CN106519006B - Sox2-CDP protein binding domain and its identification method - Google Patents
Sox2-CDP protein binding domain and its identification method Download PDFInfo
- Publication number
- CN106519006B CN106519006B CN201611108011.7A CN201611108011A CN106519006B CN 106519006 B CN106519006 B CN 106519006B CN 201611108011 A CN201611108011 A CN 201611108011A CN 106519006 B CN106519006 B CN 106519006B
- Authority
- CN
- China
- Prior art keywords
- pbifc
- cdp
- sox2
- expression vector
- carrier
- 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.)
- Expired - Fee Related
Links
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 28
- 230000000295 complement effect Effects 0.000 claims abstract description 23
- 238000005516 engineering process Methods 0.000 claims abstract description 20
- 238000010276 construction Methods 0.000 claims abstract description 8
- 239000013598 vector Substances 0.000 claims abstract description 8
- 239000013604 expression vector Substances 0.000 claims description 42
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 claims description 35
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 claims description 27
- 102100024270 Transcription factor SOX-2 Human genes 0.000 claims description 27
- 230000029087 digestion Effects 0.000 claims description 20
- 230000003993 interaction Effects 0.000 claims description 15
- 238000000749 co-immunoprecipitation Methods 0.000 claims description 13
- 238000013461 design Methods 0.000 claims description 13
- 238000012408 PCR amplification Methods 0.000 claims description 9
- 239000002299 complementary DNA Substances 0.000 claims description 8
- 238000004043 dyeing Methods 0.000 claims description 3
- 108700026244 Open Reading Frames Proteins 0.000 claims 2
- 230000003321 amplification Effects 0.000 claims 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims 2
- 238000001114 immunoprecipitation Methods 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000000969 carrier Substances 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 21
- 210000004027 cell Anatomy 0.000 description 18
- 206010028980 Neoplasm Diseases 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000005267 amalgamation Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 208000007276 esophageal squamous cell carcinoma Diseases 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- YMHOBZXQZVXHBM-UHFFFAOYSA-N 2,5-dimethoxy-4-bromophenethylamine Chemical compound COC1=CC(CCN)=C(OC)C=C1Br YMHOBZXQZVXHBM-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 241000545067 Venus Species 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 1
- ADSGHMXEAZJJNF-DCAQKATOSA-N Ala-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N ADSGHMXEAZJJNF-DCAQKATOSA-N 0.000 description 1
- OEVCHROQUIVQFZ-YTLHQDLWSA-N Ala-Thr-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](C)C(O)=O OEVCHROQUIVQFZ-YTLHQDLWSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- FSNVAJOPUDVQAR-AVGNSLFASA-N Arg-Lys-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FSNVAJOPUDVQAR-AVGNSLFASA-N 0.000 description 1
- RIIVUOJDDQXHRV-SRVKXCTJSA-N Arg-Lys-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O RIIVUOJDDQXHRV-SRVKXCTJSA-N 0.000 description 1
- CIBWFJFMOBIFTE-CIUDSAMLSA-N Asn-Arg-Gln Chemical compound C(C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N)CN=C(N)N CIBWFJFMOBIFTE-CIUDSAMLSA-N 0.000 description 1
- WUQXMTITJLFXAU-JIOCBJNQSA-N Asn-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N)O WUQXMTITJLFXAU-JIOCBJNQSA-N 0.000 description 1
- ZAESWDKAMDVHLL-RCOVLWMOSA-N Asn-Val-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O ZAESWDKAMDVHLL-RCOVLWMOSA-N 0.000 description 1
- DGKCOYGQLNWNCJ-ACZMJKKPSA-N Asp-Glu-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O DGKCOYGQLNWNCJ-ACZMJKKPSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- HGBHRZBXOOHRDH-JBACZVJFSA-N Gln-Tyr-Trp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O HGBHRZBXOOHRDH-JBACZVJFSA-N 0.000 description 1
- JVSBYEDSSRZQGV-GUBZILKMSA-N Glu-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O JVSBYEDSSRZQGV-GUBZILKMSA-N 0.000 description 1
- WPLGNDORMXTMQS-FXQIFTODSA-N Glu-Gln-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O WPLGNDORMXTMQS-FXQIFTODSA-N 0.000 description 1
- LGYCLOCORAEQSZ-PEFMBERDSA-N Glu-Ile-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O LGYCLOCORAEQSZ-PEFMBERDSA-N 0.000 description 1
- PJBVXVBTTFZPHJ-GUBZILKMSA-N Glu-Leu-Asp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)O)N PJBVXVBTTFZPHJ-GUBZILKMSA-N 0.000 description 1
- VPKIQULSKFVCSM-SRVKXCTJSA-N Leu-Gln-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O VPKIQULSKFVCSM-SRVKXCTJSA-N 0.000 description 1
- KAFOIVJDVSZUMD-DCAQKATOSA-N Leu-Gln-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-DCAQKATOSA-N 0.000 description 1
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-N 0.000 description 1
- HGFGEMSVBMCFKK-MNXVOIDGSA-N Leu-Ile-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O HGFGEMSVBMCFKK-MNXVOIDGSA-N 0.000 description 1
- VCHVSKNMTXWIIP-SRVKXCTJSA-N Leu-Lys-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O VCHVSKNMTXWIIP-SRVKXCTJSA-N 0.000 description 1
- 102000006830 Luminescent Proteins Human genes 0.000 description 1
- 108010047357 Luminescent Proteins Proteins 0.000 description 1
- WALVCOOOKULCQM-ULQDDVLXSA-N Lys-Arg-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O WALVCOOOKULCQM-ULQDDVLXSA-N 0.000 description 1
- YRAWWKUTNBILNT-FXQIFTODSA-N Met-Ala-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YRAWWKUTNBILNT-FXQIFTODSA-N 0.000 description 1
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- 206010061534 Oesophageal squamous cell carcinoma Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- LLGTYVHITPVGKR-RYUDHWBXSA-N Phe-Gln-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O LLGTYVHITPVGKR-RYUDHWBXSA-N 0.000 description 1
- PEFJUUYFEGBXFA-BZSNNMDCSA-N Phe-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=CC=C1 PEFJUUYFEGBXFA-BZSNNMDCSA-N 0.000 description 1
- 101710188306 Protein Y Proteins 0.000 description 1
- YUSRGTQIPCJNHQ-CIUDSAMLSA-N Ser-Arg-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O YUSRGTQIPCJNHQ-CIUDSAMLSA-N 0.000 description 1
- HEYZPTCCEIWHRO-IHRRRGAJSA-N Ser-Met-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 HEYZPTCCEIWHRO-IHRRRGAJSA-N 0.000 description 1
- 208000036765 Squamous cell carcinoma of the esophagus Diseases 0.000 description 1
- BKVICMPZWRNWOC-RHYQMDGZSA-N Thr-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)[C@@H](C)O BKVICMPZWRNWOC-RHYQMDGZSA-N 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of Sox2-CDP protein binding domain and its identification method, the binding structural domain sequence are as follows: CDP Δ C (101-1516), present invention determine that binding structural domain fineness it is high, therefore, the great application value of the present invention.Beneficial effects of the present invention (1) obtain multiple bimolecular fluorescence complementary carriers (2) determined for binding structural domain respectively and pass through bimolecular fluorescence complementary technology, it is determined that Sox2-CDP protein binding domain by way of vector construction；(3) Immunoprecipitation is utilized, the correctness of the Sox2-CDP protein binding domain determined by bimolecular fluorescence complementary technology is demonstrated.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to Sox2-CDP protein binding domain and its identification side
Method.
Background technique
Bimolecular fluorescence complementary technology is that one kind is intuitive, quickly judges that target protein is positioned and interacted in living cells
Technology, the technology dexterously by two complementary fragments of fluorescent protein molecule respectively with target protein amalgamation and expression.If glimmering
Photoprotein activation recovering then shows that two target proteins are interacted.The technology has been used for different Protein-protein interactions
The research work of strong and weak comparison and protein ubiquitination etc..
Based on Immunoprecipitation is the specificity effect between antibody and antigen, for mutual between protein
The classical way of Effect study.The technology is based under solution state or in cell, and albumin X is somebody's turn to do as can with protein Y interaction
Compound can combine the principle precipitated by the antibody of any albumen, have been widely used for interacting between protein
Identification experiment.
Sox2 albumen is a powerful transcription factor, maintains the evil with self-renewing, multiple tumours in stem cell stemness
Property process (such as tumour occur, tumor cell proliferation, migration, invasion transfer, tumor stem cell characteristic maintain, chemotherapeutic drug resistance
Deng) many aspects have facilitation.Sox2 and CDP not only plays a very important role in the development tool of histoorgan,
And they are all in close relations with malignancy of tumor process.Sox2 has function Chong Die with CDP in malignancy of tumor process, i.e. cell
Proliferation, migration, invasion and transfer, leaf conversion etc. between anti-apoptotic and epithelium.
Therefore, screening obtain can target and the small-molecule drug of the protein binding domain of Sox2 protein-interacting for
The establishment of esophageal squamous cell carcinoma therapy target has great importance.
Summary of the invention
The object of the present invention is to provide Sox2-CDP protein binding domain and its identification method, one glimmering using bimolecular
Light complementary technology quickly determines the method for binding structural domain in Sox2-CDP compound and carries out egg using Immunoprecipitation
The identification method of white matter interaction binding structural domain.
Sox2-CDP interaction binding structural domain are as follows: CDP Δ C (101-1516), sequence is as follows: MAANVGSMFQY
WKRFDLQQLQRELDATATVLANRQDESEQSRKRLIEQSREFKKNTPEDLRKQVAPLLKSFQGEIDALSKRSKEAEA
AFLNVYKRLIDVP。
Multiple expression vectors for bimolecular fluorescence complementary are obtained using vector construction, we primarily determine
Sox2-CDP interaction binding structural domain；By expression vector establishment and Immunoprecipitation, we are further demonstrated
The correctness of Sox2-CDP interaction binding structural domain.
The Sox2 overall length and truncation expression vector for bimolecular fluorescence complementary, specifically: pBiFc-VN173-
SOX2 、pBiFc-VN173-SOX2ΔN(1-167)、pBiFc-VN173-SOX2 ΔN(1-237)、 pBiFc-VN173-
SOX2 Δ N (1-257) and pBiFc-VN173-SOX2 Δ N (1-277) expression vector.
The CDP overall length and truncation expression vector for bimolecular fluorescence complementary, specifically: pBiFc-VC155-
CDP、pBiFc-VC155-CDPΔC(759-1516)、pBiFc-VC155-CDPΔC (401-1516)、pBiFc- VC155-
CDP Δ C (201-1516) and pBiFc-VC155-CDP Δ C (101-1516) expression vector.
In addition, Δ N, Δ C refer to deleting N-terminal and C-terminal to be general, Δ refers to deleting the meaning of (delete), such as
SOX2 Δ N (1-167) refers to deleting the truncation SOX2 albumen of N-terminal 1-167 amino acid sites.
FLAG label is had in pBiFcVN173 carrier, is respectively respectively truncated the CDP connecting with pBiFcVC155 carrier prominent
Variant pBiFcVC155-CDP Δ C(759-1516), pBiFcVC155-CDP Δ C(401-1516), pBiFcVC155-CDP Δ C
(201-1516), pBiFcVC155-CDP Δ C(101-1516) and pBiFcVN173-SOX2 cotransfection HEK293T cell after, mention
Take cell protein to carry out co-immunoprecipitation experiment with FLAG-Tag co-immunoprecipitation kit, coprecipitated product use respectively FLAG and
CDP antibody carries out Western blot detection.
Protein binding domain determines in Sox2-CDP compound, comprising the following steps:
1) design primer is applied, using Sox2 cDNA as template, High fidelity PCR expansion is carried out to Sox2 expression reading frame overall length
Increase；
2) Sox2 expression reading frame overall length is transferred to pBiFc-VN173 expression vector using digestion connection method, obtained
PBiFc-VN173-SOX2 expression vector；
3) design primer is applied, using CDP cDNA as template, reading frame overall length row high-fidelity PCR amplification is expressed to Sox2；
4) cdp table is transferred to pBiFcVC155 expression vector up to reading frame overall length using digestion connection method, obtained
PBiFc-VC155-CDP expression vector；
5) by cotransfection HEK293T cell, confirm the interaction between Sox2 and CDP albumen；
6) apply design primer, using pBiFc-VN173-SOX2 carrier as template, to truncated Sox2 express reading frame into
Row high-fidelity PCR amplification；
7) Sox2 is truncated into expression reading frame using digestion connection method and is transferred to pBiFc-VN173 expression vector, respectively
Obtain pBiFc-VN173-SOX2 Δ N (1-167), pBiFc-VN173-SOX2 Δ N (1-237), pBiFc-VN173-SOX2
Δ N (1-257) and pBiFc-VN173-SOX2 Δ N (1-277) expression vector；
8) design primer is applied, using pBiFc-VC155-CDP carrier as template, truncated cdp table is carried out up to reading frame
High-fidelity PCR amplification；
9) CDP truncation expression reading frame is transferred to pBiFc-VC155 expression vector using digestion connection method, obtained respectively
Obtain pBiFc-VC155-CDP Δ C (759-1516), pBiFc-VC155-CDP Δ C (401-1516), pBiFc- VC155-
CDP Δ C (201-1516) and pBiFc-VC155-CDP Δ C (101-1516) expression vector；
10) by cotransfection HEK293T cell, confirm the interaction binding structural domain between Sox2 and CDP albumen；
11) corotation dyeing method is utilized, in blocks respectively by pBiFcVN173-SOX2 and pBiFcVC155-CDP Δ C
(759-1516), pBiFcVN173-SOX2 and pBiFcVC155-CDP Δ C(401-1516), pBiFcVN173-SOX2 and
PBiFcVC155-CDP Δ C(201-1516), pBiFcVN173-SOX2 and pBiFcVC155-CDP Δ C(101-1516) transfection
Enter HEK293T cell, carries out co-immunoprecipitation using Flag antibody and combine to utilize CDP Identification of the antibodies；
Using clone technology and vector construction technology, there is specific stickiness end by what is obtained by High fidelity PCR and digestion
The open overall length of the Sox2 at end and truncate reading frame orientation and be inserted into pBiFc-VN173, obtain pBiFc-VN173-Sox2 and
pBiFc-VN173-SOX2ΔN(1-167), pBiFc-VN173-SOX2 ΔN(1-237), pBiFc-VN173-SOX2 ΔN
(1-257) and pBiFc-VN173-SOX2 Δ N (1-277) expression vector.
Using clone technology and vector construction technology, there is specific stickiness end by what is obtained by High fidelity PCR and digestion
The open overall length of the CDP at end and truncate reading frame orientation and be inserted into pBiFc-VC155, obtain pBiFc-VC155-CDP and
PBiFc-VC155-CDP, pBiFc-VC155-CDP Δ C (759-1516), pBiFc-VC155-CDP Δ C (401-1516),
PBiFc-VC155-CDP Δ C (201-1516) and pBiFc- VC155-CDP Δ C (101-1516) expression vector.
Sequencing company will be sent to be sequenced after all purposes plasmid extraction and confirms the correctness and consistency of sequence.
Using bimolecular fluorescence complementary technology, all expression vectors are distributed into 25 combinations and carry out cotransfection HEK293T
Cell after 24 hours, detects the appearance of fluorescence, primarily determines its protein binding domain.
Expression vector is distributed into four combinations and carries out cotransfection HEK293T cell, group unification are as follows: pBiFcVN173-
SOX2 and pBiFcVC155-CDP Δ C(759-1516)；Combination two are as follows: pBiFcVN173-SOX2 and pBiFcVC155-CDP Δ C
(401-1516)；Combination three are as follows: pBiFcVN173-SOX2 and pBiFcVC155-CDP Δ C(201-1516)；Combination four are as follows:
PBiFcVN173-SOX2 and pBiFcVC155-CDP Δ C(101-1516).After 48 hours, carried out using Flag antibody immune total
It precipitates and combines and utilize CDP Identification of the antibodies.
The beneficial effects of the present invention are:
(1) Sox2 overall length and truncated expression vector, respectively pBiFc-VN173-SOX2, pBiFc- are constructed
VN173-SOX2ΔN(1-167), pBiFc-VN173-SOX2 ΔN(1-237), pBiFc-VN173- SOX2 ΔN(1-
257) and pBiFc-VN173-SOX2 Δ N (1-277) expression vector, the 1-172 of Sox2 overall length and truncated protein and VENUS albumen
Amino acid residue carries out amalgamation and expression；
(2) CDP overall length and truncated expression vector are constructed, pBiFc-VC155-CDP, pBiFc-VC155- are respectively as follows:
CDP Δ C (759-1516), pBiFc-VC155-CDP Δ C (401-1516), pBiFc- VC155-CDP Δ C (201-1516)
With pBiFc-VC155-CDP Δ C (101-1516) expression vector, the C155 ammonia of Sox2 overall length and truncated protein and VENUS albumen
Base acid residue carries out amalgamation and expression；
(3) bimolecular fluorescence complementary technology is utilized, it is CDP Δ C that preliminary screening, which obtains Sox2 and CDP binding structural domain,
(101-1516) ；
(4) on the basis of sequencing result is correct, using go endotoxin plasmid extraction kit by corresponding expression vector
Be stripped, by they by four combination in the way of cotransfection HEK293T cell, co-immunoprecipitation is the result shows that demonstrate Sox2
With the binding structural domain of CDP protein-interacting, which is CDP Δ C (101-1516).
Detailed description of the invention
Fig. 1 is the clone of Sox2 and CDP overall length and corresponding truncated mutant and the structure of bimolecular fluorescence complementary carrier
Build schematic diagram.The overall length of A:SOX2 and CPD and the design of truncated mutant.B: sample 1 is DNA Marker；2,4,6 points of sample
SOX2 overall length, SOX Δ N(1-167 are not represented) and SOX Δ N(1-237)；Sample 8,10,12 respectively represents CDP overall length, CDP Δ C
(759-1516) and CDP Δ C(401-1516)；Sample 3,5,7 is that SOX2 overall length and truncated mutant and pBiFcVN173 connect
Vector construction identification after connecing；Sample 9,11,13 is the carrier structure after CDP overall length and truncated mutant are connect with pBiFcVC155
Build identification.C: sample 1,3 respectively represents CDP Δ C(201-1516) and CDP Δ C(101-1516)；Sample 5,7 respectively represents
SOX Δ N(1-277) and SOX Δ N(1-257)；Sample 2,4 is the carrier structure after CDP truncated mutant is connect with pBiFcVC155
Build identification；Sample 6,8 is the vector construction identification after SOX2 truncated mutant is connect with pBiFcVN173.
Fig. 2 is that bimolecular fluorescence complementary experiment determines Sox2-CDP binding structural domain schematic diagram (red fluorescence is internal reference).
A: overall length and truncated Sox2 and overall length and truncated cdp table reach carrier and pass through fluorescence after combination cotransfection HEK293T cell
Figure.B:Sox2-CDP compound forms illustraton of model.
Fig. 3 is the interaction schematic diagram between each truncated mutant of co-immunoprecipitation experiment verifying CDP and Sox2 albumen.
Western blott as the result is shown swimming lane 1Control as blank control both without FLAG or without CDP band；FLAG antibody test
There is band near 35KD to 4 experimental group samples；At swimming lane 2-5 with CDP antibody detect respectively about 100KD, 70KD,
35KD and 25KD size strip.Show that each CDP truncated mutant can be got off by the SOX2 albumen precipitation with FLAG label, explanation
All there is interaction between each truncated mutant of CDP and SOX2 albumen, determines generation phase interaction between CDP albumen and SOX2 albumen
Structural domain is present in CDP Δ C (101-1516), i.e., between the 1-100 amino acid sites of CDP albumen.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Experimental material in following embodiments is unless otherwise specified conventional life
Change reagent store to obtain.Quantitative experiment in following embodiment is disposed as independent three repeated experiments, is as a result averaged
Value.
Embodiment 1
Bimolecular fluorescence complementary technology determine Sox2-CDP protein-interacting binding structural domain the following steps are included:
Step 1) pBiFc-VN173-Sox2 expression vector establishment: it is inverted with the RNA of Human esophageal squamous cell cancer cell line KYSE450
The cDNA of record is template, and design carries out high-fidelity PCR amplification using 1 primer pair Sox2 cDNA of table, and uses restriction enzyme
After EcoRI and XbaI carries out digestion to amplified production, it is inserted into pBiFcVN173 carrier, to form pBiFcVN173-Sox2
Expression vector.Amplimer sequence is as shown in table 1.Show through digestion identification and sequencing result: obtaining expected expression Sox2
Target and the carrier (Fig. 1, table 2) that can be used for double fluorescence complementaries.
PCR reaction condition is as follows:
Step 2 Sox2 truncated protein expression vector establishment: using pBiFc-VN173-Sox2 expression vector as template, respectively
Design truncates Sox2 using 1 primer pair of table and carries out high-fidelity PCR amplification, and using restriction enzyme EcoRI and XbaI to expansion
After increasing production object progress digestion, it is inserted into pBiFcVN173 carrier, to form pBiFc-VN173-SOX2 Δ N (1-167),
PBiFc-VN173-SOX2 Δ N (1-237), pBiFc-VN173- SOX2 Δ N (1-257) and pBiFc-VN173-SOX2 Δ N
(1-277) expression vector.Amplimer sequence is as shown in table 1.Show through digestion identification and sequencing result: obtaining expected table
Up to Sox2 truncated protein and it can be used for the carriers (Fig. 1, table 2) of double fluorescence complementaries.
PCR reaction condition is as follows:
Step 3) pBiFc-VC155-CDP expression vector establishment: it is inverted with the RNA of Human esophageal squamous cell cancer cell line KYSE450
The cDNA of record is template, and design carries out high-fidelity PCR amplification using 1 primer pair CDP cDNA of table, and uses restriction enzyme
After SalI and KpnI carries out digestion to amplified production, it is inserted into pBiFcVC155 carrier, to form pBiFc-VC155-CDP table
Up to carrier.Amplimer sequence is as shown in table 1.Show through digestion identification and sequencing result: obtaining expected expression CDP target
And it can be used for the carrier (Fig. 1, table 2) of double fluorescence complementaries.
PCR reaction condition is as follows:
Step 4) CDP truncated protein expression vector establishment: it using pBiFc-VC155-CDP as template, separately designs using table 1
Primer pair truncates CDP and carries out high-fidelity PCR amplification, and carries out digestion to amplified production using restriction enzyme SalI and KpnI
Afterwards, it is inserted into pBiFcVC155 carrier, to form pBiFc-VC155-CDP, pBiFc-VC155-CDP Δ C (759-1516),
PBiFc-VC155-CDP Δ C (401-1516), pBiFc- VC155-CDP Δ C (201-1516) and pBiFc- VC155-
CDP Δ C (101-1516) expression vector.Amplimer sequence is as shown in table 1.Show through digestion identification and sequencing result: obtaining
Expected expression CDP target and the carrier (Fig. 1, table 2) that can be used for double fluorescence complementaries.
PCR reaction condition is as follows:
Step 5) use goes endotoxin plasmid extraction kit extracting sequencing correctly above for bimolecular fluorescence complementary
Carrier and pDsRED2-C1 carrier, using 2000 transfection reagent of Lipofectamine, by 200ng pBiFc-VN173-SOX2
, pBiFc-VN173-SOX2ΔN(1-167), pBiFc-VN173-SOX2 ΔN(1-237), pBiFc-VN173- SOX2
Δ N (1-257) and pBiFc-VN173-SOX2 Δ N (1-277) expression vector, 200ng pBiFc-VC155-CDP, pBiFc-
VC155-CDP Δ C (759-1516), pBiFc-VC155-CDP Δ C (401-1516), pBiFc- VC155-CDP Δ C
(201-1516) and pBiFc-VC155-CDP Δ C (101-1516) expression vector and 50ng pDsRED2-C1(pDsRed2-C1
The red fluorescence of transfection is as internal reference) carrier is respectively formed 25 various combination cotransfections in the HEK293T cell of 12 orifice plates.
25 kinds of combinations are as follows respectively:
PBiFc-VN173-Sox2 and pBiFc-VC155-CDP, pBiFc-VN173-Sox2 and pBiFc-VC155-CDP Δ
C (759-1516), pBiFc-VN173-Sox2 and pBiFc-VC155-CDP Δ C (401-1516), pBiFc-VN173-Sox2
With pBiFc-VC155-CDP Δ C (201-1516), pBiFc-VN173-Sox2 and pBiFc-VC155-CDP Δ C (101-
1516), pBiFc-VN173-SOX2 Δ N (1-167) and pBiFc-VC155-CDP, pBiFc-VN173-SOX2 Δ N (1-167)
With pBiFc-VC155-CDP Δ C (759-1516), pBiFc-VN173-SOX2 Δ N (1-167) and pBiFc-VC155-CDP Δ
C (401-1516), pBiFc-VN173-SOX2 Δ N (1-167) and pBiFc- VC155-CDP Δ C (201-1516), pBiFc-
VN173-SOX2 Δ N (1-167) and pBiFc-VC155-CDP Δ C (101-1516), pBiFc-VN173-SOX2 Δ N (1-
And pBiFc-VC155-CDP, pBiFc-VN173-SOX2 Δ N (1-237) and pBiFc-VC155-CDP Δ C (759- 237)
1516), pBiFc-VN173-SOX2 Δ N (1-237) and pBiFc-VC155-CDP Δ C (401-1516), pBiFc-VN173-
SOX2 Δ N (1-237) and pBiFc- VC155-CDP Δ C (201-1516), pBiFc-VN173-SOX2 Δ N (1-237) and
PBiFc-VC155-CDP Δ C (101-1516), pBiFc-VN173-SOX2 Δ N (1-257) and pBiFc-VC155-CDP,
PBiFc-VN173-SOX2 Δ N (1-257) and pBiFc-VC155-CDP Δ C (759-1516), pBiFc-VN173-SOX2 Δ N
(1-257) and BiFc-VC155-CDP Δ C (401-1516), pBiFc-VN173-SOX2 Δ N (1-257) and pBiFc-
VC155-CDP Δ C (201-1516), pBiFc-VN173-SOX2 Δ N (1-257) and pBiFc-VC155-CDP Δ C (101-
1516), pBiFc-VN173-SOX2 Δ N (1-277) and pBiFc-VC155-CDP, pBiFc-VN173-SOX2 Δ N (1-
And pBiFc-VC155-CDP Δ C (759-1516), pBiFc-VN173-SOX2 Δ N (1-277) and BiFc-VC155- 277)
CDP Δ C (401-1516), pBiFc-VN173-SOX2 Δ N (1-277) and pBiFc- VC155-CDP Δ C (201-1516),
PBiFc-VN173-SOX2 Δ N (1-277) and pBiFc-VC155-CDP Δ C (101-1516).
The HEK293T cell of transfection is placed in by step 6) cultivated 24 hours in cell incubator after, in fluorescence microscope
Under observe the appearance of fluorescence.PRELIMINARY RESULTS shows: the zone location of SOX2 and CDP interaction is in CDP Δ C (101-1516)
With SOX2 Δ N (1-257), i.e., the 1-100 amino acid of CDP albumen and the 278-317 amino acid of SOX2 albumen are two albumen phases
The region (Fig. 2) mutually combined.
Amplimer used in table 1:PCR
The CDP and SOX2 overall length and truncated mutant that table 2:PCR is obtained
Embodiment 2
Immunoprecipitation verifying Sox2-CDP protein-interacting binding structural domain includes the following steps
Step 1) transfection: according to following four combination: pBiFc-VN173-Sox2 and pBiFc-VC155-CDP Δ C
(759-1516), pBiFc-VN173-Sox2 and pBiFc-VC155-CDP Δ C (401-1516), pBiFc-VN173-Sox2 and
PBiFc-VC155-CDP Δ C (201-1516), pBiFc-VN173-Sox2 and pBiFc-VC155-CDP Δ C (101-1516),
It can extract cell protein by plasmid transfection to HEK293T cell, after 48h and be used for co-immunoprecipitation.
Step 2 co-immunoprecipitation: using SIGMA FLAG- co-immunoprecipitation kit, carries out standard behaviour to specifications
Make, harvest albumen, because Sox2 and Flag tag fusion are expressed in pBiFc-VN173-Sox2, uses Flag tag antibody
Do co-immunoprecipitation.
Step 3) Western blot verifies Sox2 and truncates the interaction of CDP: the sample of co-immunoprecipitation runs SDS electricity
Swimming is incubated for transfer film using Flag primary antibody (source of mouse) and CDP primary antibody (rabbit source) respectively, and uses dilution secondary antibody (goat-anti rabbit) and two
Anti- (sheep anti mouse) is identified.
The correctness of step 4) co-immunoprecipitation verifying Sox2-CDP protein-interacting binding structural domain: as the result is shown
Control is as blank control both without FLAG or without CDP band；FLAG antibody test is to 4 experimental group samples near 35KD
There is band；CDP antibody test has band (figure to 4 experimental group samples near 100KD, 70KD, 35KD and 25KD respectively
3).Show that each CDP truncated mutant can be got off by the SOX2 albumen precipitation with FLAG label, illustrates each truncated mutant of CDP
All there is interaction between SOX2 albumen, determines that the structural domain to interact between CDP albumen and SOX2 albumen exists
In CDP Δ C (101-1516), i.e., between the 1-100 amino acid sites of CDP albumen.
SEQUENCE LISTING
<110>Fuzhou General Hospital, Nanjing Military Area, PLA
<120>Sox2-CDP protein binding domain and its identification method
<130> 21
<160> 21
<170> PatentIn version 3.3
<210> 1
<211> 100
<212> PRT
<213> CDPΔC(101-1516)
<400> 1
Met Ala Ala Asn Val Gly Ser Met Phe Gln Tyr Trp Lys Arg Phe Asp
1 5 10 15
Leu Gln Gln Leu Gln Arg Glu Leu Asp Ala Thr Ala Thr Val Leu Ala
20 25 30
Asn Arg Gln Asp Glu Ser Glu Gln Ser Arg Lys Arg Leu Ile Glu Gln
35 40 45
Ser Arg Glu Phe Lys Lys Asn Thr Pro Glu Asp Leu Arg Lys Gln Val
50 55 60
Ala Pro Leu Leu Lys Ser Phe Gln Gly Glu Ile Asp Ala Leu Ser Lys
65 70 75 80
Arg Ser Lys Glu Ala Glu Ala Ala Phe Leu Asn Val Tyr Lys Arg Leu
85 90 95
Ile Asp Val Pro
100
<210> 2
<211> 29
<212> DNA
<213>artificial sequence
<400> 2
cggaattcaa tgtacaacat gatggagac 29
<210> 3
<211> 27
<212> DNA
<213>artificial sequence
<400> 3
tgctctagac atgtgtgaga ggggcag 27
<210> 4
<211> 28
<212> DNA
<213>artificial sequence
<400> 4
cggaattcaa acggcagcta cagcatga 28
<210> 5
<211> 27
<212> DNA
<213>artificial sequence
<400> 5
tgctctagac atgtgtgaga ggggcag 27
<210> 6
<211> 27
<212> DNA
<213>artificial sequence
<400> 6
cggaattcag gctccatggg ttcggtg 27
<210> 7
<211> 27
<212> DNA
<213>artificial sequence
<400> 7
tgctctagac atgtgtgaga ggggcag 27
<210> 8
<211> 27
<212> DNA
<213>artificial sequence
<400> 8
cggaattcat cctcccactc cagggcg 27
<210> 9
<211> 27
<212> DNA
<213>artificial sequence
<400> 9
tgctctagac atgtgtgaga ggggcag 27
<210> 10
<211> 26
<212> DNA
<213>artificial sequence
<400> 10
cggaattcac tccccggcgc cgaggt 26
<210> 11
<211> 27
<212> DNA
<213>artificial sequence
<400> 11
tgctctagac atgtgtgaga ggggcag 27
<210> 12
<211> 30
<212> DNA
<213>artificial sequence
<400> 12
acgcgtcgac catggcggcc aatgtgggat 30
<210> 13
<211> 27
<212> DNA
<213>artificial sequence
<400> 13
ggggtaccga actcccattc gataggt 27
<210> 14
<211> 30
<212> DNA
<213>artificial sequence
<400> 14
acgcgtcgac catggcggcc aatgtgggat 30
<210> 15
<211> 26
<212> DNA
<213>artificial sequence
<400> 15
ggggtaccgg acagaagctt gggggt 26
<210> 16
<211> 30
<212> DNA
<213>artificial sequence
<400> 16
acgcgtcgac catggcggcc aatgtgggat 30
<210> 17
<211> 29
<212> DNA
<213>artificial sequence
<400> 17
ggggtaccgt tcttctccag caacagcac 29
<210> 18
<211> 30
<212> DNA
<213>artificial sequence
<400> 18
acgcgtcgac catggcggcc aatgtgggat 30
<210> 19
<211> 28
<212> DNA
<213>artificial sequence
<400> 19
ggggtaccct ttgaggtggt ggacatct 28
<210> 20
<211> 30
<212> DNA
<213>artificial sequence
<400> 20
acgcgtcgac catggcggcc aatgtgggat 30
<210> 21
<211> 29
<212> DNA
<213>artificial sequence
<400> 21
ggggtacctg ggacgtcaat caatctttt 29
Claims (3)
1.Sox2-CDP protein binding domain, it is characterised in that: the binding structural domain is CDP Δ C (101-1516), sequence
As shown in SEQ ID NO.1.
2. a kind of Sox2-CDP protein binding domain identification method as described in claim 1, it is characterised in that: utilize carrier
Intact proteins expression cassette and truncated protein expression cassette are built into double fluorescence complementary carrier systems by building, by combination,
Interaction protein binding structural domain is primarily determined using bimolecular fluorescence complementary method；
Using clone technology and vector construction technology, by by High fidelity PCR and digestion obtain with specific cohesive terminus,cohesive termini
Sox2 overall length and truncation open reading frame orientation are inserted into pBiFc-VN173, obtain pBiFc-VN173-SOX2, pBiFc-
VN173-SOX2ΔN(1-167), pBiFc-VN173-SOX2 ΔN(1-237), pBiFc-VN173-SOX2 ΔN(1-
And pBiFc-VN173-SOX2 Δ N (1-277) expression vector 257)；
Using clone technology and vector construction technology, by by High fidelity PCR and digestion obtain with specific cohesive terminus,cohesive termini
CDP overall length and truncation open reading frame orientation are inserted into pBiFc-VC155, obtain pBiFc-VC155-CDP, pBiFc-
VC155-CDPΔC(759-1516)、pBiFc-VC155-CDPΔC(401-1516) 、pBiFc- VC155-CDPΔC(201-
And pBiFc-VC155-CDP Δ C (101-1516) expression vector 1516).
3. Sox2-CDP protein binding domain identification method according to claim 2, it is characterised in that: first pass through double points
Sub- fluorescence complementary technology primarily determines protein-binding domains, then is verified with co-immunoprecipitation method, specifically include with
Lower step:
1) design primer is applied, using the cDNA of Sox2 as template, high-fidelity PCR amplification is carried out to Sox2；
2) Sox2 is transferred to pBiFc-VN173 expression vector using digestion connection method, obtains expression vector pBiFc-
VN173-SOX2；
3) design primer is applied, using the Sox2 in step 2 as template, high-fidelity amplification obtains Sox2 truncated mutant respectively,
And digestion connection method is utilized, it is built into pBiFc-VN173 carrier respectively, obtains pBiFc-VN173-SOX2 Δ N (1- respectively
167), pBiFc-VN173-SOX2 Δ N (1-237), pBiFc-VN173- SOX2 Δ N (1-257) and pBiFc-VN173-
SOX2 Δ N (1-277) expression vector；
4) design primer is applied, using the cDNA of CDP as template, high-fidelity PCR amplification is carried out to CDP；
5) CDP is transferred to pBiFc-VC155 expression vector using digestion connection method, obtains pBiFc-VC155-CDP expression
Carrier；
6) design primer is applied, using the CDP in step 5) as template, high-fidelity amplification obtains CDP truncated mutant respectively, and
Using digestion connection method, it is built into pBiFc-VC155 carrier respectively, obtains pBiFc-VC155-CDP Δ C (759- respectively
1516), pBiFc-VC155-CDP Δ C (401-1516), pBiFc- VC155-CDP Δ C (201-1516) and pBiFc-
VC155-CDP Δ C (101-1516) expression vector；
7) corotation dyeing method is utilized, in blocks respectively by overall length and truncated SOX2 expression vector and overall length and truncation
Cdp table enter HEK293T cell up to carrier cotransfection, and pDsRED2-C1 is internal reference, observes the appearance of its fluorescence respectively, really
Whether the albumen expressed surely has interaction；
8) utilize corotation dyeing method, in blocks respectively by be used for bimolecular fluorescence complementary overall length SOX2 expression vector and
Overall length and truncated cdp table reach carrier cotransfection and enter HEK293T cell, carry out co-immunoprecipitation using Flag antibody and combine
Utilize CDP Identification of the antibodies.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611108011.7A CN106519006B (en) | 2016-12-06 | 2016-12-06 | Sox2-CDP protein binding domain and its identification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611108011.7A CN106519006B (en) | 2016-12-06 | 2016-12-06 | Sox2-CDP protein binding domain and its identification method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106519006A CN106519006A (en) | 2017-03-22 |
| CN106519006B true CN106519006B (en) | 2019-06-25 |
Family
ID=58341629
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611108011.7A Expired - Fee Related CN106519006B (en) | 2016-12-06 | 2016-12-06 | Sox2-CDP protein binding domain and its identification method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106519006B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104844691A (en) * | 2015-05-22 | 2015-08-19 | 中国人民解放军南京军区福州总医院 | Sox2 protein peptide aptamer and authenticating method thereof |
| CN105018564A (en) * | 2015-07-22 | 2015-11-04 | 华东师范大学 | G protein-coupled receptor (GPCR) in-vivo tracking method and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7259256B2 (en) * | 2002-10-02 | 2007-08-21 | National Chunghsing University | Primers useful in polymerase chain reaction for the identification of subtypes C1, C2 and C3 of staphylococcal enterotoxin type C |
-
2016
- 2016-12-06 CN CN201611108011.7A patent/CN106519006B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104844691A (en) * | 2015-05-22 | 2015-08-19 | 中国人民解放军南京军区福州总医院 | Sox2 protein peptide aptamer and authenticating method thereof |
| CN105018564A (en) * | 2015-07-22 | 2015-11-04 | 华东师范大学 | G protein-coupled receptor (GPCR) in-vivo tracking method and application |
Non-Patent Citations (2)
| Title |
|---|
| Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation;Hu CD ET AL;《Mol Cell》;20021231;摘要 |
| 转录因子SOX2的研究进展;陈艳玫等;《生命科学》;20040630;第16卷(第3期);129-133 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106519006A (en) | 2017-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103459415B (en) | Designed repeat proteins that bind to serum albumin | |
| CN106478809B (en) | Identify the TCR of PRAME antigen small peptide | |
| US20190202887A1 (en) | Humanized Variable Lymphocyte Receptors (VLR) and Compositions and Uses Related Thereto | |
| CN106459177B (en) | High affinity ny-eso T cell receptor | |
| CN110713522B (en) | Use of extracellular domain of low pH insertion peptide as antigen | |
| Cao et al. | Acetyl-Coenzyme A acyltransferase 2 attenuates the apoptotic effects of BNIP3 in two human cell lines | |
| CN106478808B (en) | Identify the T cell receptor of NY-ESO-1 antigen small peptides | |
| WO2023108803A1 (en) | Fusion protein, and preparation method therefor and use thereof | |
| US20180265552A1 (en) | Nepovirus coat protein fusion polypeptides and their use | |
| US11453886B2 (en) | Virus-like particles and uses thereof | |
| CN108117596A (en) | For the high-affinity TCR of NY-ESO | |
| CN107223136B (en) | Method for introducing antibody into cell | |
| CN111569065B (en) | Composition for targeted ubiquitination degradation of endogenous Survivin of cells by using nano antibody and application of composition | |
| CN104844691B (en) | Sox2 protein peptides aptamer and its identification | |
| CN101830972B (en) | Fluorescence complementary system based on green fluorescent protein sfGFP | |
| Dao et al. | A TCR mimic monoclonal antibody reactive with the “public” phospho-neoantigen pIRS2/HLA-A* 02: 01 complex | |
| CN106519006B (en) | Sox2-CDP protein binding domain and its identification method | |
| CN106432475B (en) | high affinity NY-ESO T cell receptor | |
| CN110903402A (en) | Bispecific fusion protein and construction method and application thereof | |
| CN106589061A (en) | Sox2-CDP binding domain candidate peptide aptamer | |
| CN108484770B (en) | Recombinant rat anti-mouse CD4 monoclonal antibody, preparation method and application | |
| US20220411472A1 (en) | Self-assembling circular tandem repeat proteins with increased stability | |
| CN113999306B (en) | Method for obtaining antibody for recognizing space conformation epitope | |
| Honfozo et al. | The luminal domain of Toxoplasma gondii sortilin adopts a ring-shaped structure exhibiting motifs specific to apicomplexan parasites | |
| Iurova et al. | Use of Natural Peptides with a Cysteine Knot of Arthropods as a Carrier of a Peptide Tropic to Glutamate Carboxypeptidase II. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190625 |