CN103951743B - Human SFRP 1 variant and application thereof - Google Patents
Human SFRP 1 variant and application thereof Download PDFInfo
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- CN103951743B CN103951743B CN201410172978.6A CN201410172978A CN103951743B CN 103951743 B CN103951743 B CN 103951743B CN 201410172978 A CN201410172978 A CN 201410172978A CN 103951743 B CN103951743 B CN 103951743B
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- 108010020277 WD repeat containing planar cell polarity effector Proteins 0.000 title abstract description 4
- 101000864743 Homo sapiens Secreted frizzled-related protein 1 Proteins 0.000 claims abstract description 11
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 4
- 239000003814 drug Substances 0.000 claims abstract description 3
- 230000001629 suppression Effects 0.000 claims abstract description 3
- 102000055203 human SFRP1 Human genes 0.000 claims abstract 3
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 235000018102 proteins Nutrition 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000000259 anti-tumor effect Effects 0.000 claims 1
- 201000011510 cancer Diseases 0.000 abstract description 3
- 230000002018 overexpression Effects 0.000 abstract description 2
- 230000004614 tumor growth Effects 0.000 abstract 1
- 238000000034 method Methods 0.000 description 12
- 229920001184 polypeptide Polymers 0.000 description 10
- 108090000765 processed proteins & peptides Proteins 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 102100030058 Secreted frizzled-related protein 1 Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 230000009471 action Effects 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 108050003627 Wnt Proteins 0.000 description 4
- 102000013814 Wnt Human genes 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 3
- 201000007270 liver cancer Diseases 0.000 description 3
- 208000014018 liver neoplasm Diseases 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 101150046427 Sfrp1 gene Proteins 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 102100030053 Secreted frizzled-related protein 3 Human genes 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 1
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000011098 chromatofocusing Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000225 tumor suppressor protein Substances 0.000 description 1
Classifications
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- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of human SFRP 1 protein variant, and the application in the medicine of preparation suppression tumor growth.Make human SFRP 1 variant overexpression in cancerous cell of the present invention, or by external supply human SFRP 1 protein variant, to control cancer cell multiplication and to kill cancerous cell.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to tumor suppressor protein variant.
Background technology
SFRP, secreting type Frz associated protein (secreted frizzled-related proteins), containing one rich in
The domain (cysteine rich domain, CRD) of cysteine, but lack seven membrane-spanning domains, it may be with volume
Bent albumen (frizzled, Frz) competition binding Wnt albumen, thus suppress the overexpression of Wnt albumen.Wnt
It is a class secreting type glycoprotein, human normal cell has been bred adjustment facilitation.Recent experimental finds, presses down
When making the gene SFRP generation exception of this protein effect, the function of Wnt protein can occur exception therewith,
Cancerous cell is constantly bred, causes colorectal cancer.After normal SFRP gene injection cancerous cell,
Cancer cell multiplication will be controlled and kill cancerous cell.Research shows, the PATIENTS WITH LARGE BOWEL of 90% and SFRP gene
Abnormal relevant.
In the prior art, in order to improve the activity of albumen, the avtive spot for albumen carries out specific prominent
Become and replace, thus finding active higher variant is conventional way.In order to improve the life of this albumen further
Thing activity, applicant, by substantial amounts of experiment, is carried out for site specific in SFRP1 aminoacid sequence
Point mutation, thus obtain the variant than SFRP1 albumen itself with higher inhibitory activity.
Detailed Description Of The Invention
The present invention relates to the variant of the separation of Parent Protease, it corresponds to SEQ ID NO:1's at least one
The position 34Y of mature polypeptide, 39F, 42D, 50R, 58C, 63A, 89E, 100L, 122L, 137R,
The position of 151W, 174A, 198I, 220N, 242D, 271M, 286K or 293E comprises modification.Tool
For body, the variant of the present invention has the inhibitory activity of improvement.
Preferably, the method that is applied to the present invention, the protein variant of the inhibitory activity presenting improvement are following arbitrary
Modify: 34Y/A, 39F/D, 42D/R, 50R/S, 58C/G, 63A/H, 89E/A, 100L/D,
122L/R、137R/S、151W/G、174A/R、198I/A、220N/S、242D/G、271M/D、
286K/R or 293E/L.
The method that the invention still further relates to produce the protein variant of the present invention, cultivates host cell including (a)；(b)
Reclaim described protein variant.
In the production method of the present invention, use method well known in the art in the battalion being suitable for producing described polypeptide
Support and culture medium is cultivated cell.For example, it is possible to by expressing with permission and/or separate institute in suitable culture medium
Small-scale in the shake-flask culture carried out under conditions of stating polypeptide, and laboratory or industrial fermentation tank or send out on a large scale
Ferment (include continuously, in batches, fed-batch or solid fermentation) cultivates cell.Use methods known in the art
Cultivating in suitable Nutrient medium, described Nutrient medium comprises carbon source and nitrogen source and inorganic salt.Close
Suitable culture medium can obtain from commercial supplier or can prepare (such as, in U.S.'s allusion quotation according to disclosed composition
In the catalogue at type culture collection center).If polypeptide is secreted in Nutrient medium, this polypeptide can be from institute
State in culture medium and directly reclaim.If polypeptide is not secreted, it can reclaim from cell lysate (1ysate).
Gained polypeptide can use methods known in the art to reclaim.Such as, polypeptide can by conventional method from
Nutrient medium reclaims, described conventional method includes but not limited to be centrifuged, filters, extracts, is spray-dried,
Evaporation or precipitation.
The polypeptide of the present invention can be included but not limited to by multiple methods known in the art purification, described method
Chromatography (such as, ion exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis method (such as, system
Standby type (preparative) isoelectrofocusing), differential solubility (such as, ammonium sulfate precipitation), SDS-PAGE or carry
Take and (see, e.g., Protein Purification, J.-C.Janson and Lars Ryden to compile, VCH
Publishers,New York,1989)。
The polypeptide of the present invention is used for preparing corresponding medicine, goes for treating cancer.
Detailed description of the invention
The acquisition of 1.SFRP1 gene
ORF district totally 314 aminoacid of PCR amplification SFRP1, template is containing human genome
Sample.Two ends introduce restriction enzyme site BamHI and EcoRI, after enzyme action with as the pET of enzymic digestion
Carrier connects, and chooses monoclonal after converting escherichia coli, order-checking.Record its aminoacid sequence such as sequence 1
Shown in.
Primer sequence used is as follows, and the primer of SFRP1 is (not removing termination codon without his-myc):
Sfrpl3.1B (-): F:5 '-CGGGATCCATGGGCATCGGG-3 ':
R:5 '-CGGAATTCCGTCACTTAAACACGGACT-3 '.
The structure of 2.SFRP1 variant
The introducing of catastrophe point
(1) design corresponding mutagenic primer according to corresponding mutational site, use PCR fixed-point mutation method to exist
On SFRP1 gene, amino acid sites corresponding for wild type is suddenlyd change；
(2) above-mentioned PCR primer reclaims after purification through glue, carries out enzyme action with restricted enzyme, with process
After the plasmid PCDNA3.1 fragment of identical enzyme action connects, convert bacillus coli DH 5 alpha competence thin
Born of the same parents；
3.2. recombiant plasmid is identified: by enzyme action, PCR method, recombiant plasmid is identified, and inspection of checking order,
Nucleotide sequence after thus being suddenlyd change.
4.PCDNA3.1-SFRP1 variant plasmid construction
By second step build obtain SFRP1 variant gene two ends introduce restriction enzyme site BamHI and
EcoRI, after enzyme action with as enzymic digestion PCDNA3.1 connect, after qualification, plasmid is turned
Dye cell strain: 7721 hepatoma carcinoma cell, the effect of the suppression liver cancer cell growth of inspection SFRP1 variant energy
Really.Concrete outcome is as follows:
Table 1SFRP1 variant process LAN cell growth inhibiting situation
By the above results it can be seen that some mutants to the inhibitory activity of liver cancer cell growth relative to wild
Type SFRP1 albumen has increased significantly, and can further apply liver cancer treatment.
Claims (3)
1. a human SFRP 1 protein variant, it is characterised in that its aminoacid sequence compared with SEQ ID NO:1,
Differ only in and the Y of the 34th is replaced with A.
2. the protein variant as claimed in claim 1 purposes in preparation suppression tumour medicine.
3. an anti-tumor medicinal preparation, it contains the protein variant described in claim 1 and the most suitable
Carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410172978.6A CN103951743B (en) | 2014-04-25 | 2014-04-25 | Human SFRP 1 variant and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410172978.6A CN103951743B (en) | 2014-04-25 | 2014-04-25 | Human SFRP 1 variant and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103951743A CN103951743A (en) | 2014-07-30 |
| CN103951743B true CN103951743B (en) | 2016-11-23 |
Family
ID=51329116
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410172978.6A Active CN103951743B (en) | 2014-04-25 | 2014-04-25 | Human SFRP 1 variant and application thereof |
Country Status (1)
| Country | Link |
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| CN (1) | CN103951743B (en) |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106349367A (en) * | 2016-11-15 | 2017-01-25 | 李露青 | Human SFRP1 varient and application thereof |
| CN106349369A (en) * | 2016-11-15 | 2017-01-25 | 李露青 | Human SFRP1 varient and application thereof |
| CN106349368A (en) * | 2016-11-15 | 2017-01-25 | 李露青 | Human SFRP1 varient and application thereof |
| CN106349371A (en) * | 2016-11-16 | 2017-01-25 | 李露青 | Human SFRP1 varient and application thereof |
| CN106349370A (en) * | 2016-11-16 | 2017-01-25 | 李露青 | Human SFRP1 varient and application thereof |
| CN106349372A (en) * | 2016-11-16 | 2017-01-25 | 李露青 | Human SFRP1 varient and application thereof |
| CN106349374A (en) * | 2016-11-17 | 2017-01-25 | 李露青 | Human SFRP1 (secreted frizzled-related protein 1) variant and application thereof |
| CN106349375A (en) * | 2016-11-17 | 2017-01-25 | 李露青 | Human SFRP1 (secreted frizzled-related protein 1) variant and application thereof |
| CN106349373A (en) * | 2016-11-17 | 2017-01-25 | 李露青 | Human SFRP1 (secreted frizzled-related protein 1) variant and application thereof |
| CN106349376A (en) * | 2016-11-18 | 2017-01-25 | 李露青 | Human SFRP1 (secreted frizzled-related protein 1) variant and application thereof |
| CN106349377A (en) * | 2016-11-18 | 2017-01-25 | 李露青 | Human SFRP1 (secreted frizzled-related protein 1) variant and application thereof |
| CN106336454A (en) * | 2016-11-18 | 2017-01-18 | 李露青 | Human SFRP1 variant and application thereof |
| CN106349381A (en) * | 2016-11-21 | 2017-01-25 | 李露青 | Human SFRP1 (secreted frizzled-related protein 1) variant and application thereof |
| CN106349382A (en) * | 2016-11-21 | 2017-01-25 | 李露青 | Human SFRP1 (secreted frizzled-related protein 1) variant and application thereof |
| CN106349383A (en) * | 2016-11-21 | 2017-01-25 | 李露青 | Human SFRP1 (secreted frizzled-related protein 1) variant and application thereof |
| CN106349380A (en) * | 2016-11-22 | 2017-01-25 | 李露青 | Human SFRP1 (secreted frizzled-related protein 1) variant and application thereof |
| CN106349379A (en) * | 2016-11-22 | 2017-01-25 | 李露青 | Human SFRP1 (secreted frizzled-related protein 1) variant and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1962865A (en) * | 2005-11-08 | 2007-05-16 | 上海人类基因组研究中心 | Human SFRP1 gene, its coded protein and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2389199B1 (en) * | 2011-04-08 | 2013-09-17 | Consejo Superior De Investigaciones Científicas (Csic) | METHOD OF DIAGNOSIS OF ALZHEIMER'S DISEASE THAT USES SFRP1 AS A BIOMARCATOR. |
-
2014
- 2014-04-25 CN CN201410172978.6A patent/CN103951743B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1962865A (en) * | 2005-11-08 | 2007-05-16 | 上海人类基因组研究中心 | Human SFRP1 gene, its coded protein and application |
Non-Patent Citations (2)
| Title |
|---|
| Structure-function analysis of secreted frizzled-related protein-1 for its wnt antagonist function;Ramesh A Bhat et al;《Journal of Cellular Biochemistry》;20071231;第102卷;1519-1528 * |
| 分泌型卷曲相关蛋白在肿瘤中的研究进展;郑芸等;《癌症进展杂志》;20090531;第7卷(第3期);290-295 * |
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| CN103951743A (en) | 2014-07-30 |
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Effective date of registration: 20161018 Address after: 315113 Zhejiang city of Ningbo province Yinzhou District east Wu Town Road No. 58 Applicant after: Li Luqing Address before: Yu Cai Lu Heng Jie Zhen Yinzhou District 315181 Zhejiang city of Ningbo province No. 25 Applicant before: Chen Xiuding |
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Inventor after: Yi Jihui Inventor after: Xu Chunlian Inventor after: Mao Kanlang Inventor before: Chen Xiuding |
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Effective date of registration: 20170323 Address after: Room 1008, building 1A, software industrial base, No. 518000 Nanshan District Road, Shenzhen, Guangdong, China Patentee after: SHENZHEN GENTARGET MEDICAL TECHNOLOGY CO., LTD. Address before: 315113 Zhejiang city of Ningbo province Yinzhou District east Wu Town Road No. 58 Patentee before: Li Luqing |
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