CN102558299B

CN102558299B - Synthetic polypeptide and application thereof

Info

Publication number: CN102558299B
Application number: CN201210087011.9A
Authority: CN
Inventors: 任娇艳; 顾龙建; 崔春; 赵谋明; 李文治
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2012-03-29
Filing date: 2012-03-29
Publication date: 2014-04-02
Anticipated expiration: 2032-03-29
Also published as: CN102558299A

Abstract

The invention discloses synthetic polypeptide and application thereof. The synthetic polypeptide has the following amino acid sequence: Tyr-Glu-Cys-Gly. The polypeptide is synthesized by using a polypeptide synthesizer by a solid-phase synthesis method. Through in-vitro antioxidant activity detectioon of the polypeptide, the polypeptide has high oxidation resistance; and when the synthetic polypeptide is applied to protection of nerve cells which are subjected to oxidative damage, cell survival rate is improved greatly, and an obvious effect is achieved.

Description

A kind of synthetic polypeptide and application thereof

Technical field

The present invention relates to field of biological pharmacy, be specifically related to a kind of synthetic polypeptide and application thereof.

Background technology

Ageing-related, and neurodegenerative disease mainly contains alzheimer's disease (AD), diffusivity LeWy body sick (DLBD), Parkinsons sick (PD), amyotrophic lateral sclerosis sick (ALS).The pathogenesis of these illnesss is still very not clear, and free-radical oxidn stress damage theory is the focus of biological study in recent years.

In aging neurodegenerative disease, there are 3 kinds of common pathological changes in cerebral tissue: oxidative damage, albumen are built up, neuronal degeneration is downright bad, about three's relation, not yet set forth clearly, and Free radicals injury theory may provide contact tie for three.In regular, the generation of oxyradical and removing are in running balance, mainly have benefited from the antioxidant system in health, comprise enzyme material: SOD, CAT, Selenoperoxidase (GSH-Px), glutathione reductase (GRH) and non-enzyme material: the hydrophobicity oxidizing substance on gsh (GSH), xitix, wetting ability antioxidant and film.But in aging neurodegenerative disease, because ROS in body gathers, running balance is broken, and brings out a series of free radical chain reactionses, causes cell injury.

Take Parkinson's disease as example, and Parkinson's disease are the 4th modal neurodegenerative diseases in the elderly, and in >=65 years old crowd, 1% people suffers from this disease.Oxidative stress is considered to the principal element of disturbances in patients with Parkinson disease substantia nigra neuron death, and Dopamine HCL, under the existence of oxygen and water, is subject to monoamine oxidase effect Hydrogen Peroxide, aldehyde and ammonia.Hydrogen peroxide can cause at black substance position Fe ²⁺under catalysis, further generate the larger hydroxy radical qiao of toxicity, and now the complex activity of black substance mitochondrial respiratory chain declines, polyphenoils (particularly gsh) disappears, cannot remove free radical, therefore, free radical is by oxidation neu lipoid, destruction DA neuron membrane function or directly destroy cell DNA, finally causes substantia nigra dopaminergic neuron sex change.Due to the regression of substantia nigra dopaminergic neuron, disturbances in patients with Parkinson disease shows serious dyskinesia, as tremble, bradykinesia, tetanic and attitudinal reflex obstacle.

Lipid on the senile plaque inducing cell film that the pathogenesis of Alzheimer's disease has comprised amyloid-beta (A β) formation of deposits and protein oxidation are modified, and cause R0S to generate and increase, and have changed the permeability of film, Ca ²⁺interior stream increases, and activates Ca ²⁺deopendent protein kinase, lipase, synthesize kytoplasm free radical and increase, damaging cells device.Amyotrophic lateral sclerosis (ALS) morbidity mainly causes because nmda receptor or ampa receptor activate, and plastosome overload Ca2+ and ROS generate has pushing effect to it.

Sum up the pathogenesis of above nerve degenerative diseases, antioxidant is for the generation that can reduce this type of illness with scavenging(action) of catching of free radical, for antioxidant provides theoretical basis in the application aspect treatment nerve degenerative diseases.The application of antioxidant to nerve degenerative diseases at present, major part still rests on the experimental phase, what through clinical verification, have a therapeutic activity only has a vitamin-E, but the immunotherapy targeted autoantibody effect of antioxidant and without the characteristic of bad toxic side effect has boundless application prospect.

Anti-oxidation peptide is as an important composition of antioxidant, and relevant research is more and more.The method cost that the means separation of separation and purification after proteolysis obtains high anti-oxidation bioactive peptide is high, and disengaging time is long, and quality is difficult to control, and yields poorly and has restricted scale operation and the application of anti-oxidation peptide.Expedite the emergence of thus chemosynthesis and prepared the method for anti-oxidation peptide.The chemically synthesized polypeptide present stage solid-phase synthesis that adopt, the technology of solid-phase synthesis is comparatively ripe at present, can be widely used in the synthetic of polypeptide more.The polypeptide with strong anti-oxidation character that chemical method synthesizes, as medicine, possesses following advantage: molecular weight is little, is easy to synthetic, and cost is low, high specificity, and side effect is low, and non-immunogenicity is more safe and reliable; And artificial synthetic polypeptide purity is high, there is not pyrogen problem, the treatment that is applied to nerve degenerative diseases has boundless application prospect.Gsh can be applicable to the early treatment of Parkinson disease, has comparatively significant curative effect.Gsh has been brought into play important effect and has been promoted infant curative ratio in the therapeutic process of hypoxic ischemic encephalopathy of newborn simultaneously, becomes the active drug for the treatment of hypoxic ischemic encephalopathy of newborn.

The cell strain of PC12 cell mouse adrenal medullary pheochromocytoma cloning, the PC12 cell of differentiation has typical neurocyte characteristic, extremely similar to neurone on cellular form, structure and function, has high consistency with the neurocyte of former culture.Therefore as external model, be widely used in studying multiple nervous system disorders, as Parkinson's disease, alzheimer's disease etc.The present invention uses H ₂o ₂the PC12 cell injury of induction is evaluated the neuroprotective effect of synthetic polypeptide as nerve injury external model.

Summary of the invention

The object of this invention is to provide a kind of synthetic polypeptide with protection PC12 cellular oxidation damage, the neurocyte that can be applicable to oxidative damage is protected, and cell survival rate is improved.

The sequence of synthetic polypeptide of the present invention is: Tyr-Glu-Cys-Gly,

Wherein, Tyr represents that English name is Tyrosine, the amino acid whose corresponding residue that Chinese is tyrosine;

Glu represents that English name is Glutamic acid, the amino acid whose corresponding residue that Chinese is L-glutamic acid;

Cys represents that English name is Cysteine, the amino acid whose corresponding residue that Chinese is halfcystine.

Gly represents that English name is Glycine, the amino acid whose corresponding residue that Chinese is glycine.

The existing mature technology that the described aminoacid sequence of invention adopts, by the screening of resin, obtains rational polypeptide synthesis method: adopt standard Fmoc scheme, select Wang resin to carry out solid phase synthesis.

The invention provides the application of described synthetic polypeptide in the protection of neurocyte Hydroperoxide injury; by concentration, be specifically that the described synthetic polypeptide of 0.001-0.1 mM adds in neuronal cell cultures liquid and hatches; after oxidative damage 2 h, make cell survival rate improve 20%-35%.

Compared with prior art; tool of the present invention has the following advantages and technique effect: the present invention has adopted external chemical anti-oxidation method to detect the anti-oxidant activity of synthetic polypeptide; the synthetic polypeptide hydrogen supply capacity providing is strong; there is higher ORAC activity and hydroxy radical qiao and remove ability; the neurocyte that is simultaneously applied to oxidative damage is protected; cell survival rate is improved, and effect is better.

Accompanying drawing explanation

Fig. 1 is the ESI collection of illustrative plates of synthetic peptide T yr-Glu-Cys-Gly.

Fig. 2 is for synthesizing the PC12 cell survival rate change curve that carries out oxidative damage after polypeptide adds.

Embodiment

Solid-phase Polypeptide is synthetic:

1, resin type selecting

(1) adopt standard Fomc scheme; select 0.0125 mmol; 2-chlorotrityl chloride resin resin (Tianjin Southern is opened Compositech Inc.); according to the sequence signature of aminoacid sequence Tyr-Glu-Cys-Gly; add first Fmoc protected amino acid of 0.3 mol; DCC and 5 % (massfraction) DMAP are joined to reactor oscillatory reaction, with NMP, rinse resin and remove redundant protection amino acid.The joint efficiency of measuring first amino acid and resin is coupling rate (as shown in table 1).

(2) adopt standard Fomc scheme; select 0.0125 mmol; Wang resin; according to the sequence signature of aminoacid sequence Tyr-Glu-Cys-Gly; add first Fmoc protected amino acid of 0.3 mol; DCC and 5 % (massfraction) DMAP are joined to reactor oscillatory reaction, with NMP, rinse resin and remove redundant protection amino acid.The joint efficiency of measuring first amino acid and resin is coupling rate (as shown in table 1).

Table 1

Resinous type	Coupling rate (%)
		2-chlorotrityl chloride resin resin	95.07±0.34
Wang resin	98.01±0.62

2, building-up process

Employing standard Fomc scheme; select the resin that coupling rate is higher; according to the sequence signature of aminoacid sequence Tyr-Glu-Cys-Gly; peptide chain is extended to N end one by one from C end, and each amino acid whose consumption is 0.1mol, adds 0.3 mol Fmoc protected amino acid; every step condensation all adds the amino acid whose carboxyl of HOBT activates relay; every step condensation adopts 20% piperidines DMF solution (15ml/g), processes 20min, removes Fmoc protecting group.After peptide side chain is synthetic, the peptide chain that contains resin is joined in following reaction solution: methylene dichloride (99%) and trifluoroacetic acid (1%) (volume fraction), cut down peptide chain from resin.Again polypeptide is joined to reaction solution: trifluoroacetic acid (94.5%), ethylenediamine tartrate (2.5%), distilled water (2%), TIS(1%) (volume fraction) middle reaction 2 h, slough Side chain protective group.Above process all completes in SYMPHONY type 12 passage Peptide synthesizers, and the polypeptide of synthesized is through SHIMADZU high performance liquid chromatograph purifying, and purity reaches more than 95%, and identifies structure (as shown in Figure 1) through ESI-MS.

The ORAC determination of activity of synthetic polypeptide:

ORAC reacts in the environment of 37 ℃ and carries out, and synthetic polypeptide, fluorescein, AAPH free radical are all dissolved in 75 Mm, and in the phosphate buffered saline buffer of pH 7.4, its final concentration is respectively 6 mM, 70 nM, 12 mM.Using Trolox as standard substance, and concentration gradient is 1-8 μ M.To synthesize polypeptide and fluorescein mixed solution and after 15 minutes, add azo-compound AAPH 37 ℃ of insulations, in excitation wavelength 485 nm, absorbing wavelength 520 nm place fluorescence intensity, detect duration 2 h, obtain fluorescence decline curve.The oxygen radical removing ability ORAC value (ORAC) of antioxidant is compared and is obtained with the protected area of standard antioxidant (Trolox) by the protected area of fluorescence decline curve.ORAC value is expressed (as shown in table 2) with Trolox equivalent.

The hydroxy radical qiao removing ability of synthetic peptide is measured:

Hydroxyl radical free radical (OH) is removed the mensuration of power and is selected o-phenanthroline, and reaction is with H ₂o ₂/ Fe ²⁺system is reacted and is produced hydroxy radical qiao by Fenton, and total reaction can be represented by the formula: Fe ²⁺+ H ₂o ₂→ Fe ³⁺+ OH ^-+ OH.Phenanthroline-Fe ²⁺the aqueous solution is oxidized to phenanthroline-Fe by hydroxy radical qiao ³⁺after, its 536nm maximum absorption band disappears, according to above principle, with A ₅₃₆change the oxygenizement of reflection hydroxy radical qiao.The solution that preparation protein concn is 7.5mM,

Concrete measuring method is as follows:

Sample hose: get 0.6 mL phenanthroline solution (5mmol/L) and add 0.4 mL phosphate buffered saline buffer (0.2M, pH 7.4) mix after, add the synthetic polypeptide solution of 0.6 mL and 0.6 mL EDTA (15mmol/L), again mix, after add 0.6 mL FeSO ₄solution (5mmol/L), mends to volume 2.8mL with deionized water, adds 0.8 mL H after fully mixing ₂o ₂(0.1%), after shaking up, in 37 ℃ of insulation 1 h, survey the 536 light absorption value A536 of nm place (sample);

Damage pipe: with deionized water, replace synthetic polypeptide, the same sample hose of all the other steps, the light absorption value of surveying is counted A _{536 (damages)}

Damage is not managed: with deionized water, replace H ₂o ₂, all the other steps are with damage pipe, and the light absorption value of surveying is counted A _{536 (not damages)};

Calculate OH clearance rate (as shown in table 2):

OH clearance rate (%)=(A _{536 samples}-A _{536 damages}) * 100%/(A _{536 not damages}-A _{536 damages})

Table 2

Sample	GSH	TQCG
			Scavenging action to hydroxyl free radical (%)	78.74±0.06	80.16±0.10
ORAC value (mM Trolox/ mM)	0.77	2.17

Synthetic polypeptide is to neurocyte Hydroperoxide injury protection application:

Application Example 1

PC12 cell cultures (containing 10% foetal calf serum, penicillin and Streptomycin sulphate 100U/ml) in DMEM substratum is placed in 37 ℃, CO ₂volume fraction is in 5% saturated humidity incubator.Every 3 d, change liquid 1 time.Get in the PC12 of logarithmic phase cell and be inoculated in 96 well culture plates, every hole 100 μ l, concentration is 5 * 10 ⁴/ ml, after Growth of Cells 24 h, adds PBS, 10 μ l, and after preincubate 12 h, adding concentration is 600 mM H ₂o ₂, zero-dose replaces (being normal control) with PBS, in 37 ℃ of incubators, acts on 2 h.Adopt mtt assay to detect cell survival rate (as shown in Figure 2).

Application Example 2

PC12 cell cultures (containing 10% foetal calf serum, penicillin and Streptomycin sulphate 100U/ml) in DMEM substratum is placed in 37 ℃, CO ₂volume fraction is in 5% saturated humidity incubator.Every 3 d, change liquid 1 time.Get in the PC12 of logarithmic phase cell and be inoculated in 96 well culture plates, every hole 100 μ l, concentration is 5 * 10 ⁴/ ml, after Growth of Cells 24 h, adding 10 μ l concentration is 0.01 mM synthetic peptide sample (gsh is in contrast), after preincubate 12 h, adding concentration is 600 mM H ₂o ₂, zero-dose replaces (being normal control) with PBS, in 37 ℃ of incubators, acts on 2 h.Adopt mtt assay to detect cell survival rate (as shown in Figure 2).

Application Example 3

PC12 cell cultures (containing 10% foetal calf serum, penicillin and Streptomycin sulphate 100U/ml) in DMEM substratum is placed in 37 ℃, CO ₂volume fraction is in 5% saturated humidity incubator.Every 3 d, change liquid 1 time.Get in the PC12 of logarithmic phase cell and be inoculated in 96 well culture plates, every hole 100 μ l, concentration is 5 * 10 ⁴/ ml, after Growth of Cells 24 h, adding 10 μ l concentration is 0.05 mM synthetic peptide sample (gsh is in contrast), after preincubate 12 h, adding concentration is 600 mM H ₂o ₂, zero-dose replaces (being normal control) with PBS, in 37 ℃ of incubators, acts on 2 h.Adopt mtt assay to detect cell survival rate (as shown in Figure 2).

Application Example 4

PC12 cell cultures (containing 10% foetal calf serum, penicillin and Streptomycin sulphate 100U/ml) in DMEM substratum is placed in 37 ℃, CO ₂volume fraction is in 5% saturated humidity incubator.Every 3 d, change liquid 1 time.Get in the PC12 of logarithmic phase cell and be inoculated in 96 well culture plates, every hole 100 μ l, concentration is 5 * 10 ⁴/ ml, after Growth of Cells 24 h, adding 10 μ l concentration is 0.1 mM synthetic peptide sample (gsh is in contrast), after preincubate 12 h, adding concentration is 600 mM H ₂o ₂, zero-dose replaces (being normal control) with PBS, in 37 ℃ of incubators, acts on 2 h.Adopt mtt assay to detect cell survival rate (as shown in Figure 2).

Synthetic polypeptide shows in the protection application result of neurocyte oxidative damage, and synthetic polypeptide neurocyte for oxidative damage within the scope of concentration range 0.001-0.01 mM has good protection effect, and along with the increase of concentration, protection effect strengthens gradually.Tyr-Glu-Cys-Gly protection successful with concentration is better than GSH, has more wide application prospect.

Claims

1. a synthetic polypeptide, its aminoacid sequence is Tyr-Glu-Cys-Gly, described synthetic polypeptide be take concentration and is hatched as 0.001-0.1 mM adds in neuronal cell cultures liquid, after oxidative damage 2 h, can make cell survival rate improve 20%-35%.