CN108017711A - A kind of anticalcium small peptide and its application - Google Patents
A kind of anticalcium small peptide and its application Download PDFInfo
- Publication number
- CN108017711A CN108017711A CN201711210235.3A CN201711210235A CN108017711A CN 108017711 A CN108017711 A CN 108017711A CN 201711210235 A CN201711210235 A CN 201711210235A CN 108017711 A CN108017711 A CN 108017711A
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- small peptide
- anticalcium
- calcification
- pharmaceutical composition
- amino acid
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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
- C07K14/81—Protease inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The present invention relates to a kind of anticalcium small peptide, more particularly to a kind of anticalcium small peptide and its application, its amino acid sequence is as shown in SEQ ID NO.1.The experiment proves that small peptide provided by the invention can significantly inhibit warfarin and high calcium, the valve interstitial cell calcification of high phosphorus induction, and suppress the valve interstitial cell apoptosis of low sugar culture induction.Therefore, small peptide provided by the invention is likely to become good low side effect anticalcium chemical drug thing.
Description
Technical field
The invention belongs to biomedicine field, more particularly to a kind of anticalcium small peptide and its application.
Background technology
Calcified disease is that clinically very common one kind is occurred similar to osteogenetic process and with calcium salt with non-bone tissue
The disease mainly showed is deposited as, it is flexible that calcification is most often apt to occur in cardiac valves, big blood vessel, synovial membrane, cartilagines tracheales etc.
Soft tissue.In recent years, more and more researchs show for example most common cardiac valves of heterotopic calcification and blood vessel is one in external cause
Process is actively adjusted with internal cause collective effect.Clinically heterotopic calcification venereal disease change main cause has chronic inflammation disease at present
Disease such as long-term atherosclerosis, alcium and phosphor metabolization obstacle caused by renal insufficiency, alcium and phosphor metabolization caused by endocrine system disease hinder
Hinder, long-term use can cause medicine such as warfarin of calcification etc..Unfortunately, at present clinically still without a kind of effective
Prevention or the means for the treatment of calcification, existing treatment are all the tissues that man-made organ can only be utilized to substitute severe calcification, such as manually
Valve replaces the cardiac valves of calcification, and artificial blood vessel substitutes blood vessel of calcification etc..For the patient of renal insufficiency, do not have
A kind of effective method can delay the relevant cardiovascular calcifications of long-term dialysis, and it is outstanding that urgent need will develop some effective treatment methods
It is long-term treatment method to alleviate the harm of Calcified disease.Research group's early period is in National Natural Science base where applicant
Under golden project (81100162,81570247) is subsidized, it have studied peptase first and suppress albumen PI16 suppression calcific aortic valves
Effect, the albumen that we prove to be overexpressed first people's PI16 gene codes can heart that significantly warfarin and high calcium high phosphorus induce
Valvular calcification.Studied by the analysis to PI16 protein structure domains and protein-interacting, I has found one section of ammonia of PI16 first
Base acid sequence can play anti-calcification as complete albumen, we by carrying out mutation transformation to this section of amino acid,
We have invented a kind of anticalcium small peptide that physicochemical property is more stable.
The content of the invention
Suppress albumen PI16 from people's peptase the object of the present invention is to provide one kind, one section after point mutation is short
Peptide, the small peptide have notable stability and anti-calcification.
It is a further object of the present invention to provide the medicinal usage of this anticalcium small peptide.
The anticalcium small peptide of the present invention, its amino acid sequence such as SEQ ID NO.1 (Val-Ala-Leu-His-Asn-Leu-
Tyr-Arg-Ala-Gln-Val-Ser-Pro-Thr shown in), which is made of 14 amino acid.
Present invention additionally comprises the preparation method of the small peptide of the present invention, the method is selected from conventional solid phase synthesis process, molten
Synthetic method in liquid, genetic recombination synthetic method or chemical synthesis process.
Medicinal usage of the present invention is the medicinal usage for treating Calcified disease.Calcified disease described in it is the heart
Dirty valvular calcification, annulus calcification, ectopic soft tissue calcification caused by kidney trouble include but not limited to cardiac valves, blood vessel, gas
Cardiac valves, angiosteosis caused by pipe etc., atherosclerotic or calcium and phosphorus metabolism disorder.
Present invention additionally comprises the drug regimen containing small peptide of the present invention.The drug regimen of the present invention, can containing medicine
All kinds of carriers received.The drug regimen of the present invention can exist with any dosage form, such as injection or oral formulations, be preferably
Freeze drying injection.
Polypeptide with amino acid sequence of the present invention both can with polypeptide form individualism, can also with other albumen or
Polypeptide is merged, or by or by carry out such as acetylation, methylate modification, carry out Individual amino acids replacement or mutation,
Or the privileged site of insertion protein surface, or it is connected use with other materials.No matter exist in what manner, also have this amino acid
The polypeptides matter of structure may show anti-calcification property.
Material containing this amino acid sequence can have a variety of application forms, including internal and external anticalcium
Activity, the disease treatment that purpose is turned to anticalcium is realized with various administering modes.
Beneficial effect:
Small peptide of the present invention can suppress the cell and tissue calcification of its mediation specifically with p53 protein bindings, can be with
Other unknown target spots are acted on, are used to prepare the medicine for the treatment of Calcified disease.
The polypeptide of the present invention has length short, the characteristics of being readily synthesized, and stablize under acid-base environment.
Brief description of the drawings
The present invention is further described with reference to the accompanying drawings and examples.
Fig. 1 small peptides suppress the Alizarin red staining of the valve interstitial cell calcification of warfarin induction
Fig. 2 small peptides suppress the intracellular calcium concentration measure of the valve interstitial cell calcification of warfarin induction
Fig. 3 small peptides suppress the Alizarin red staining of the valve interstitial cell calcification of high calcium high phosphorus induction
Fig. 4 small peptides suppress the intracellular calcium concentration measure of the valve interstitial cell calcification of high calcium high phosphorus induction
Fig. 5 small peptides suppress serum-free induction valve interstitial cell apoptosis flow cytometer schematic diagram
The statistic analysis result of Fig. 6 drain cells instrument detection
Fig. 7 co-immunoprecipitations and protein blot Faxian show small peptide specifically with p53 protein bindings
Fig. 8 small peptides degradation rate in ph2.5 and ph8.0 aqueous solutions
Embodiment
Below by way of example, the present invention is further illustrated, but does not do limitation of the present invention.
Embodiment 1, the operating procedure of the preparation method of small peptide：
The main operational steps of solid phase small peptide are as follows：
(1) loading for weighing 0.5mmol is Fmoc-Thr (tBu) wang resin of 0.35mmol/g, is placed in reaction
In column, suitable DCM immersions 30min is added, DCM is pumped, is washed 3 times with DMF.
(2) deprotect, add suitable deprotection liquid (DMF solution of 20% piperidines) reaction 20min, pump deprotection
Liquid, is washed 6 times with DMF.
(3) coupling of amino acid, weighs the Fmoc-Pro-OH of 3eq with TBTU, adds in reaction column, using DMF as molten
Whether agent, adds the DIEA of 3eq, reacts 1h, complete by ninhydrin detection reaction, after reaction, pumps reaction solution, uses
DMF is washed three times.
(4) repeat step (2) and (3), until the reaction of last amino acid terminates.
(5) deprotection is shunk, and is added suitable deprotection liquid (DMF solution of 20% piperidines) reaction 20min, is pumped de-
Liquid is protected, is washed three times with DMF, DCM, methanol respectively, dry adsorbent.
(6) suitable lysate (TFA95%/2.5% water/2.5%Tis) is added, 30 ° of constant temperature shakes 2h in shaking table,
Resin is removed in worry, and suitable no water-ice ether is added into filtrate, separates out solid, and centrifugation makes solid precipitating, supernatant removed, with nothing
Water-ice ether washs three times, drains to obtain crude product.
(7) amino acid sequence to sample after purification using mass spectrography detection small peptide, it is correct to be determined as amino acid sequence
Small peptide product.
Embodiment 2, small peptide suppress the experiment of the valve interstitial cell calcification of warfarin induction
The primary porcine aortic valve interstitial cell of DMEM medium cultures containing 10% hyclone, is about 1* with cell density
105A cells/well cell concentration is added in 12 orifice plates, is separately added into the warfarin hydroponics of 1.6mM 7 days, is separately added at the same time
Small peptide and the negative control peptide of the present invention (SEQ ID NO.3) of various concentrations, 4% poly first was used at the 7th day to cell respectively
Aldehyde uses 1% Alizarin red staining after fixing, show doped calcium situation, in addition with the diluted hydrochloric acid dissolution intracellular Ca2+ of 0.6M, use
Colorimetric method calcium concentration assay kit detects the level of intracellular calcium content.
The result shows that small peptide of the invention can significantly inhibit the valve interstitial cell calcium ion deposition of warfarin induction,
The inhibition has dose-effect relationship, and negative control peptide is then without obvious effect.Alizarin red staining (as shown in Figure 1) and calcium content
The valve interstitial that measure (as shown in Figure 2) the results show 200-400ug/ml concentration small peptide can significantly inhibit warfarin induction is thin
Born of the same parents' calcification.
Embodiment 3, small peptide suppress the experiment of the valve interstitial cell calcification of high calcium high phosphorus induction
The primary porcine aortic valve interstitial cell of DMEM medium cultures containing 10% hyclone, is about 1* with cell density
105A cells/well cell concentration is added in 12 orifice plates, is separately added into the calcium chloride of 2.0mM, 2.0mM (dibastic sodium phosphates+phosphoric acid hydrogen two
Sodium) hydroponics 5 days, while small peptide and the negative control peptide of the present invention (SEQ ID NO.3) of various concentrations is separately added into,
Respectively to cell using the Alizarin red staining after the fixation of 4% paraformaldehyde with 1%, show doped calcium situation, in addition use 0.6M within 5 days
Diluted hydrochloric acid dissolution intracellular Ca2+, using colorimetric method calcium concentration assay kit detection intracellular calcium content level.As a result table
It is bright, small peptide of the invention can notable high calcium high phosphorus induction valve interstitial cell calcium ion deposition, which has amount
Effect relation, and negative control peptide is then without obvious effect.Alizarin red staining (as shown in Figure 3) and determination of calcium content (as shown in Figure 4)
The result shows that 400ug/ml concentration small peptide can significantly inhibit the valve interstitial cell calcification of high calcium high phosphorus induction.
Embodiment 4, small peptide suppress the experiment of serum-free induction valve interstitial cell apoptosis
The primary porcine aortic valve interstitial cell of DMEM medium cultures containing 10% hyclone, is about 1* with cell density
105A cells/well cell concentration is added in 12 orifice plates, replaces low sugar culture medium after cell is completely adherent, while be separately added into not
With small peptide and the negative control peptide of the present invention (SEQ ID NO.3) of concentration, when culture 12 is small, 0.25% trypsin digestion cell, after
Immunostaining, flow cytomery Annexin V are carried out to cell with the fluorescence antibody of Apoptosis marker Annexin V
Positive cell ratio, flow cytomery show that 200-400ug/ml small peptides can significantly inhibit the valve of low sugar culture medium induction
Theca-titerstitial cells apoptosis.And negative control peptide is then without obvious effect.As seen in figs. 5-6.
Embodiment 5, small peptide specificity and p53 protein bindings
Be respectively adopted the plasmid transfection 293T cells containing small peptide and negative control peptide (SEQ ID NO.3), 48 it is small when after,
Cracking extraction albumen.Respectively with anti-Flag antibody and anti-p53 antibody incubations, after being precipitated using paramagnetic particle method, using conventional western
Blot methods, respectively hybridize albumen after transferring film with p53 and Flag antibody.The result shows that small peptide of the present invention is tied with p53
Close, and negative control peptide is without combination.As shown in Figure 7.
Embodiment 6, the ph stability of small peptide
The mutation small peptide (SEQ ID NO.1) of original short peptide sequence (SEQ ID NO.2) and the present invention is configured to respectively
1mg/ml aqueous solutions, it is 2.5 and 8.0 that dilute hydrochloric acid and hydrogen peroxide, which adjust pH value, respectively.Under 37 degree of waters states, to it is different when
Between the sample put concentration mensuration is carried out using high performance liquid chromatography.The result shows that mutation small peptide of the invention has preferably acid
Alkaline stability.As shown in Figure 8.
Part that the present invention does not relate to is same as the prior art or can be realized using the prior art.
The present invention obtains the subsidy of national project.
Sequence table
<110>Sun Wei
<120>A kind of anticalcium small peptide and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Val Ala Leu His Asn Leu Tyr Arg Ala Gln Val Ser Pro Thr
1 5 10
<210> 2
<211> 14
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 2
Val Glu Leu His Asn Leu Tyr Arg Ala Gln Val Ser Pro Thr
1 5 10
<210> 3
<211> 14
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Val Ala Leu Ala Asn Leu Ala Ala Ala Ala Val Ala Pro Ala
1 5 10
Claims (10)
1. a kind of anticalcium small peptide, it is characterised in that its amino acid sequence is as shown in SEQ ID NO.1.
2. the preparation method of small peptide described in claim 1, it is characterised in that：Selected from conventional solid phase synthesis process, closed in solution
Into method, genetic recombination synthetic method or chemical synthesis process.
3. the pharmaceutical composition containing the anticalcium small peptide described in claim 1.
4. pharmaceutical composition according to claim 3, it is characterised in that pharmaceutical dosage form in any form exist.
5. pharmaceutical composition according to claim 3, it is characterised in that the pharmaceutical composition is freeze-drying injection
Agent or other any type of oral formulations.
6. pharmaceutical composition according to claim 3, it is characterised in that further include other auxiliary materials pharmaceutically allowed.
7. application of the anticalcium small peptide in the medicine for preparing treatment Calcified disease described in claim 1.
8. application according to claim 7, it is characterised in that the Calcified disease is cardiac valves, annulus calcification,
Angiosteosis caused by ectopic soft tissue calcification caused by kidney trouble, atherosclerotic or calcium and phosphorus metabolism disorder.
9. application according to claim 8, it is characterised in that ectopic soft tissue calcification bag caused by the kidney trouble
Include but be not limited to cardiac valves, blood vessel, calcification of trachea.
10. application according to claim 7, it is characterised in that the anticalcium small peptide is when preparing medicine with polypeptide
Form be used alone, or with other albumen and peptide fusion, or carried out such as acetylation, methylate modification, or carry out individually
The replacement of amino acid or mutation, or the privileged site of insertion protein surface, or it is connected use with other materials.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711210235.3A CN108017711B (en) | 2017-11-28 | 2017-11-28 | Anti-calcification short peptide and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711210235.3A CN108017711B (en) | 2017-11-28 | 2017-11-28 | Anti-calcification short peptide and application thereof |
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| Publication Number | Publication Date |
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| CN108017711A true CN108017711A (en) | 2018-05-11 |
| CN108017711B CN108017711B (en) | 2020-05-26 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117247446A (en) * | 2023-09-08 | 2023-12-19 | 江苏省人民医院（南京医科大学第一附属医院） | Restructured short peptide and application thereof |
| CN117247446B (en) * | 2023-09-08 | 2024-05-14 | 江苏省人民医院（南京医科大学第一附属医院） | Restructured short peptide and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102119224A (en) * | 2008-05-30 | 2011-07-06 | 不列颠哥伦比亚大学 | Methods of diagnosing rejection of a kidney allograft using genomic or proteomic expression profiling |
| WO2011127543A1 (en) * | 2010-04-16 | 2011-10-20 | Transbio Ltd | Proteins that bind pi16 and uses thereof |
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2017
- 2017-11-28 CN CN201711210235.3A patent/CN108017711B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102119224A (en) * | 2008-05-30 | 2011-07-06 | 不列颠哥伦比亚大学 | Methods of diagnosing rejection of a kidney allograft using genomic or proteomic expression profiling |
| WO2011127543A1 (en) * | 2010-04-16 | 2011-10-20 | Transbio Ltd | Proteins that bind pi16 and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| ARUNESH MOHANDAS等: "Peptidase inhibitor 16 identifies a unique subset of memory T helper cells with hyperproliferative and proinflammatory properties (IRC8P.477)", 《J IMMUNOL》 * |
| NCBI DATABASE: "Accession number：EAX03923.1", 《GENBANK》 * |
| 李名鹏; 孙伟; 赵蓉; 杨杨; 王惠; 孔祥清: "PI16重组腺病毒表达载体的构建及其在人主动脉瓣间质细胞中的表达", 《江苏医药》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117247446A (en) * | 2023-09-08 | 2023-12-19 | 江苏省人民医院（南京医科大学第一附属医院） | Restructured short peptide and application thereof |
| CN117247446B (en) * | 2023-09-08 | 2024-05-14 | 江苏省人民医院（南京医科大学第一附属医院） | Restructured short peptide and application thereof |
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| CN108017711B (en) | 2020-05-26 |
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Inventor after: Sun Wei Inventor after: Kong Xiangqing Inventor after: Qiu Ming Inventor after: Lu Yan Inventor after: Ji Yue Inventor before: Sun Wei Inventor before: Kong Xiangqing Inventor before: Qiu Ming Inventor before: Ji Yue |
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