CN109280657A - A kind of the epimerase family protein SDR39U1 mutation and application of people - Google Patents
A kind of the epimerase family protein SDR39U1 mutation and application of people Download PDFInfo
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Abstract
The present invention relates to a kind of epimerase family protein SDR39U1 of people mutation and applications, by using a large amount of liver cancer patients as research case, genetic test is carried out to case and is analyzed, determine the mutain of the epimerase family protein SDR39U1 of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the mutain of the epimerase family protein SDR39U1 of the people, impulse is provided for the gene diagnosis of liver cancer, and certain theoretical basis is provided for the diagnosing and treating of kinds cancer.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to the epimerase family protein SDR39U1 of people a kind of
Mutation and application.
Background technique
Epimerase (epimerase), also known as epimerase, mutarotase.One kind catalysis monosaccharide molecule (contains 2 or more
Asymmetric carbon atom) in some asymmetric carbon atom occur change of configuration enzyme.Such as aldose -1- epimerism enzymatic α -
Interconversion between D-Glucose and β-D-Glucose.
Epimerase family protein (the epimerase family protein) mutation of people can be to human health
It causes to seriously affect.During the peripheral blood to certain liver cancer patients carries out gene sequencing, discovery patient's is poor to different
Structure enzyme family Protein S DR39U1 is mutated, therefore, the detection to the epimerase family protein SDR39U1 mutation of people
To judging whether human body suffers from related disease with certain impulse.
Summary of the invention
It is an object of that present invention to provide a kind of epimerase family protein SDR39U1 of people mutation and applications.
Technical solution of the present invention includes:
In a first aspect, providing the epimerase family protein SDR39U1 mutain of people a kind of, amino acid sequence is such as
Shown in SEQ ID NO:1.
Second aspect provides a kind of encoding gene of the epimerase family protein SDR39U1 mutain of people, core
Nucleotide sequence is as shown in SEQ ID NO:2.
The third aspect provides a kind of genetic chip, comprising: solid phase carrier and fixed nucleotide probe on this carrier；
The nucleotide probe according to the nucleotide sequence of the epimerase family protein SDR39U1 mutain of people described above,
It is determined with the comparison result of the nucleotide sequence of the normal albumen of epimerase family protein SDR39U1 of people.
Preferably, the nucleotide probe is base sequence shown in SEQ ID NO:3；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:4；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:5.
Fourth aspect provides a kind of reagent, contains the epimerism enzyme family egg for detecting above-mentioned people in the reagent
The primer pair of white SDR39U1 mutain.
Preferably, the primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream
Primer is the base sequence as shown in any in SEQ ID NO:8~NO:17；Downstream primer is such as SEQ ID NO:24~NO:
29, base sequence shown in any in SEQ ID NO:34~NO:40；
Or,
Its described primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream primer is such as
In SEQ ID NO:8~NO:23 it is any shown in base sequence；Downstream primer is as any in SEQ ID NO:34~NO:40
Shown in base sequence；
Or,
Its described primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream primer is such as
In SEQ ID NO:8~NO:23, SEQ ID NO:30~NO:33 it is any shown in base sequence；Downstream primer is such as SEQ ID
In NO:38~NO:40 it is any shown in base sequence.
5th aspect, provides a kind of list of the epimerase family protein SDR39U1 mutain of specific recognition people
Clonal antibody is prepared by mentioned reagent, can be in conjunction with amino acid sequencespecific shown in SEQ ID NO:1.
6th aspect, provides a kind of for detecting the ELISA of the epimerase family protein SDR39U1 mutain of people
Kit, the ELISA kit include: be coated with said monoclonal antibody ELISA ELISA Plate, ELIAS secondary antibody, sample it is dilute
Release liquid, coating buffer, ELISA ELISA Plate cleaning solution, 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 a kind of epimerase family protein SDR39U1 of people mutation and applications, and a large amount of liver cancer are suffered from
Person carries out genetic test to case and analyzes, determine the epimerase family protein SDR39U1's of people as research case
Mutain, according to the epimerase family protein SDR39U1 mutain of the people prepare genetic chip, monoclonal antibody and
ELISA kit provides impulse for the gene diagnosis of liver cancer, provides certain theory for the diagnosing and treating of kinds cancer
Basis.
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 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 diagram that the embodiment of the present invention five 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 that the epimerase family protein SDR39U1 of people is prominent
The white encoding gene of a kink of preserved egg, nucleotide sequence is as shown in SEQ ID NO:2；Correspondingly, people is determined according to the encoding gene
Epimerase family protein SDR39U1 mutain, amino acid sequence is as shown in SEQ ID NO:1.
Secondly, according to the epimerase family protein SDR39U1 mutain and its encoding gene of people, according to such as lower section
The preparation of formula realization genetic chip.
1, the design of nucleotide probe
(1) design of nucleotide probe: nucleotide probe is mutated egg according to the epimerase family protein SDR39U1 of people
White nucleotide sequence, the comparison result with the nucleotide sequence of the normal albumen of epimerase family protein SDR39U1 of people
It determines, and according to the design principle of following probe, the epimerase family protein SDR39U1 designed for people is mutated egg
The nucleotide probe of white specificity.
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 difference of the corresponding amino acid sequence of epimerase family protein SDR39U1 mutain of people and people to
The comparison result of the corresponding amino acid sequence of the normal albumen of isomerase family protein SDR39U1 is referring to FIG. 1, wherein, in Fig. 1
Query sequence is the corresponding amino acid sequence of epimerase family protein SDR39U1 mutain of people, and Sbjct sequence is
The corresponding amino acid sequence of the normal albumen of epimerase family protein SDR39U1 of people, Query sequence and Sbjct sequence it
Between sequence be comparison result, as can be seen from FIG. 1, the epimerase family protein SDR39U1 mutain of people is relative to people
The normal albumen of epimerase family protein SDR39U1, there is amino acid sequence at one to be lacked, several places have occurred prominent
Become.It is to be detected in order to specific recognition according to nucleotide sequence shown in SEQ ID NO:2 and the comparison result of Fig. 1
Whether the epimerase family protein SDR39U1 of object is mutated, can be according to then when choosing nucleotide probe
Any mode in following several modes designs nucleotide probe:
1. will include a nucleotide sequence of mutated site nucleotide as nucleotide probe.
2. respectively selecting several nucleotide sequence (sequences of base sequence before deletion sites and after deletion sites
It is constant), generate nucleotide probe.
In the present embodiment, it the nucleotide sequence according to shown in SEQ ID NO: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:
The nucleotide probe as shown in SEQ ID NO:3 are as follows: ctgtgagcta tg；
The nucleotide probe as shown in SEQ ID NO:4 are as follows: gagctatggc ttacta；
The nucleotide probe as shown in SEQ ID NO:5 are as follows: cagaccttcg gt.
(2) above-mentioned designed nucleotide probe the synthesis of nucleotide probe: is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether the epimerase family protein SDR39U1 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 2.In Fig. 2, is blank pair
According to, zero is negative control,For positive control,For experimental group.
Wherein, the position of experimental group is the point sample position of nucleotide probe.
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 with are as follows:
tgatgctgat aattgcat。
Positive internal control Quality Control probe: being one section of other genetic fragment for having homology with detection gene, in the poor to different of people
In structure enzyme family Protein S DR39U1 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 with are as follows: tgccttaaag ga.
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, in detected sample processing tube
The middle 300 μ L of blood that test object is added, adds 700 μ L of Trizol, mixes 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 being cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min, 4 DEG C of centrifugation 15min carefully suck all supernatants,
10 μ LDEPC processing water dissolution is added in the dry 15min in super-clean bench.
(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:
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 StrandB 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, note: 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
| 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 LTranscription 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 LBuffer 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 LRNase 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 cRNA4 μ g and is concentrated into 6.6 μ L.
(2) plus 10 μ LDMSO 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.
| Cy3 cRNA 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 2 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 andDrying 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 epimerase family protein of test object is determined according to scanning result
Whether SDR39U1 albumen is mutated.Please refer to Fig. 3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence,
Positive control is green fluorescence, shows that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence,
So show that the epimerase family protein SDR39U1 albumen of test object is mutated.In result shown in Fig. 4, yin
Property control be colorless fluorescent, positive control is 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 epimerase family protein SDR39U1 albumen of test object does not mutate.
Embodiment three, specific recognition people epimerase family protein SDR39U1 mutain monoclonal antibody
Preparation
1, the determination of reagent
Contain the primer for detecting the epimerase family protein SDR39U1 mutain of above-mentioned people in the reagent
It is right.
Wherein, according to the ORF of the normal albumen of epimerase family protein SDR39U1 of people (such as SEQ ID NO:6 institute
Show), it can determine corresponding base sequence (such as SEQ of ORF of the normal albumen of epimerase family protein SDR39U1 of people
Shown in ID NO:7).
It is possible to further according to the base sequence of the epimerase family protein SDR39U1 mutain of people (such as
Shown in SEQ ID NO:2), and it is corresponding according to the ORF of the normal albumen of epimerase family protein SDR39U1 of the people of people
Base sequence (as shown in SEQ ID NO:7), the primer pair designed include following any upstream primers with it is following any
The combination of downstream primer: upstream primer is the base sequence as shown in any in SEQ ID NO:8~NO:17；Downstream primer is
The base sequence as shown in any in SEQ ID NO:24~NO:29, SEQ ID NO:34~NO:40；Or, the primer designed
To the combination including following any upstream primers and following any downstream primers: upstream primer is such as SEQ ID NO:8~NO:23
In it is any shown in base sequence；Downstream primer is the base sequence as shown in any in SEQ ID NO:34~NO:40；Or,
The primer pair designed includes the combination of following any upstream primers Yu following any downstream primers: upstream primer is such as SEQ ID
In NO:8~NO:23, SEQ ID NO:30~NO:33 it is any shown in base sequence；Downstream primer is such as SEQ ID NO:38
In~NO:40 it is any shown in base sequence:
As shown in SEQ ID NO:8: atgcgtgtgc ttgtgggtgg cgggacaggc
As shown in SEQ ID NO:9: ttcattggga cagccctaac ccagctgctg
As shown in SEQ ID NO:10: aatgccagag gccacgaagt gacgttggtc
As shown in SEQ ID NO:11: tcccgaaagc ccgggcccgg ccggatcacg
As shown in SEQ ID NO:12: tgggatgagc tcgctgcatc ggggctgccg
As shown in SEQ ID NO:13: agctgcgatg ccgccgtcaa cctggccgga
As shown in SEQ ID NO:14: gagaacatcc tcaaccctct ccgaaggtca
As shown in SEQ ID NO:15: gcccgggccc taaagctgat
As shown in SEQ ID NO:16: acccactaga gcacagggag
As shown in SEQ ID NO:17: gacagtgccc cactgatgag aaccta
As shown in SEQ ID NO:18: ctgctgccct ttcgcctggg cctggggggc
As shown in SEQ ID NO:19: cccatcggct caggccacca attcttcccc
As shown in SEQ ID NO:20: tggatacaca tcggggacct ggcaggaatc
As shown in SEQ ID NO:21: ctgacccatg cccttgaagc aaaccacgtg
As shown in SEQ ID NO:22: cacggggtcc tgaatggagt ggctccatcc
As shown in SEQ ID NO:23: tccgccacta atgctgagtt tgcccagacc
As shown in SEQ ID NO:24: gcccatggca ccacccccac ggcccagcac
As shown in SEQ ID NO:25: aacccctgag cgcaccacca cctggcgtgt
As shown in SEQ ID NO:26: agaatctcca ggaagcctgg ctgcagcttc
As shown in SEQ ID NO:27: ccatttggtt acgaggttgg agaaaaagtc
As shown in SEQ ID NO:28: aaagtcccct cctgggctgt cttcatcata
As shown in SEQ ID NO:29: ctccgcagtc agactgggct ggtagtaagc
As shown in SEQ ID NO:30: ggtgctgccc tgggccgccg agccttcatc
As shown in SEQ ID NO:31: cctctcccca gcgctgtggt gcaagctgtc
As shown in SEQ ID NO:32: tttgggcgac agcgtgccat catgctgctg
As shown in SEQ ID NO:33: gagggccaga aggtgatccc a
As shown in SEQ ID NO:34: tgggatcacc ttctggccct ccagcagcat
As shown in SEQ ID NO:35: gatggcacgc tgtcgcccaa agacagcttg
As shown in SEQ ID NO:36: caccacagcg ctggggagag ggatgaaggc
As shown in SEQ ID NO:37: tcggcggccc agggcagcac c
As shown in SEQ ID NO:38: ttaggctaca atttccttta aggcagcccc
As shown in SEQ ID NO:39: tagctctggg aaggaatact
As shown in SEQ ID NO:40: ggtagccagt ggccagtgtt cg
In the present embodiment, it is used to detect the epimerase family protein SDR39U1 of above-mentioned people in reagent containing one
The primer pair of mutain.
For the primer pair (primer (F) and primer (R)) of each combination producing, following PCR amplification steps are executed.
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 epimerase family protein SDR39U1 for obtaining people is prominent
Become the complete segment of protein gene, and connect pMD19-TVector (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, epimerase family protein SDR39U1 mutain for detecting people antibody ELISA examination
Agent box
In the present embodiment, the composition of ELISA kit are as follows: be coated with the ELISA enzyme of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, the epimerase family protein SDR39U1 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
In embodiments of the present invention, using liver cancer patient as test object, and the liver cancer is detected using ELISA kit and is suffered from
Whether the epimerase family protein SDR39U1 albumen of person is mutated, and this method at least may include following one kind:
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer, 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 epimerase family protein SDR39U1 albumen of liver cancer patient is diluted to using sample diluting liquid different dense
Gradient is spent, and the epimerase family protein SDR39U1 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, make standard curve: 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 ELISACalc
The Software on Drawing standard curve can know regression coefficient R according to the ELISACalc software2=0.99948, therefore, this survey
It is fixed effective.The wavelength that will test is that the light absorption value substitution standard curve at 450nm can acquire the poor to different of liver cancer patient
The concentration of structure enzyme family Protein S DR39U1 albumen.Using difference in the ELISA method detection liver cancer patient blood serum sample of foundation to different
The content of structure enzyme family Protein S DR39U1 albumen.And it is identified with Westernblot.Referring to FIG. 5, being the mark of light absorption value
Directrix curve.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/LTris(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 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 (being diluted with 3% milk with 1:5000-1:10000) is incubated 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 the epimerase family protein SDR39U1 mutain monoclonal antibody of the standby people of corporation,
Secondary antibody is the Goat anti-Human IgG of horseradish peroxidase-labeled.
J, Western blot Analysis of test results: showing according to Western blot experimental procedure as a result, such as Fig. 6 institute
Show, wherein the Marker in Fig. 6 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 68KD
There is obvious band, wherein epimerase family protein SDR39U1 mutant protein molecules amount is about 68KD, shows the liver
The epimerase family protein SDR39U1 albumen of cancer 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 epimerase family protein SDR39U1 mutation and application of a kind of people
<160> 40
<170> SIPOSequenceListing 1.0
<210> 1
<211> 273
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 1
Gly Gly Gly Thr Gly Phe Ile Gly Thr Ala Leu Thr Gln Leu Leu Asn
1 5 10 15
Ala Arg Gly His Glu Val Thr Leu Val Ser Arg Lys Pro Gly Pro Gly
20 25 30
Arg Ile Thr Trp Asp Glu Leu Ala Ala Ser Gly Leu Pro Ser Cys Asp
35 40 45
Ala Ala Val Asn Leu Ala Gly Glu Asn Ile Leu Asn Pro Leu Arg Arg
50 55 60
Ser Ala Arg Ala Leu Lys Leu Ile Pro Thr Arg Ala Gln Gly Gly Gln
65 70 75 80
Cys Pro Thr Asp Glu Asn Leu Ala Met Ala Tyr Tyr Gln Pro Ser Leu
85 90 95
Thr Ala Glu Tyr Asp Glu Asp Ser Pro Gly Gly Asp Phe Asp Phe Phe
100 105 110
Ser Asn Leu Val Thr Lys Trp Glu Ala Ala Ala Arg Leu Pro Gly Asp
115 120 125
Ser Thr Arg Gln Val Val Val Arg Ser Gly Val Val Leu Gly Arg Gly
130 135 140
Gly Gly Ala Met Gly His Met Leu Leu Pro Phe Arg Leu Gly Leu Gly
145 150 155 160
Gly Pro Ile Gly Ser Gly His Gln Phe Phe Pro Trp Ile His Ile Gly
165 170 175
Asp Leu Ala Gly Ile Leu Thr His Ala Leu Glu Ala Asn His Val His
180 185 190
Gly Val Leu Asn Gly Val Ala Pro Ser Ser Ala Thr Asn Ala Glu Phe
195 200 205
Ala Gln Thr Phe Gly Ala Ala Leu Gly Arg Arg Ala Phe Ile Pro Leu
210 215 220
Pro Ser Ala Val Val Gln Ala Val Phe Gly Arg Gln Arg Ala Ile Met
225 230 235 240
Leu Leu Glu Gly Gln Lys Val Ile Pro Arg Arg Thr Leu Ala Thr Gly
245 250 255
Tyr Gln Tyr Ser Phe Pro Glu Leu Gly Ala Ala Leu Lys Glu Ile Val
260 265 270
Ala
<210> 2
<211> 822
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 2
ggtggcggga caggcttcat tgggacagcc ctaacccagc tgctgaatgc cagaggccac 60
gaagtgacgt tggtctcccg aaagcccggg cccggccgga tcacgtggga tgagctcgct 120
gcatcggggc tgccgagctg cgatgccgcc gtcaacctgg ccggagagaa catcctcaac 180
cctctccgaa ggtcagcccg ggccctaaag ctgataccca ctagagcaca gggaggacag 240
tgccccactg atgagaacct gtgagctatg gcttactacc agcccagtct gactgcggag 300
tatgatgaag acagcccagg aggggacttt gactttttct ccaacctcgt aaccaaatgg 360
gaagctgcag ccaggcttcc tggagattct acacgccagg tggtggtgcg ctcaggggtt 420
gtgctgggcc gtgggggtgg tgccatgggc cacatgctgc tgccctttcg cctgggcctg 480
gggggcccca tcggctcagg ccaccaattc ttcccctgga tacacatcgg ggacctggca 540
ggaatcctga cccatgccct tgaagcaaac cacgtgcacg gggtcctgaa tggagtggct 600
ccatcctccg ccactaatgc tgagtttgcc cagaccttcg gtgctgccct gggccgccga 660
gccttcatcc ctctccccag cgctgtggtg caagctgtct ttgggcgaca gcgtgccatc 720
atgctgctgg agggccagaa ggtgatccca cggcgaacac tggccactgg ctaccagtat 780
tccttcccag agctaggggc tgccttaaag gaaattgtag cc 822
<210> 3
<211> 12
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ctgtgagcta tg 12
<210> 4
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gagctatggc ttacta 16
<210> 5
<211> 12
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cagaccttcg gt 12
<210> 6
<211> 316
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 6
Met Arg Val Leu Val Gly Gly Gly Thr Gly Phe Ile Gly Thr Ala Leu
1 5 10 15
Thr Gln Leu Leu Asn Ala Arg Gly His Glu Val Thr Leu Val Ser Arg
20 25 30
Lys Pro Gly Pro Gly Arg Ile Thr Trp Asp Glu Leu Ala Ala Ser Gly
35 40 45
Leu Pro Ser Cys Asp Ala Ala Val Asn Leu Ala Gly Glu Asn Ile Leu
50 55 60
Asn Pro Leu Arg Arg Ser Ala Arg Ala Leu Lys Leu Ile Pro Thr Arg
65 70 75 80
Ala Gln Gly Gly Gln Cys Pro Thr Asp Glu Asn Leu Trp Asn Glu Thr
85 90 95
Phe Gln Lys Glu Val Ile Gly Ser Arg Leu Glu Thr Thr Gln Leu Leu
100 105 110
Ala Lys Ala Ile Thr Lys Ala Pro Gln Pro Pro Lys Ala Trp Val Leu
115 120 125
Val Thr Gly Val Ala Tyr Tyr Gln Pro Ser Leu Thr Ala Glu Tyr Asp
130 135 140
Glu Asp Ser Pro Gly Gly Asp Phe Asp Phe Phe Ser Asn Leu Val Thr
145 150 155 160
Lys Trp Glu Ala Ala Ala Arg Leu Pro Gly Asp Ser Thr Arg Gln Val
165 170 175
Val Val Arg Ser Gly Val Val Leu Gly Arg Gly Gly Gly Ala Met Gly
180 185 190
His Met Leu Leu Pro Phe Arg Leu Gly Leu Gly Gly Pro Ile Gly Ser
195 200 205
Gly His Gln Phe Phe Pro Trp Ile His Ile Gly Asp Leu Ala Gly Ile
210 215 220
Leu Thr His Ala Leu Glu Ala Asn His Val His Gly Val Leu Asn Gly
225 230 235 240
Val Ala Pro Ser Ser Ala Thr Asn Ala Glu Phe Ala Gln Thr Leu Gly
245 250 255
Ala Ala Leu Gly Arg Arg Ala Phe Ile Pro Leu Pro Ser Ala Val Val
260 265 270
Gln Ala Val Phe Gly Arg Gln Arg Ala Ile Met Leu Leu Glu Gly Gln
275 280 285
Lys Val Ile Pro Gln Arg Thr Leu Ala Thr Gly Tyr Gln Tyr Ser Phe
290 295 300
Pro Glu Leu Gly Ala Ala Leu Lys Glu Ile Val Ala
305 310 315
<210> 7
<211> 951
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 7
atgcgtgtgc ttgtgggtgg cgggacaggc ttcattggga cagccctaac ccagctgctg 60
aatgccagag gccacgaagt gacgttggtc tcccgaaagc ccgggcccgg ccggatcacg 120
tgggatgagc tcgctgcatc ggggctgccg agctgcgatg ccgccgtcaa cctggccgga 180
gagaacatcc tcaaccctct ccgaaggtca gcccgggccc taaagctgat acccactaga 240
gcacagggag gacagtgccc cactgatgag aacctatgga atgaaacctt ccaaaaagag 300
gtaatcggca gccgcctaga gaccacccaa ttgctggcta aagccatcac caaagcccca 360
caacccccca aggcctgggt cttagtcaca ggtgtagctt actaccagcc cagtctgact 420
gcggagtatg atgaagacag cccaggaggg gactttgact ttttctccaa cctcgtaacc 480
aaatgggaag ctgcagccag gcttcctgga gattctacac gccaggtggt ggtgcgctca 540
ggggttgtgc tgggccgtgg gggtggtgcc atgggccaca tgctgctgcc ctttcgcctg 600
ggcctggggg gccccatcgg ctcaggccac caattcttcc cctggataca catcggggac 660
ctggcaggaa tcctgaccca tgcccttgaa gcaaaccacg tgcacggggt cctgaatgga 720
gtggctccat cctccgccac taatgctgag tttgcccaga ccttgggtgc tgccctgggc 780
cgccgagcct tcatccctct ccccagcgct gtggtgcaag ctgtctttgg gcgacagcgt 840
gccatcatgc tgctggaggg ccagaaggtg atcccacagc gaacactggc cactggctac 900
cagtattcct tcccagagct aggggctgcc ttaaaggaaa ttgtagccta a 951
<210> 8
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
atgcgtgtgc ttgtgggtgg cgggacaggc 30
<210> 9
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ttcattggga cagccctaac ccagctgctg 30
<210> 10
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
aatgccagag gccacgaagt gacgttggtc 30
<210> 11
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tcccgaaagc ccgggcccgg ccggatcacg 30
<210> 12
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
tgggatgagc tcgctgcatc ggggctgccg 30
<210> 13
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
agctgcgatg ccgccgtcaa cctggccgga 30
<210> 14
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gagaacatcc tcaaccctct ccgaaggtca 30
<210> 15
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
gcccgggccc taaagctgat 20
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
acccactaga gcacagggag 20
<210> 17
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gacagtgccc cactgatgag aaccta 26
<210> 18
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
ctgctgccct ttcgcctggg cctggggggc 30
<210> 19
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
cccatcggct caggccacca attcttcccc 30
<210> 20
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
tggatacaca tcggggacct ggcaggaatc 30
<210> 21
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ctgacccatg cccttgaagc aaaccacgtg 30
<210> 22
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
cacggggtcc tgaatggagt ggctccatcc 30
<210> 23
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
tccgccacta atgctgagtt tgcccagacc 30
<210> 24
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
gcccatggca ccacccccac ggcccagcac 30
<210> 25
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
aacccctgag cgcaccacca cctggcgtgt 30
<210> 26
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
agaatctcca ggaagcctgg ctgcagcttc 30
<210> 27
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
ccatttggtt acgaggttgg agaaaaagtc 30
<210> 28
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
aaagtcccct cctgggctgt cttcatcata 30
<210> 29
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
ctccgcagtc agactgggct ggtagtaagc 30
<210> 30
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
ggtgctgccc tgggccgccg agccttcatc 30
<210> 31
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
cctctcccca gcgctgtggt gcaagctgtc 30
<210> 32
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
tttgggcgac agcgtgccat catgctgctg 30
<210> 33
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
gagggccaga aggtgatccc a 21
<210> 34
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
tgggatcacc ttctggccct ccagcagcat 30
<210> 35
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
gatggcacgc tgtcgcccaa agacagcttg 30
<210> 36
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
caccacagcg ctggggagag ggatgaaggc 30
<210> 37
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
tcggcggccc agggcagcac c 21
<210> 38
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
ttaggctaca atttccttta aggcagcccc 30
<210> 39
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
tagctctggg aaggaatact 20
<210> 40
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
ggtagccagt ggccagtgtt cg 22
Claims (9)
1. the mutain of the epimerase family protein SDR39U1 of people a kind of, which is characterized in that its amino acid sequence is such as
Shown in SEQ ID NO:1.
2. a kind of encoding gene of the epimerase family protein SDR39U1 mutain of people, which is characterized in that its nucleotide
Sequence is as shown in SEQ ID NO:2.
3. a kind of genetic chip characterized by comprising solid phase carrier and fixed nucleotide probe on this carrier；It is described
The nucleotide sequence of the nucleotide probe epimerase family protein SDR39U1 mutain of people according to claim 2,
It is determined with the comparison result of the nucleotide sequence of the normal albumen of epimerase family protein SDR39U1 of people.
4. genetic chip according to claim 3, which is characterized in that
The nucleotide probe is base sequence shown in SEQ ID NO:3；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:4；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:5.
5. a kind of reagent, which is characterized in that contain the epimerase man for detecting people described in claim 1 in the reagent
The primer pair of race's Protein S DR39U1 mutain.
6. reagent according to claim 5, which is characterized in that
The primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream primer is such as SEQ ID
In NO:8~NO:17 it is any shown in base sequence；Downstream primer is such as SEQ ID NO:24~NO:29, SEQ ID NO:34
In~NO:40 it is any shown in base sequence；
Or,
The primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream primer is such as SEQ ID
In NO:8~NO:23 it is any shown in base sequence；Downstream primer is the alkali as shown in any in SEQ ID NO:34~NO:40
Basic sequence；
Or,
The primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream primer is such as SEQ ID
In NO:8~NO:23, SEQ ID NO:30~NO:33 it is any shown in base sequence；Downstream primer is such as SEQ ID NO:38
In~NO:40 it is any shown in base sequence.
7. a kind of monoclonal antibody of the epimerase family protein SDR39U1 mutain of specific recognition people, feature
It is, is prepared by reagent such as described in claim 5 or 6, it can be special with amino acid sequence shown in SEQ ID NO:1
Property combine.
8. a kind of for detecting the ELISA kit of the epimerase family protein SDR39U1 mutain of people, feature exists
In the ELISA kit includes: ELISA ELISA Plate, ELIAS secondary antibody, the sample for being coated with monoclonal antibody described in claim 7
Product dilution, coating buffer, ELISA ELISA Plate cleaning solution, developing solution and terminate liquid.
9. being used to detect the antibody of the epimerase family protein SDR39U1 mutain of people according to claim 8
ELISA kit, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human of diluted HRP horseradish peroxidase-labeled
IgG。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811055147.5A CN109280657A (en) | 2018-09-11 | 2018-09-11 | A kind of the epimerase family protein SDR39U1 mutation and application of people |
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|---|---|---|---|
| CN201811055147.5A CN109280657A (en) | 2018-09-11 | 2018-09-11 | A kind of the epimerase family protein SDR39U1 mutation and application of people |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109280657A true CN109280657A (en) | 2019-01-29 |
Family
ID=65178363
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| CN201811055147.5A Pending CN109280657A (en) | 2018-09-11 | 2018-09-11 | A kind of the epimerase family protein SDR39U1 mutation and application of people |
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