CN108546292A - A kind of 7 family's C mutains of chronic B cell leukemia/lymthoma and application - Google Patents
A kind of 7 family's C mutains of chronic B cell leukemia/lymthoma and application Download PDFInfo
- Publication number
- CN108546292A CN108546292A CN201810256000.6A CN201810256000A CN108546292A CN 108546292 A CN108546292 A CN 108546292A CN 201810256000 A CN201810256000 A CN 201810256000A CN 108546292 A CN108546292 A CN 108546292A
- Authority
- CN
- China
- Prior art keywords
- chronic
- lymthoma
- family
- cell leukemia
- elisa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4747—Apoptosis related proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57426—Specifically defined cancers leukemia
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Toxicology (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of 7 family's C mutains of chronic B cell leukemia/lymthoma and applications, by regarding a large amount of chronic lymphocytic leukemias and patients with lung cancer as research case, genetic test is carried out to case and is analyzed, determine 7 family's C mutains of chronic B cell leukemia/lymthoma of people, genetic chip is prepared according to 7 family's C mutains of chronic B cell leukemia/lymthoma of the people, monoclonal antibody and ELISA kit, gene diagnosis for chronic lymphocytic leukemia and lung cancer provides impulse, and to realize that the diagnosing and treating of relevant disease provides theoretical foundation.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to a kind of 7 family C of chronic B cell leukemia/lymthoma are prominent
Kink of preserved egg bletilla application.
Background technology
Human body chronic B cell leukemia/7 gene family of lymthoma (B-cell CLL/lymphoma 7gene family,
Bcl-7) include Bcl-7A, Bcl-7B and Bcl-7C.A large amount of clinical researches show 7 base of chronic B cell leukemia/lymthoma
Because family participates in the generation and development of cancer.Bcl-7 plays key effect in the maintenance of nuclear structure, simultaneously participates in Wnt accesses
With the number of ways such as apoptosis.
During the peripheral blood to certain chronic lymphocytic leukemias and patients with lung cancer carries out gene sequencing, send out
The 7 family C of chronic B cell leukemia/lymthoma of existing patient is mutated, therefore, chronic B cell leukemia/leaching to people
The detection of Ba Liu 7 family C mutation has certain impulse to judging whether human body suffers from relevant disease.
Invention content
Present invention aims at 7 family's C mutains of chronic B cell leukemia/lymthoma of people of offer a kind of and its answer
With.
Technical solution of the present invention includes：
In a first aspect, providing a kind of 7 family's C mutains of chronic B cell leukemia/lymthoma of people, amino acid sequence
Row such as SEQ ID NO:Shown in 1.
Second aspect provides a kind of encoding gene of 7 family's C mutains of chronic B cell leukemia/lymthoma of people,
Its nucleotide sequence such as SEQ ID NO:Shown in 2.
The third aspect provides a kind of genetic chip, including：Solid phase carrier and fixed nucleotide probe on this carrier；
The nucleotide probe according to the nucleotide sequences of 7 family's C mutains of chronic B cell leukemia/lymthoma of the people, with
The comparison result of the nucleotide sequence of the 7 normal albumen of family C of chronic B cell leukemia/lymthoma of people determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of 7 family's C mutains of chronic B cell leukemia/lymthoma of specific recognition people
The amino acid sequence of monoclonal antibody, coding is SEQ ID NO:Shown in 1.
5th aspect provides a kind of for detecting the anti-of 7 family's C mutains of chronic B cell leukemia/lymthoma of people
The ELISA kit of body, the ELISA kit include：It is coated with ELISA ELISA Plates, the enzyme mark two of said monoclonal antibody
Anti-, detection object 7 family's C protein of chronic B cell leukemia/lymthoma, sample diluting liquid, coating buffer solution, ELISA enzyme marks
Plate cleaning solution, developing solution and terminate liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
6th aspect, provide it is a kind of based on any of the above-described ELISA kit detect the chronic B cell leukemia of people/
The method of the antibody of 7 family's C mutains of lymthoma, including：
A, said monoclonal antibody is diluted using the coating buffer solution, and the monoclonal after dilution is resisted
Body is loaded into the hole of ELISA ELISA Plates, incubation at room temperature；
B, the 7 family's C protein of chronic B cell leukemia/lymthoma for detecting object is diluted using the sample diluting liquid
At various concentration gradient, and by 7 family's C protein of chronic B cell leukemia/lymthoma of various concentration gradient be loaded respectively to
In the hole of ELISA ELISA Plates, incubation at room temperature；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature is protected from light incubation, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
Preferably, further comprise：Blank control is tested.
The present invention provides 7 family's C mutains of chronic B cell leukemia/lymthoma of people a kind of and its applications, will be big
Chronic lymphocytic leukemia is measured with patients with lung cancer as research case, genetic test is carried out to case and is analyzed, determines people
7 family C of chronic B cell leukemia/lymthoma mutain, according to chronic B cell leukemia/lymthoma 7 of the people
Race's C mutains prepare genetic chip, monoclonal antibody and ELISA kit, are chronic lymphocytic leukemia and lung cancer
Gene diagnosis provides impulse, to realize that the diagnosing and treating of relevant disease provides theoretical foundation.
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention and after coordinating attached drawing to be described in detail such as.
Description of the drawings
Fig. 1 is a kind of comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 2 is a kind of genetic chip layout that the embodiment of the present invention one provides；
Fig. 3 is a kind of colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 4 is another colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 5 is the standard curve that the offer of the embodiment of the present invention five is protein content；
Fig. 6 is the Western blot testing result schematic diagrames that the embodiment of the present invention five provides；
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 diagrames that the embodiment of the present invention six provides.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment one, genetic chip
It is possible, firstly, to be a kind of " detection side of mutain according to application No. is " 201710429915.8 ", patent name
Detection method described in the Chinese invention patent of method and device " determines 7 family of chronic B cell leukemia/lymthoma of people
The encoding gene of C mutains, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, it is determined according to the encoding gene
7 family's C mutains of chronic B cell leukemia/lymthoma of people, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the 7 family's C mutains of chronic B cell leukemia/lymthoma and its encoding gene of people, according to such as
Under type realizes the preparation of genetic chip.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is prominent according to the 7 family C of chronic B cell leukemia/lymthoma of people
Become the nucleotide sequence of albumen, the ratio with the nucleotide sequence of the 7 normal albumen of family C of chronic B cell leukemia/lymthoma of people
Result is determined, and according to the design principle of following probe, designs chronic B cell leukemia/lymthoma 7 for people
The nucleotide probe of the specificity of race's C mutains.
Wherein, the principle of nucleotide probe design is as follows：
1. nucleotide probe Tm values should be close to the average Tm values of whole gene group, 5 DEG C of fluctuation up and down；
2. the single base that nucleotide probe intramolecular repeats continuously is no more than 4；
3. G+C contents are 40%-60%, the specificity that non-specific hybridization ensures hybridization is reduced；
4. it should be less than 4bp with the bases longs of probe molecule internal stability secondary structure pairing in nucleotide probe sequences,
Can ensure 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 7 family's C mutains of chronic B cell leukemia/lymthoma of people and people's is slow
Property the corresponding amino acid sequence of the 7 normal albumen of family C of B cell leukemia/lymthoma comparison result please refer to Fig.1, wherein figure
Query sequences in 1 are the corresponding amino acid sequences of 7 family's C mutains of chronic B cell leukemia/lymthoma of people,
Sbjct sequences are the corresponding amino acid sequence of the 7 normal albumen of family C of chronic B cell leukemia/lymthoma of people, Query sequences
Sequence between Sbjct sequences is comparison result, as can be seen from FIG. 1, the 7 family C of chronic B cell leukemia/lymthoma of people
Chronic B cell leukemia/lymthoma 7 family C normal albumen of the mutain relative to people, amino acid sequence has occurred scarce at one
It loses, many places amino acid is mutated, according to SEQ ID NO:The comparison result of nucleotide sequence and Fig. 1 shown in 2 is
Whether chronic B cell leukemia/lymthoma 7 family the C for capableing of specific recognition object to be detected is mutated, then
When choosing nucleotide probe, nucleotide probe can be designed according to any one of following several modes mode：
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, according to SEQ ID NO:It nucleotide sequence shown in 2 and is set according to above-mentioned nucleotide probe
Principle is counted, 1. designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, the nucleotide probe
For：acgggaacct ggaa
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether 7 family C of chronic B cell leukemia/lymthoma of detection people mutates
In order to ensure to detect the quality of subject sample, blank control, positive control and the moon need to be also designed on genetic chip
Property control.Wherein, the layout of the genetic chip at least can be a kind of layout as shown in Figure 2.In fig. 2, is blank pair
According to, zero is negative control,For positive control,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：Be one section does not have other genetic fragments of homology with detection gene, as hybridizing
The monitor control index of non-specific hybridization in journey, the nucleotide number that negative internal reference Quality Control probe includes can be with nucleotide probes
Base number is identical, can also be different.In the embodiment of the present invention, the negative internal reference probe sequence needed for genetic chip can be：
tgatgctgat aattgcat。
Positive internal control Quality Control probe：It is one section of other genetic fragment for having homology with detection gene, it is thin in the chronic B of people
In 7 family's C mutains of born of the same parents' leukaemia/lymthoma, select sequence corresponding from nucleotide probe different, and can be with nucleotide
The number of probe base is identical, and one section of nucleotide sequence that can also be different from the number of nucleotide probe base is as in the positive
Join Quality Control probe.Preferably, nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen.
In the embodiment of the present invention, the positive internal control probe sequence needed for genetic chip can be：tgggtgattt tctt.
It should be noted that in the deposition process of genetic chip, clicks and enters negative internal reference according to the layout of genetic chip and visit
Needle and positive internal control probe solution；The spotting buffer (10% aqueous trehalose) that reagent used in blank control is 1 times.
The application of embodiment two, genetic chip
1, sample treatment
(1) the blood 1-3mL of acquisition testing object.
(2) the 1.5mL EP pipes that DEPC is handled are taken, as detected sample processing tube, in detected sample processing tube
The middle 300 μ L of blood that detection object is added, add 700 μ L of Trizol, mix well, be placed at room temperature for 10min.
(3) chloroform of 140 μ L is added, covers tightly pipe lid, firmly shakes, be placed in a centrifuge, 12000r/min, 4 DEG C of centrifugations
15min, it is careful to draw supernatant in centrifuge tube, the supernatant of absorption is transferred in clean centrifuge tube.
(4) isometric isopropanol is added in the centrifuge tube of supernatant in storing step (3), it is abundant gently overturns centrifuge tube
Mixing liquid is placed at room temperature for 10min, 12000r/min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(5) it being cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min, 4 DEG C of centrifugation 15min carefully suck all supernatants,
The dry 15min in super-clean bench is added 10 μ L DEPC and handles water dissolution.
(6) products therefrom is RNA can influence the labeling effciency and chip hybridization knot of probe if the purity of total serum IgE is not high
Fruit.So using QIAGENKit purifies total serum IgE.
2, the first chains of cDNA and the second chain one-step synthesis method
(1) 2 μ g RNA is taken to configure following reaction solution in the centrifuge tube of 1.5mL：
| Total serum IgE | The most 6.5 μ L of 2 μ g |
| T7 Promotor primer | 5μL |
| RNase-free Water | X μL |
| Total volume | 11.5μL |
Wherein, the addition X μ L of RNase-free Water are subtracted according to 11.5 μ L of total volume
The 5 μ L of addition of T7Promotorprimer, then subtract the addition of total serum IgE and calculate and get.
(2) 10min, and ice bath 5min are kept the temperature at 65 DEG C, in advance by 5 × First Strand B μ ffer at 65 DEG C
Preheat 5min.
(3) following cDNA synthetic systems are configured：
| 5×First Strand Buffer | 4μL |
| 0.1M DTT | 2μL |
| 10mM dNTP mix | 1μL |
| MMLV RT | 1μL |
| RNase OUT | 0.5μL |
| Total volume | 8.5μL |
(4) above-mentioned 8.5 μ L are added after mixing after being denaturalized in the RNA of ice bath.
(5) pipette tips mixing is used to centrifuge later.
(6) 40 DEG C of reaction 2h.
3, aaUTP marks cRNA synthesis
The configuration of NTP：
| 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, notes：40 DEG C of heat preservation 1min before 50%PEG (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcription mix；
(1) configuration Transcription mix
| RNase-free Water | 5.7μL |
| 4×Transcription Buffer | 20μL |
| NTP | 16μL |
| 0.1M DTT | 6μL |
| 50%PEG | 6.4μL |
| aa-UTP(25mM) | 4μL |
| Inorganic Pyrophosphatase | 0.6μL |
| T7 RNA Polymerase | 0.8μL |
| Total volume | 60μL |
(2) 60 μ L Transcription mix and mixing is added.
(3) 60 DEG C of hot lid in PCR instrument, 40 DEG C of reaction 2h.
4, cRNA is purified
QIAGEN RNeasy Mini kit purify cRNA, and specific method can be found in what QIAGEN companies provided with kit
Operation manual.
(1) 20 μ L RNase free water are added, 350 μ L Buffer RLT are added and mix well.
(2) 250 μ L absolute ethyl alcohols are added, Tip mix well.
(3) total 700 solution of the μ L containing total serum IgE are transferred in the RNeasy pillars being sleeved in 2mL centrifuge tubes >=8000g from
Heart 15-30s, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=8000g centrifuge washings 15-30s discards filter
Cross liquid discard the casing of filtered solution and 2mL in >=8000g centrifuge washings 2min with 500 μ L Buffer RPE again will
RNeasymini pillars are transferred in a new 1.5mL Eppendorf pipes.
(5) water for drawing 30 μ L RNase free stands 1min, >=8000g centrifugation elutions 1min.
(6) it is primary to repeat step (5).
5, cRNA concentration mensurations
With spectrophotometric analysis cRNA concentration.It needs to measure the light absorption value at 260nm and 280nm to determine the dense of sample
Degree and purity, A260/A280 should be purer cRNA (ratio 1.9-2.1 also can) close to 2.0.
6, cRNA fluorescents mark；
(1) above-mentioned cRNA4 μ g are taken and are concentrated into 6.6 μ L.
(2) add 10 μ L DMSO mixings.
(3) add the sodium bicarbonate (NaHCO that the 0.3M pH of 3.4 μ L are 9.03) and mixing.
(4) above-mentioned 20 μ L cRNA mixtures are added in fluorescent dye Cy3 simultaneously mixing.25 DEG C of heat preservation 1h.
(5) plus 9 μ L 4M Hydroxylamine mixings after 25 DEG C heat preservation 15min.
7, fluorescent marker cRNA Sample Purification on Single
The process that specific steps are purified with cRNA in the step 4 of the present embodiment, this step are not repeating.
8, cRNA sample fragments and chip hybridization 4x44Kmicroarrays
(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 fluorescences | 875ng |
| 10×Blocking Agent | 11μL |
| 25×Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ 2 × GEx of L HybridizationBuffer are added.
(3) 100 μ L hybridization solutions is taken to be added drop-wise on chip ware, at the same be added dropwise respectively as shown in Figure 2 by chip layout blank,
On negative, positive, experimental group position.It covers chip and is sealed in hybridizing box, 60 DEG C roll hybridization 16h.
9, chip washs
Washing lotion 1 (1L) configures：
| DEPC-H2O | 700mL |
| 20×SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 2 (1L) configures：
| DEPC-H2O | 997mL |
| 20×SSPE | 3.0mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 3：Stabilization and Drying Solution
(1) it takes out chip and washs 1min in washing lotion 1；
(2) chip is put into washing lotion 2 again and washs 1min (37 DEG C)；
(3) chip is finally washed into 30s in washing lotion 3.
10, chip scanning
Chip is scanned in scanner, chronic B cell leukemia/leaching of detection object is determined according to scanning result
Whether 7 family's C proteins of Ba Liu are mutated.It please refers 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 7 family's C protein of chronic B cell leukemia/lymthoma for detecting 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 7 family's C protein of chronic B cell leukemia/lymthoma for detecting object is not sent out
Raw mutation.
Embodiment three, the monoclonal of 7 family's C mutains of chronic B cell leukemia/lymthoma of specific recognition people are anti-
The preparation of body
1, according to base sequence (such as SEQID NO of 7 family's C mutains of chronic B cell leukemia/lymthoma of people:2
It is shown) design sense primer such as SEQ ID NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (F)：atgaaaatgatattagac
Downstream primer (R)：gactttttca ccttgcca
2, the DNA of detection 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 |
DNA to detect object carries out PCR amplification as template, obtains the 7 family C of chronic B cell leukemia/lymthoma of people
The complete segment of mutating protein gene, and pMD19-T Vector (Takara companies) are 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, 7 family's C mutains of chronic B cell leukemia/lymthoma for detecting people
ELISA kit
In the present embodiment, the group of ELISA kit becomes：It is coated with the ELISA enzymes of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, the 7 family's C protein of chronic B cell leukemia/lymthoma for detecting object, sample diluting liquid, coating buffering
Liquid, ELISA ELISA Plates cleaning solution, developing solution and terminate liquid.
Wherein, the parameter of above-mentioned each composition at least can be a kind of following parameter：
ELISA ELISA Plates can be the ELISA enzyme marks in 96 holes；
ELIAS secondary antibody can be the Goat anti-Human IgG of diluted HRP (horseradish peroxidase) labels；Wherein, dilution times
Number can be 8000 times.
It can be 1 × PBS, pH to be coated with buffer solution:7.4；
ELISA ELISA Plates cleaning solution can be 1 × PBS solution containing 0.05%Tween-20；
Developing solution can be 3,3 ', 5,5 '-tetramethyl benzidines；
Terminate liquid can be the sulfuric acid solution of 2mol/L.
Embodiment five
In embodiments of the present invention, it using patients with lung cancer as detection object, and detects the lung cancer using ELISA kit and suffers from
Whether 7 family's C protein of chronic B cell leukemia/lymthoma of person is mutated, and this method at least may include as next
Kind：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h.
B, 7 family's C protein of chronic B cell leukemia/lymthoma of patients with lung cancer is diluted to not using sample diluting liquid
Same concentration gradient, and 7 family's C protein of chronic B cell leukemia/lymthoma 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 Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions are added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated 10-20min, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
H, standard curve is made：Using standard concentration as abscissa, the light absorption value that standard items measure is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measures effective when ＞ 0.99；Wherein it is possible to use ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc softwares2=0.99841, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire the chronic B of patients with lung cancer
The concentration of 7 family's C protein of chronic myeloid leukemia/lymthoma.It is detected using the ELISA method of foundation slow in Serum of Patients with Lung Cancer sample
The content of property 7 family's C protein of B cell leukemia/lymthoma.It is used in combination Westernblot to be identified.Referring to FIG. 5, being extinction
The standard curve of value.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Westernblot 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)
| 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 after gelling to be separated is solid with filter paper, and upper layer is then added and concentrates glue, after being inserted into comb
Wait for upper layer concentration gelling solid.
2, albumen loading electrophoresis：
(1) comb is pulled up, duct label is carried out, 5 × SDS electrophoretic buffers is added, then to being added in protein sample
SDS-PAGE albumen sample-loading buffer (5 ×), boiling water bath heat 3-5min, loading；
(2) electrophoresis first is carried out with 80V voltages, after waiting for that band ran concentration glue, uses 100V voltages instead and run about 100min.
3, transferring film and incubation：
(1) first pvdf membrane is activated in methyl alcohol about 30s.Respectively by gel, foam-rubber cushion and pvdf membrane in advance in electrotransfer
30min is balanced in buffer solution.It is clipped by the sequence of filter paper-glue-film-filter paper, transferring film device is put into the principle of black flour according to black flour
In, refrigerator (ice bag) is added, about 120min is run with the electric current of 150mA in ice；
(2) it takes the film out after the completion of, is put into 5% skim milk at once, gently room temperature closes 1h on shaking table；
(3) PBST cleans closed film, cleans 3 times, each 10min；
(4) primary antibody for being diluted to corresponding multiple is added, 4 DEG C of incubator overnights are incubated；
(5) primary antibody is recycled, PBST cleans film 3 times, each 10min；
(6) secondary antibody is (with 3% milk with 1:5000-1:10000 dilutions) incubation at room temperature film 2h；
(7) film is cleaned 3 times with PBST, each 10min；
(8) it after the isometric mixing of Pierce ECLWestern Blotting Substrate kit A, B liquid, drips dropwise
It is added on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment Image J analyses.
Wherein, primary antibody is that the 7 family C mutain monoclonals of chronic B cell leukemia/lymthoma of the standby people of corporation are anti-
Body, secondary antibody are the Goat anti-Human IgGs of horseradish peroxidase-labeled.
J, Western blot Analysis of test results：It is shown according to Western blot experimental procedures as a result, such as Fig. 6 institutes
Show, wherein the Marker in Fig. 6 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 27KD
There is apparent band, wherein 7 family C mutant protein molecules amounts of chronic B cell leukemia/lymthoma are about 27KD, show this
7 family's C protein of chronic B cell leukemia/lymthoma of patients with lung cancer is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, using patients with chronic lymphocytic as detection object, and ELISA reagents are utilized
Box detect the patients with chronic lymphocytic 7 family's C protein of chronic B cell leukemia/lymthoma whether have occurred it is prominent
Become, this method at least may include following one kind：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h；
B, the 7 family's C protein of chronic B cell leukemia/lymthoma for detecting object is diluted to not using sample diluting liquid
Same concentration gradient, and 7 family's C protein of chronic B cell leukemia/lymthoma 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 Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions are added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated 10-20min, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
H, standard curve is made：Using standard concentration as abscissa, the light absorption value that standard items measure is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measures effective when ＞ 0.99；Wherein it is possible to use ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc softwares2=0.99866, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire chronic lymphatic in sample
The concentration of 7 family's C protein of chronic B cell leukemia/lymthoma of chronic myeloid leukemia patient.It is examined using the ELISA method of foundation
Survey the content of 7 family's C protein of chronic B cell leukemia/lymthoma in patients with chronic lymphocytic blood serum sample.It is used in combination
Westernblot is identified.Referring to FIG. 7, for the standard curve of light absorption value.Wherein, X-axis is light absorption value, and Y-axis is corresponding
Concentration.
I, Westernblot is identified
J, Western blot Analysis of test results
Wherein, step i and step j are 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 apparent band in 27KD, wherein 7 family C mutant protein molecules amounts of chronic B cell leukemia/lymthoma are about
It is prominent to show that 7 family's C protein of chronic B cell leukemia/lymthoma of the patients with chronic lymphocytic is implicitly present in by 27KD
Become.
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 7 family's C mutains of chronic B cell leukemia/lymthoma and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 110
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Trp Val Ile Phe Leu Phe Lys Phe Lys Ile Gly His Lys Ala Val Glu
1 5 10 15
Thr Thr His Asn Ile Asn Asn Ala Phe Gly Pro Gly Ala Ala Asn Lys
20 25 30
His Thr Val Gln Trp Ser Leu Lys Lys Phe Cys Lys Gly Asp Gly Asn
35 40 45
Leu Glu Asp Glu Glu His Ser Gly Trp Pro Leu Lys Val Asp Asn Asn
50 55 60
Gln Met Gly Ala Ile Ile Glu Ala Asp Pro Leu Thr Thr Thr Phe Arg
65 70 75 80
Thr Gln His Arg Pro Phe Tyr Gly His Leu Thr Phe Glu Ala Asn Trp
85 90 95
Gln Gly Glu Lys Ala Leu Tyr Lys Trp Val Pro His Glu Leu
100 105 110
<210> 2
<211> 339
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atgaaaatga tattagacaa aaagcaaatt tgggtgattt tcttattcaa gttcaaaata 60
ggtcataaag cagtggagac aactcataac atcaacaacg catttggccc aggagctgct 120
aacaaacata cagtgcagtg gtcattaaag aagttttgca aaggagacgg gaacctggaa 180
gatgaggagc atagtggctg gccattaaaa gttgacaaca accaaatggg agcaatcatc 240
gaagctgatc ctcttaccac tacattctga agaactcaac atcgaccatt ctatggtcat 300
ttgacatttg aagcaaattg gcaaggtgaa aaagctctc 339
<210> 3
<211> 14
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
acgggaacct ggaa 14
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atgaaaatga tattagac 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gactttttca ccttgcca 18
Claims (9)
1. 7 family's C mutains of chronic B cell leukemia/lymthoma of people a kind of, which is characterized in that its amino acid sequence is such as
SEQ ID NO:Shown in 1.
2. a kind of encoding gene of 7 family's C mutains of chronic B cell leukemia/lymthoma of people, which is characterized in that its core
Nucleotide sequence such as SEQ ID NO:Shown in 2.
3. a kind of genetic chip, which is characterized in that including：Solid phase carrier and fixed nucleotide probe on this carrier；It is described
Nucleotide probe is according to the nucleotide sequences of 7 family's C mutains of chronic B cell leukemia/lymthoma of the people, with people's
The comparison result of the nucleotide sequence of the 7 normal albumen of family C of chronic B cell leukemia/lymthoma determines.
4. genetic chip according to claim 3, which is characterized in that the nucleotide probe is SEQ ID NO:Shown in 3
Base sequence.
5. a kind of monoclonal antibody of 7 family's C mutains of chronic B cell leukemia/lymthoma of specific recognition people, special
Sign is that the amino acid sequence of coding is SEQ ID NO:Shown in 1.
6. a kind of ELISA reagents for detecting the antibody of 7 family's C mutains of chronic B cell leukemia/lymthoma of people
Box, which is characterized in that the ELISA kit includes：Be coated with monoclonal antibody described in claim 5 ELISA ELISA Plates,
ELIAS secondary antibody, detect 7 family's C protein of chronic B cell leukemia/lymthoma of object, sample diluting liquid, coating buffer solution,
ELISA ELISA Plates cleaning solution, developing solution and terminate liquid.
7. being used to detect the antibody of 7 family's C mutains of chronic B cell leukemia/lymthoma of people according to claim 6
ELISA kit, which is characterized in that the ELIAS secondary antibody be diluted HRP horseradish peroxidase-labeleds Goat anti-Human
IgG。
8. a kind of 7 family C of chronic B cell leukemia/lymthoma detecting people based on the ELISA kit of claim 6 or 7
The method of the antibody of mutain, which is characterized in that including：
A, monoclonal antibody described in claim 5 is diluted using the coating buffer solution, and by the list after dilution
Clonal antibody is loaded into the hole of ELISA ELISA Plates, incubation at room temperature；
B, the 7 family's C protein of chronic B cell leukemia/lymthoma for detecting object is diluted to not using the sample diluting liquid
Same concentration gradient, and 7 family's C protein of chronic B cell leukemia/lymthoma of various concentration gradient is loaded respectively to ELISA
In the hole of ELISA Plate, incubation at room temperature；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature is protected from light incubation, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
9. the 7 family C of chronic B cell leukemia/lymthoma of ELISA kit detection people is mutated egg according to claim 8
The method of white antibody, which is characterized in that further comprise：Blank control is tested.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810256000.6A CN108546292A (en) | 2018-03-27 | 2018-03-27 | A kind of 7 family's C mutains of chronic B cell leukemia/lymthoma and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810256000.6A CN108546292A (en) | 2018-03-27 | 2018-03-27 | A kind of 7 family's C mutains of chronic B cell leukemia/lymthoma and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108546292A true CN108546292A (en) | 2018-09-18 |
Family
ID=63517133
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810256000.6A Pending CN108546292A (en) | 2018-03-27 | 2018-03-27 | A kind of 7 family's C mutains of chronic B cell leukemia/lymthoma and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108546292A (en) |
-
2018
- 2018-03-27 CN CN201810256000.6A patent/CN108546292A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108676785A (en) | A kind of ATP dependent forms RNA helicase DHX3 mutains and application | |
| CN108424888A (en) | 1 mutain of serine palmitoyltransferase of people a kind of and its application | |
| CN108424447A (en) | 1 mutain of cytochrome b-c1 complex subunits of people a kind of and application | |
| CN108559741A (en) | The imidazolone propionase mutain of people a kind of and its application | |
| CN108866024A (en) | ATP dependenc RNA unwindase DDX12 mutain and its application | |
| CN108546689A (en) | A kind of 1 mutain of vitamin K epoxy reductase complex subunit and application | |
| CN108359662A (en) | The uroporphyrinogen decarboxylase mutain of people a kind of and its application | |
| CN108546292A (en) | A kind of 7 family's C mutains of chronic B cell leukemia/lymthoma and application | |
| CN107828748B (en) | The NDUFA13 protein mutations albumen of people a kind of and its application | |
| CN108588048A (en) | The transaldolase mutain of people a kind of and its application | |
| CN108300709A (en) | A kind of 2 mutain of protein arginine N- diformazans based transferase and its application | |
| CN108424887A (en) | 5 mutain of transmethylase sample albumen of people a kind of and its application | |
| CN108362880A (en) | A kind of mutain of the PARP1 albumen of people and its application | |
| CN108424903A (en) | The uroporphyrinogen original III synzyme mutain of people a kind of and its application | |
| CN108546293A (en) | - 36 mutain of sperm antigen of people a kind of and its application | |
| CN108486083A (en) | A kind of structure-specific nuclease inscribe enzyme subunit SLX1 mutains and application | |
| CN108424884A (en) | The glutathione S-transferase κ 1b mutains of people a kind of and its application | |
| CN108559736A (en) | The 3 related protein kinase mutain of phosphatidyl inositol kinase of people a kind of and application | |
| CN108410828A (en) | 1 mutain of dehydrogenase SDR family members of people a kind of and its application | |
| CN108424895A (en) | 1 mutain of maintenance mitochondrial genomes exonuclease of people a kind of and application | |
| CN108101977A (en) | The ACP protein mutations albumen of people a kind of and its application | |
| CN108611335A (en) | A kind of interferon-induced gtp binding protein Mx2 mutains and its application | |
| CN108300706A (en) | The anaphase-promoting complex subunit 5a mutains of people a kind of and its application | |
| CN108362886A (en) | The guanylate kinase b mutains of people a kind of and its application | |
| CN108148820A (en) | The glutathione S transferase M1 c mutains of people a kind of and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180918 |