CN108949708A - A kind of mutain of the class Hep27 albumen of people and its application - Google Patents
A kind of mutain of the class Hep27 albumen of people and its application Download PDFInfo
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Abstract
The present invention relates to a kind of mutain of the class Hep27 albumen of people and its applications, by using a large amount of liver cancer, patient with breast cancer as research case, genetic test is carried out to case and is analyzed, determine the mutain of the class Hep27 albumen of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the mutain of the class Hep27 albumen of the people, impulse is provided for the gene diagnosis of liver cancer, breast cancer, and certain theoretical basis is provided for the diagnosing and treating of cancer.
Description
Technical field
The present invention relates to the mutain of a kind of genetic engineering field more particularly to the class Hep27 albumen of people a kind of and its
Using.
Background technique
Class Hep27 albumen (Hep27-like protein) is a member of short-chain dehydrogenase/reductase enzyme family, be
The NADPH dependence dicarboxylic acids reductase expressed in vascular endothelial cell tissue.
The class Hep27 albumen mutation of people can cause to seriously affect to human health.To certain cancers, including liver
During the peripheral blood of the patients such as cancer, breast cancer carries out gene sequencing, the class Hep27 albumen of discovery patient has occurred prominent
Become, therefore, to the detection of the class Hep27 protein mutation of people to judge whether human body suffers from cancer and have certain impulse.
Summary of the invention
It is an object of that present invention to provide a kind of mutain of the class Hep27 albumen of people and its applications.
Technical solution of the present invention includes:
In a first aspect, the class Hep27 protein mutation albumen of people a kind of is provided, amino acid sequence such as SEQ ID NO:1 institute
Show.
Second aspect provides a kind of encoding gene of the class Hep27 protein mutation albumen of people, nucleotide sequence such as SEQ
Shown in ID NO:2.
The third aspect provides a kind of genetic chip, comprising: solid phase carrier and fixed nucleotide probe on this carrier；
Class Hep27 egg of the nucleotide probe according to the nucleotide sequence of the class Hep27 protein mutation albumen of people described above, with people
The comparison result of the nucleotide sequence of Bai Zhengchang albumen determines.
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.
Fourth aspect provides a kind of monoclonal antibody of the class Hep27 protein mutation albumen of specific recognition people, can be with
Amino acid sequencespecific shown in SEQ ID NO:1 combines.
5th aspect, provide it is a kind of for detecting the ELISA kit of the antibody of the class Hep27 protein mutation albumen of people,
The ELISA kit include: the ELISA ELISA Plate for being coated with said monoclonal antibody, ELIAS secondary antibody, test object class
Hep27 albumen, sample diluting 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 mutain of the class Hep27 albumen of people and its applications, and a large amount of liver cancer, breast cancer are suffered from
Person carries out genetic test to case and analyzes, determine the mutain of the class Hep27 albumen of people as research case, according to
The class Hep27 protein mutation albumen of the people prepares genetic chip, monoclonal antibody and ELISA kit, is liver cancer, breast cancer
Gene diagnosis provides impulse, provides certain theoretical basis for the diagnosing and treating of cancer.
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 a kind of genetic chip layout that the embodiment of the present invention one provides；
Fig. 4 is a kind of colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 5 is another colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 6 is the standard curve that the offer of the embodiment of the present invention five is protein content；
Fig. 7 is the Western blot testing result schematic diagram that the embodiment of the present invention five provides；
Fig. 8 is the standard curve that the offer of the embodiment of the present invention six is protein content；
Fig. 9 is the 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 coding base of the class Hep27 protein mutation albumen of people
Cause, nucleotide sequence is as shown in SEQ ID NO:2；Correspondingly, determine that the class Hep27 albumen of people is prominent according to the encoding gene
A kink of preserved egg is white, and amino acid sequence is as shown in SEQ ID NO:1.
Secondly, realizing genetic chip as follows according to the class Hep27 protein mutation albumen and its encoding gene of people
Preparation.
1, the design of nucleotide probe
(1) design of nucleotide probe: nucleotide probe is according to the nucleotides sequence of the class Hep27 protein mutation albumen of people
Column determine with the comparison result of the nucleotide sequence of the class Hep27 protein normal albumen of people, and according to the design of following probe
Principle designs the nucleotide probe of the specificity of the class Hep27 protein mutation albumen for people.
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 corresponding amino acid sequence of class Hep27 protein mutation albumen of people and the class Hep27 protein normal albumen of people
The comparison result of corresponding amino acid sequence is referring to FIG. 1, wherein, the Query sequence in Fig. 1 is that the class Hep27 albumen of people is prominent
The white corresponding amino acid sequence of a kink of preserved egg, Sbjct sequence are the corresponding amino acid sequences of class Hep27 protein normal albumen of people,
Sequence between Query sequence and Sbjct sequence is comparison result, as can be seen from FIG. 1, the class Hep27 protein mutation albumen of people
Relative to the class Hep27 protein normal albumen of people, there is amino acid sequence at one to be lacked, be mutated at one.According to
The comparison result of nucleotide sequence shown in SEQ ID NO:2 and Fig. 1, in order to specific recognition object to be detected
Whether class Hep27 albumen is mutated, then when choosing nucleotide probe, it can be according to any in the following two kinds mode
Kind mode 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: gtgtgccatg tg；
The nucleotide probe as shown in SEQ ID NO:4 are as follows: acaagggctt cagt.
(2) above-mentioned designed nucleotide probe the synthesis of nucleotide probe: is synthesized by nucleotide sequence.
When the class Hep27 protein mutation albumen of people is compared in NCBI, the dehydrogenase/reductase of chimpanzee in comparison
The normal albumen of enzyme SDR family member 4, comparison result is referring to FIG. 2, Query sequence is that the class Hep27 albumen of people is prominent in Fig. 2
The white corresponding amino acid sequence of a kink of preserved egg, Sbjct sequence are the normal albumen pair of dehydrogenase/reductase enzyme SDR family member 4 of chimpanzee
The amino acid sequence answered, the sequence between Query sequence and Sbjct sequence are comparison result.As can be seen from FIG. 2, the class of people
The homology of Hep27 protein mutation albumen and the normal albumen of dehydrogenase/reductase enzyme SDR family member 4 of chimpanzee is 98%, and
It is 85% according to the homology of the class Hep27 protein mutation albumen of people and the class Hep27 protein normal albumen of people, with chimpanzee
The homology of the normal albumen of dehydrogenase/reductase enzyme SDR family member 4 is apparently higher than and the class Hep27 protein normal albumen of people
Homology.Thus illustrate, test object atavism occurs after the mutation of class Hep27 albumen.
2, the preparation for the genetic chip whether the class Hep27 albumen 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 3.In Fig. 3, 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 class Hep27 of people
In protein mutation albumen, select sequence corresponding from nucleotide probe different, and can be with the number phase of nucleotide probe base
Together, one section of nucleotide sequence that can also be different from the number of nucleotide probe base is as positive internal control Quality Control probe.It is preferred that
Nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen on ground.In the embodiment of the present invention,
Positive internal control probe sequence needed for genetic chip can be with are as follows: aggcctctgt gc.
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 L DEPC 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:
| Total serum IgE | The most 6.5 μ L of 2 μ g |
| T7 Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
Wherein, the additional amount X μ L of RNase-free Water is to subtract T7 Promotor 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, 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 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 L RNase 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
Cross liquid again with 500 μ L Buffer RPE the casing that >=8000g centrifuge washing 2min discards filtered solution and 2mL will
RNeasymini 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 cRNA4 μ g 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.
| 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 3 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, determines whether the class Hep27 albumen of test object is sent out according to scanning result
Mutation is given birth to.Please refer to Fig. 4 and Fig. 5, in result shown in Fig. 4, negative control redgreen fluorescence, positive control is that green is glimmering
Light shows that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence, then showing test object
Class Hep27 albumen be mutated.In result shown in fig. 5, negative control is colorless fluorescent, and positive control is that green is glimmering
Light shows that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, then showing test object
Class Hep27 albumen do not mutate.
Embodiment three, specific recognition people class Hep27 protein mutation albumen monoclonal antibody preparation
1, upstream is designed according to the base sequence (as shown in SEQ ID NO:2) of the class Hep27 protein mutation albumen of people to draw
Object as shown in SEQ ID NO:5, and, downstream primer is as shown in SEQ ID NO:6:
Upstream primer (F): atgcacaagg cggggctgct
Downstream primer (R): gaggcgggac ggggttcctc
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 class Hep27 protein mutation protein gene for obtaining people is complete
Segment, and pMD19-T Vector (Takara company) is connected, it is sequenced.Then antibody is prepared by special biotech firm,
It is a kind of humanization or Chimeric antibodies.Wherein, the monoclonal antibody prepared can be with amino acid sequence shown in SEQ ID NO:1
Column specific binding.The antibody of preparation is measured into content using ELISA method.
Example IV, class Hep27 protein mutation albumen for detecting people antibody ELISA kit
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 class Hep27 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 class Hep27 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 class Hep27 albumen of liver cancer patient is diluted to various concentration gradient using sample diluting liquid, and will be different dense
The class Hep27 albumen of degree gradient is loaded respectively into the hole of ELISA ELISA Plate, and 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 ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc software2=0.99813, 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 class of liver cancer patient
The concentration of Hep27 albumen.Utilize the content of class Hep27 albumen in the ELISA method detection liver cancer patient blood serum sample of foundation.And
It is identified with Westernblot.Referring to FIG. 6, being the standard curve of light absorption value.Wherein, X-axis is light absorption value, and Y-axis is to correspond to
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(PH8.8) | 3.8mL |
| 10%SDS | 0.15mL |
| 10% ammonium persulfate | 0.15mL |
| TEMED | 0.006mL |
B. upper layer spacer gel concentration preparation system (Total Volum:8mL)
(3) lower layer's separation gel of respective volume is added in each offset plate, adds the sterile ddH of 1mL2O flattens separation
Glue blots remaining moisture in plastic plate 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) it after Pierce ECLWestern Blotting Substrate kit A, B liquid mixes in equal volume, drips dropwise
It is added on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment are analyzed with Image J.
Wherein, primary antibody is the class Hep27 protein mutation protein monoclonal antibody of the standby people of corporation, and secondary antibody is horseradish peroxide
The Goat anti-Human IgG of compound enzyme label.
J, Western blot Analysis of test results: showing according to Western blot experimental procedure as a result, such as Fig. 7 institute
Show, wherein the Marker in Fig. 7 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 57KD
There is obvious band, wherein class Hep27 protein mutation molecular weight of albumen is about 57KD, shows the class Hep27 of the liver cancer patient
Albumen is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, using patient with breast cancer as test object, and the mammary gland is detected using ELISA kit
Whether the class Hep27 albumen of cancer 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, 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, it is diluted to various concentration gradient using the class Hep27 albumen that sample diluting liquid will test object, and will be different dense
The class Hep27 albumen of degree gradient is loaded respectively into the hole of ELISA ELISA Plate, and 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 ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc software2=0.99910, therefore, this
Measurement is effective.The wavelength that will test is that the light absorption value substitution standard curve at 450nm can acquire breast cancer trouble in sample
The concentration of the class Hep27 albumen of person.Utilize class Hep27 albumen in the ELISA method detection blood serum of patients with human breast carcinoma sample of foundation
Content.And it is identified with Western blot.Referring to FIG. 8, being the standard curve of light absorption value.Wherein, X-axis is extinction
Value, Y-axis are 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. 9, in Fig. 9
Marker is used to characterize the size of labelled protein.The experimental display result of the present embodiment, compared with blank control, only in experimental group
Nearby there is obvious band in 57KD, wherein class Hep27 protein mutation molecular weight of albumen is about 57KD, shows that the breast cancer is suffered from
The class Hep27 albumen of person 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>a kind of mutain of the class Hep27 albumen of people and its application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 237
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 1
Met His Lys Ala Gly Leu Leu Gly Leu Cys Ala Arg Ala Trp Asn Ser
1 5 10 15
Val Arg Met Ala Ser Ser Gly Met Thr Arg Arg Asp Pro Leu Ala Asn
20 25 30
Lys Val Ala Leu Val Thr Ala Ser Thr Asp Gly Ile Gly Phe Ala Ile
35 40 45
Ala Arg Arg Leu Ala Gln Asp Gly Ala His Val Val Val Ser Ser Arg
50 55 60
Lys Gln Gln Asn Val Asp Gln Ala Val Ala Thr Leu Gln Gly Glu Gly
65 70 75 80
Leu Ser Val Thr Gly Thr Val Cys His Val Gly Lys Ala Glu Asp Arg
85 90 95
Glu Arg Leu Val Ala Thr Ala Val Lys Leu His Gly Gly Ile Asp Ile
100 105 110
Leu Val Ser Asn Ala Ala Val Asn Pro Phe Phe Gly Ser Ile Met Asp
115 120 125
Val Thr Glu Glu Val Trp Asp Lys Gly Phe Ser Pro Tyr Asn Val Ser
130 135 140
Lys Thr Ala Leu Leu Gly Leu Thr Lys Thr Leu Ala Ile Glu Leu Ala
145 150 155 160
Pro Arg Asn Ile Arg Val Asn Cys Leu Ala Pro Gly Leu Ile Lys Thr
165 170 175
Ser Phe Ser Arg Met Leu Trp Met Asp Lys Glu Lys Glu Glu Ser Met
180 185 190
Lys Glu Thr Leu Arg Ile Arg Arg Leu Gly Glu Pro Glu Asp Cys Ala
195 200 205
Gly Ile Val Ser Phe Leu Cys Ser Glu Asp Ala Ser Tyr Ile Thr Gly
210 215 220
Glu Thr Val Val Val Gly Gly Gly Thr Pro Ser Arg Leu
225 230 235
<210> 2
<211> 711
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 2
atgcacaagg cggggctgct aggcctctgt gcccgggctt ggaattcggt gcggatggcc 60
agctccggga tgacccgccg ggacccgctc gcgaataagg tggccctggt aacggcctcc 120
accgacggga tcggcttcgc catcgcccgg cgtttggccc aggacggggc ccatgtggtc 180
gtcagcagcc ggaagcagca gaatgtggac caggcggtgg ccacgctgca gggggagggg 240
ctgagcgtga cgggcaccgt gtgccatgtg gggaaggcgg aggaccggga gcggctggtg 300
gccacggctg tgaagcttca tggaggtatc gatatcctag tctccaatgc tgctgtcaac 360
cctttctttg gaagcataat ggatgtcact gaggaggtgt gggacaaggg cttcagtcct 420
tacaatgtca gtaaaacagc cttgctgggc ctgaccaaga ccctggccat agagctggcc 480
ccaaggaaca ttagggtgaa ctgcctagca cctggactta tcaagactag cttcagcagg 540
atgctctgga tggacaagga aaaagaggaa agcatgaaag aaaccctgcg gataagaagg 600
ttaggcgagc cagaggattg tgctggcatc gtgtctttcc tgtgctctga agatgccagc 660
tacatcactg gggaaacagt ggtggtgggt ggaggaaccc cgtcccgcct c 711
<210> 3
<211> 12
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtgtgccatg tg 12
<210> 4
<211> 14
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
acaagggctt cagt 14
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
atgcacaagg cggggctgct 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gaggcgggac ggggttcctc 20
Claims (7)
1. a kind of mutain of the class Hep27 albumen of people, which is characterized in that its amino acid sequence is as shown in SEQ ID NO:1.
2. a kind of encoding gene of the class Hep27 protein mutation albumen of people, which is characterized in that its nucleotide sequence such as SEQ ID
Shown in 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 class Hep27 protein mutation albumen of people according to claim 2, the class Hep27 with people
The comparison result of the nucleotide sequence of protein normal albumen determines.
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.
5. a kind of monoclonal antibody of the class Hep27 protein mutation albumen of specific recognition people, which is characterized in that it can be with SEQ
Amino acid sequencespecific shown in ID NO:1 combines.
6. a kind of for detecting the ELISA kit of the antibody of the class Hep27 protein mutation albumen of people, which is characterized in that described
ELISA kit includes: the ELISA ELISA Plate for being coated with monoclonal antibody described in claim 5, ELIAS secondary antibody, test object
Class Hep27 albumen, sample diluting liquid, coating buffer, ELISA ELISA Plate cleaning solution, developing solution and terminate liquid.
7. according to claim 6 for detecting the ELISA kit of the antibody of the class Hep27 protein mutation albumen of people,
It is characterized in that, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeled.
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|---|---|---|---|
| CN201810747395.XA CN108949708A (en) | 2018-07-10 | 2018-07-10 | A kind of mutain of the class Hep27 albumen of people and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810747395.XA CN108949708A (en) | 2018-07-10 | 2018-07-10 | A kind of mutain of the class Hep27 albumen of people and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108949708A true CN108949708A (en) | 2018-12-07 |
Family
ID=64482574
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810747395.XA Pending CN108949708A (en) | 2018-07-10 | 2018-07-10 | A kind of mutain of the class Hep27 albumen of people and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108949708A (en) |
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2018
- 2018-07-10 CN CN201810747395.XA patent/CN108949708A/en active Pending
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Application publication date: 20181207 |