CN105838799A - New application of KCNK2 gene - Google Patents
New application of KCNK2 gene Download PDFInfo
- Publication number
- CN105838799A CN105838799A CN201610284031.3A CN201610284031A CN105838799A CN 105838799 A CN105838799 A CN 105838799A CN 201610284031 A CN201610284031 A CN 201610284031A CN 105838799 A CN105838799 A CN 105838799A
- Authority
- CN
- China
- Prior art keywords
- kcnk2
- gene
- application
- thyroid
- cell
- 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
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
-
- 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
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- 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
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- C12Q2600/158—Expression 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)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to new application of a KCNK2 gene, in particular to application of the KCNK2 gene and an expression product thereof to treatment of papillary thyroid carcinoma as tumor markers .In order to diagnose thyroid carcinoma more accurately and avoid missed diagnosis and misdiagnosis, tumor molecular markers are screened based on sequencing and data analysis, the result shows that the KCNK2 gene can be used for preparing a papillary thyroid carcinoma auxiliary diagnosis and treatment preparation, and quite good clinical application value is achieved.
Description
Technical field
The present invention relates to biomedicine field, the concrete present invention relates to the new application of KCNK2 gene, is more particularly to KCNK2 gene and expression product thereof as tumor markers application in diagnosis and treatment papillary thyroid carcinoma.
Background technology
Papillary carcinoma is the one of the most common thyroid carcinoma, accounts for the 90% of all thyroid carcinomas.Diagnosis of Thyroid Carcinoma inspection relies primarily on ultrasonic examination, is one of the method for thyroid carcinoma primary lesion diagnosis most worthy.Ultrasonic examination also has certain misdiagnosis rate, and clinicist is it is to be understood that the ultrasonic findings of specific type thyroid carcinoma, and such as capsule thyroid papillary carcinoma, diffusivity thyroid papillary carcinoma, clinic is easily obscured mutually with thyroid cyst or lymphocytic thyroiditis.Fine needle aspiration cytopathology biopsy (FNA) is the method that assessment thyroid nodule is the most accurate and cost performance is the highest.But there is also and fail to pinpoint a disease in diagnosis, be summarized as during the causes of misdiagnosis of FNA: puncture needle is too thin, and the specimen amount of cytolgical examination is not enough；Relevant with thyroid tumor size and interior tissue situation, thyroid tumor volume is the least, positions the most difficult, Puncture cytology checks that diagnosis is the lowest, has the thyroid tumor of liquefaction especially for inside, and puncture needle penetrates after in capsule and only extracts liquid out, inorganization cell, meaningless to diagnosis；Cytodiagnosis changes prominent disease for cellular morphology and can play a role, but then has any problem to changing into main pathological changes with organizational structure.
How Precise Diagnosis thyroid carcinoma, it is to avoid fail to pinpoint a disease in diagnosis, the phenomenon of mistaken diagnosis, is problem demanding prompt solution in current clinic.The rise of molecular biology, it is provided that a new solution route, assists Diagnosis of Thyroid Carcinoma by Molecular Detection, to reach the purpose of accurate diagnosis and treatment.In consideration of it, thyroid papillary carcinoma (PTC) case sample and healthy population are checked order by the HiSeq 2500 that inventor uses advanced person, and the data obtained are carried out deep processing, filter out the KCNK2 gene that significant difference is expressed.In order to verify the relation of KCNK2 and thyroid papillary carcinoma, carry out again quantitative experiment and slow virus carrier has built and functional verification, result shows that KCNK2 can be used for preparing thyroid papillary carcinoma assisting in diagnosis and treatment preparation, the assisting in diagnosis and treatment that this research is clinical thyroid papillary carcinoma provides basis, has good clinical value.
Summary of the invention
It is an object of the invention to provide detection KCNK2 gene or albumen application in preparing diagnosis of thyroid cancer preparation.
Further, thyroid carcinoma is thyroid papillary carcinoma.
For achieving the above object, invention combines bioinformatics method by high-flux sequence and screens gene KCNK2, and then demonstrate KCNK2 and thyroid carcinoma by RT-PCR there is good dependency, can be used for preparing thyroid carcinoma assisting in diagnosis and treatment preparation, there is important clinical value.
Further, thyroid diagnostic preparation includes with the expression of KCNK2 gene in fluorescence quantifying PCR method, method for gene chip detection human thyroid carcinoma.
In fluorescence quantifying PCR method detection thyroid carcinoma, the product of KCNK2 gene contains the primer of a pair specific amplification KCNK2 gene；Gene chip includes the probe of the nucleic acid array hybridizing with KCNK2 gene.
Preferably, fluorescence quantifying PCR method detection gene KCNK2, using special forward primer and downstream primer, forward primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.
Further, the ELISA method of detection KCNK2 albumen is for using ELISA detection kit.Antibody in test kit can use commercially available KCNK2 monoclonal antibody.Further, described test kit includes: be coated the solid phase carrier of KCNK2 monoclonal antibody, enzyme labelled antibody, the substrate of enzyme, protein standard substance, negative controls, diluent, cleaning mixture, enzyme reaction stop buffer etc..
Further, the colloidal gold method of detection KCNK2 albumen is for using detection kit, and antibody can use commercially available KCNK2 monoclonal antibody.Further, gold-immunochromatographyreagent reagent for assay box uses colloidal gold immunochromatographimethod technology or gold colloidal percolation.Further, detection zone (T) specking on gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-KCNK2 monoclonal antibody, quality control region (C) specking to have immunoglobulin IgG.
Fluorescence quantitative PCR method is by fluorescent dye or fluorescently-labeled specific probe, PCR primer is marked tracking, real time and on line monitoring course of reaction, can be analyzed product in conjunction with corresponding software, calculates the initial concentration of testing sample template.The appearance of quantitative fluorescent PCR, greatly simplifies the process of detection by quantitative, and is truly realized absolute quantitation.The appearance of multiple detecting system, the selectivity making experiment is higher.Automation mechanized operation improves work efficiency, reacts quick, reproducible, highly sensitive, high specificity, result are clear.
Gene chip is also called DNA microarray (DNA microarray), three kinds of main Types can be divided into: 1) it is fixed on polymer matrix film (nylon membrane, nitrocellulose membrane etc.) nucleic probe on surface or cDNA fragment, generally it is hybrid with it with isotope-labeled target gene, is detected by radiography technology.2) with the fixing DNA probe array on a glass of point sample method, by detecting with the hybridization of fluorescently-labeled target gene.3) oligonucleotide probe array being directly synthesized on the hard surfaces such as glass, detects with the hybridization of fluorescently-labeled target gene.Gene chip, as a kind of advanced person, extensive, high throughput testing technology, is applied to the diagnosis of disease, and its advantage has the following aspects: one is susceptiveness and the accuracy of height；Two is fast and convenient；Three is can to detect multiple disease simultaneously.
Further, the diagnosis and treatment preparation of thyroid carcinoma includes with the expression of KCNK2 albumen in immunization method detection thyroid papillary carcinoma.The most described immunologic detection method detection thyroid papillary carcinoma in KCNK2 protein expression for western blot and/or ELISA and/gold colloidal detection method.
Enzyme-linked immunosorbent assay (ELISA) will known antigen or antibody adsorb at surface of solid phase carriers, makes the technology that the antigen antibody reaction of enzyme labelling is carried out at solid phase surface.This technology can be used for detecting macromole antigen and specific antibody etc., has quick, sensitive, easy, carrier and is prone to the advantages such as standardization.ELISA detection kit can be divided into indirect method, double-antibody method, competition law, dibit point one-step method, prize law to survey IgM antibody, application Avidin and the ELISA of biotin according to testing goal and operating procedure.In ELISA detection kit, chromogenic substrate may select horseradish peroxidase (HRP) or alkali phosphatase (AP).
Conventional immune colloid gold detection technique: (1) immune colloid gold light microscopic staining cell suspension smear or tissue slice, the antibody of available colloid gold label dyes, also can be on the basis of colloid gold label, labelling is strengthened with silver developer solution, make the silver atoms being reduced be deposited on marked gold grain surface, the sensitivity of colloid gold label can be remarkably reinforced.(2) immune colloid gold staining method for electron microscopy can be combined with negative staining Virus Sample or tissue ultrathin section with the antibody of colloid gold label or anti antibody, then carries out negative staining.Can be used for observation and the Viral diagnosis of morphology of virus.(3) dot immunogold filtration assay application microporous filter membrane makees carrier, first by antigen or antibody point on film, adds sample to be checked after closing, with the corresponding antigen of antibody test or the antibody of colloid gold label after washing.(4) specific antigen or antibody are fixed on film by colloidal gold immunity chromatography with ribbon, colloid gold label reagent (antibody or monoclonal antibody) adsorbs on pad, after in the sample pad that sample to be checked is added to test strips one end, moved forward by capillarity, react to each other after dissolving the colloid gold label reagent on pad, when the region of the most fixing mobile antigen or antibody, thing to be checked and the conjugate of gold marked reagent occur specific binding the most therewith and are trapped, it is gathered on detection band, colour developing result can be observed by the naked eye.This method has developed into diagnosis test paper, uses very convenient.
It is an object of the invention to provide a kind of gene detecting kit detecting thyroid papillary carcinoma, test kit detection gene KCNK2, using special forward primer and downstream primer, forward primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.
Further, this PCR kit is suitable for all types fluorescence quantitative gene extender that presently, there are on market, highly sensitive, the most quick and precisely, good stability, has a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component includes: specific primer, internal reference primer, fluorescence quantitative PCR reaction solution.Wherein said specific primer includes that forward primer and downstream primer, forward primer sequence are SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.Described internal reference primer is GAPDH internal reference primer, and forward primer sequence is SEQ ID NO.3, and downstream primer sequence is SEQ ID NO.4.
A kind of thyroid papillary carcinoma protein detection kit, detection kit detection KCNK2 albumen are it is an object of the present invention to provide.Further, test kit also includes other detectable.
It is an object of the present invention to provide a kind of gene chip detecting thyroid papillary carcinoma, gene chip includes the probe of the nucleic acid array hybridizing with KCNK2 gene.
It is an object of the invention to provide promotion KCNK2 gene or the application in preparing antithyroid cancer preparation of the protein expression reagent.
Further, thyroid carcinoma is thyroid papillary carcinoma.
Further, the propagation of antithyroid cancer preparation suppression thyroid carcinoma cell.
Further, the migration of antithyroid cancer preparation suppression thyroid carcinoma cell.
Preferably, methods such as building over-express vector is used to promote KCNK2 gene or protein expression.
It is an object of the invention to provide a kind of antithyroid papillary carcinoma preparation, antithyroid papillary carcinoma preparation promotes the expression of KCNK2 gene in papillary thyroid carcinoma cells.
Accompanying drawing explanation
Fig. 1 is KCNK2 gene relative expression's spirogram (KCNK2mRNA relative expression's situation) in cancerous tissue and normal structure
Fig. 2 is cell proliferation curve chart (two groups of cell proliferation curve)
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and is not considered as limiting the invention.It will be understood by those skilled in the art that: these embodiments can carry out in the case of without departing from the principle of the present invention and objective multiple change, revise, replace and modification, the scope of the present invention is limited by claim and equivalent thereof.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition or according to the condition examinations proposed by manufacturer.
The collection of embodiment 1 case
Take 5 example thyroid papillary carcinoma case samples and 8 example normal healthy controls tissues.Case sample is the patient that in art, frost rapid pathology and the slow pathology of postoperative paraffin confirm as thyroid papillary carcinoma, selects through first merit eight and the confirmation of thyroid color ultrasound examination.
Embodiment 2 high-flux sequence and analysis
UseReagent extracts sample rna, agarose gel electrophoresis after RNA extraction, and then is detected the extraction situation of RNA sample by NanoDrop1000 spectrophotometer, the sample requirement of RNA-seq order-checking: OD260/OD280 is 1.8-2.2.
The HiSeq 2500 high-flux sequence platform using Illumina company carries out the order-checking of the high flux transcript profile degree of depth, use Fast-QC software that the quality of sequencing data is carried out total evaluation after order-checking, mass value including base is distributed, the position distribution of mass value, G/C content situation, PCR duplication content situation, the frequency etc. of kmer.When differential genes expression analysis, according to the FPKM value obtained, internationally recognized algorithm EBSeq is used to carry out differential screening.Wherein, during screening, LOG2FC>1 or<-1, FDR<0.05.In order to be better understood from the function of difference expression gene, we have carried out Gene Onlogy and signal path analysis to difference expression gene, and difference expression gene carries out functional annotation and protein interaction analysis of network.Finally, comprehensive data above analysis result, in conjunction with searching document situation, the present invention have chosen to lower in thyroid papillary carcinoma group and expresses significant gene KCNK2 as object of study.
Embodiment 3 thyroid papillary carcinoma group and matched group KCNK2 expression conditions
One, material and method
1, material
Take during in November, 2015 in May, 2013 in hospital because of thyroid tumor hospitalisation for surgery, choose 45 example Papillary Thyroid Carcinomas and 18 example normal healthy controls tissues, it is grouped and numbers.
2, method
2.1 thyroid papillary carcinoma groups and the extraction of matched group total serum IgE
UseReagent extracts sample rna, operates reference description, specific as follows:
After collecting sample, frozen mortar tissue being put into pre-cooling after liquid nitrogen, taking-up is ground, after tissue samples is powdered:
1. adding Trizol, room temperature stands 5min；
2. every milliliter of Trizol adds 0.2ml chloroform, uses forced oscillation centrifuge tube, fully mixes, and ambient temperatare puts 5-10min；
3. EP pipe point 3 layers after 4 DEG C of 12000rpm high speed centrifugation 15min, in absorption upper strata aqueous phase to another new centrifuge tube pipe, is careful not to the protein substance being drawn onto between two-layer aqueous phase.Move into new pipe, add isopyknic-20 DEG C of precooled isopropanols, the most reverse mixing, stand 10min on ice；
4. 4 DEG C of 12000rpm carefully discard at a high speed supernatant after 10min, add 75%DEPC ethanol and wash precipitation, vibration mixing, 4 DEG C of 12000rpm high speed centrifugation 5min；
5. discard ethanol liquid, ambient temperatare put 5 minutes fully to dry precipitation, add 20-40 microlitre DEPC processed water dissolution precipitation；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet spectrophotometer, frozen in-70 DEG C.
2.2 one-step method Real-Time PCR
2.2.1 instrument and the method for analysis
With ABI 7500 type quantitative real time PCR Instrument, 2-Δ Δ CT method is used to carry out the relative quantitative assay of data.
2.2.2 design of primers
Using online primer-design software, gene order, with reference to NCBI:NM_001017424, is synthesized by invitrogen company after design of primers.Concrete primer sequence is as follows:
KCNK2 gene primer:
5’-ATTCGCATCATCTCAACAAT-3’(SEQ ID NO.1)
5’-ACTCCAGCCTTCTATGTG-3’(SEQ ID NO.2)
Amplification length is 96bp.
GAPDH gene primer:
5’-TCTCTGATTTGGTCGTAT-3’(SEQ ID NO.3)
5’-TTGATGGCAACAATATCC-3’(SEQ ID NO.4)
Amplification length is 78bp.
UltraSYBR one-step method PCR kit for fluorescence quantitative (article No.: CW2624S) operating procedure:
1. RNA template, primer, 2 × UltraSYBR One Step Buffer, UltraSYBR One Step EnzymeMix and RNase-Free Water dissolving is placed in the most standby.
2. reaction system:
3. vortex concussion mixing, of short duration centrifugal, solution is collected at the bottom of pipe.
4.RT-qPCR reaction condition (quantitative fluorescent PCR is two-step method), 45 DEG C of 10min of reverse transcription；96 DEG C of 5min of denaturation；30 circulations (95 DEG C of 10s of degeneration anneal and extend 60 DEG C of 45s)；Reaction terminates rear 4 DEG C of constant temperature.
Two, experimental result
Real-time quantitative PCR amplification curve entirety collimation is good, shows that the amplification efficiency of each reaction tube is close, and amplification curve flex point understands, the limit is flat and nothing raises up now, and exponent phase slope is relatively big, illustrates that amplification efficiency is higher；Sample amplified production solubility curve is unimodal, shows that amplified production is unique, is specific amplification；Relative quantification formula according to qRT-PCR: 2-Δ Ct × 100%, compares KCNK2 gene expression in thyroid papillary carcinoma group and normal group.Result shows that (being specifically shown in Fig. 1): KCNK2 expression in cancerous tissue is substantially less than matched group, being about 1/5th of matched group, result above demonstrates high flux transcript profile and expresses confluence analysis KCNK2 result of low expression in Papillary Thyroid Carcinoma of data.
Embodiment 4 builds recombinant slow virus expression vector
One, material
TPC-1 cell strain, 293T cell strain are purchased from Hui Ying bio tech ltd, Shanghai.
Two, experimental technique
1. the structure of recombiant plasmid
For the sequence of gene KCNK2 (NM_001017424), the primer sequence of design amplifying target genes, primer contains exchange pairing base and Age I restriction enzyme site.Carrying out PCR amplification, purified pcr product, carry out the structure of recombiant plasmid after PCR primer exchanges linear expression vector, the molal quantity ratio of the linearized vector DNA of addition and the PCR primer of purification is 1:3.By T4DNA ligase will through double digestion linearizing slow virus empty carrier GV208 empty carrier and annealing formed double chain DNA fragment be attached in suitable buffer reaction, 16 DEG C overnight connect, it is thus achieved that recombined lentivirus vector named GV208-KCNK2.
Being transformed in E. coli competent by connection product, be coated in the LB culture medium of AMP resistance after conversion, be inverted in incubator for 37 DEG C and hatch, the bacterium colony that picking grows carries out PCR qualification.The universal primer using slow virus carrier carries out bacterium colony PCR and identifies that transformant and order-checking are identified.
2. slow virus carrier packaging
Cell recovery
Taking out rapidly and be put in the cryopreservation tube of TPC-1,293T cell in liquid nitrogen container, put in 37 DEG C of thermostat water baths, quickly shake, in making pipe in two minutes, frozen stock solution melts completely.Putting in centrifuge by two cryopreservation tubes, after 1000rpm/min is centrifuged 3min, fast transfer is to Cytology Lab, after carrying out alcohol disinfecting, inhales and abandons old liquid, be separately added into the RPMI-1640 containing serum, each 1ml of DMEM culture medium, blow and beat cell gently in aseptic super-clean bench.Proceed to 50T culture bottle, be subsequently adding appropriate culture fluid and cultivate.Second day observation of cell state also carries out changing liquid.
Cell is cultivated
TPC-1,293T cell respectively with the RPM1640 culture fluid containing 10% hyclone and DMEM culture fluid, size be 50ml culture bottle in cultivate, and be positioned over the CO that condition is 5%2Concentration, constant temperature 37 DEG C incubator in cultivate, carry out after two to three days changing liquid.
Cell cryopreservation
When cell be in exponential phase and be paved with a bottle wall reach 80%-90% time, wash three times with PBS, be subsequently adding appropriate pancreatin and digest, when slightly coming off occurs in cell, add lml culture fluid immediately and terminate digestion, piping and druming cell is until dispelling into single cell suspension the most gently, is then transferred in centrifuge tube, and 800rpm/min is centrifuged 5min, old liquid is abandoned in suction, adding the cells frozen storing liquid of just preparation, piping and druming cell is until mixing gently, be distributed in cryopreservation tube carry out sealing, labelling.Blunting of attention cooling time frozen: 30min under the conditions of 4 DEG C, 2h under the conditions of proceeding to-20 DEG C, then under the conditions of-80 DEG C overnight.Within second day, proceed to the medium-term and long-term preservation of liquid nitrogen container.
DNA vector cotransfection 293 delta cell
(1) day before transfection, washes the 293T cell PBS of exponential phase twice, is subsequently adding pancreatin and digests, and adjusts cell concentration to 6 × 10 by the culture medium containing 10%FBS under mirror after cell counting5Individual/ml, is transferred in Tissue Culture Dish, is subsequently placed in 37 DEG C, 5%CO2Incubator is cultivated.When observing that cell density can be used for transfecting when reaching about 80%.
(2) when cell reach transfection require time, 2h need to be shifted to an earlier date cell culture medium is replaced by serum-free antibiotic-free culture medium.
(3) when transfecting, prepared GV208-KCNK2 carrier 20ug is added in an aseptic Ep pipe, pHelperl.0 carrier 15ug, the DNA of pHelper2.0 carrier l0ug connects liquid, mix with the Opti-MEM culture medium of same volume, adjust cumulative volume to 2.5ml, mixing mixing, incubated at room 5min.
(4) in a sterile centrifugation tube, add 100ug Lipofeetamine2000, mix gently after mixing with 2.4m1Opti-MEM culture medium, incubated at room temperature 5min.
(5) next within 5 minutes, two pipe liquid in (3) and (4) being carried out mix homogeneously lightly, then room temperature incubates 20min.
(6) DNA Yu the Lipofeetamine2000 mixed liquor formed in (5) is added in the culture dish that cultivation has 293T cell, mixed liquor is the most fully mixed, is placed in 37 DEG C, 5%CO2Incubator is cultivated.
(7) outwell culture medium after cultivating 8h, add PBS to every bottle of cell and wash, come again, be subsequently adding the fresh culture 25m1 containing 10%FBS, continue to cultivate 48 hours in incubator.
The results of virus, concentration
(1) from incubator, take out culture dish after 48h, collect supernatant.
(2) by supernatant at 4 DEG C, after 4000rpm is centrifuged l0min, filtering with 0.45um filter, the supernatant after then filtering with pipettor is transferred in 40m1 ultracentrifugation pipe.
(3) from centrifuge tube, draw filtrate in filter cup, cover tightly lid, then filter cup is carefully inserted in filtrate collecting pipe.
(4) it is placed directly within centrifuge after combining collecting pipe, adjusts to 4000rpm and be centrifuged 10min.
(5) after centrifuge stops operating, centrifugal device and collecting pipe are taken out, by filtered solution collection cups separately.
(6) after filter cup separates with collecting pipe, filter cup is tipped upside down on sample collection cup, 1000rpm is centrifuged 2min, removing filter cup afterwards, the residual liquid observed on sample collection cup is viral concentration liquid, preserves after subpackage in-80 DEG C of low temperature in refrigerator.
Virus titer measures
(1) mensuration titre the previous day, 293T cell being inoculated in preprepared 96 orifice plates, selects 10 holes to inoculate, making every porocyte is 4 × 104Expanding individual, volume is 100ul.
(2), when virus titer being measured, get out 10 aseptic Ep pipes, then add the DMEM culture medium of 90u1 serum-free with pipettor to each Ep pipe.
(3) from-80 DEG C of refrigerators, take out a pipe GV208 slow virus stock solution rapidly, in ready first Ep pipe, add 10u1, blow and beat mixing gently, in second Ep pipe, add 10u1 the most again, by that analogy until the tenth Ep manages.
(4) from each cell culture well, carefully sop up 90u1 culture medium with pipettor, be then numbered successively by 1-10, and add the own viral solution diluted of corresponding number, confirm that coding is errorless and is placed in incubator cultivation 24 hours.
Within (5) 24 hours, backward each cell culture well adds the complete medium of 100u1 gently, is careful not to blow afloat cell.
When (6) the 5th days, the luciferase expression situation of cell in each culture hole of fluorescence microscopy Microscopic observation, and fluorecyte is counted, virus titer can be calculated according to the extension rate in corresponding hole, then carry out record and compare.
3. recombiant plasmid slow virus carrier expression effect measures
(1) the good purpose cell of accurate assurance changes the liquid time, makes to carry out cell state when virus infects and is in exponential phase.
(2) collection status is optimal cell is also centrifuged, and is subsequently adding the fresh culture containing 10%FBS, and is inoculated in 6 orifice plates, makes every porocyte number be about 5 × 10 according to cell counting situation4。
(3) according to the MOI value (MOI value=1) of slow virus infection TPC-1 cell, calculate required addition virus liquid, by the culture medium containing 10%FBS, every hole cumulative volume is complemented to 2m1, put into after incubator is cultivated 12 hours and carry out changing liquid.
(4) by taking out from incubator after metainfective cell 72h, under inverted fluorescence microscope, GFP expression is observed, contrast with cell number under the white light visual field simultaneously, estimate transfection efficiency.
(5) experimentation there is provision of the blank group being not added with virus liquid and the negative control group adding negative control virus liquid, and three multiple holes are respectively set.
The expression of 4.RT-PCR detection KCNK2mRNA
(1) collecting the TPC-1 cell after the slow virus 48h of transfection empty vector control and transfection KCNK2, extract each group of cell total rna, then, one-step method Real-Time PCR with Trizol respectively, concrete operations are with reference to embodiment 3.
Three, experimental result
Recombiant plasmid, after PCR qualification, enzyme action identify dual confirmation, send order-checking company to check order, sequencing result and the sequence alignment of Genbank KCNK2 (NM_001017424), and result is completely the same, shows construction of recombinant plasmid success.
Choose slow virus carrier pGV-208, pHelperl.0, pHelper2.0 tri-plasmid jointly transfect 293T cell, the green fluorescence sent after transfection 48h at fluorescence microscopy Microscopic observation 293T cell.Slow virus after concentrating is measured, counts under fluorescence microscope and each hole sends the cell number conversion of fluorescence obtain virus titer and be about 5 × 108TU/ml。
The Ct value of corresponding gene detected by Real-Time PCR, be computed genes of interest KCNK2 consistent with the amplification efficiency of reference gene GAPDH.Calculate the relative expression levels of each group of cell KCNK2mRNA, with negative control group as normalized sample, remaining group is this group KCNK2 gene expression amount multiple proportion relative to criterion group, with negative control group for 1, and the process LAN effect of the ratio reflection KCNK2 of remaining experimental group and negative control group.The relative expression quantity being computed learning negative control group KCNK2mRNA is 1, and experimental group is 15.12, and statistical result showed carries the experimental group expression of KCNK2 gene apparently higher than negative control group (P < 0.0001).
The impact on TPC-1 cell biological function of the embodiment 5KCNK2 gene overexpression
One, experimental technique
1. detection cell proliferation
The each group cell of trophophase of taking the logarithm becomes single cell suspension through trypsinization, and adjusting TPC-1 cell density is 1 × 103Individual/ml.Each hole in two groups of each 4 96 orifice plates adds cell suspension, makes purpose cell number reach 2 × 103Individual.After cell attachment, take 0h, the time period after 24h, 48h, 72h, super-clean bench adds in every hole 10 microlitre CCK-8 solution, continues in cell culture incubator to hatch 2h.Set the wavelength of enzyme-linked immunosorbent assay instrument as 450nm, measure each hole absorbance (OD value).With OD value as vertical coordinate, time point is abscissa, draws experimental group and the growth curve of negative control group cell, and the proliferative conditions of two groups of cells is carried out contrast statistics.
2.Transwe ll Cell migration assay
(1) after being cultivated 24 hours by each group cell of own packet, carrying out digesting, being centrifuged and collect, with the culture fluid diluting cells of serum-free after counting cell, making cell number is 2 × 105Individual, then to draw 200 l of cell suspension and add on Transwell cell in room, lower room all adds the RPMI1640 culture medium containing 10% hyclone of 600 microlitres, and often group cell is respectively provided with 3 multiple holes.It is placed in into constant temperature 37 DEG C, the CO of 5%2The incubator of concentration hatches 24h.
(2) cell hatched is taken out, wipe cell and the liquid of upper indoor with medical cotton stick carefully away, dry in super-clean bench.Cell is placed in 4% paraformaldehyde immersion 30 minutes, the effect that cell is fixing can be made.Cell rinses 3 times with PBS after having fixed, and the crystal violet solution adding 0.1% after drying dyes, and the time is 30 minutes.Dyeing is taken out cell PBS liquid after terminating and is again rinsed 3 times, observe under inverted microscope after drying, first take off the overall condition of cell inner cell, then randomly select skilful the visual field under high power lens, the cell concentration in each visual field is counted and the place of calculating goes out average cell number.
Two, experimental result
Negative control group and experimental group cell after inoculation the 1-5 days, operate according to CCK-8 detection kit description, then density (OD) value of each group of cell is detected by microplate reader, add up each group of cell OD value and draw the growth curve of cell, two groups of results show that experimental group growth curve is shallower after contrasting, vitro growth rates relatively negative control group significantly reduces (p < 0.05), this shows that the process LAN of KCNK2 gene has the effect of suppression TPC-1 cell proliferation, is specifically shown in Fig. 2.
Transwell cell migration results shows, compares in experimental group and negative control group and finds that experimental group penetrates filter membrane cell quantity and significantly reduces.Two groups of differences of statistical result showed have statistical significance (P < 0.01).Result shows the travel motion reduced capability of TPC-1 cell after process LAN KCNK2 gene.
The present invention uses high-flux sequence to filter out thyroid papillary carcinoma pathogenic related gene KCNK2, and binding molecule Cell Biology Experiment checking is it was confirmed the KCNK2 mark that to be thyroid papillary carcinoma close.The present invention is that thyroid papillary carcinoma clinic diagnosis provides new target, has good potential applicability in clinical practice.
Claims (10)
1. the preparation of detection KCNK2 gene or albumen application in preparing diagnosis of thyroid cancer preparation.
Application the most according to claim 1, it is characterised in that thyroid carcinoma is thyroid papillary carcinoma.
Application the most according to claim 1, it is characterised in that it is fixed that diagnosis of thyroid cancer preparation includes with fluorescence
Amount PCR method, the expression of method for gene chip detection KCNK2 gene.
Application the most according to claim 3, it is characterised in that detect first shape for fluorescence quantifying PCR method
In adenocarcinoma, the product of KCNK2 gene contains the primer of a pair specific amplification KCNK2 gene；Gene chip
Including the probe with the nucleic acid array hybridizing of KCNK2 gene, it is preferred that specific amplification KCNK2 gene
Primer sequence be SEQ ID NO.1 and SEQ ID NO.2.
Application the most according to claim 1, it is characterised in that thyroid carcinoma diagnosis and treatment preparation includes with immunity side
The expression of method detection KCNK2 albumen, it is preferred that detect KCNK2 albumen by ELISA or gold colloidal method
Express.
6. promote KCNK2 gene or the application in preparing antithyroid cancer preparation of the protein expression reagent.
Application the most according to claim 6, it is characterised in that thyroid carcinoma is thyroid papillary carcinoma.
Application the most according to claim 6, it is characterised in that antithyroid cancer preparation suppression thyroid carcinoma is thin
The propagation of born of the same parents.
Application the most according to claim 6, it is characterised in that antithyroid cancer preparation suppression thyroid carcinoma is thin
The migration of born of the same parents.
Application the most according to claim 6, it is characterised in that use the method building over-express vector
Promote KCNK2 gene or protein expression.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610284031.3A CN105838799A (en) | 2016-04-29 | 2016-04-29 | New application of KCNK2 gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610284031.3A CN105838799A (en) | 2016-04-29 | 2016-04-29 | New application of KCNK2 gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105838799A true CN105838799A (en) | 2016-08-10 |
Family
ID=56590672
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610284031.3A Pending CN105838799A (en) | 2016-04-29 | 2016-04-29 | New application of KCNK2 gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105838799A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652552A (en) * | 2018-07-27 | 2019-04-19 | 四川大学华西医院 | A kind of ARMS-PCR detection kit of the 961st bit base mutated gene of mankind MAP2K5 |
| CN109762902A (en) * | 2018-07-27 | 2019-05-17 | 四川大学华西医院 | A kind of ARMS-PCR detection kit of the 1100th bit base mutated gene of mankind MAP2K5 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020028485A1 (en) * | 1998-01-27 | 2002-03-07 | Meadows Helen Jane | Novel compounds |
| WO2005054866A2 (en) * | 2003-11-25 | 2005-06-16 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with two-pore domain potassium channel (kcnk2) |
| WO2007050706A2 (en) * | 2005-10-27 | 2007-05-03 | University Of Missouri-Columbia | Dna methylation biomarkers in lymphoid and hematopoietic malignancies |
| CN101443771A (en) * | 2004-12-16 | 2009-05-27 | 新星筛生物科技公司 | Method for identification and functional characterization of agents which modulate ion channel activity |
| CN102844036A (en) * | 2009-07-16 | 2012-12-26 | 生物时代股份有限公司 | Methods and compositions for in vitro and in vivo chondrogenesis |
| CN104093706A (en) * | 2011-12-30 | 2014-10-08 | 克莱蒙费朗国立高等化工学校 | Pain relief compounds |
-
2016
- 2016-04-29 CN CN201610284031.3A patent/CN105838799A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020028485A1 (en) * | 1998-01-27 | 2002-03-07 | Meadows Helen Jane | Novel compounds |
| WO2005054866A2 (en) * | 2003-11-25 | 2005-06-16 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with two-pore domain potassium channel (kcnk2) |
| CN101443771A (en) * | 2004-12-16 | 2009-05-27 | 新星筛生物科技公司 | Method for identification and functional characterization of agents which modulate ion channel activity |
| WO2007050706A2 (en) * | 2005-10-27 | 2007-05-03 | University Of Missouri-Columbia | Dna methylation biomarkers in lymphoid and hematopoietic malignancies |
| US20090264306A1 (en) * | 2005-10-27 | 2009-10-22 | Curators Of The University Of Missouri | Dna methylation biomarkers in lymphoid and hematopoietic malignancies |
| CN102844036A (en) * | 2009-07-16 | 2012-12-26 | 生物时代股份有限公司 | Methods and compositions for in vitro and in vivo chondrogenesis |
| CN104093706A (en) * | 2011-12-30 | 2014-10-08 | 克莱蒙费朗国立高等化工学校 | Pain relief compounds |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652552A (en) * | 2018-07-27 | 2019-04-19 | 四川大学华西医院 | A kind of ARMS-PCR detection kit of the 961st bit base mutated gene of mankind MAP2K5 |
| CN109762902A (en) * | 2018-07-27 | 2019-05-17 | 四川大学华西医院 | A kind of ARMS-PCR detection kit of the 1100th bit base mutated gene of mankind MAP2K5 |
| CN109762902B (en) * | 2018-07-27 | 2022-07-05 | 四川大学华西医院 | ARMS-PCR detection kit for 1100 th base mutant gene of human MAP2K5 |
| CN109652552B (en) * | 2018-07-27 | 2022-07-05 | 四川大学华西医院 | ARMS-PCR detection kit for 961 th base mutant gene of human MAP2K5 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105779618A (en) | Novel target gene for diagnosing and treating tongue squamous carcinoma and application thereof | |
| CN106191067B (en) | Circular rna circ-NFATC3 and application thereof | |
| CN105524924A (en) | Cyclic RNA circ-ZKSCAN1 use | |
| CN106834486A (en) | Osteosarcoma molecule diagnosis and treatment mark and its application | |
| CN105838799A (en) | New application of KCNK2 gene | |
| CN103969452B (en) | The classification diagnosis kit of BAY 43-9006 personalized treatment liver cancer | |
| CN105838820A (en) | Novel application of transcription factor FOXD1 | |
| CN105288659A (en) | Application of TENM1 gene and its expression product on diagnosis and treatment of papillary adenocarcinoma | |
| CN106399485A (en) | Genes highly expressed in tongue squamous carcinoma para-carcinoma tissue and applications of genes | |
| CN105132547B (en) | The application of STAC2 genes and its encoding proteins in pelvic prolapse is suppressed | |
| CN114002431B (en) | Application of TNNT1 in preparation of virus type hepatocellular carcinoma diagnosis kit and preparation or screening of anti-liver cancer drugs | |
| CN107083428B (en) | Application of PAK5 in cancer diagnosis prognosis treatment and drug screening | |
| CN105233290B (en) | The application of C22orf26 genes and its expression product in Parkinson's diagnosis and treatment reagent is prepared | |
| CN106702015A (en) | Application of IPCEF1 (Interaction Protein For Cytohesin Exchange Factors 1) in diagnosing and treating osteosarcoma | |
| CN105695617A (en) | Tumor marker of papillary carcinoma and application of tumor marker | |
| JP5316749B2 (en) | Cisplatin resistance gene diagnosis method and cisplatin therapeutic effect gene diagnosis kit | |
| CN104740649A (en) | Application of PLEKHA5 in preparation of tumor diagnosis reagent | |
| CN104721836B (en) | Applications of the chaperonin CCT γ in tumour diagnostic reagent is prepared | |
| CN110835648B (en) | Application of NSrp70 gene in preparation of tumor metastasis related pharmaceutical preparation | |
| CN105506171A (en) | LYRM2 (LYR motif-containing 2) gene and new use of expression product thereof | |
| CN113249474A (en) | Application of PCDH20 in prediction of liver cancer chemotherapy sensitivity | |
| de Mello | The role of laboratory diagnosis of influenza | |
| CN117431322A (en) | Application of biomarker TMX2 in diagnosis and treatment of hepatocellular carcinoma | |
| CN105039576B (en) | Pelvic prolapse cance high-expression gene and its application | |
| CN114164272A (en) | BREA2 gene detection kit and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Applicant after: BEIJING MEDINTELL BIOMED Co.,Ltd. Address before: 100080 Beijing city Haidian District Shanyuan Street No. 1 cubic court building room 3103 Applicant before: BEIJING MEDINTELL BIOMED Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200401 Address after: Room 2503, Qianshan building, building D2, Qingdao International Innovation Park Phase II, No. 1, Keyuan Weiyi Road, Laoshan District, Qingdao, Shandong Province Applicant after: Qingdao Yangshen biomedical Co.,Ltd. Address before: Room 1210, Building 3, Ronghua Xintai Building, 10 Ronghua South Road, Yizhuang Economic and Technological Development Zone, Daxing District, Beijing Applicant before: BEIJING MEDINTELL BIOMED Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160810 |
|
| RJ01 | Rejection of invention patent application after publication |