CN108484734A - A kind of polypeptide with anti-tumor activity and its application - Google Patents
A kind of polypeptide with anti-tumor activity and its application Download PDFInfo
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- CN108484734A CN108484734A CN201810242993.1A CN201810242993A CN108484734A CN 108484734 A CN108484734 A CN 108484734A CN 201810242993 A CN201810242993 A CN 201810242993A CN 108484734 A CN108484734 A CN 108484734A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
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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
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Abstract
The present invention relates to a kind of polypeptide, preparation method and their applications with anti-tumor activity.
Description
It is related to sequence table
The application contains the sequence table of computer-reader form, is incorporated herein by carrying stating.
Technical field
The present invention relates to a kind of polypeptide with anti-tumor activity, the polynucleotides for encoding it and their applications.
Technical background
Cervical carcinoma (Cervical cancer) is common one of gynecological tumor, and the incidence as gynecological disease exists
Developed country is only second to breast cancer, carcinoma of endometrium, then ranks first in developing country.About the treatment of cervical carcinoma, currently, can
Select the distinct methods such as operation, radiotherapy and chemotherapy.Above-mentioned therapy is for improving patient's long term prognosis, improving survival rate and paying no attention to
Think.Many researchers by attention be transferred to it is active it is high, adverse reaction is small, the antineoplastic polypeptide that is not likely to produce drug resistance is ground
Study carefully.But the stability of antineoplastic polypeptide, selectivity are to limit two principal elements of its application.Due to the filtration of kidney,
The effects that degradation of protease, lead to that the poor stability of antineoplastic polypeptide, half-life short, blood concentration be low, biological utilisation
It spends low.Simultaneously because the poor selectivity of antineoplastic polypeptide, can lead to cell and the tissue damage of human normal, have centainly to human body
Toxic side effect.
Scientist is dedicated to the research of targeting anti-tumor peptide.Have now been found that a variety of and relevant protease of tumour swollen
Be overexpressed in oncocyte or extracellular matrix, it has been found that organized enzyme have matrix metalloproteinase (MMP) and at fiber finer
Born of the same parents' activator protein (FAPot) etc., these are specific expressed or small point by tumor cell specific secretion in tumor cell surface
The common feature of sub- antigenic substance is to be provided simultaneously with two kinds of characteristics of tumor-localizing and proteolytic enzyme.In general, will have cell toxicant
Property parent drug and polypeptide be condensed into the hydrolysis substrate of the fermentoid, so that drug is preferably targeted to tumor tissues.
Therefore, it is antineoplastic polypeptide medicament research and development to work out effective method and improve the selectivity of polypeptide drugs and stability
Middle urgent problem is of great significance to its clinical application.
Summary of the invention
The present invention relates to a kind of polypeptides, are selected from the group：
(a) include SEQ ID NO:1、SEQ ID NO:2 or SEQ ID NO:Amino acid sequence shown in 3 or by SEQ ID
NO:1、SEQ ID NO:2 or SEQ ID NO:The polypeptide that amino acid sequence shown in 3 forms；
(b) amino acid sequence in (a) is by replacing, missing or adding one or several amino acid derived from (a)
Polypeptide, with antitumor activity.
The invention further relates to the polynucleotides of encoding such polypeptides, the carrier comprising the polynucleotides, host cells.
The invention further relates to the pharmaceutical compositions comprising aforementioned polypeptides and aforementioned polypeptides to prepare anti-tumor drug, especially
It is in the drug that integrin (Intigrin) or matrix metalloproteinase 2/9 (MMP2/9) relevant disease is secreted in treatment expression
Using.
Description of the drawings
Fig. 1 is the ESI mass spectrograms for the LSAP-1 polypeptides of the present invention prepared by synthesis in solid state.
Fig. 2 is the ESI mass spectrograms for the LSAP-2 polypeptides of the present invention prepared by synthesis in solid state.
Fig. 3 is the ESI mass spectrograms for the LSAP-3 polypeptides of the present invention prepared by synthesis in solid state.
Fig. 4 is the TEM figures of polypeptide LSAP-3 self assembly forms of the present invention.
Fig. 5 be polypeptide LSAP-3 of the present invention and its in the presence of MMP2/9 inhibitor SB-3CT to the activity of Hela cells
Figure.
Detailed description of the invention
The present invention relates to a kind of polypeptides, are selected from the group：
(a) include SEQ ID NO:1、SEQ ID NO:2 or SEQ ID NO:Amino acid sequence shown in 3 or by SEQ ID
NO:1、SEQ ID NO:2 or SEQ ID NO:The polypeptide that amino acid sequence shown in 3 forms；
(b) amino acid sequence in (a) is by replacing, missing or adding one or several amino acid derived from (a)
Polypeptide, with antitumor activity.
In the other embodiment of the present invention, polypeptide provided by the invention is comprising SEQ ID NO.1, SEQ ID NO:
2 or SEQ ID NO:The polypeptide of amino acid sequence shown in 3 and its modified polypeptide or its homeopeptide.
In other embodiments of the present invention, " include SEQ ID NO：1、SEQ ID NO:2 or SEQ ID NO:3 institutes
The polypeptide of the amino acid sequence shown " includes, for example, by SEQ ID NO：Shown in 1 amino acid sequence form polypeptide, and by
SEQ ID NO：1、SEQ ID NO:2 or SEQ ID NO:The more of signal peptide sequence composition are added in amino acid sequence shown in 3
Peptide, and by SEQ ID NO：1、SEQ ID NO:2 or SEQ ID NO:The N-terminal and/or C of amino acid sequence shown in 3
Add the polypeptide that amino acid sequence obtained by appropriate flag sequence is constituted in end.
" modified polypeptide " in the present invention is referred to included in SEQ ID NO：1、SEQ ID NO:2 or SEQ ID NO:3
Shown in lack, replace, be inserted into or add amino acid sequence obtained from one or several amino acid in amino acid sequence, and have
There is the protein of antitumor activity.
In the preferred embodiment of the present invention, the modification to amino acid in the modified polypeptide or its homeopeptide
It is " conservative sex modification ".Such as " conservative replaces " refer under conditions of not material alterations protein active, by one
Or other amino acid substitutions of the more amino acid to be chemically similar.It can enumerate, by certain hydrophobic residue with other hydrophobic
Property residue substitution situation, the situation that certain polar residues is replaced with other polar residues with identical charges.
The functionally similar amino acid that can carry out this conservative replaces is all public in the corresponding technical field of each amino acid
Know.Specifically, as nonpolar (hydrophobicity) amino acid, can enumerate, alanine, valine, isoleucine, bright ammonia
Acid, proline, tryptophan, phenylalanine, methionine etc..It as polarity (neutrality) amino acid, can enumerate, glycine, silk ammonia
Acid, threonine, tyrosine, glutamine, asparagine, cysteine etc..It, can as the amino acid with positive charge (alkalinity)
To enumerate, arginine, histidine, lysine etc..In addition, as negative electrical charge (acidity) amino acid, can enumerate, aspartic acid,
Glutamic acid etc..
" homeopeptide " in the present invention, which refers to, includes and SEQ ID NO：1、SEQ ID NO:2 or SEQ ID NO:3
The amino acid sequence of expression have at least 85%, at least preferably 90%, at least preferably 92%, at least preferably 93%, at least preferably
94%, at least preferably 95%, at least preferably 96%, at least preferably 97%, at least preferably 98%, at least preferably 99%, more preferably
The amino acid sequence of 100% homology (sequence identity).
For the present invention, the degree of sequence identity between two amino acid sequences uses such as EMBOSS software packages
(EMBOSS:The European Molecular Biology Open Software Suite, Rice etc., 2000, Trends
Genet.16:276-277), Needleman-Wunsch performed in the Needle programs of preferably 3.0.0 editions or more highest version
Algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) measure.The optional parameters used is notch
Open point penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5 He
EBLOSUM62 (the EMBOSS versions of BLOSUM62) substitution matrix.It is labeled as " highest identity (longest using Needle
Identity output result (- nobrief options is used to obtain)) " is used as homogeneity percentage, and calculates as follows：
(same residue × 100)/(sum for comparing notch in length-comparison)
Polypeptide of the present invention can be that natural, synthesis, semi-synthetic or recombination generates.The polypeptide of the present invention can lead to
Cross genetic engineering, by known peptide synthesis or by being generated with the polypeptide of the peptidase digestion present invention appropriate.
In the preferred embodiment of the invention, the biological engineering method that polypeptide of the present invention can be routinely is by host cell
Recombinant DNA sequence coding generates polypeptide product, also can according to synthesis in solid state or liquid phase synthesis, such as can according to Steward and
Young (Steward, J.M. and Young, J.D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce
Chemical Company, Rockford, I11., (1984)) description method Applied Biosystem synthesizers or
PioneerTM peptide synthesizers are synthesized by Solid phase chemistry technology.In the preferred embodiment of the present invention, polypeptide of the present invention can be consolidated
Phase synthesis resin is starting material, is prepared by Fmoc chemical synthesis.
Invention further provides the polynucleotides of encoding such polypeptides.The polynucleotides of the present invention can be used in vivo
Or polypeptide present invention as described above is generated in vitro, or can be used for being attributable to encoding hereditary different in the gene of polypeptide of the present invention
The gene therapy of normal disease.Any form of polynucleotides of the present invention all can be used, as long as it encodes the polypeptide of the present invention, packet
Include mRNA, RNA, cDNA, genomic DNA and chemically synthesized polynucleotides.The polynucleotides of the present invention include comprising specified core
The DNA of nucleotide sequence and its cylinder and sequence, as long as gained DNA encoding polypeptide of the present invention.
Polynucleotides of the present invention can by those skilled in the art will know that method prepare.For example, the multinuclear of the present invention
Thuja acid can be prepared as follows：CDNA library is prepared from the cell for expressing polypeptide of the present invention, and using the partial sequence of its DNA as probe
Hybridized.The preparation of cDNA library can be used, and be write for example, with reference to Sambrook etc.《Molecular cloning》, referring to Cold SpringHarbor
Method described in laboratory Press (1989)；Or commercially available cDNA library can be used.
In addition, as the nucleotide sequencing of the cDNA to obtained by, the translated region encoded by the cDNA, and energy can be routinely determined
It is readily derived the amino acid sequence of the polypeptide of the present invention.In addition, using obtained cDNA or part thereof as probe, screening-gene
Group DNA library, can isolated genomic DNA.
The present invention also provides a kind of engineering carriers containing the polynucleotides for encoding polypeptide of the present invention.The gene work
Cheng Zaiti can be general carrier or expression vector.Specifically, the commercially available expression vector generally band suitable for prokaryotic cell
Have and mark and cellular replication origin may be selected, with promoters and the known cloning vector such as lacI, T7, λ PL and trp
Other genetic elements of pBR322 (ATCC37017).Such commercial vector includes pGEM (Promega) and pKK223-3
(Pharmacia).It can select to be derived from pBR322 according to selected appropriate promoter and structural gene sequence to be expressed
Suitable carrier.GST prokaryotic expression systems can also be used for the present invention.Carrier suitable for eukaryocyte starts with eukaryocyte
Son such as CMV, SV40, such carrier include pMT-hIL-3 (horse big dragon, the communication of Di Chunhui, Pang Jian etc. (1991) high-tech
11：26-29)、pQE-9(Qiagen)、pD10、pNH18A(Stratagene)、pKK233-3、pDR540、pRIT5
(Pharmacia) and pcDNA3, pCI, pWLNEO, pSG (Stratagene), pSVL (Pharmacia).
The present invention also provides a kind of host cell of the carrier containing the present invention, which can be used for expressing this hair
Bright polypeptide, the host include but not limited to：Prokaryotic hosts, Escherichia coli, bacillus, streptomyces etc.；Eukaryon
Host, such as：Saccharomyces, aspergillus, insect cell such as drosophila S2 and fall army worm Sf 9, zooblast such as CHO, COS (monkey kidney
Fibroblast, Gluzman (Cell 23：175,1981) Human cell line such as PC-3 cell lines, DU145 cell lines and its
It can express the cell line of compatible vehicle.
The present invention also provides a kind of pharmaceutical composition, polypeptide, polynucleotides containing therapeutically effective amount of the present invention, loads
Body, host cell；Pharmaceutically acceptable carrier or excipient when necessary.Pharmaceutically acceptable carrier or excipient refer to nothing
Malicious solid-state, semisolid or liquid filler, diluent, coating material or other pharmaceutical adjuncts.
Polypeptide application in preparation of anti-tumor drugs of the present invention, especially treatment expression are utilized the present invention also provides a kind of
Secrete the application in the drug of integrin (Intigrin) or matrix metalloproteinase 2/9 (MMP2/9) relevant disease.With table
It is, for example, cervical disease up to secretion integrin (Intigrin) or matrix metalloproteinase 2/9 (MMP2/9) relevant disease, more
Preferably, the disease is cervical carcinoma.
In the preferred embodiment of the invention, for example, as needed, drug can take orally, such as sugar coated tablet, capsule is made
With microcapsules etc.；Or it is non-oral, such as the sterile vehicle or suspension of injection water or any other pharmaceutics acceptable liquid
Form.For example, compound can be mixed into acceptable drug unit dose in execution with pharmaceutics acceptable carriers or medium
Form specifically has aqua sterilisa, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, aromatic, figuration
Agent, catalytic agent, preservative, binder etc..The quantity of active constituent is in the suitable agent in shown required range in these preparations
Amount.
Can be made into the example of tablet or capsule additive therefor is, binder such as gelatin, cornstarch and Arabic gum,
Excipient such as avicel cellulose, swelling agent such as cornstarch, gelatin and alginic acid；Lubricant such as magnesium stearate；Sweet taste
Agent such as sucrose, lactose or saccharin；Aromatic such as peppermint.When unit dosage form is microcapsules, in mentioned component
It may also include liquid phase carrying agent, it is such as oily.Injectable sterile composition can follow conventional drug execution and such as be injected with catalytic agent
It is prepared with distilled water.Physiological saline, glucose and other isotonic liquids include adjuvant, such as D- D-sorbites, D-MANNOSE, D-
Mannitol and sodium chloride, can be used as aqueous solution for injection.These can be used in combination with solubilizer appropriate, such as alcohols, tool
Body such as ethyl alcohol, polyalcohols such as propylene glycol and polyethylene glycol, nonionic surfactant such as polysorbate80.Sesame oil
Or soybean oil can be used as oil-based liquid, and can be used in combination with methyl benzoate, the benzyl alcohol as solubilizer, and can be with buffering
Liquid such as phosphate buffer, sodium acetate buffer；Analgesic, such as procaine hydrochloride；Stabilizer, such as benzyl alcohol；With
Antioxidant is prepared together.The injection of preparation can be fitted into suitable ampoule.
Method well known to those skilled in the art can be used that the medicinal compound of the present invention is applied to patient, such as artery
It is interior, intravenously, percutaneous injection and intranasal, through bronchus, intramuscular or oral administration.Dosage and method of administration are according to patient
Weight and age and method of administration and change；And those skilled in the art can routinely select.If described chemical combination
The DNA can be inserted into the carrier for gene therapy and be applied the carrier to treat by object by DNA encoding.Dosage
It is different because of the weight of patient, age and symptom with method of administration, but can be made appropriate choice by those skilled in the art.
Described herein and claimed invention is not limited to the range of specific embodiment disclosed herein, these realities
The scheme of applying is intended only to illustrate several aspects of the present invention.
Embodiment 1：The preparation of LSAP-1 polypeptides
The synthesis of LSAP-1 polypeptides of the present invention uses solid phase synthesis process, and to synthesize 0.25 mM of polypeptide, step is such as
Under：
(1) activated resin：The Wang resins for connecting fmoc-protected glycine (are purchased from the limited public affairs in gill biochemistry Shanghai
Department) it weighs up, it pours into clean anhydrous solid phase reactor, 5ml DCM (dichloromethane) is added and dissolve activation, overnight；
(2) resin is cleaned：The liquid in reactor is drained, 4ml DMF (n,N-Dimethylformamide) fully shaking is added
1min is drained, and is operated 8 times repeatedly；The resin after a small amount of activation is collected to remain to do Kaiser detections；
(3) Fmoc protections are taken off：After draining solvent, the DMF solution that 4ml contains 20% piperidines is added, is placed in shaking table, shakes
Solvent is drained after 5min；The DMF solution that 4ml contains 20% piperidines is added, shaking table is placed in, shakes 20min；
(4) it cleans resin, remove piperidines：After draining solvent, 4ml DMF solutions are added, are placed in shaking table, shake 1min, after
It drains；Repeatedly, it is repeated 8 times until piperidines is completely removed；
(5) electronic balance weighs amino acid to be accessed (i.e. fmoc-protected valine) and coupling reagent：4 times are measured
Amino acid, 3.9 times of HBTU (O- benzotriazole-tetramethylurea hexafluorophosphate), 4 times of HOBT (1- hydroxy benzos three measured
Azoles) it is dissolved in 4ml DMF, it mixes to after being completely dissolved, is added in solid phase reactor and is sufficiently mixed with resin, shaking table oscillation five
Minute；
(6) after then, 8 times of the DIEA (n,N-diisopropylethylamine) that mole is resin is added, after being sufficiently mixed, sets
In shaking table, clock reaction 2h；
(7) the follow-up proline for being sequentially ingressed into Fomc protections, leucine, leucine, arginine, serine, leucine, third
Propylhomoserin, arginine, phenylalanine, leucine, alanine, glycine, isoleucine, phenylalanine (are purchased from gill biochemistry Shanghai
Co., Ltd), often connecing an amino acid needs repetitive operation step 2-6；
(8) Kaiser is detected, and ninhydrin generates aubergine complex with ammonia or level-one amine, and Kaiser reagents include：6%
Ethanol solution of ninhydrin；80% phenol ethanol solution；The KCN pyridine solutions of 2%0.001M take reaction in a small amount of (6) to complete
When resin, and the resin in (2) adds each 2-3 drops of three kinds of ingredients in Kaiser reagents, 100 DEG C of heating 1-2min, if presenting
Blue or bronzing show the amino also to dissociate, then indicate that connection is complete on the contrary；
(9) when peptide chain engagement finishes, after cleaning resin, twice with piperidines deprotection；
(10) resin is cleaned 10 times with DMF, each 4ml；Resin is cleaned with DCM 10 times, each 4ml again；
(11) vacuum dried sample；
(12) it after waiting for that sample drying finishes, transfers a resin into heart bottle, installs magnetic stirring apparatus, heart bottle is consolidated
It sets, is slowly added to the cutting reagent (trifluoroacetic acid mixed：Ultra-pure water：Thioanisole：Phenol：Dithioglycol=82.5:5:
5:5:2.5) magneton, is added to be sufficiently stirred, reacts 12h at room temperature；
(13) it waits for that reaction is completed, reactant is transferred in solid phase reactor, do not shifted in solid phase reactor with reacting
Resin so stands 10min, rinses heart bottle with TFA (trifluoroacetic acid), all resins and solution are poured into solid phase reactor
In, after mixture is filtered under nitrogen flowing, filtrate is placed in round-bottomed flask, dries up under nitrogen flowing；
(14) it is sticky to wait for that the sample in round-bottomed flask is blown to, removes nitrogen, about 20ml ice ether is poured into round-bottomed flask
Precipitated polypeptide fully breaks up insoluble matter, and then trim is placed in refrigerated centrifuge, and 8000rpm/min is centrifuged at 4 DEG C
15min abandons supernatant, is re-dissolved in 20ml ice ether and breaks up, centrifugation；Centrifugation 3 times is repeated operation, precipitation is dried in vacuo,
Obtain polypeptide crude product；
(15) polypeptide crude product carries out purity analysis using analytic type HPLC, after purified with preparative HPLC；
(16) target polypeptides after purification are identified using ESI high resolution mass spectrums, mass spectrogram as shown in Figure 1, by
The molecular weight that figure measures synthesized LSAP-1 polypeptides is 1729.70, illustrates its amino acid sequence such as SEQ ID NO of the present invention:1
It is shown.
Embodiment 2：The preparation of LSAP-2 polypeptides
The synthesis of LSAP-2 polypeptides of the present invention uses solid phase synthesis process, and to synthesize 0.25 mM of polypeptide, step is such as
Under：
(1) activated resin：The Wang resins for connecting fmoc-protected lysine (are purchased from the limited public affairs in gill biochemistry Shanghai
Department) it weighs up, it pours into clean anhydrous solid phase reactor, 5ml DCM (dichloromethane) is added and dissolve activation, overnight；
(2) resin is cleaned：The liquid in reactor is drained, 4ml DMF (n,N-Dimethylformamide) fully shaking is added
1min is drained, and is operated 8 times repeatedly；The resin after a small amount of activation is collected to remain to do Kaiser detections；
(3) Fmoc protections are taken off：After draining solvent, the DMF solution that 4ml contains 20% piperidines is added, is placed in shaking table, shakes
Solvent is drained after 5min；The DMF solution that 4ml contains 20% piperidines is added, shaking table is placed in, shakes 20min；
(4) it cleans resin, remove piperidines：After draining solvent, 4ml DMF solutions are added, are placed in shaking table, shake 1min, after
It drains；Repeatedly, it is repeated 8 times until piperidines is completely removed；
(5) electronic balance weighs amino acid to be accessed (i.e. fmoc-protected aspartic acid) and coupling reagent：4 times are measured
Amino acid, 3.9 times of HBTU (O- benzotriazole-tetramethylurea hexafluorophosphate), 4 times amount HOBT (1- hydroxy benzos
Triazole) it is dissolved in 4ml DMF, it mixes to after being completely dissolved, is added in solid phase reactor and is sufficiently mixed with resin, shaking table oscillation
Five minutes；
(6) after then, 8 times of the DIEA (n,N-diisopropylethylamine) that mole is resin is added, after being sufficiently mixed, sets
In shaking table, clock reaction 2h；
(7) the follow-up glycine for being sequentially ingressed into Fomc protections, arginine, glycine, isoleucine, leucine, glycine,
Valine, proline, leucine, leucine, arginine, serine, leucine, alanine, arginine, phenylalanine, bright ammonia
Acid, alanine, glycine, isoleucine, phenylalanine (being purchased from gill biochemistry Shanghai Co., Ltd), often connecing an amino acid needs
Repetitive operation step 2-6；
(8) Kaiser is detected, and ninhydrin generates aubergine complex with ammonia or level-one amine, and Kaiser reagents include：6%
Ethanol solution of ninhydrin；80% phenol ethanol solution；The KCN pyridine solutions of 2%0.001M take reaction in a small amount of (6) to complete
When resin, and the resin in (2) adds each 2-3 drops of three kinds of ingredients in Kaiser reagents, 100 DEG C of heating 1-2min, if presenting
Blue or bronzing show the amino also to dissociate, then indicate that connection is complete on the contrary；
(9) when peptide chain engagement finishes, after cleaning resin, twice with piperidines deprotection；
(10) resin is cleaned 10 times with DMF, each 4ml；Resin is cleaned with DCM 10 times, each 4ml again；
(11) vacuum dried sample；
(12) it after waiting for that sample drying finishes, transfers a resin into heart bottle, installs magnetic stirring apparatus, heart bottle is consolidated
It sets, is slowly added to the cutting reagent (trifluoroacetic acid mixed：Ultra-pure water：Thioanisole：Phenol：Dithioglycol=82.5:5:
5:5:2.5) magneton, is added to be sufficiently stirred, reacts 12h at room temperature；
(13) it waits for that reaction is completed, reactant is transferred in solid phase reactor, do not shifted in solid phase reactor with reacting
Resin so stands 10min, rinses heart bottle with TFA (trifluoroacetic acid), all resins and solution are poured into solid phase reactor
In, after mixture is filtered under nitrogen flowing, filtrate is placed in round-bottomed flask, dries up under nitrogen flowing；
(14) it is sticky to wait for that the sample in round-bottomed flask is blown to, removes nitrogen, about 20ml ice ether is poured into round-bottomed flask
Precipitated polypeptide fully breaks up insoluble matter, and then trim is placed in refrigerated centrifuge, and 8000rpm/min is centrifuged at 4 DEG C
15min abandons supernatant, is re-dissolved in 20ml ice ether and breaks up, centrifugation；Centrifugation 3 times is repeated operation, precipitation is dried in vacuo,
Obtain polypeptide crude product；
(15) polypeptide crude product carries out purity analysis using analytic type HPLC, after purified with preparative HPLC；
(16) target polypeptides after purification are identified using ESI high resolution mass spectrums, mass spectrogram as shown in Fig. 2, by
The molecular weight that figure measures synthesized LSAP-2 polypeptides is 2469.45, illustrates its amino acid sequence such as SEQ ID NO of the present invention:2
It is shown.
Embodiment 3：The preparation of LSAP-3 polypeptides
The synthesis of LSAP-3 polypeptides of the present invention uses solid phase synthesis process, and to synthesize 0.25 mM of polypeptide, step is such as
Under：
(1) activated resin：The Wang resins for connecting fmoc-protected lysine (are purchased from the limited public affairs in gill biochemistry Shanghai
Department) it weighs up, it pours into clean anhydrous solid phase reactor, 5ml DCM (dichloromethane) is added and dissolve activation, overnight；
(2) resin is cleaned：The liquid in reactor is drained, 4ml DMF (n,N-Dimethylformamide) fully shaking is added
1min is drained, and is operated 8 times repeatedly；The resin after a small amount of activation is collected to remain to do Kaiser detections；
(3) Fmoc protections are taken off：After draining solvent, the DMF solution that 4ml contains 20% piperidines is added, is placed in shaking table, shakes
Solvent is drained after 5min；The DMF solution that 4ml contains 20% piperidines is added, shaking table is placed in, shakes 20min；
(4) it cleans resin, remove piperidines：After draining solvent, 4ml DMF solutions are added, are placed in shaking table, shake 1min, after
It drains；Repeatedly, it is repeated 8 times until piperidines is completely removed；
(5) electronic balance weighs amino acid to be accessed (i.e. fmoc-protected aspartic acid) and coupling reagent：4 times are measured
Amino acid, 3.9 times of HBTU (O- benzotriazole-tetramethylurea hexafluorophosphate), 4 times amount HOBT (1- hydroxy benzos
Triazole) it is dissolved in 4ml DMF, it mixes to after being completely dissolved, is added in solid phase reactor and is sufficiently mixed with resin, shaking table oscillation
Five minutes；
(6) after then, 8 times of the DIEA (n,N-diisopropylethylamine) that mole is resin is added, after being sufficiently mixed, sets
In shaking table, clock reaction 2h；
(7) the follow-up glycine for being sequentially ingressed into Fomc protections, arginine, aspartic acid, leucine, leucine, glycine,
Isoleucine, leucine, glycine, valine, proline, leucine, leucine, arginine, serine, leucine, the third ammonia
Acid, arginine, phenylalanine, leucine, alanine, glycine, isoleucine, phenylalanine (have purchased from gill biochemistry Shanghai
Limit company), often connecing an amino acid needs repetitive operation step 2-6；
(8) Kaiser is detected, and ninhydrin generates aubergine complex with ammonia or level-one amine, and Kaiser reagents include：6%
Ethanol solution of ninhydrin；80% phenol ethanol solution；The KCN pyridine solutions of 2%0.001M take reaction in a small amount of (6) to complete
When resin, and the resin in (2) adds each 2-3 drops of three kinds of ingredients in Kaiser reagents, 100 DEG C of heating 1-2min, if presenting
Blue or bronzing show the amino also to dissociate, then indicate that connection is complete on the contrary；
(9) when peptide chain engagement finishes, after cleaning resin, twice with piperidines deprotection；
(10) resin is cleaned 10 times with DMF, each 4ml；Resin is cleaned with DCM 10 times, each 4ml again；
(11) vacuum dried sample；
(12) it after waiting for that sample drying finishes, transfers a resin into heart bottle, installs magnetic stirring apparatus, heart bottle is consolidated
It sets, is slowly added to the cutting reagent (trifluoroacetic acid mixed：Ultra-pure water：Thioanisole：Phenol：Dithioglycol=82.5:5:
5:5:2.5) magneton, is added to be sufficiently stirred, reacts 12h at room temperature；
(13) it waits for that reaction is completed, reactant is transferred in solid phase reactor, do not shifted in solid phase reactor with reacting
Resin so stands 10min, rinses heart bottle with TFA (trifluoroacetic acid), all resins and solution are poured into solid phase reactor
In, after mixture is filtered under nitrogen flowing, filtrate is placed in round-bottomed flask, dries up under nitrogen flowing；
(14) it is sticky to wait for that the sample in round-bottomed flask is blown to, removes nitrogen, about 20ml ice ether is poured into round-bottomed flask
Precipitated polypeptide fully breaks up insoluble matter, and then trim is placed in refrigerated centrifuge, and 8000rpm/min is centrifuged at 4 DEG C
15min abandons supernatant, is re-dissolved in 20ml ice ether and breaks up, centrifugation；Centrifugation 3 times is repeated operation, precipitation is dried in vacuo,
Obtain polypeptide crude product；
(15) polypeptide crude product carries out purity analysis using analytic type HPLC, after purified with preparative HPLC；
(16) target polypeptides after purification are identified using ESI high resolution mass spectrums, mass spectrogram as shown in figure 3, by
The molecular weight that figure measures synthesized LSAP-3 polypeptides is 2811.30, illustrates its amino acid sequence such as SEQ ID NO of the present invention:3
It is shown.
Embodiment 4：The self assembly form of polypeptide LSAP-3
By the form after transmission electron microscope observing polypeptide LSAP-3 self assemblies, operating procedure is as follows：
(1) the LSAP-3 solution that compound concentration is 40 μM, stand overnight makes its self assembly at room temperature, prepares 20mg/mL
Phosphotungstic acid (PTA), phosphotungstic acid dye liquor need to 0.1M sodium hydroxides adjust pH be 6.0-7.0；
(2) the special copper mesh of TEM is taken out, polypeptide sample is dripped on copper mesh, forms water droplet, after standing 1min, use filter paper
Draw a small amount of liquid at copper mesh edge, when copper mesh at semi-moist state, a drop phosphotungstic acid dye liquor is then added dropwise again, dyes 1.5min
Left and right, is blotted with filter paper, then copper mesh is positioned in culture dish dry；
(3) form after transmission electron microscope observing LSAP-3 self-assembling polypeptides is utilized, as shown in figure 4, containing LSAP- of the present invention
The solution of 3 polypeptides can be self-assembly of nanosized micelles in left at room temperature over night.
Referring to invention LSAP-3 polypeptides amino acid sequence, N-terminal is hydrophobic, and C-terminal is hydrophilic, the polypeptide have it is amphipathic, and this
Self assembly polypeptide micella is formed conducive to LSAP-3 polypeptides of the present invention, the polypeptide LSAP-3 with self assembly ability has in human serum
Higher stability.
Embodiment 5：The activity experiment of LSAP-1 polypeptides, LSAP-2 polypeptides and LSAP-3 polypeptides
The cell activity experiment of polypeptide is detected using mtt assay, and steps are as follows for experiment：
(1) solvent is prepared：The preparation of MTT solution：5mg is prepared with PBS (phosphate buffer is purchased from Gibco companies)
mL-1MTT (tetrazolium bromide, be purchased from Sigma companies), wrapped with masking foil, 30 minutes hydrotropies be stirred at room temperature, are completely dissolved
Afterwards with 0.22 μM of membrane filtration, finally it is kept in dark place at 4 DEG C；The preparation of three lysates：SDS (lauryl sodium sulfate)
Dosage for 10g, isobutanol 5mL, 10M hydrochloric acid is 0.12mL, and 100mL solution is made into distilled water dissolving；The preparation of culture medium：
90% DMEM culture mediums (being purchased from Gibco companies) and 10% FBS；
DMEM culture mediums are for cultivating Hela cells (Human cervical cancer cell lines are purchased from Chinese Academy of Sciences's Shanghai cell bank)
(2) the Hela cells in culture bottle grow to 80% or more, and 1mL pancreatin (being purchased from Sigma companies) digestion is added, places
At 37 DEG C, 5% carbon dioxide incubator culture, microscopic observation cell dissociation is completed, and is added after 4mL culture mediums terminate and is centrifuged, from
Heart rotating speed, time are respectively 900rpm/min, 4min；Supernatant is abandoned, culture medium is added and gently blows and beats, cell is made to be uniformly dispersed
In the medium；
(3) number of cells is calculated with cell counter, and adds culture medium and adjusts cell concentration to 5 × 104mL-1；
(4) cell suspension bed board is taken, it is then 100 μ L are placed per hole cell number 5000 that volume is added in each hole
5%CO2, it is incubated 16-18 hours in 37 DEG C of incubators；
(5) after being incubated, the polypeptide drugs of various concentration are prepared with serum free medium, 96 orifice plates are added, with cell
It is incubated 24 hours.Blank group only adds culture medium (acellular), and culture medium is only added in cellular control unit, and experimental group is added different dense
The polypeptide drugs of degree；
(6) after reaction, culture medium is sucked out.Pre-arranged MTT is diluted ten times with without phenol red full culture medium, so
100 μ L are added per hole afterwards, wherein blank group need not addition MTT.96 orifice plates are put into cell incubator；
(7) after reacting 4 hours, the three joint-trial agent (10%SDS+5% isobutanols+0.012mol/L of 100 μ L is added per hole
HCl), at room temperature, 12-24 hours mixings on shaking table are placed on, the OD values in 570nm are detected with microplate reader, with SPSS software meters
Calculate the IC of various cells50Value, as shown in table 1.
Toxicity of table 1.LSAP-1, LSAP-2, LSAP-3 polypeptide to Hela cells
Embodiment 6：Specificity of the LSAP-3 polypeptides to MMP2/9
The specificity of polypeptide LSAP-3 is detected using mtt assay, and steps are as follows for experiment：
(1) solvent is prepared：The preparation detailed in Example 5 of MTT solution, three lysates, culture medium.
MMP2/9s of the MMP2/9 inhibitor SB-3CT (being purchased from Sigma-Aldrich companies) for inhibiting Hela cells to secrete
Enzymatic activity.
(2) the Hela cells in culture bottle grow to 80% or more, and 1mL pancreatin (being purchased from Sigma companies) digestion is added, places
At 37 DEG C, 5% carbon dioxide incubator culture, microscopic observation cell dissociation is completed, and is added after 4mL culture mediums terminate and is centrifuged, from
Heart rotating speed, time are respectively 900rpm/min, 4min；Supernatant is abandoned, culture medium is added and gently blows and beats, cell is made to be uniformly dispersed
In the medium；
(3) number of cells is calculated with cell counter, and adds culture medium and adjusts cell concentration to 5 × 104mL-1；
(4) cell suspension bed board is taken, it is then 100 μ L are placed per hole cell number 5000 that volume is added in each hole
5%CO2, it is incubated 16-18 hours in 37 DEG C of incubators；
(5) after being incubated, the polypeptide drugs of various concentration are prepared with serum free medium, 96 orifice plates are added, with cell
It is incubated 24 hours.Blank group only adds culture medium (acellular), and culture medium is only added in polypeptide group control cell, and polypeptide group is added not
With the polypeptide drugs of concentration, inhibition group control cell is added 10 μM of SB-3CT, inhibition group be added various concentration polypeptide and 10 μM
SB-3CT；
(6) after reaction, culture medium is sucked out.Pre-arranged MTT is diluted ten times with without phenol red full culture medium, so
100 μ L are added per hole afterwards, wherein blank group need not addition MTT.96 orifice plates are put into cell incubator；
(7) after reacting 4 hours, the three joint-trial agent (10%SDS+5% isobutanols+0.012mol/L of 100 μ L is added per hole
HCl), at room temperature, 12-24 hours mixings on shaking table are placed on, with microplate reader detection in the OD values of 570nm, calculate cell survival
Rate.
Cell survival rate (%)=(ODPolypeptide group-ODBlank group)/(ODControl group-ODBlank group)
As shown in figure 5, under low concentration (<8 μM), in the case of MMP2/9 activity is suppressed, the activity of polypeptide LSAP-3
Also it is suppressed, and then proves that the activity of polypeptide LSAP-3 can further be activated by MMP2/9, improve it to tumour cell
Activity.
Claims (10)
1. polypeptide selected from the group below：
(a) include SEQ I D NO:1、SEQ I D NO:2 or SEQ I D NO:Amino acid sequence shown in 3 or by SEQ I D
NO:1、SEQ I D NO:2 or SEQ I D NO:The polypeptide that amino acid sequence shown in 3 forms；
(b) amino acid sequence in (a), which passes through, replaces, misses or adds one or several amino acid polypeptide derived from (a),
It is with antitumor activity.
2. polypeptide described in claim 1, by SEQ I D NO:1、SEQ I D NO:2 or SEQ I DNO:Ammonia shown in 3
Base acid sequence forms.
3. encoding the polynucleotides of polypeptide described in claims 1 or 2.
4. including the carrier of polynucleotides described in claim 3.
5. including the host cell of carrier described in polynucleotides or claim 4 described in claim 3.
6. the preparation method of polypeptide described in claims 1 or 2, using synthesis in solid state resin as starting material, by Fmocization
Synthetic method is learned to prepare.
7. pharmaceutical composition, polypeptide is as active constituent and pharmaceutics described in claims 1 or 2 it includes pharmaceutical effective amount
Acceptable carriers.
8. polypeptide application in preparation of anti-tumor drugs described in claims 1 or 2.
9. polypeptide described in claims 1 or 2 is being prepared and the relevant disease of expression secretion integrin or matrix metalloproteinase 2/9
Application in drug.
10. the application described in claim 8 or 9, wherein the disease is cervical carcinoma.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016143904A1 (en) * | 2015-03-12 | 2016-09-15 | 三井化学株式会社 | Method for disrupting exosomes, exosome disruption kit, and method for separating exosomes derived from normal cells |
| CN106831953A (en) * | 2017-01-19 | 2017-06-13 | 北京化工大学 | A kind of polypeptide and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016143904A1 (en) * | 2015-03-12 | 2016-09-15 | 三井化学株式会社 | Method for disrupting exosomes, exosome disruption kit, and method for separating exosomes derived from normal cells |
| CN106831953A (en) * | 2017-01-19 | 2017-06-13 | 北京化工大学 | A kind of polypeptide and its application |
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| Title |
|---|
| LONG CHEN: "Peptide fibrils with altered stability, activity, and cell selectivity", 《BIOMACROMOLECULES》 * |
| 周希蕊: "自组装抗肿瘤多肽的设计合成、表征和作用机制研究", 《中国优秀博硕士学位论文全文数据库(博士) 工程科技Ⅰ辑》 * |
| 尹会伟: "多重靶向自组装多肽的设计与活性机理研究", 《中国优秀博硕士学位论文全文数据库(硕士) 医药卫生科技辑》 * |
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