CN109852595A - Mediate rna plymerase ii transcripton subunit 11 mutain and its application - Google Patents
Mediate rna plymerase ii transcripton subunit 11 mutain and its application Download PDFInfo
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- CN109852595A CN109852595A CN201711231911.5A CN201711231911A CN109852595A CN 109852595 A CN109852595 A CN 109852595A CN 201711231911 A CN201711231911 A CN 201711231911A CN 109852595 A CN109852595 A CN 109852595A
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Abstract
The present invention relates to mediate rna plymerase ii transcripton subunit 11 mutain and its application, by using a large amount of cancer patients as research case, genetic test is carried out to case and is analyzed, determine the mutain of the mediation rna plymerase ii transcripton subunit 11 of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the mediation rna plymerase ii transcripton subunit 11 mutain of the people, for the impulse that the gene diagnosis of cancer provides, the early diagnosis, early discovery and associated treatment of cancer are realized.
Description
Technical field
The present invention relates to a kind of genetic engineering fields, more particularly to mediate rna plymerase ii transcripton subunit 11 and be mutated egg
Its application of bletilla.
Background technique
Mediate the enzyme that rna plymerase ii transcripton subunit 11 is core during gene expression, the transcription participated in
It is basic, hight coordinate a biological process of eukaryocyte chromosome.The mediation RNA polymerase II transcripton of people
Subunit 11 mutation human health can be impacted, the study found that certain cancers (cancer include lung cancer, liver cancer,
Cancer of pancreas and leukaemia) patient peripheral blood carry out gene sequencing during, discovery patient mediation rna plymerase ii turn
It records sub- subunit 11 to be mutated, therefore, to the detection of the mediation rna plymerase ii transcripton subunit 11 of people mutation to judging people
Whether body suffers from cancer with certain impulse.
Summary of the invention
It is an object of that present invention to provide the mediation rna plymerase ii transcripton subunit 11 mutain of people a kind of and its answer
With.
Technical solution of the present invention includes:
In a first aspect, providing the mediation rna plymerase ii transcripton subunit 11 mutain of people a kind of, amino acid sequence
As shown in SEQ ID NO:1.
Second aspect provides a kind of encoding gene of the mutain of the mediation rna plymerase ii transcripton subunit 11 of people,
Its 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 people mediation rna plymerase ii transcripton subunit 11 mutain nucleotide sequence, with
The comparison result of the nucleotide sequence of the mediation normal albumen of rna plymerase ii transcripton subunit 11 of people determines.
Preferably, the nucleotide probe is base sequence shown in SEQ ID NO:3.
Fourth aspect provides the mediation rna plymerase ii transcripton subunit 11 mutain of specific recognition people a kind of
Monoclonal antibody, the amino acid sequence of coding are shown in SEQ ID NO:1.
5th aspect provides a kind of for detecting the anti-of the mediation rna plymerase ii transcripton subunit 11 mutain of people
The ELISA kit of body, the ELISA kit include: the ELISA ELISA Plate for being coated with said monoclonal antibody, enzyme mark two
Anti-, the mediation rna plymerase ii transcripton subunit 11 albumen of test object, sample diluting liquid, coating buffer, ELISA enzyme mark
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.
6th aspect provides a kind of mediation RNA polymerase II based on any of the above-described ELISA kit detection people
The method of the antibody of transcripton subunit 11 mutain, comprising:
A, said monoclonal antibody is diluted using the coating buffer, and the monoclonal after dilution is resisted
Body is loaded into the hole of ELISA ELISA Plate, and is incubated at room temperature；
B, it is diluted to using the mediation rna plymerase ii transcripton subunit 11 albumen that the sample diluting liquid will test object
Various concentration gradient, and by the mediation rna plymerase ii transcripton subunit 11 albumen of various concentration gradient be loaded respectively to
In the hole of ELISA ELISA Plate, and it is incubated at room temperature；
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；
E, the liquid in each hole is got rid of, the ELISA ELISA Plate cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature is protected from light incubation, and the terminate liquid is added and terminates reaction；
G, 450nm measures OD value in microplate reader.
Preferably, further comprise: blank control experiment.
The present invention provides the mediation rna plymerase ii transcripton subunit 11 mutain of people a kind of and its applications, will be big
Cancer patient is measured as research case, genetic test is carried out to case and is analyzed, determines the mediation rna plymerase ii of people
The mutain of transcripton subunit 11 prepares gene according to the mediation rna plymerase ii transcripton subunit 11 mutain of the people
Chip, monoclonal antibody and ELISA kit, for the impulse that provides of gene diagnosis of cancer, realize cancer early diagnosis,
Early discovery and associated treatment.
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；
Fig. 7 is the standard curve that the offer of the embodiment of the present invention six is protein content；
Fig. 8 is the Western blot testing result schematic diagram that the embodiment of the present invention six provides；
Fig. 9 is the standard curve that the offer of the embodiment of the present invention seven is protein content；
Figure 10 is the Western blot testing result schematic diagram that the embodiment of the present invention seven 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 mediation RNA polymerase II transcripton subunit of people
The encoding gene of 11 mutains, nucleotide sequence is as shown in SEQ ID NO:2；Correspondingly, it is determined according to the encoding gene
The mediation rna plymerase ii transcripton subunit 11 mutain of people out, amino acid sequence is as shown in SEQ ID NO:1.
Secondly, according to the mediation rna plymerase ii transcripton subunit 11 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 mediation rna plymerase ii transcripton subunit 11 of people
The nucleotide sequence of albumen, the comparison with the nucleotide sequence of the mediation normal albumen of rna plymerase ii transcripton subunit 11 of people
As a result it determines, and according to the design principle of following probe, designs the mediation rna plymerase ii transcripton subunit 11 for people
The nucleotide probe of the specificity of mutain.
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%-65%, the specificity that non-specific hybridization guarantees hybridization is reduced；
4. nucleotide probe intramolecule, which stablizes secondary structure pairing bases longs, is less than 4bp, can guarantee in this way will not
Hybridization efficiency is influenced because of the secondary structure of nucleotide probe internal stability；
5. the similitude through Homology search and other sequences is less than 40%；
6. being continuously no more than 20 bases with the homologous fragment of non-probe alternative sequence by contrast.
Wherein, the tune for mediating rna plymerase ii transcripton subunit 11 mutain corresponding amino acid sequence and people of people
Stop the comparison result of the corresponding amino acid sequence of the normal albumen of rna plymerase ii transcripton subunit 11 referring to FIG. 1, wherein, Fig. 1
In Query sequence be people the corresponding amino acid sequence of mediation rna plymerase ii transcripton subunit 11 mutain, Sbjct
Sequence is the corresponding amino acid sequence of the normal albumen of mediation rna plymerase ii transcripton subunit 11 of people, Query sequence with
Sequence between Sbjct sequence is comparison result, and as can be seen from FIG. 1, the mediation rna plymerase ii transcripton subunit 11 of people is mutated
Mediation rna plymerase ii transcripton subunit 11 normal albumen of the albumen relative to people, a part of amino acid sequence has 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 mediation rna plymerase ii transcripton subunit 11 of object is mutated, can be with then when choosing nucleotide probe
It will include a nucleotide sequence of mutated site nucleotide as 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, designing one kind in the manner described above, preferably nucleotide probe is as shown in SEQ ID NO:3, the nucleotide probe are as follows:
aggtcggtgt g
(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 mediation rna plymerase ii transcripton subunit 11 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 digit that negative internal reference Quality Control probe includes can be with nucleotide probes
Digit is identical.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 mediation RNA of people
In polymerase II transcripton subunit 11 mutain, select sequence corresponding from nucleotide probe different, and digit can be with core
The digit of thuja acid probe is identical, and one section of nucleotide sequence that can also be different from nucleotide probe digit is as positive internal control Quality Control
Probe.Preferably, nucleotide digit positive internal control Quality Control probe identical with nucleotide probe digit is chosen.The embodiment of the present invention
In, positive internal control probe sequence needed for genetic chip can be with are as follows: atggctacct a.
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 1.5mLEP pipe for taking DEPC to handle is added in being detected sample processing tube to be detected sample processing tube
The 300 μ L of blood of test object, adds 700 μ L of Trizol, mixes well, be placed at room temperature for 10 Min.
(3) chloroform of 200 μ L is added, covers tightly centrifuge tube lid, firmly shakes centrifuge tube, be placed at room temperature for 3-5min,
12000r/min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(4) isometric isopropanol is added, gently overturns centrifuge tube and mixes well liquid, be placed at room temperature for 10min, 12000r/
Min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(5) one time, 7500r/min, 4 DEG C centrifugation 15min is washed with 1mL75% ethyl alcohol, all supernatants is carefully sucked, ultra-clean
10 μ L DEPC processing water dissolution is added in dry 15min in 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 1.5mL centrifuge tube:
| Total serum IgE | The most 6.5 μ L of 2 μ g |
| T7Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
(2) 65 DEG C keep the temperature ice bath 5 minutes 10 minutes, and 5 × First Strand B μ ffer is preheated 5 points at 65 DEG C in advance
Clock.
(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 mix is added 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 use.Following operative configuration is pressed simultaneously
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 |
| T7RNAPolymerase | 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 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 15-30sec, discards filtered solution.
(4) >=8000g centrifuge washing 15-30sec abandoning is drawn in 500 μ L Buffer RPE to RNeasy mini pillars
Go filtered solution again with 500 μ L Buffer RPE the casing that >=8000g centrifuge washing 2min discards filtered solution and 2mL will
RNeasy 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 RNA concentration.Need 260 and 280nm measure absorbance come determine sample concentration and
Purity A260/A280 should be purer RNA (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 0.3M sodium bicarbonate (NaHCO3) pH9.0 of 3.4 μ L 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 chip hybridization 4x44K microarrays
(1) according to the form below prepares fragmentation mixed liquor and then carries out fragmentation in 60 DEG C of warm bath 30min.
| Cy3cRNA green fluorescence | 875ng |
| 10XBlocking Agent | 11μL |
| 25X Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ L 2X 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
(1 liter) of washing lotion 1 configuration:
| DEPC-H2O | 700m L |
| 20*SSPE | 300m L |
| 20%N-Lauroylsarcosine | 0.25m L |
(1 liter) of washing lotion 2 configuration:
Washing lotion 3:Stabilization and Drying Solution
(1) chip is taken out to wash 1 minute in washing lotion 1；
(2) chip is put into washing lotion 2 again and is washed 1 minute (37 DEG C)；
(3) finally chip is washed 30 seconds in washing lotion 3.
10, chip scanning
Chip is scanned in scanner, determines that the mediation rna plymerase ii of test object turns according to scanning result
Record whether sub- subunit 11 albumen is mutated.Please refer to Fig. 3 and Fig. 4, in result shown in Fig. 3, 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 mediation rna plymerase ii transcripton subunit 11 albumen of test object is mutated.Result shown in Fig. 4
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 mediation rna plymerase ii transcripton subunit 11 albumen of test object does not occur
Mutation.
Embodiment three, the monoclonal for mediating rna plymerase ii transcripton subunit 11 mutain of specific recognition people are anti-
The preparation of body
1, according to base sequence (such as SEQ ID NO:2 of the mediation rna plymerase ii transcripton subunit 11 mutain of people
It is shown) upstream primer is designed as shown in SEQ ID NO:4, and, downstream primer is as shown in SEQ ID NO:5:
Upstream primer (P): atggctacct acagcctg
Downstream primer (F): cacaccgacc tgggtgag
2, the DNA of test object is that template carries out PCR amplification
PCR amplification is carried out by template of the DNA of test object, obtains the mediation rna plymerase ii transcripton subunit 11 of people
The complete segment of mutating protein gene, and pMD19-T Vector (Takara company) is connected, it is sequenced.Then by special life
Object corporation is a kind of humanization or Chimeric antibodies for antibody.The antibody of preparation is measured into content using ELISA method.
The antibody of example IV, mediation rna plymerase ii transcripton subunit 11 mutain for detecting people
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 mediation rna plymerase ii transcripton subunit 11 albumen of test object, sample diluting liquid, coating buffering
Liquid, 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 patients with lung cancer as test object, and the lung cancer is detected using ELISA kit and is suffered from
Whether the mediation rna plymerase ii transcripton subunit 11 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 mediation rna plymerase ii transcripton subunit 11 albumen of patients with lung cancer is diluted to difference using sample diluting liquid
Concentration gradient, and the mediation rna plymerase ii transcripton subunit 11 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, 450nm measures OD value in microplate reader.
H, make standard curve: using standard concentration as abscissa, the OD value of standard items measurement is ordinate, makes standard
Curve；Calculate standard curve regression coefficient R2, work as R2This measurement is effective when > 0.99；Wherein it is possible to soft using ELISACalc
Part draws the standard curve, can know regression coefficient R according to the ELISACalc software2=0.9971, therefore, this measurement
Effectively.The OD450 value that will test, which brings standard curve into, can acquire the mediation rna plymerase ii transcripton Asia of patients with lung cancer
The concentration of 11 albumen of base.It is transcribed using rna plymerase ii is mediated in the ELISA method detection Serum of Patients with Lung Cancer sample of foundation
The content of sub- subunit 11 albumen.And it is identified with Western blot.Referring to FIG. 5, being the standard curve of OD value.Wherein, X
Axis is OD 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. upper layer spacer gel concentration preparation system (Total Volum:8mL)
| ddH2O | 5.5mL |
| 30% acrylamide mixed liquor | 1.3mL |
| 1.0mol/L Tris(PH 6.8) | 1.0mL |
| 10%SDS | 0.08mL |
| 10% ammonium persulfate | 0.08ml |
| TEMED | 0.008mL |
(3) lower layer's separation gel of respective volume is added in each offset plate, adds the sterile ddH of 1mL2O flattens separation
Glue blots remaining moisture in plastic plate 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 close membrane 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 cleans film three times, each 10min；
(6) secondary antibody (being diluted with 3% milk with 1:5000-10000) is incubated at room temperature film 2h；
(7) film is cleaned three times 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 mediation rna plymerase ii transcripton subunit 11 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: 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 18KD
There is obvious band, wherein the molecular weight for mediating rna plymerase ii transcripton subunit 11 mutain is about 18KD, is shown
The mediation rna plymerase ii transcripton subunit 11 albumen of the patients with lung cancer is implicitly present in mutation.
Embodiment six
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 mediation rna plymerase ii transcripton subunit 11 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, difference is diluted to using the mediation rna plymerase ii transcripton subunit 11 albumen that sample diluting liquid will test object
Concentration gradient, and the mediation rna plymerase ii transcripton subunit 11 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, 450nm measures OD value in microplate reader.
H, make standard curve: using standard concentration as abscissa, the OD value of standard items measurement is ordinate, makes standard
Curve；Calculate standard curve regression coefficient R2, work as R2This measurement is effective when > 0.99；Wherein it is possible to soft using ELISACalc
Part draws the standard curve, can know regression coefficient R according to the ELISACalc software2=0.9963, therefore, this measurement
Effectively.The OD450 value that will test, which brings standard curve into, can acquire the mediation rna plymerase ii turn of liver cancer patient in sample
Record the concentration of sub- subunit 11 albumen.It is detected in liver cancer patient blood serum sample using the ELISA method of foundation and mediates rna plymerase ii
The content of transcripton subunit 11 albumen.And it is identified with Western blot.Referring to FIG. 7, being the standard curve of OD value.Its
In, X axis is OD 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. 8, in Fig. 8
Marker is used to characterize the size of labelled protein.The experimental display result of the present embodiment, compared with blank control, only in experimental group
Nearby there is obvious band in 18KD, wherein the molecular weight for mediating rna plymerase ii transcripton subunit 11 mutain is about
18KD shows that the mediation rna plymerase ii transcripton subunit 11 albumen of the liver cancer patient is implicitly present in mutation.
Embodiment seven
In embodiments of the present invention, using Pancreas cancer patients as test object, and the pancreas is detected using ELISA kit
Whether the mediation rna plymerase ii transcripton subunit 11 albumen of cancer patient is mutated, and this method at least may include as follows
It is a kind of:
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 mediation rna plymerase ii transcripton subunit 11 albumen of Pancreas cancer patients is diluted to not using sample diluting liquid
Same concentration gradient, and the mediation rna plymerase ii transcripton subunit 11 albumen of various concentration gradient is loaded respectively to ELISA
In the hole of ELISA 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, 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, 450nm measures OD value in microplate reader.
H, standard curve is made, using standard concentration as abscissa, the OD value of standard items measurement is ordinate, makes standard
Curve；Calculate standard curve regression coefficient R2, work as R2This measurement is effective when > 0.99；Wherein it is possible to soft using ELISACalc
Part draws the standard curve, can know regression coefficient R according to the ELISACalc software2=0.9961, therefore, this measurement
Effectively.The OD450 value that will test, which brings standard curve into, can acquire the mediation rna plymerase ii of Pancreas cancer patients in sample
The concentration of transcripton subunit 11 albumen.It is polymerize using RNA is mediated in the ELISA method detection Pancreas cancer patients blood serum sample of foundation
The content of enzyme II transcripton subunit 11 albumen.And it is identified with Western blot.Referring to FIG. 9, the standard for OD value is bent
Line.Wherein, X-axis is OD 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 18KD, wherein the molecular weight for mediating rna plymerase ii transcripton subunit 11 mutain is about
18KD shows that the mediation rna plymerase ii transcripton subunit 11 albumen of the Pancreas cancer patients 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>rna plymerase ii transcripton subunit 11 mutain and its application are mediated
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 75
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 1
Met Ala Thr Tyr Ser Leu Ala Asn Glu Arg Leu Arg Ala Leu Glu Asp
1 5 10 15
Ile Glu Arg Glu Ile Gly Ala Ile Leu Gln Asn Ala Gly Thr Val Ile
20 25 30
Leu Glu Leu Ser Lys Glu Lys Thr Asn Glu Arg Leu Leu Asp Arg Gln
35 40 45
Ala Ala Ala Phe Thr Ala Ser Val Gln His Val Glu Ala Glu Leu Ser
50 55 60
Ala Gln Ile Arg Tyr Leu Thr Gln Val Gly Val
65 70 75
<210> 2
<211> 225
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 2
atggctacct acagcctggc gaacgagaga ctacgcgctc tggaagacat tgaacgggaa 60
atcggcgcca tccttcagaa tgcaggtact gtgatcctag aattgtccaa ggaaaaaact 120
aacgagcggc tcctagaccg gcaggcggcg gccttcaccg cttcagtgca acacgtggag 180
gcggagctgt cagctcagat ccgctacctc acccaggtcg gtgtg 225
<210> 3
<211> 11
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aggtcggtgt g 11
<210> 4
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
atggctacct acagcctg 18
<210> 5
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cacaccgacc tgggtgag 18
Claims (9)
1. the mediation rna plymerase ii transcripton subunit 11 mutain 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 mediation rna plymerase ii transcripton subunit 11 mutain of people, which is characterized in that its nucleosides
Acid 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
Nucleotide probe according to the people mediation rna plymerase ii transcripton subunit 11 mutain nucleotide sequence, with people's
The comparison result for mediating the nucleotide sequence of the normal albumen of rna plymerase ii transcripton subunit 11 determines.
4. genetic chip according to claim 3, which is characterized in that the nucleotide probe is shown in SEQID NO:3
Base sequence.
5. a kind of monoclonal antibody of the mediation rna plymerase ii transcripton subunit 11 mutain of specific recognition people, special
Sign is that the amino acid sequence of coding is shown in SEQ ID NO:1.
6. it is a kind of for detecting the ELISA kit of the antibody of the mediation rna plymerase ii transcripton subunit 11 mutain of people,
It is characterized in that, the ELISA kit includes: ELISA ELISA Plate, the enzyme for being coated with monoclonal antibody described in claim 5
Mark secondary antibody, the mediation rna plymerase ii transcripton subunit 11 albumen of test object, sample diluting liquid, coating buffer, ELISA
ELISA Plate cleaning solution, developing solution and terminate liquid.
7. according to claim 6 for detecting the antibody of the mediation rna plymerase ii transcripton subunit 11 mutain of people
ELISA kit, which is characterized in that the ELIAS secondary antibody be diluted HRP horseradish peroxidase-labeled Goat anti-Human
IgG。
8. a kind of mediation rna plymerase ii transcripton subunit 11 based on the ELISA kit detection people of claim 6 or 7
The method of the antibody of mutain characterized by comprising
A, monoclonal antibody described in claim 5 is diluted using the coating buffer, and by the list after dilution
Clonal antibody is loaded into the hole of ELISA ELISA Plate, and is incubated at room temperature；
B, difference is diluted to using the mediation rna plymerase ii transcripton subunit 11 albumen that the sample diluting liquid will test object
Concentration gradient, and the mediation rna plymerase ii transcripton subunit 11 albumen of various concentration gradient is loaded respectively to ELISA enzyme mark
In the hole of plate, and it is incubated at room temperature；
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；
E, the liquid in each hole is got rid of, the ELISA ELISA Plate cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature is protected from light incubation, and the terminate liquid is added and terminates reaction；
G, 450nm measures OD value in microplate reader.
9. the mediation rna plymerase ii transcripton subunit 11 mutain of ELISA kit detection people according to claim 8
Antibody method, which is characterized in that further comprise: blank control experiment.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795542A (en) * | 2021-02-07 | 2021-05-14 | 华中农业大学 | Hybridoma cell of monoclonal antibody for producing pig RNAP II and application |
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2017
- 2017-11-30 CN CN201711231911.5A patent/CN109852595A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795542A (en) * | 2021-02-07 | 2021-05-14 | 华中农业大学 | Hybridoma cell of monoclonal antibody for producing pig RNAP II and application |
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