CN106397575A - Preparation method and application of polypeptide with ectoenzyme histone toxicity inhibition function - Google Patents
Preparation method and application of polypeptide with ectoenzyme histone toxicity inhibition function Download PDFInfo
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- CN106397575A CN106397575A CN201610912769.XA CN201610912769A CN106397575A CN 106397575 A CN106397575 A CN 106397575A CN 201610912769 A CN201610912769 A CN 201610912769A CN 106397575 A CN106397575 A CN 106397575A
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- polypeptide
- histone
- ectoenzyme
- extracellular
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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
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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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 invention discloses a preparation method and application of polypeptide with an ectoenzyme histone toxicity inhibition function, and belongs to the technical field of biology. The amino acid sequence of polypeptide A is Lys-Phe-Gln-Asn-Ala-Leu-Leu-Val-Arg-Tyr. The invention relates to a solid-phase synthesis method of the polypeptide A. In addition, the HUVECs cell experiments are utilized for proving the cytotoxic effect of exogenously given polypeptide A restrainable ectoenzyme histone on HUVECs cells. Therefore, the synthesized human-derived polypeptide A can be used for preparing medicine for treating ectoenzyme histone related diseases such as septicemia.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention describes there is extracellular histone toxicity inhibition work
Polypeptide A -- humanized's albumin polypeptide, and the aminoacid sequence of this polypeptide.The invention still further relates to the preparation side of this polypeptide
Method and application.
Background technology
Histone is chromatinic structural protein in eukaryotic cells, including H1, H2A, H2B, H3 and H4.Wherein H2A,
H2B, H3, H4 combine to form nucleosome as core histones and DNA, and H1 then combines on the DNA between nucleosome.Histone
Once by intracellular dystopy to extracellular, becoming extracellular histone.Histone can be multiple as a kind of nucleoprotein of generally existing
It is released to extracellular under pathologic condition by actively and passively means of transportation.Extracellular histone is essentially from following two aspects：One
It is by dead parenchymal tissue's cell release.Now, chromatin degradation, membranolysises, cell dissolving, histone in a large amount of cores
Release to extracellular；Two is by leukocyte, particularly neutrophilic granulocyte release.Sharply increase in a large number in acute phase response neutrophilic granulocyte
And form the extracellular trap baiting net of neutrophilic granulocyte（NETs）When, histone is with DNA and high mobility group protein B（HMGB1）Deng together
Discharge to extracellular.Research to histone is related to various clinical disease, including cancer, diseases associated with inflammation, ischemic stroke, from
Body immune disease etc., the staging diagnosis, prognosis and the monitoring that are mainly used in disease judge the aspects such as curative effect.Early in 1958
The toxic action of histone is just pointed out in the research of James.Increasing research shows that extracellular histone is unfavorable to body
Essentially consist in induced tissue cell especially vascular endothelial cell damage, activated leukocyte and platelet, the abnormal blood coagulation of promotion
Journey and thrombosiss, it is caused directly or indirectly inflammatory reaction and immunoreation, lead to disseminated inravascular coagulation, acute respiration embarrassed
Urgent syndrome or even multiple organ dysfunction syndrome.Therefore, intervened for extracellular histone phase with histone for target spot
The prevention of related disorders has important clinical value with treatment.
Albumin is the most protein of content in the blood plasma that hepatic parenchymal cellses synthesize, and accounts for the 40 ~ 60% of total protein.This
Bright foundation can be suppressed on the dead discovery of the vascular endothelial cell of extracellular histone induction in albumin.Additionally, root of the present invention
According to this albumin sequence information, identification one polypeptide of chemosynthesis, i.e. polypeptide A, have studied it to extracellular histone toxicity
Inhibitory action is it is proposed that carry out mankind's histone relevant disease such as medical value of septicemia diagnosis and treatment and clinic using polypeptide A
Using value.
Content of the invention
First purpose of the present invention is to disclose a kind of white egg of serum human suppressing extracellular histone cytotoxicity
The polypeptide A in white source.
Described polypeptide A, its aminoacid sequence as shown in SEQ ID NO.1, specially：Lys-Phe-Gln-Asn-Ala-
Leu-Leu-Val-Arg-Tyr.
Second object of the present invention is to provide a kind of preparation method of described humanized's polypeptide A.
It comprises the steps：（1）Peptide systhesis order is from C-terminal to N-terminal.By 2-Chlorotrityl Chloride
Resin puts in reaction tube, plus 15 ml/g（Ml/g）DCM（Dichloromethane）, shake 30 minutes.
（2）Connect first aminoacid.Solvent is leached out by husky core, add the Fmoc-Thr (tBu) of 3 times of molar excess-
OH aminoacid, adds DMF（Dimethylformamide）Dissolving, adds the DIEA of 10 times of molar excess（N, N- diisopropyl second
Amine）, shake 60 minutes.Closed with methanol.
（3）Deprotection.Remove DMF, plus 15 ml/g 20% Piperidine/DMF solution, after washing 5 minutes, remove, then 15
Ml/g 20% Piperidine/DMF solution, washs 15 minutes.
（4）Detection.Transfer piperidine solution, take more than ten grainy resins, with washing with alcohol three times, add detectable detection.105
C-110 C heats 5 minutes, and color is changed into navy blue and is positive reaction.
（5）Rinse resin.Use DMF respectively successively（10 ml/g）, DCM（10 ml/g）, DMF（10 ml/g）Rinse resin
Twice.
（6）Condensation.Protected amino acid three times are excessive, HBTU（BTA-N, N, N', N'- tetramethylurea hexafluorophosphoric acid
Ester）Three times are excessive, all dissolved with a small amount of DMF, add reaction tube, be added immediately ten times of excess of DIEA, react 30 minutes.
（7）Detection.Take more than ten grainy resins, with washing with alcohol three times, add detectable detection, 105 C-110 C heating 5
Minute, colourless as negative reaction.
（8）Rinse resin.Use DMF respectively successively（10 ml/g）, DCM（10 ml/g）, DMF（10 ml/g）Rinse resin
Twice.
（9）Repeat（3）Extremely（6）Operation, is sequentially connected the aminoacid in sequence from right to left.
（10）Drain, and wash resin in following manner：DMF（10 ml/g）Twice, methanol（10 ml/g）Twice, DMF
（10 ml/g）Twice, DCM（10 ml/g）Twice, 10 minutes are drained.
（11）Polypeptide is cut from resin.Cutting liquid composition used is：95% TFA（Trifluoroacetic acid）, 1% water, 2% EDT
（Mercaptoethanol）, 2%TIS（Tri isopropyl silane）；Clipping time is：120 minutes.
（12）Dry up washing.Lysate nitrogen is dried up, ether washs six times, then places room temperature volatilization.
（13）Analysis purification lyophilizing.Purify polypeptide crude product with high performance liquid chromatography；Collect polypeptide solution to put in freeze dryer
Concentrated, and white powder is made in lyophilizing.
Third object of the present invention is to provide described humanized's polypeptide A answering in extracellular histone toxicity inhibition
With.Polypeptide A can directly suppress HUVEC cell death and the HUVEC cell membrane damage of extracellular histone induction, thus can be used for
Develop polypeptide related drugs, thus preventing and treating the diseases such as the related inflammation of extracellular histone, ischemia, immunity, tumor（As sepsis,
Septicemia etc.）.Further, since polypeptide A is the small molecule segment of human serum albumin, there is no the secondary work of poison of general chemicalses
With but having biocompatibility and the blood compatibility that chemicalses are not replaced.Polypeptide A can effectively suppress HUVEC cell
The activation of middle Inflammatory Pathway, thus playing its inhibitory action to extracellular histone toxicity, improves extracellular histone relevant disease
Long-term efficacy and safety.Therefore, polypeptide A has larger medical value and is widely applied prospect.
Brief description
Fig. 1 is the result figure that the present invention detects cell survival rate.### represents compared with blank group, p<0.001, have notable
Sex differernce；* represent compared with 200 μ g/ml histone treatment groups, p<0.05, there is significant difference；* represents and 200 μ g/ml
Histone treatment group is compared, p<0.01, there is significant difference.
Fig. 2 is the result figure of observation of cell membrane damage under scanning electron microscope of the present invention.
Fig. 3 is Western Blot method detection I κ B, p38 and the phosphorylated p38 of the present invention（p-p38）Protein expression
Level.
Fig. 4 is the ELISA method detection inflammatory factor TNFalpha of the present invention, the expression of IL-6.## represents and blank group
Compare, p<0.01, there is significant difference；### represents compared with blank group, p<0.001, there is significant difference；* represent and 200
μ g/ml histone treatment group is compared, p<0.05, there is significant difference；* represents compared with 200 μ g/ml histone treatment groups, p
<0.01, there is significant difference.
Sequence table is polypeptide A sequence.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Embodiment 1：The solid phase synthesis of polypeptide A.
1. Peptide systhesis order is from C-terminal to N-terminal.2-Chlorotrityl Chloride Resin is put into reaction tube
In, plus 15 ml/g（Ml/g）DCM（Dichloromethane）, shake 30 minutes.
2. connect first aminoacid.Solvent is leached out by husky core, adds Fmoc-Thr (the tBu)-OH of 3 times of molar excess
Aminoacid, adds DMF（Dimethylformamide）Dissolving, adds the DIEA of 10 times of molar excess（N, N- diisopropylethylamine）,
Concussion 60 minutes.Closed with methanol.
3. deprotection.Remove DMF, plus 15 ml/g 20% Piperidine/DMF solution, after washing 5 minutes, remove, then in 15 ml/
G 20% Piperidine/DMF solution, washs 15 minutes.
4. detect.Transfer piperidine solution, take more than ten grainy resins, with washing with alcohol three times, add detectable detection.105º
C-110 C heats 5 minutes, and color is changed into navy blue and is positive reaction.
5. rinse resin.Respectively successively using DMF（10 ml/g）Twice, DCM（10 ml/g）Twice, DMF（10 ml/g）
Rinse resin twice.
6. it is condensed.Protected amino acid three times are excessive, HBTU（BTA-N, N, N', N'- tetramethylurea hexafluorophosphoric acid
Ester）Three times are excessive, all dissolved with a small amount of DMF, add reaction tube, be added immediately ten times of excess of DIEA, react 30 minutes.
7. detect.Take more than ten grainy resins, with washing with alcohol three times, add detectable detection, 105 C-110 C heating 5
Minute, colourless as negative reaction.
8. rinse resin.Respectively successively using DMF（10 ml/g）, DCM（10 ml/g）, DMF（10 ml/g）Rinse resin
Twice.
9. repeat（3）Extremely（6）Operation, is sequentially connected the aminoacid in sequence from right to left.
10. drain, and wash resin in following manner：DMF（10 ml/g）Twice, methanol（10 ml/g）Twice, DMF
（10 ml/g）Twice, DCM（10 ml/g）.Twice, 10 minutes are drained.
11. cut polypeptide from resin.Cutting liquid composition used is：95% TFA（Trifluoroacetic acid）, 1% water, 2% EDT（Mercapto
Base ethanol）, 2%TIS（Tri isopropyl silane）；Clipping time is：120 minutes.
12. dry up washing.Lysate nitrogen is dried up, ether washs six times, then places room temperature volatilization.
13. analysis purification lyophilizing.Purify polypeptide crude product with high performance liquid chromatography；Collect polypeptide solution to put in freeze dryer
Row concentrates, and white powder is made in lyophilizing.
Embodiment 2：Polypeptide A suppresses the cytotoxic effect to HUVEC cell for the extracellular histone.
1. primary human endothelial cell HUVECs is purchased from ATCC, is incubated at LSGS-M200 culture medium.Experimental group is acceptance 5
The HUVECs cell that mg/ml or 10 mg/ml polypeptide As and 200 μ g/ml histones from calf thymus are processed；Matched group is to connect respectively
The HUVECs cell being processed by 5 mg/ml or 10 mg/ml polypeptide As, 100 μ g/ml or 200 μ g/ml histones from calf thymus,
After accepting 1 hour to process, by counting cell survival rate, under finding that histone process leads to HUVECs cell survival rate notable
Fall, and assume concentration dependent effect, and accept after polypeptide A is processed, to suppress the cell survival rate that extracellular histone leads to decline
Phenomenon.
2. primary human endothelial cell HUVECs is purchased from ATCC, is incubated at LSGS-M200 culture medium.Experimental group is acceptance 10
Mg/ml polypeptide A and the HUVECs cell of 200 μ g/ml histones from calf thymus process；Matched group is to accept 200 μ g/ml respectively
Histones from calf thymus or the HUVECs cell of 200 μ g/ml histones from calf thymus joint 20 mg/ml albumin process, connect
After being processed by 1 hour, scanned Electronic Speculum it has been observed that histone is processed leads to the major injury of HUVECs cell membrane, and connect
The cell membrane damage phenomenon that after being processed by the polypeptide A of albumin or albumin sources, all energy inhibition of histone leads to.
Data above（See Fig. 1, Fig. 2）Show polypeptide A as the potentiality of histone toxic inhibitor.
3. it is directed to each group HUVECs cell in step 1, extracts total protein further, and it is outer to carry out Western Blot checking
Source gives the impact of the inflammatory reaction to extracellular histone induces for the polypeptide A.Test result indicate that, extracellular histone may result in I κ B
Expression decline, p38 phosphorylation level raises, and external source gives the NF- κ that polypeptide A can significantly reduce the induction of extracellular histone
The activation of B/p38 inflammatory signals path（Fig. 3）；Extracellular histone can induce the expression of downstream inflammatory factor TNF-α and IL-6, and
External source gives this effect that polypeptide A can significantly inhibit extracellular histone（Fig. 4）.
4. it is directed to each group HUVECs cell serum culture medium in step 1, carry out ELISA and detect in further verification step 3
External source give the impact of inflammatory factor TNF-α that polypeptide A induces and IL-6 release to histone.Test result indicate that, extracellular
Histone can induce the release of inflammatory factor TNF-α and IL-6, and external source gives this that polypeptide A can significantly inhibit extracellular histone
Plant effect.
Illustrate based on the above results, the exogenous polypeptide A giving to synthesize can substantially suppress extracellular protein to HUVEC cell
Cytotoxic effect, damages and inflammatory reaction including histone inducing cell, for clinical treatment extracellular histone relevant disease
There is good potential applicability in clinical practice.
[0001]
<110>Shanghai Yi Beirui biological medicine Science and Technology Ltd.
<120>A kind of polypeptide production methods with extracellular histone toxicity inhibition effect and purposes
<130> 2016
<160> 1
<210> 1
<211> 10
<212> PRT
<213>Artificial sequence
<400> 1
Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr
1 5 10
Claims (3)
1. a kind of polypeptide A suppressing extracellular histone toxicity is it is characterised in that this aminoacid sequence is：Lys-Phe-Gln-
Asn-Ala-Leu-Leu-Val-Arg-Tyr.
2. the preparation method of the polypeptide A of the extracellular histone toxicity of suppression described in claim 1.
3. the purposes of the polypeptide A of the extracellular histone toxicity of suppression described in claim 1 is it is characterised in that be used for suppressing extracellular
The inflammatory reaction of histone induction and cytotoxicity, and treat extracellular histone relevant disease such as septicemia for producing
Medicine.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610912769.XA CN106397575A (en) | 2016-10-20 | 2016-10-20 | Preparation method and application of polypeptide with ectoenzyme histone toxicity inhibition function |
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| CN201610912769.XA CN106397575A (en) | 2016-10-20 | 2016-10-20 | Preparation method and application of polypeptide with ectoenzyme histone toxicity inhibition function |
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| CN106397575A true CN106397575A (en) | 2017-02-15 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003017920A2 (en) * | 2001-08-29 | 2003-03-06 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Protective factors against inflammation, burns and noxious stimuli |
| WO2005090387A2 (en) * | 2004-03-23 | 2005-09-29 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Histone h2a peptide derivatives and analogs and methods of use thereof |
| US20070117824A1 (en) * | 2005-11-23 | 2007-05-24 | Berk Scott C | Spirocyclic compounds |
| EP2209485B1 (en) * | 2007-11-06 | 2016-03-30 | Oklahoma Medical Research Foundation | Extracellular histones as biomarkers for prognosis and molecular targets for therapy |
| CN104388538B (en) * | 2014-11-18 | 2017-01-18 | 中国人民解放军第三军医大学 | Method for screening histone deacetylase inhibitor |
-
2016
- 2016-10-20 CN CN201610912769.XA patent/CN106397575A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003017920A2 (en) * | 2001-08-29 | 2003-03-06 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Protective factors against inflammation, burns and noxious stimuli |
| WO2005090387A2 (en) * | 2004-03-23 | 2005-09-29 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Histone h2a peptide derivatives and analogs and methods of use thereof |
| US20070117824A1 (en) * | 2005-11-23 | 2007-05-24 | Berk Scott C | Spirocyclic compounds |
| EP2209485B1 (en) * | 2007-11-06 | 2016-03-30 | Oklahoma Medical Research Foundation | Extracellular histones as biomarkers for prognosis and molecular targets for therapy |
| CN104388538B (en) * | 2014-11-18 | 2017-01-18 | 中国人民解放军第三军医大学 | Method for screening histone deacetylase inhibitor |
Non-Patent Citations (2)
| Title |
|---|
| FAN,J等: "primase/helicase [Enterobacteria phage ECGD1]GenBank: AMM43496.1", 《NCBI GENBANK DATABASE》 * |
| 李铭新: "血浆透析滤过在脓毒症中的应用", 《万方学位论文数据库》 * |
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Application publication date: 20170215 |