CN101054418B - Fusion polypeptide and application thereof in preparing scald medicament - Google Patents
Fusion polypeptide and application thereof in preparing scald medicament Download PDFInfo
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- CN101054418B CN101054418B CN2007100408580A CN200710040858A CN101054418B CN 101054418 B CN101054418 B CN 101054418B CN 2007100408580 A CN2007100408580 A CN 2007100408580A CN 200710040858 A CN200710040858 A CN 200710040858A CN 101054418 B CN101054418 B CN 101054418B
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Abstract
The invention relates to biomedicine technology field, Thymic peptide alpha1 is polypeptide with immune function with amino acid sequence indicated by SEQ ID NO:1. MCD peptide is a polypeptide with strong anti-inflammatory function and has amino acid sequence indicated by SEQ ID NO:2. The present invention provides a polypeptide, fused by thymic peptide alpha1 and MCD peptide which has the function of facilitating tissue rehabilitation of scald wound surface, anti-inflammatory, anti-extravasate and anti-shock. The inventive fusion polypeptide(M-Talpha1) possesses amino acid sequence indicated by SEQ ID NO:3 has the function of facilitating scald wound surface healing testified by pharmacodynamic test.
Description
Technical field
The present invention relates to the biological medicine technology field, is a kind of fusion polypeptide and application in the preparation scald medicament thereof that promotes scald wound tissue repair, anti-inflammatory, impervious and Antishock function that have.
Background technology
Thymosin alpha 1 (thymosin α 1, be called for short T α 1) be a kind of polypeptide with immunologic function, its relative molecular weight is 3108, form by 28 amino acid, has the aminoacid sequence shown in the SEQ ID NO:1, clinically as a kind of immunostimulant, be used for the assisting therapy of viral hepatitis B, malignant tumour, immune deficiency disorder at present.In addition, T α 1 also has the stimulation migration of vascular endothelial cells, promotes effects such as vasculogenesis and wound healing.(referring to document GoldsteinA L, Low TL, McAdoo M, et al.Thymosin alpha 1:isolation and sequenceanalysis of an immunological active thymic peptide.Proc Natl Acad SciUSA, 1977,74 (2): 725-729.).
Mastoparan (Mast Cell Degranulating Peptide, be called for short the MCD-peptide), it is the stronger polypeptide of a kind of anti-inflammatory, molecular weight 2593, form by 22 amino-acid residues, have the aminoacid sequence shown in the SEQ ID NO:2, the MCD-Toplink reduces capillary permeability, stops leukoplania and suppresses the synthetic of prostaglandin E2.Be used for the treatment of rheumatic arthritis as a kind of effective constituent in the bee venom clinically, rheumatoid arthritis, peripheral neuropathy and neurodynia etc.(referring to document Buku A, Reibman J, Pistelli A, et al.Mast celldegranulating (MCD) peptide analogs with reduced ring structure.J ProtChem 1992,11:275-280.).
Summary of the invention
The object of the present invention is to provide a kind of fusion polypeptide, it is merged by Thymosin alpha 1 and mastoparan and forms.This fusion polypeptide has the scald wound of promotion tissue repair, anti-inflammatory, impervious and Antishock function.
The invention provides a kind of fusion polypeptide (M-T α 1), have the aminoacid sequence of Thymosin alpha 1 (SEQ ID NO:1) and the aminoacid sequence of mastoparan (SEQ ID NO:2), and the connection peptides sequence between SEQ ID NO:1 and SEQ ID NO:2.
Above-mentioned connection peptides sequence is made up of 4 Gly or Ser.
Fusion polypeptide of the present invention (M-T α 1) has the aminoacid sequence shown in the SEQ ID NO:3.
The preparation method of fusion polypeptide of the present invention (M-T α 1): utilize the artificial solid phase synthesis fusogenic peptide of Fmoc protection amino acid M-T α 1; synthetic fusogenic peptide M-T α 1 crude product is carried out the HPLC purifying; HPLC adopts the Waters600 pump; Waters2487 detector and HS (V4.0) chromatographic data workstation processing data; collect active peak composition, the packing postlyophilization promptly gets the fusogenic peptide sample.
Fusion polypeptide of the present invention (M-T α 1) confirms effectively to promote really the effect of wound tissue healing through the pharmacodynamics experimentation on animals.
Embodiment
Below in conjunction with embodiment the present invention is further described:
Reagent and material:
The product analysis of Piperidine Shanghai preparation company of pharmaceuticals is pure
The product analysis of DMF Shanghai preparation company of pharmaceuticals is pure
The product analysis of MeOH Shanghai preparation company of pharmaceuticals is pure
The product analysis of CH2Cl2 Shanghai preparation company of pharmaceuticals is pure
The biochemical 100-200mesh 1%DVB of company limited of Fmoc-Ile-Wang Resin Shanghai gill
The biochemical company limited 98% of DIC Shanghai gill
The biochemical company limited 98% of HOBt (anhydrous) Shanghai gill
The biochemical company limited 95% of trifluoroacetic acid (TFA) Shanghai gill
Embodiment 1: preparation and the separation and purification of fusogenic peptide M-T α 1
1. preparation fusogenic peptide: synthetic fusogenic peptide M-T α 1, synthetic method is that Fmoc protection amino acid carries out solid phase synthesis, contracts and reagent is DIC, HOBt (anhydrous).Reaction solvent is DMF, and reacted washing reagent is DMF, MeOH, CH2Cl2.Building-up process: the Fmoc-Ile-Wang Resin that drops into 0.6g drains then with the CH2Cl2 swollen resin in connecing the peptide bottle, again with Fmoc-Pro with contract and reagent D IC, HOBt (anhydrous), and add DMF reaction 40min, drain, wash 3 times, do not develop the color up to Kaiser test.Take off Fmoc (20%piperidine), connect next amino acid after the washing, by that analogy.After connecting last amino acid, take off Fmoc, drain, take out resin.Pour cutting liquid (TFA 95%) cutting 2-3 hour into, washing is also used nitrogen protection, drains at last.Liquid chromatography mass combined instrument (LCMS-2010A type) detects MS.
2. purifying fusogenic peptide: synthetic fusogenic peptide M-T α 1 crude product is dissolved in respectively in the aseptic Milli-Q water of 10ml, divide 2ml successively sample introduction carry out the HPLC purifying.HPLC adopts Waters600 pump, Waters2487 detector and HS (V4.0) chromatographic data workstation processing data.Adsorb and adopt Symmetry
TM3.9 the C18 post of * 150mm.Mobile phase A is the Milli-Q water that contains 0.1% trifluoroacetic acid (TFA), and Mobile phase B is the acetonitrile that contains 0.1% trifluoroacetic acid (TFA).
Elution requirement: time mobile phase A Mobile phase B
0.01min 100％ 0％
35.00min 0％ 100％
With mobile phase A with the system balance after, in 35 minutes by above-mentioned condition with the elution flow rate linear gradient elution of 1ml/min, collect active peak composition, the liquid mixing is collected at each active peak, every pipe 2ml packing promptly gets the fusogenic peptide sample after the lyophilize.
Embodiment 2: the pharmacodynamics experimentation on animals of fusogenic peptide M-T α 1
Select 60 of kunming mices about the about 20g of body weight for use, male and female half and half are divided into two groups at random.After the slight anesthesia of ether, 80 ± 2 ℃ in constant temperature and pressure permanent hair styling instrument 10 seconds, is set up the dark II ° of scalding model that diameter is 2.5cm at mouse skin.Be subjected to the reagent thing respectively at each group mouse wound surface smearing the same day from scalding after the debridement, the 1st group of negative control group only smeared physiological saline 100 μ l/; Smear 500g/ M-T α 1 for the 2nd group; Every day 1 time, successive administration 21 days, the response situation of record wound.Respectively at after the medication the 10th day and the 14th day, cut the healing wound tissue, routinely fixation of tissue is drawn materials, embedding, film-making, HE dyeing and immunohistochemical staining observe, and simultaneously Evans Blue detected the content that scalded skin tissue sample vascular permeability and enzyme linked immunological absorption test (ELISA) detect blood preparation TNF-α.
Wound tissue pathological observation result
1. dermal pathology HE dyeing result of variations
After the medication 10 days, the skin shallow-layer reticular tissue of the visible medication group treatment of HE stained only has a small amount of granulation hyperplasia to change, and in the deep skin reticular tissue blood vessel hyperplasia is arranged, and beginning changes to normal weave construction, deep layer necrocytosis is also less relatively, and inflammatory reaction is also light than control group; Control group surface of a wound high dermis has obvious granulation tissue hyperplasia, and deep layer still has large stretch of non-viable non-apoptotic cell, and inflammatory reaction is violent.
After the medication 14 days, medication was formed face and is healed, and main tissue morphology recovers near normal, and the deep layer cell boundaries is clear, near the normal skin form; And control group corium deep layer cellularstructure is unclear, still has the partially denaturing necrosis.
Illustrate that 1 pair of wound tissue healing of synthetic fusogenic peptide M-T α has promoter action.
2. immunohistochemical analysis result
Medication 10 days, immunohistochemical analysis result: the surface of a wound inoblast and the cell Zhou Jizhi of (1) M-T α 1 treatment show type i collagen strong positive result, and control group is negative; (2) surface of a wound basal layer of epidermis epithelial cell proliferation cell nuclear antigen (PCNA) of M-T α 1 treatment is positive, and inoblast and cells of vascular wall are also seen positive reaction, and the basal layer of epidermis cell expressing of control group is negative; (3) surface of a wound epidermic cell Keratin sulfate (CK) of M-T α 1 treatment is positive, and the rarely seen minority basal layer cell of control group surface of a wound epidermis is the weak positive; (4) surface of a wound vascular endothelial growth factor (VEGF) of M-T α 1 treatment is expressed and is positive, and control group does not see that obvious VEGF positive expression is arranged.Illustrate that synthetic fusogenic peptide M-T α 1 can promote the proteic expression relevant with the scald wound tissue repair, thereby promote the reparation of the surface of a wound.
3. Evans Blue detects scalded skin tissue sample vascular permeability analytical results
After the medication 1,3,5,7,10,14,21 day, 1 group of institute of collection of illustrative plates explanation M-T α OD value of surveying that measured result is drawn significantly descends in early days significantly at organized renewing, showing has than the obvious suppression effect scalding the tissue blood vessel permeability, and not medication group is surveyed the then relatively slowly level and smooth change of OD value.The scald mouse surface of a wound 1 group of the 14th day M-T α heals, and its OD value continues up to the 21st day, and all floating ground is very little; The OD value of medication control group did not still continue to descend between the 14th day to the 21st day, was tending towards approaching up to the 21st talent with 1 group of M-T α and M-T508 group OD value.
Illustrate that fusogenic peptide M-T α 1 possesses the effect that reduces capillary permeability, can play impervious, antishock effect.
4. enzyme linked immunological absorption test (ELISA) detects the content of blood preparation TNF-α.
After the medication 1,3,5,7,10,14,21 day, in the chart that the TNF-α concentration of being surveyed and minute plot, can see that 1 group of fusogenic peptide M-T α rises to some extent in early stage the 1st day to the 3rd day TNF-α concentration of organized renewing, TNF-α concentration significantly decreased in the 5th day, illustrate that scald phase beginning inflammatory reaction is strong, but under M-T α 1 effect, begin gradually to descend, show that 1 couple of M-T α scalds tissue inflammation reaction restraining effect is arranged.And not medication group is surveyed TNF-α concentration early stage lasting rising of scald, just begins to descend slowly after a week.This lags behind the medication group in when healing on mutually, compares with the control group normal healing, and M-T α 1 plays antiphlogistic effect in scalding tissue.This is from the 1 group of healing that can accelerate scald wound really of a side illustration M-T α.
Therefore, under the effect of fusogenic peptide M-T α 1, the various factors of tissue repair such as epidermis angiogenic growth strengthen, collagen expression strengthens cell nuclear antigen and expresses raising, surface of a wound vascular permeability reduces, inflammatory reaction reduces, this fusogenic peptide illustrates that M-T α 1 has the effect that promotes the scald wound tissue repair, so can be used to prepare the medicine that treatment is scalded according to the ordinary method of pharmaceutical field.
SEQUENCE?LISTING
＜110〉Second Military Medical University, PLA
＜120〉a kind of fusion polypeptide and the application in the preparation scald medicament thereof
＜130〉specification sheets, claims
<160>3
<170>PatentIn?version?3.1
<210>1
<211>28
<212>PRT
＜213〉artificial sequence
<400>1
Ser?Asp?Ala?Ala?Val?Asp?Thr?Ser?Ser?Glu?Ile?Thr?Thr?Lys?Asp?Leu
1 5 10 15
Lys?Glu?Lys?Lys?Glu?Val?Val?Glu?Glu?Ala?Glu?Asn
20 25
<210>2
<211>22
<212>PRT
＜213〉artificial sequence
<400>2
Ile?Lys?Cys?Asn?Cys?Lys?Arg?His?Val?Ile?Lys?Pro?His?Ile?Cys?Arg
1 5 10 15
Lys?Ile?Cys?Gly?Lys?Asn
20
<210>3
<211>54
<212>PRT
＜213〉artificial sequence
<400>3
Ile?Lys?Cys?Asn?Cys?Lys?Arg?His?Val?Ile?Lys?Pro?His?Ile?Cys?Arg
1 5 10 15
Lys?Ile?Cys?Gly?Lys?Asn?Gly?Gly?Gly?Gly?Ser?Asp?Ala?Ala?Val?Asp
20 25 30
Thr?Ser?Ser?Glu?Ile?Thr?Thr?Lys?Asp?Leu?Lys?Glu?Lys?Lys?Glu?Val
35 40 45
Val?Glu?Glu?Ala?Glu?Asn
50
Claims (2)
1. fusion polypeptide, the aminoacid sequence that it is characterized in that it is shown in SEQ ID NO:3.
2. the application of the described fusion polypeptide of claim 1 in the preparation scald medicament.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100408580A CN101054418B (en) | 2007-05-18 | 2007-05-18 | Fusion polypeptide and application thereof in preparing scald medicament |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100408580A CN101054418B (en) | 2007-05-18 | 2007-05-18 | Fusion polypeptide and application thereof in preparing scald medicament |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101054418A CN101054418A (en) | 2007-10-17 |
| CN101054418B true CN101054418B (en) | 2011-08-03 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2007100408580A Expired - Fee Related CN101054418B (en) | 2007-05-18 | 2007-05-18 | Fusion polypeptide and application thereof in preparing scald medicament |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112316110B (en) * | 2020-11-12 | 2023-06-23 | 温州大学 | Pharmaceutical preparation for promoting skin wound repair and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1375500A (en) * | 2001-03-21 | 2002-10-23 | 中国科学院上海生物化学研究所 | Thymic peptide fusion protein as one new interferon and its prepn. and use |
-
2007
- 2007-05-18 CN CN2007100408580A patent/CN101054418B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1375500A (en) * | 2001-03-21 | 2002-10-23 | 中国科学院上海生物化学研究所 | Thymic peptide fusion protein as one new interferon and its prepn. and use |
Non-Patent Citations (4)
| Title |
|---|
| 王先远等.胸腺肽 原融合蛋白基因表达、纯化与生物学活性.中国生物化学和分子生物学报18 4.2002,18(4),495-498. |
| 王先远等.胸腺肽 原融合蛋白基因表达、纯化与生物学活性.中国生物化学和分子生物学报18 4.2002,18(4),495-498. * |
| 陈亮等.神经肽P物质促进增生性瘢痕中肥大细胞组胺释放的定量检测.中国临床康复10 4.2006,10(4),113-115. |
| 陈亮等.神经肽P物质促进增生性瘢痕中肥大细胞组胺释放的定量检测.中国临床康复10 4.2006,10(4),113-115. * |
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| CN101054418A (en) | 2007-10-17 |
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Granted publication date: 20110803 Termination date: 20130518 |