2=0.99904, therefore, this
Measurement is effective.The wavelength that will test, which substitutes into standard curve for the light absorption value at 450nm, can acquire the silk of patient in sample
Propylhomoserin/Serineprotein kinase PINK1 albumen concentration.Utilize silk ammonia in the ELISA method detection patient serum sample of foundation
Acid/Serineprotein kinase PINK1 albumen content.And it is identified with Western blot.Referring to FIG. 9, being light absorption value
Standard curve.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot is identified
J, Western blot Analysis of test results
Wherein, step i and step j is identical as in embodiment five, and details are not described herein, referring to FIG. 10, in Figure 10
Marker is used to characterize the size of labelled protein.The experimental display result of the present embodiment, compared with blank control, only in experimental group
Nearby there is obvious band in 60KD, wherein the molecular weight of serine/threonine protein kitase PINK1 mutain is about
60KD shows that the serine/threonine protein kitase PINK1 albumen of the Parkinsonian is implicitly present in mutation.
According to the studies above method, serine/threonine protein kitase PINK1 albumen mutates general in 42 patients
Rate is 70%, it is possible thereby to determine, the abrupt climatic change of serine/threonine protein kitase PINK1 albumen is to Parkinson's disease
Gene diagnosis has certain impulse.
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 serine/threonine protein kitase PINK1 mutain of people a kind of and its application
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 240
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Glu Ile Gln Ala Ile Phe Thr Gln Lys Ser Lys Pro Gly Pro Asp Pro
1 5 10 15
Leu Asp Thr Arg Arg Leu Gln Gly Phe Arg Leu Glu Glu Tyr Leu Ile
20 25 30
Gly Gln Ser Ile Gly Lys Gly Cys Ser Ala Ala Val Tyr Glu Ala Thr
35 40 45
Met Pro Thr Leu Pro Gln Asn Leu Glu Val Thr Lys Ser Thr Gly Leu
50 55 60
Leu Pro Gly Arg Gly Pro Arg Tyr Gln Cys Thr Arg Arg Arg Ala Gly
65 70 75 80
Ala Ser Ser Gly Gly Pro Cys Leu Pro Leu Gly His Gln Asp Asp Val
85 90 95
Glu His Leu Gly Arg Phe Leu Gln Arg Ser His Leu Glu His Asn Glu
100 105 110
Pro Gly Ala Gly Pro Ser Glu Pro Ser Gly Leu Gly Trp Gly Val Trp
115 120 125
Ser Ser His Leu Gln Lys Ile Gln Glu Arg Ser Lys Gln Leu Ala Pro
130 135 140
His Pro Asn Ile Ile Arg Val Leu Arg Ala Phe Thr Ser Ser Val Pro
145 150 155 160
Leu Leu Pro Gly Ala Leu Val Asp Tyr Pro Asp Val Leu Pro Ser Arg
165 170 175
Leu His Pro Glu Gly Leu Gly His Gly Arg Thr Leu Phe Leu Val Met
180 185 190
Lys Asn Tyr Pro Cys Thr Leu Arg Gln Tyr Leu Cys Val Asn Thr Pro
195 200 205
Ser Pro Arg Leu Ala Thr Met Met Leu Leu Gln Leu Leu Glu Gly Val
210 215 220
Asp His Leu Val Gln Gln Gly Ile Ala His Arg Asp Leu Lys Ser Asp
225 230 235 240
<210> 2
<211> 720
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
gagatccagg caatttttac ccagaaaagc aagccggggc ctgacccgtt ggacacgaga 60
cgcttgcagg gctttcggct ggaggagtat ctgatagggc agtccattgg taagggctgc 120
agtgctgctg tgtatgaagc caccatgcct acattgcccc agaacctgga ggtgacaaag 180
agcaccgggt tgcttccagg gagaggcccc aggtaccagt gcaccaggag aagggcagga 240
gcgagctccg ggggcccctg ccttcccctt ggccatcaag atgatgtgga acatctcggc 300
aggttcctcc agcgaagcca tcttgaacac aatgagccag gagctggtcc cagcgagccg 360
agtggccttg gctggggagt atggagcagt cacttacaga aaatccaaga gaggtccaag 420
caactagccc ctcaccccaa catcatccgg gttctccgcg ccttcacctc ttccgtgccg 480
ctgctgccag gggccctggt cgactaccct gatgtgctgc cctcacgcct ccaccctgaa 540
ggcctgggcc atggccggac gctgttcctc gttatgaaga actatccctg taccctgcgc 600
cagtaccttt gtgtgaacac acccagcccc cgcctcgcca ccatgatgct gctgcagctg 660
ctggaaggcg tggaccatct ggttcaacag ggcatcgcgc acagagacct gaaatccgac 720
<210> 3
<211> 12
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ccaggtacca gt 12
<210> 4
<211> 12
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tgccttcccc tt 12
<210> 5
<211> 11
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
agaggtccaa g 11
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gagatccagg caattttta 19
<210> 7
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gtcggatttc aggtctctg 19
Claims (7)
1. the serine/threonine protein kitase PINK1 mutain 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 serine/threonine protein kitase PINK1 mutain of people, which is characterized in that its nucleosides
Acid 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
The nucleotides sequence of the nucleotide probe serine/threonine protein kitase PINK1 mutain of people according to claim 2
Column are determined with the comparison result of the nucleotide sequence of the normal albumen of serine/threonine protein kitase PINK1 of people.
4. genetic chip according to claim 3, which is characterized in that
The nucleotide probe is SEQ ID NO:Base sequence shown in 3；
Or, the nucleotide probe is SEQ ID NO:Base sequence shown in 4；
Or, the nucleotide probe is SEQ ID NO:Base sequence shown in 5.
5. a kind of monoclonal antibody of the serine/threonine protein kitase PINK1 mutain of specific recognition people, feature
It is, it can be with SEQ ID NO:Amino acid sequencespecific shown in 1 combines.
6. it is a kind of for detecting the ELISA kit of the antibody of the serine/threonine protein kitase PINK1 mutain of people,
It is characterized in that, the ELISA kit includes：It is coated with ELISA ELISA Plate, the enzyme of monoclonal antibody described in claim 5
Mark secondary antibody, the serine/threonine protein kitase PINK1 albumen of test object, sample diluting liquid, coating buffer, ELISA enzyme
Target cleaning solution, developing solution and terminate liquid.
7. being used to detect the antibody of the serine/threonine protein kitase PINK1 mutain of people according to claim 6
ELISA kit, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human of diluted HRP horseradish peroxidase-labeled
IgG。
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| CN117777297A (en) * | 2023-01-09 | 2024-03-29 | 暨南大学 | Monoclonal antibody for resisting endogenous PINK1 protein and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117777297A (en) * | 2023-01-09 | 2024-03-29 | 暨南大学 | Monoclonal antibody for resisting endogenous PINK1 protein and application thereof |
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