CN101153056A - Regrouped anticancer peptide, producing method and application of the same - Google Patents

Abstract

The present invention discloses an anti-cancer peptide with an amino acid sequence of SEQ ID NO.1, an expression body, a preparation method and the application thereof in preparing anti-cancer drugs. The method comprises that: a. the plasmid pTWIN2 carrier is used; a 13-peptide containing RGD sequence of OPN is inserted into the viscous end after treatment of endonuclease BspQI; the homocysteine TGC is added in designing of primer; the primer and the carrier after treatment of endonuclease are connected for a night at a temperature of 16 DEG C according to a ratio of 1 to 10 (molar ratio), through the denaturalization and annealing method of the primer; the recombinant plasmid pTWIN2-RGD can be prepared; b. the recombinant plasmid is digested by colicin and transferred into the colicin or Escherichia to induce and collect purified protein. The anti-cancer peptide of the present invention has good anti-cancer activity and is easy for synthesis.

Description

A kind of regrouped anticancer peptide and preparation method thereof and its application

Technical field

The present invention relates to recombinate amino acid and preparation method thereof and its application specifically relate to a kind of recombinant polypeptide and preparation method thereof and its application.

Background technology

Along with developing rapidly of tumour molecules and peptide combinatorial chemistry, the derivative of peptide and peptide acts as focus for people research in oncotherapy.People have isolated many small-molecular peptides or cyclic peptide with antitumour activity from marine organisms at present.From the Dolabellaauriculara sea hare, isolate a series of small-molecular peptides (dolastatins1--15) as State of Arizona, US ICR Pettir etc. and (see Pettit GR for details with antitumour activity, Kamano Y, Herald L L.et al.The isolation and structure of a remarkablemarine animal antmeoplastic consrituent.J Am Chem Soc.1987,109 (22): 6883-6885; Document 1-3); Rinerhart etc. isolate five kinds of cyclic peptide didemninA, B, C, D, E with antitumour activity and (see Rinehart KL Jr for details from Caribbean Ascidian (Caribbean tunicates), Gloer JB, CookJC.et al.Structure of the didemnins, antiviral and cytotoxic depsipeptides from aCaribbean tunicate.J Am Chem Soc, 1981,103 (7): 1857-1859).But regrettably, institute's its content of isolating peptide matters is very low at present, and synthetic difficulty, has seriously restricted its application in the preparation cancer therapy drug thus.

Summary of the invention

One of purpose of the present invention provides a kind of anticancer peptide of reorganization.

Two of purpose of the present invention provides a kind of preparation method of this anticancer peptide.

Three of purpose of the present invention provides a kind of expression vector that contains this anticancer peptide.

Four of purpose of the present invention provides the application of this anticancer peptide in the preparation cancer therapy drug.

The object of the present invention is achieved like this:

Regrouped anticancer peptide provided by the present invention is called for short the reorganization tridecanoic peptide, and its aminoacid sequence is Gly-Arg-Gly-Asp-Ser-Val-Val-Tyr-Gly-Leu-Arg-Ser-Lys (SEQ ID NO.1).

The preparation method of regrouped anticancer peptide of the present invention may further comprise the steps:

A, usefulness plasmid pTWIN2 carrier, after restriction endonuclease BspQ I handles, insert the tridecanoic peptide of the RGD sequence that contains OPN of sticky end, when the design primer, added halfcystine TGC, carrier after making primer and enzyme is cut processing through primer sex change, annealed method is connected for 16 ℃ by 1: 10 (mol ratio) and spends the night, and obtains recombinant plasmid pTWIN2-RGD.

B, recombinant plasmid is digested through intestinal bacteria, change colibacillus or escherichia coli over to, induce, collection, purifying target protein.

In expression vector, insert the present invention clone's anticancer peptide and import host cell such as colibacillus or escherichia coli express recombinant tridecanoic peptide.

Anticancer peptide of the present invention is to belong to the little peptide of small molecules ring-type, has the effect of significant inhibition tumor cell invasion and transfer, is convenient to synthetic simultaneously, is easy to industrialization.Therefore anticancer peptide of the present invention can be applied in the preparation cancer therapy drug.Be particularly useful for preparation treatment intestinal cancer, mammary cancer, ovarian cancer, liver cancer or lung-cancer medicament.

Anticancer peptide of the present invention or the expression vector that contains this anticancer peptide can determine its sequence through 5.7mol/L hydrochloric acid hydrolysis 16 hours by automatic analyzer for amino acids and aminoacid sequence instrument.

Anticancer peptide of the present invention is when being used to prepare cancer therapy drug, can anticancer peptide be active constituents of medicine, and mix with pharmaceutically acceptable carrier such as physiological saline, injection liquid, emulsion (as triglyceride level emulsion, oil-in-water/water-in-oil emulsion) or medicinal conventional assistant agent such as vehicle, disintegrating agent, tamanori, lubricant, antioxidant, Drug coating, tinting material, perfume compound, tensio-active agent etc., be made into pharmaceutical dosage forms such as injection liquid, infusion solution, controlled release agent, granule, capsule, tablet according to the preparation technique of routine.

Anticancer peptide of the present invention can adopt oral administration, also can adopt injection etc. non-oral by way of administration.Dosage can be 1ng-10g/kg, and concrete dosage can be adjusted according to different administering modes in good time.The doctor also can take the circumstances into consideration to determine according to the state of an illness.

Description of drawings

Fig. 1 is a pTWIN2 carrier collection of illustrative plates.

Embodiment

The following examples, experimental example and example of formulations can illustrate in greater detail the present invention, but do not limit the present invention in any form.

Reorganization, evaluation, purifying and the collection of embodiment 1 anticancer peptide of the present invention.

Utilize the IMPACT-TWIN system of NEB (New England Biolabs) company, realize the preparation of the little peptide of reorganization ring-type by chitin pearl affinity chromatography and intein (intein) from splicing function.

One, construction recombination plasmid pTWIN2-RGD

PTWIN2 carrier collection of illustrative plates, as shown in Figure 1.

PTWIN2 carrier part sequence is as follows:

Polylinker Region：pTW IN2

5’...AC TGG GAC TCC ATC GTT TCT ATT ACG GAG ACT GGA GTC GAA GAG GTT TTT

Ssp DnaB Intein Forward Primer→

←Intein

...Ssp DnaB Intein... Val Ala Asn Asp Ile Ile Val His Asn

GAT TTG ACT GTG CCA GGA CCA CAT AAC TTT G TC GCG AAT GAC ATC ATT GTA CAC AAC

Nru I

Intein→

Gly Arg Ala Het Gly Gly Arg Glu Phe Leu Glu Gly Ser Ser Cys Val Ser Gly Asp Thr

G GA AGA GCC ATG GGC GGC CGC GAA TTC CTC GAG G GC TCT TCC TGC GTA TCC GGT GAC ACC ATT

SapI NcoI NotI EcoRI XhoI SapI

...Mth RIR1 Intein...

GTA ATG ACT AGT GGC GGT CCG CGC ACT GTG GCT GAA CTG GAG GGC AAA CCG TTC ACC...3’

SpeI

←Mth RI R1 Intein Reverse Primer

The pTWIN2 carrier contains two inteins, the intein2 of the intein1 of N end and C end, multiple clone site then is positioned in the middle of two inteins, in order to be beneficial to proteic cyclisation, utilize restriction endonuclease Sap I cut vector, because of it has two restriction enzyme sites that are positioned at the multiple clone site two ends, and the restriction enzyme site of design just in time is reverse complemental, so the purpose fragment only can directed be inserted and can oppositely not insert.

The recognition sequence of restriction endonuclease Sap I is 5 ' ... GCTCTTC (N) ₁ ... 3 '

3′…CGAGAAG(N) ₄▲…5′

N represents the base number that cuts behind the recognition site, according to the carrier sequence as can be known, the sticking end of pTWIN2 carrier after restriction endonuclease Sap I cutting be 5 ' ... ATT GTA CAC 3 '

3′…TAA CAT GTG TTG 5′

And 5 ' TGC GTA TCC ... 3 '

3′ CAT AGG…5′

So sticky end and experiment needs that the inventor exposes after Sap I handles according to carrier, design has the tridecanoic peptide of the RGD sequence that contains OPN of corresponding sticky end, does not comprise stop code, and its sequence is as follows

5′…AAC TGC GGC CGA GGT GAT AGT GTG GTT TAT GGA CTG AGG TCAAAA…3′

5′…GCA TTT TGA CCT CAG TCC ATAAAC CAC ACT ATC ACC TCG GCC…3′

In order to make target protein be beneficial to cyclisation, the inventor has added halfcystine TGC when the design primer, carrier after making primer and enzyme is cut processing through primer sex change, annealed method is connected for 16 ℃ by 1: 10 (mol ratio) and spends the night, and obtains recombinant plasmid pTWIN2-RGD.

Because cutting and the connection characteristics of Sap I cause losing of Sap I restriction enzyme site after fragment and carrier connect,, the inventor connects product so identifying with the method for PCR.Because of Sap I very unstable in transportation, so recognition site and the on all four isoschizomers BspQI of cleavage site with Sap I that the inventor adopts NEB company to produce substitute Sap I as restriction endonuclease.

Two, the conversion of recombinant plasmid and evaluation

With recombinant expression plasmid (promptly connecting product) transformed into escherichia coli DH5 α, concrete grammar is as follows: 10 μ l are connected product add in the 100 μ l DH5 α competent cells, ice bath is 30 minutes behind the abundant mixing, 42 ℃ of heat shocks 90 seconds, ice bath 2-3 minute rapidly, be coated with bacterium (the solid LB substratum that contains the ammonia benzyl), plate is inverted in 37 ℃ of incubator overnight incubation.Choose the mono-clonal bacterium colony next day, send order-checking further to identify earlier after the method preliminary evaluation with bacterium colony PCR.

Three, the abduction delivering of target protein and purifying:

Chitin (but a few fourth) the affinity post of utilization 15ml carries out the method (being applicable to the inducing culture bacterium of 1L) of protein purification

1. microbial culture:

Bacteria plasmid is carried in the correct reorganization of order-checking, changed over to intestinal bacteria ER2566 (being the expression bacterium that the pTWIN2 carrier carries), method for transformation is the same, and just 1 μ l pure plasmid changes in the 50 μ l ER2566 competent cells.Choose the mono-clonal bacterium colony evening next day, 37 ℃ of shaking bath shaking culture are spent the night.Ratio in 1: 100 was inoculated in 10ml liquid LB substratum (containing ammonia benzyl 100 μ g/ml) in second day, 37 ℃ of shaking culture 3-4h, after be inoculated in 1L liquid nutrient medium (containing ammonia benzyl 100 μ g/ml), 37 ℃ are cultured to OD600 and reach 0.5-0.7.

2. inducible protein is expressed: adding final concentration is the IPTG of 0.1mM-1.0mM, expresses 2-20h in 12-37 ℃ of inducible protein, to establish best inductive condition and to set up negative control (not inducing).Sampling 10-20 μ l carries out the SDS-PAGE electrophoresis, and sampling 1-2 μ l carries out Western blot to be identified.

3. cell harvesting: 5000 * g, 4 ℃ centrifugal 10 minutes, abandon supernatant.The cell freeze thawing is beneficial to fragmentation.

Annotate: following steps 4-9 is except that having special indicating, all 4 ℃ of operations.

4. smudge cells: (20mM Tris-HCl, pH8.5,0.5MNaCl or Buffer B1) is resuspended with thalline with the suitable lysis buffer of 50ml, ice bath, and the ultrasonication cell discharges albumen.Centrifugal 30 minutes of 19000 * g, supernatant is clarifying cell crude extract.Sampling 2-5 μ l carries out the SDS-PAGE electrophoresis and identifies (Western blot identifies: dilution in 1: 10, last sample 2-5 μ l).

5.Chitin the balance of post: 24ml chitin beads (15ml column volume) is poured in 2.5 * 10cm post, and the corresponding damping fluid of 10 column volumes (with step 4 liquid) carries out column equilibration for 4 ℃.Annotate: the consumption of Chitin (but a few fourth) is decided by the total amount of fusion rotein, and every milliliter of column volume approximately can be in conjunction with 3mg albumen, therefore cultivates the resin that about 45mg fusion rotein that bacterium obtained approximately needs the 15ml column volume for one liter.

6.Chitin post: clarifying cell crude extract is slowly added the Chitin post, and flow velocity is about 0.5-1.0ml/min.Take a morsel and flow through liquid and carry out the SDS-PAGE electrophoresis, and compare, observe the protein binding efficient of affinity column with the electrophoresis of cell crude extract.Can be with cell crude extract and Column Buffer in proportion 1: 2-1 before the upper prop: 5 mixed dilutings, to improve protein binding efficient.

7. wash post: the damping fluid with 800mL (at least 20 times of column volumes) is thoroughly washed post, because the bonding force of CBD (chitin binding domain) and Chitin beads (but a few fourth pearl) is very strong, can be with higher flow velocity (as: 2-3ml/min) and the harsher column condition (as high salt 1 M NaCl and/or nonionic stain remover) of washing when washing post.

8. induce the self cracked activity of Intein: Intein to be positioned at the C end of target protein, wash post with Buffer (damping fluid) B3 (containing 30-50 mM DTT) of 3 times of column volumes, block up with cap, 4 ℃ of placements are spent the night.The cracking that the target protein (C-terminal has thioester bond) that is used for IPL (intein mediation albumen ligation) ligation then need be induced intein with Buffer B4 (containing the MESNA mesna).Intein is positioned at target protein N end, washes post with 3 times of column volume Buffer B2, and the greenhouse is placed and spent the night.

9. the wash-out of target protein: next day, with the Buffer B1 of 3 times of column volumes or lure cracking Buffer3 (DTT), the target protein wash-out to be collected accordingly.Approximately collect the 8-10 pipe, every pipe 5-7.5ml (1/2 column volume).Target protein mostly in a column volume by wash-out (about 20ml), can detect by 280nm UV light absorption value or Bradford Protein Detection method very easily.

10.SDS-PAGE electrophoretic analysis: on the Chitin post bonded intein-CBD and the target protein of wash-out can separate observe the lysis efficiency of intein by the 1%SDS wash-out.(in addition, also has a kind of easier method: get 40 μ l chitin beads, add 20 μ l, 3 * SDS Sample Buffer mixing, draw sample on the 5 μ l, the SDS-PAGE electrophoretic analysis.

11.Chitin regeneration of resin: the Chitin resin can repeat regeneration by following steps and use 4-5 time.0.3 M NaOH (being that albumen strips off liquid Stripping Solution) rinsing Chitin post with 3 times of column volumes, make resin in this liquid, soak after 30 minutes, use the further wash-out of 0.3 MNaOH of 7 times of column volumes again, use the Column Buffer rinsing of water and 5 times of column volumes of 20 times of column volumes at last.The Chitin resin can be stored in 4 ℃.As long time stored, then need in Column Buffer (elution buffer), to add 0.02% sodium azide.

Four, target protein purifying

Adopt IMPAC ^TMSystem carries out the target protein purifying.

IMPACT ^TMSystem is a kind of protein fusion expression and the purification system that NEB company develops.IMPACT ^TMThe target protein of expressing and albumen from the montage element (be called " intein ", intein) and chitin-binding protein formation fusion rotein, by chitin post affinity purification fusion rotein.Induce the peptide bond lytic activity of intein then, on the chitin medium, target protein is discharged, and intein and chitin-binding protein still are combined on the chitin medium, reach the proteic purpose of single-column separation and purification.

Five, the authentication method of target protein

Fusion rotein is with 12% SDS-PAGE (denaturing polyacrylamide gel electrophoresis), the little peptide of the target protein of purifying is identified with the Tricine gel electrophoresis, if the albumen that needs further to distinguish polymer form, cyclisation form and linear forms then needs the laser flying mass spectroscopy to detect.

The aminoacid sequence of surveying is Gly-Arg-Gly-Asp-Ser-Val-Val-Tyr-Gly-Leu-Arg-Ser-Lys.

SEQ ID NO.1 is as follows:

SEQUENCE LISTING

＜110〉Hebei Medical University

＜120〉a kind of regrouped anticancer peptide and preparation method thereof and its application

<130>0701

<160>1

<170>PatentIn version 3.3

<210>1

<211>13

<212>PRT

＜213〉people source osteopontin (Human osteopontin)

<400>1

Gly Arg Gly Asp Ser Val Val Tyr Gly Leu Arg Ser Lys

1 5 10

Embodiment 2 anticancer peptides of the present invention (hereinafter to be referred as reorganization 13 peptides) are to the restraining effect of tumor cell migration.

With cell inoculation on slide, after cell grows to 100% fusion, take out slide, clean by behind the cell that scrapes at cut .PBS on the slide with aseptic suction nozzle, slide is placed the nutrient solution that contains 13 peptides (10,20 μ g/ml), take out after continuing to hatch 24h, fix and HE dyeing. observation of cell wound healing situation under low power lens. get 3 visuals field arbitrarily, the quantity of counting migrating cell is represented the migratory activity of cell with this. with the negative contrast of blank substratum.Detailed results sees Table 1.

Table 1: the restraining effect of the 13 peptide on cell migration abilities of recombinating (is example with 20 μ g/ml)

Cell type	Negative control group	13 peptide treatment group
			Vascular smooth muscle cell MCF-7 OVCAR HT29	320±25 573±49 431±62 332±39	76±15* 173±22* 113±28 * 89±28 *

*P＜0.05

The result shows that 13 peptides of recombinating can effectively reduce migration, the motor capacity of cell, by topical, is the disease of mechanism with the cell migration applicable to control, as the periphery infiltration of vascellum endometrial hyperplasia and tumour.

Embodiment 3 reorganization 13 peptides are to the restraining effect of tumor cell invasion

With cell inoculation on the millipore filtration of 5 microns of diameters, with filter membrane as between the upper and lower layer of a Boyden cell (inoculating cell is towards last), the nutrient solution that will contain 13 peptides (10,20 μ g/ml) adds goes up chamber (negative control is the same), following chamber all adds the substratum that contains 10% calf serum, hatches the back cell that strikes off inoculating surfaces with cotton swab of taking-up 6 hours in 37 ℃, then film is fixed, get 3 visuals field in microscopically, counting is worn to the cell count at the film back side, represents the invasive ability of cell with this.Detailed results sees Table 2.

Table 2:

Cell type	Negative control group	13 peptide treatment group
			Vascular smooth muscle cell MCF-7 OVCAR HT29	20±5 73±9 131±32 153±39	6±1.5* 17±2.2* 23±2.8 * 29±8 *

^*P＜0.05

The result shows that 13 peptides of recombinating have the ability that suppresses cell invasion, and topical can be used for reducing the invasion and attack and the transfer of tumour.

Embodiment 4 reorganization 13 peptides are to the restraining effect of metastases

Plantation knurl hepatic metastases membranous type in the spleen: the surgical exposure abdominal cavity, get 2 * 10 ⁶Cell after 1 hour, to BALB/c-nu/nu spleen coating, 15 day is got spleen and liver in postoperative with injection cell with the substratum pre-treatment that contains 13 peptides (10,20 μ g/ml), measures knurl volume and hepatic metastases tubercle number.Negative control is the same.Detailed results sees Table 3.

Table 3: recombinate 13 peptides to the influence of tumour hepatic metastases incidence (%, n=20)

The result shows that 13 peptides of recombinating can reduce the tumour liver metastasis incidence.

Embodiment 4

Prepare tablet according to methods known in the art, every contains following compositions:

Reorganization tridecanoic peptide 50mg

Lactose 70mg

Magnesium Stearate 3mg

Polyethylene is than pyrrolidone 130mg

Embodiment 5

Get with each composition once, be prepared into freeze dried injection according to methods known in the art:

Reorganization tridecanoic peptide 150mg

Gelatin hydrolysate 5mg

N.F,USP MANNITOL 10mg

Halfcystine 1.0mg

Injection liquid water 1ml

Chemicals such as solvent for use, reagent, elutriant all can be buied from professional retailer or shop among the present invention.Following content is the English name and the composition of chemicals such as solvent for use among the present invention, reagent, elutriant:

Cell Lysis and Column Buffer (Buffer B1) (a kind of cell pyrolysis liquid and elution buffer):

20mM Na-HEPES(or Tris-HCl or Na-Phosphate)(pH 8.5 or if needed，pH 6.0-9.0)

500mM NaCl(or 50-1000mM NaCl)

1mM EDTA (optional) (a kind of complexing agent)

Buffer B2 (a kind of cell pyrolysis liquid and elution buffer):

20mM Na-HEPES(alternatively，Tris-HCl or Na-Phosphate)(pH 7.0)

500mM NaCl(or 50-1000mM NaCl)

1mM EDTA(optional)

Buffer B3 (Cleavage Buffer for thiol induced cleavage) (a kind of lysis buffer):

20mM Na-HEPES(or Tris-HCl or Na-Phosphate)pH8.5(or pH 8.0-9.0)

500mM NaCl(or 50-1000mM NaCl)

1mM EDTA(optional)

40 mM DTT or β-mercaptoethanol or cysteine ^*(30-50mM)

^*2-mercaptoethanesulfonic acid (being used for the albumen ligation of intein mediation)

(Intein-meditated Protein Ligation (IPL)) (albumen of intein mediation connects) reaction Buffer B4:

The albumen ligation Intein-meditated Protein Ligation (IPL) that is used for the intein mediation

20mM Na-HEPES(or Tris-HCl or Na-Phosphate)pH8.5(or pH 8.0-9.0)

500mM NaCl(or 50-1000mM NaCl)

50mM 2-mercaptoethanesulfonic acid(or 30-50mM)

Stripping Solution I (a kind of albumen strips off liquid):

Be used on the wash-out Chitin post bonded intein-CBD and the target protein of wash-out not; The regeneration of Chitin post.

1％SDS：

20mM Na-HEPES(or Tris-HCl or Na-Phosphate)pH8.5(or pH 8.0-9.0)

500mM NaCl(or 50-1000mM NaCl)

Store at room temperature

Stripping Solution II (being used for the regeneration of Chitin post): 0.3 M NaOH.

Claims (9)

1. regrouped anticancer peptide, its aminoacid sequence is SEQ ID NO.1.

2. the preparation method of the described anticancer peptide of claim 1 may further comprise the steps:

A, usefulness plasmid pTWIN2 carrier, after restriction endonuclease BspQ I handles, insert the tridecanoic peptide of the RGD sequence that contains OPN of sticky end, when the design primer, added halfcystine TGC, carrier after making primer and enzyme is cut processing through primer sex change, annealed method is connected for 16 ℃ by 1: 10 (mol ratio) and spends the night, and obtains recombinant plasmid pTWIN2-RGD;

B, recombinant plasmid is digested through intestinal bacteria, change colibacillus or escherichia coli over to, induce and collect the purifying target protein.

3. include the expression vector of SEQ ID NO.1.

4. the application of the described anticancer peptide of claim 1 in the preparation cancer therapy drug.

5. the application of the described anticancer peptide of claim 1 in preparation treatment bowelcancer medicine.

6. the application of the described anticancer peptide of claim 1 in preparation treatment breast cancer medicines.

7. the application of the described anticancer peptide of claim 1 in preparation treatment ovarian cancer medicine.

8. the application of the described anticancer peptide of claim 1 in preparation treatment liver-cancer medicine.

9. the application of the described anticancer peptide of claim 1 in preparation treatment lung-cancer medicament.

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