CN108358999A - A kind of estrogen receptor targeting peptides and its preparation and application - Google Patents

A kind of estrogen receptor targeting peptides and its preparation and application Download PDF

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Publication number
CN108358999A
CN108358999A CN201810043625.4A CN201810043625A CN108358999A CN 108358999 A CN108358999 A CN 108358999A CN 201810043625 A CN201810043625 A CN 201810043625A CN 108358999 A CN108358999 A CN 108358999A
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seq
targeting peptides
estrogen receptor
receptor alpha
sequence
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陈礼明
张俊
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Nanjing University
Nanjing Normal University
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Nanjing Normal University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Abstract

The present invention provides a kind of estrogen receptor alpha targeting peptides, wherein the targeting peptides have such as SEQ ID No：Amino acid sequence shown in 1, the nucleotide sequence such as SEQ ID No of encoding gene：In 4 34~120 shown in；Application the present invention also provides the preparation method of the estrogen receptor alpha targeting peptides and its in preparing estrogen receptor alpha positive breast cancer drug.

Description

A kind of estrogen receptor targeting peptides and its preparation and application
Technical field
The present invention relates to a kind of polypeptide for treating tumour and preparation method and applications, more particularly to a kind of and estrogen Polypeptide of receptor alpha specific binding and preparation method thereof and its answering in preparing estrogen receptor alpha positive breast cancer drug With.
Background technology
In global female cancer, pathogenesis of breast carcinoma number and death toll are all located at front two, and incidence by Year rises, and is the primary killers for endangering women's health.About 70% patient with breast cancer due to estrogen receptor alpha (ER α) be overexpressed, It is clinically ER α positive breast cancers by parting.There is tamoxifen for the targeted drug master of ER α shot designs at present (TAM), fulvestrant (FUL) and arimedex (AI), but ER α breast cancer patients with positive receives these targeted drugs After treating a period of time, different degrees of drug resistance will produce.
In the prior art, the therapy for ER α positive breast cancers further includes immunotherapy and gene therapy, In CN201710110542.8, using CDK4/6 inhibitor and estrogen receptor antagon combination therapy breast cancer, but to immune The long-term inhibition of system be easy to cause liver renal toxicity and embryo's defect etc.；However, current gene therapy although achieve it is considerable Progress, but still will produce some serious adverse reactions over the course for the treatment of simultaneously, as haemocyte is reduced, heart failure and Embryo's defect etc..
Invention content
Goal of the invention：The first object of the present invention is to provide a kind of polypeptide with estrogen receptor alpha specific binding；This Second purpose of invention is to provide the preparation method of this polypeptide；The third object of the present invention is to provide this polypeptide and is preparing Application in estrogen receptor alpha positive breast cancer drug.
Technical solution：Estrogen receptor alpha targeting peptides of the present invention, i.e. I-CTM of α have such as SEQ ID No：Shown in 1 Amino acid sequence, specially：Lys1Phe2Glu3Arg4Gln5Lys6Ile7Leu8Asp9Gln10Arg11Phe12Phe13Glu14Se r15Ser16Glu17Tyr18Cys19Phe20Tyr21Trp22Asp23Ser 24Ala25His26Cys27Ser28Arg29；Its encoding gene Nucleotide sequence such as SEQ ID No：In 4 34~120 shown in.
Wherein, the targeting peptides also have such as SEQ ID No：Amino acid sequence shown in 2, i.e. TAT, specially： Tyr1Gly2Arg3Lys4Lys5Arg6Arg7Gln8Arg9Arg10Arg11；The nucleotide sequence of its encoding gene such as SEQ ID No：In 4 1~33 shown in.
Preferably, by SEQ ID No：Nucleotide sequence shown in 4 is cloned at the multiple cloning sites of expression plasmid, i.e., The amino acid sequence SEQ ID No of the targeting peptides：2 and amino acid sequence SEQ ID No：1 is directly connected by a peptide bond It connects, the targeting peptides, i.e. I-CTM's of TAT- α, amino acid sequence such as SEQ ID No：Shown in 3, specially： Tyr1Gly2Arg3Lys4Lys5Arg6Arg7Gln8Arg9Arg10Arg11Lys12Phe13Glu14Arg15Gln16Lys17Il e18Leu19Asp20Gln21Arg22Phe23Phe24Glu25Ser26Ser27Glu28Tyr29Cys30Phe31Tyr32Trp33As p34Ser35Ala36His37Cys38Ser39Arg40。
The expression vector of the encoding gene of the estrogen receptor alpha targeting peptides, it includes above-mentioned nucleotide sequence SEQ ID No：4.
The host cell of the encoding gene of the estrogen receptor alpha targeting peptides expresses above-mentioned estrogen receptor alpha targeting Peptide.
It is described that there is amino acid sequence SEQ ID No：1 and SEQ ID No：The preparation of 2 estrogen receptor alpha targeting peptides Method includes the following steps：
(1) plasmid construction in first structure domain：Synthesize two mononucleotide chains in first structure domain, wherein positive sequence Such as SEQ ID No：5 shown, reverse sequence such as SEQ ID No：Shown in 6, then with specific b amHI and EcoRI double digestion PET-22b carriers are attached reaction, obtain pET-22b-A carriers (i.e. pET-22b-TAT plasmids)；
The plasmid construction of (2) second structural domains：Synthesize two mononucleotide chains of the second structural domain, wherein positive sequence Such as SEQ ID No：7 shown, reverse sequence such as SEQ ID No：Shown in 8, then with the specific SalI obtained in step (1) and The pET-22b-A carriers of NotI double digestions are attached reaction, obtain pET-22b-A-B carriers (i.e. pET-22b-TAT-CTM Plasmid)；
(3) plasmid construction of third structural domain：Synthesize two mononucleotide chains of third structural domain, wherein positive sequence Such as SEQ ID No：9 shown, reverse sequence such as SEQ ID No：Shown in 10, then with the specific SalI obtained in step (1) and The pET-22b-A-B carriers of NotI double digestions are attached reaction, obtain pET-22b-A-B-C plasmids (i.e. pET-22b-TAT- α I-CTM plasmids)；
(4) the pET-22b-A-B-C plasmids obtained in step (3) are transferred to competent E.coli DH5 α, extract matter Grain is simultaneously sequenced；
(5) the correct plasmid of sequencing in step (4) is transferred to competent E.coli DE3, progress protein expression is simultaneously pure Change, obtains the estrogen receptor alpha targeting peptides.
With amino acid sequence SEQ ID No：1 or have SEQ ID No：1 and 2 estrogen receptor alpha targeting peptides are being made Application in standby estrogen receptor alpha positive breast cancer drug.
Advantageous effect：Compared with prior art, the present invention its remarkable advantage is：Small peptide α I can directly be acted on simultaneously with ER α And it specifically binds；The ER α targeting peptides α I-CTM synthesized in vitro can target that so that intracellular ER α albumen is passed through CMA approach molten Enzyme body is degraded；To influence vigor and the period of cell, the MCF-7 cell viabilities of α I-CTM-RFP are transferred to than being transferred to CTM- RFP's has dropped 6.1%, also results in the cell G0/G1 phases and blocks；Meanwhile the ER α targeting peptides TAT- α I-CTM synthesized in vitro ER α protein levels in cell can not only be reduced, and with the increase of targeting peptides dosage, the proliferation of MCF-7 Breast Cancer Cell Significantly inhibited, cell viability declines about 24.3%.It is provided newly to prepare estrogen receptor alpha positive breast cancer drug Approach.
Description of the drawings
Figure 1A is the Western Bloting for the GST pull-down experiments that α I are combined with ER alpha specifics in 293T cells Result figure；
Figure 1B is the Western for the GST pull-down experiments that α I are combined with ER alpha specifics in MCF-7 and T47D cells Bloting result figures；
Fig. 2A is ER α protein expression levels in ER α targeting peptides α I-CTM degradation ER α positive breast cancer cells MCF-7 Western Bloting result figures；
Fig. 2 B are the real-time PCR knots that ER α targeting peptides α I-CTM influence the mRNA level in-site of ER α in MCF-7 cells Fruit is schemed；
Fig. 3 A are experimental result pictures of the flow cytomery ER α targeting peptides α I-CTM to MCF-7 Cell cycle influences；
Fig. 3 B are the influence result figure that CCK8 kits detect that ER α targeting peptides α I-CTM inhibit MCF-7 cell Proliferations；
Fig. 4 A are targeting peptides TAT- α I-CTM to ER α protein expression levels in ER α positive breast cancer cells MCF-7 Western Bloting result figures；
Fig. 4 B are the influence result that CCK8 kits detection synthesis targeting peptides TAT- α I-CTM inhibit MCF-7 cell Proliferations Figure；
Fig. 5 is the high performance liquid chromatography and Mass Spectrometer Method result figure of the ER α targeting peptides TAT- α I-CTM of synthesis.
Specific implementation mode
Technical scheme of the present invention is further described below in conjunction with drawings and examples.
Biomaterial, carrier, bacterial strain, reagent and kit etc. used in the present invention can be bought by routine business and be obtained , the biological gene engineering operating technology being directed to, such as plasmid extraction, digestions, segment recycling, nucleic acid fragment and matter Grain carrier connection reaction and clone and screening etc., be routine operation in the art or with reference to corresponding commodity specification into Row operation.
Biomaterial, carrier, the source of bacterial strain or model used in the present invention are as follows：
293T cells be 293T (CRL-3216TM)；
MCF-7 cells be MCF-7 (HTB-22TM)；
T47D cells be T47D (HTB-133TM)；
DH5 α and BL21 (DE3) bacterial strain is purchased from Shanghai Wei Di Bioisystech Co., Ltd；
PEGFP-N2, pET-22b and pGEX-6P-1 plasmid are purchased from biological wind website：www.biofeng.com；
BamHI-HF, EcoRI-HF, SalI-HF and NotI-HF enzyme are purchased from New England Biolabs；
Biology, chemical reagent source used in the present invention are as shown in table 1：
1 biology of table, chemical reagent source
The primer of the present invention is as shown in table 2：
2 Primer of table and its nucleotide sequence
Embodiment 1：The chemical synthesis of estrogen receptor alpha targeting peptides
It weighs 1g resins to be put into reactor, DCM is added and is swollen half an hour, then takes out DCM, 1mmol in sequence is added First amino acid L-type glutamic acid of equivalent, the DIC of 1mmol；The DMAP of 1mmol, suitable DMF solution are bubbled anti-with nitrogen Answer 3h；Then 1ml pyridines are added, 1ml acetic anhydrides react half an hour, take out reaction solution, are cleaned with DMF, DCM.It is added appropriate Piperidines removes 9-fluorenylmethyloxycarbonyl protecting group, cleans, ninhydrin detection；Second ammonia of 3mmol in sequence is added into reactor Base acid, 3mmol HOBT and DIC, nitrogen blistering reaction one hour take out liquid, are cleaned with DMF, ninhydrin detection.According to step Rapid aforesaid way sequentially adds amino acid in sequence, until the reaction of last L-type tyrosine terminates.Add a certain amount of 95% trifluoro Acetic acid cutting liquid, concussion reaction 2h, it is therefore an objective to the side chain protecting group of amino acid be cleaved, thick peptide prod is obtained.Analysis Purification and Mass Spectrometer Method：The correctness that the acid molecules amount is detected with ESI-MS mass spectrographs is carried thick peptide with high performance liquid chromatography It is pure to 95% it is pure more than, take off tfa salt, the estrogen receptor alpha targeting peptides pET-22b-TAT- α I-CTM purified.
Embodiment 2：The biosynthesis of estrogen receptor alpha targeting peptides
1, plasmid construction
First, the plasmid construction of first structure domain pET-22b-TAT：Two mononucleotide chains of synthetic primer TAT, sequence HindIII and KpnI restriction enzyme sites are added in row, the positive sequence that BamHI and EcoRI cohesive ends are contained at sequence both ends is SEQ ID No：5, HindIII and KpnI restriction enzyme sites are added in sequence, BamHI and EcoRI cohesive ends are contained at sequence both ends Reverse sequence be SEQ ID No：6.By mononucleotide chain annealing reaction, 50 μ L reaction systems include 5 μ g forward direction monokaryon glycosides Sour chain, the reversed mononucleotide chains of 5 μ g, 5 μ L NEB buffer 2.Reagent is evenly mixed in EP pipes anti-by following conditions It answers, 95 DEG C, 5 minutes；70 DEG C, 10 minutes；Slow cooling is to room temperature.Take 8 μ L reaction products, with 50ng specific bs amHI and EcoRI double digestion pET-22b carriers are attached reaction, obtain pET-22b-TAT.
Secondly, the plasmid construction of the second structural domain pET-22b-TAT-CTM：Two mononucleotides of synthetic primer CTM Chain, the positive sequence containing SalI and NotI cohesive ends is SEQ ID No in primer：7, contain SalI and NotI viscosity end The reverse sequence at end is SEQ ID No：8.By mononucleotide chain annealing reaction, 50 μ L reaction systems include 5 μ g forward direction monokaryons Thuja acid chain, the reversed mononucleotide chains of 5 μ g, 5 μ L NEB buffer 2.Reagent is evenly mixed in EP pipes anti-by following conditions It answers, 95 DEG C, 5 minutes；70 DEG C, 10 minutes；Slow cooling is to room temperature.Take 8 μ L reaction products, with 50ng specificity SalI and NotI double digestion pET-22b-TAT carriers are attached reaction, obtain pET-22b-TAT-CTM.
Finally, the plasmid construction of third structural domain pET-22b-TAT- α I-CTM：Two mononucleotides of synthetic primer α I Chain, the positive sequence containing KpnI and EcoRI cohesive ends is SEQ ID No in primer：9, contain KpnI and EcoRI viscosity The reverse sequence of end is SEQ ID No：10.By mononucleotide chain annealing reaction, 50 μ L reaction systems include 5 μ g positive Mononucleotide chain, the reversed mononucleotide chains of 5 μ g, 5 μ L NEB buffer 2.Reagent is evenly mixed in EP pipes by following Part reacts, 95 DEG C, 5 minutes；70 DEG C, 10 minutes；Slow cooling is to room temperature.8 μ L reaction products are taken, with 50ng specificity KpnI It is attached reaction with EcoRI double digestion pET-22b-TAT-CTM carriers, obtains pET-22b-TAT- α I-CTM plasmids, it will Obtained plasmid is transferred to competent E.coli DH5 α, shakes bacterium extraction plasmid and is sequenced；.
2, expression and purification target protein
The prokaryotic expression plasmid built is transformed into protein expression Escherichia coli DE3 competent cells.First picking list Bacterium colony is placed in the triangular pyramidal bottle of the LB culture mediums containing kanamycins and is cultivated in a small amount overnight.Press 1 within second day:200 ratios Example, which is drawn to be incubated overnight bacterium solution and be added in the LB culture mediums containing kanamycins, is enlarged culture, and 37 DEG C are cultivated to OD values Up to 0.6, the IPTG that final concentration of 0.1mM is added carries out protein induced expression, and inducing temperature is 18 DEG C of overnight incubations.By from The heart collects the bacterium of induced expression overnight, with protein cleavage (50mM Tris, pH 7.5, the 100mM NaCl, 1mM of precooling EDTA, 1Mm EGTA, 1% NP-40) bacterial precipitation is resuspended.1 is pressed into the thalline of resuspension:100 ratios are added protease and inhibit After being incubated 30 minutes on ice, ultrasonication is carried out using cell Ultrasonic Cell Disruptor for agent PMSF and lysozyme.After ultrasound, 10000g, 4 DEG C of high speed centrifugations 30 minutes.After centrifugation, supernatant and PBS is taken to wash Glutathione SepHarose three times Beads carries out 4 DEG C of rotations and is incubated 4 hours.After incubation, pours the mixture into protein purification column and purified.Use wash Buffer A (50mM Tris, pH 7.5,100mM NaCl, 1 mM EDTA, 1Mm EGTA, 1mM DTT) are flushed three times, then are used Wash buffer B (50mM Tris, pH 7.5,100mM NaCl, 1mM DTT) are flushed three times, and finally use release buffer(50mM Tris, pH 7.5,150mM NaCl,1mM DTT,and 20mM reduced glutathione,pH 7.5) albumen, the estrogen receptor alpha targeting peptides purified, high performance liquid chromatography and Mass Spectrometer Method result figure such as Fig. 5 are eluted It is shown.
Embodiment 3：Verify α I small peptides specific binding ER α
GST Pull-down experiments：By the gst fusion protein of the GST zero loads albumen after purification of 5 μ g and 5 μ g respectively with The 30 μ L Glutathione SepHarose beads incubations at room temperature of 500 μ l PBS pre-wash three times combine 1 hour.It incubates After educating, flushed three times with 500 μ l PBS.Two disk of cell that 100mm culture dishes cover with is taken, is scraped and is collected with cell scraper Cell is added RIPA lysates (inhibition of 10 μ l protease has been added) lytic cell of 1ml precoolings, collects cell pyrolysis liquid, take 100 μ l supernatants are divided into two equal portions as Input groups, by remaining cell pyrolysis liquid.Respectively be combined with GST zero loads albumen and 4 DEG C of rotations of beads of gst fusion protein are incubated overnight.After incubation, supernatant 1ml wash buffer (50mM are abandoned Tris, pH 7.5,150mM NaCl, 1mM EDTA, 1mM DTT, 0.1%Nonidet P-40) it washs 3 times, 4 DEG C of rotations every time Turn 5min.Beads is collected, 50 μ L Release Buffe elutions are added.It is proportionally added into 20 μ 5 × Loading of l Buffer. 95 DEG C of heating 10min are detected result by Western Blotting experiments.
Western Bloting experiments：100mm culture dish cells are collected, the 1ml RIPA cracking being pre-chilled is added Liquid (inhibition of 10 μ l protease has been added) lytic cell, collects cell pyrolysis liquid and carries out albumen concentration quantitative, after quantifying in proportion It is added 5 × loading buffer, 95 DEG C are boiled the protein sample of 10 μ l is taken to be loaded in SDS-PAGE glue after ten minutes, are carried out Electrophoresis, then transferring film.After transferring film, takes out pvdf membrane and closed with the skim milk of 50ml 5%.It is cut after closing Purpose band is taken, is put into the centrifuge tube containing 8ml primary antibody buffer solutions, the antibody in centrifuge tube is that 8 μ l GST antibody (are purchased from CST, number 2624,1:1000 dilution), 8 μ l ER Alpha antibodies (be purchased from CST, number 8644,1:1000 dilutions) and 0.8 μ l β- Actin antibody (is purchased from Proteintech, number 60008-1-Ig, 1:10000 dilutions).4 DEG C are jiggled overnight, rear to use TBST buffer solutions wash 5 times, every time 5 minutes.Be added couple horseradish peroxidase secondary antibody horseradish peroxidase couple it is anti- Mouse IgG and anti-rabbit IgG (being purchased from Santa Cruz Biotechnology), antibody is diluted to specifications, secondary antibody and film Incubation at room temperature 1 hour is washed 5 times, every time 5 minutes after secondary antibody is incubated with TBST buffer solutions.It takes out film and developer solution is added, Albumen variation is detected by imaging of biomolecules instrument analysis system.
Plasmid is overexpressed experiment：The day before transfection takes the cell that logarithmic phase is grown, and is inoculated in 6 orifice plates after digestion, 293T Cell 1.7 × 106A/per hole, it is placed in serum free medium, 37 DEG C, 5%CO2, it is small that 18 to 20 is cultivated in cell incubator When, wait for that cell confluency rate reaches 80%-90% and transfected.It (is purchased from according to transfection reagent2000) it says Bright book prepares DNA- transfection reagent mixtures：
A：200 μ l Opti-MEM+12 μ l Lip2000, mixing are stored at room temperature 5min.
B：200 μ l Opti-MEM+4 μ g plasmids, mixing are stored at room temperature 5min.
According to 1:1 ratio is by room temperature stationary incubation 20min after A and B mixings.Old culture medium in 6 orifice plates is sucked, is added 1.5ml is added without dual anti-culture medium, and by above-mentioned transfection mixture in 6 orifice plates hole.37 DEG C, culture 24 is small under the conditions of 5% CO2 When or 48 hours, collect albumen sample and carry out Western Blotting experiment detection.
Inventor screens a small peptide α I for capableing of specific recognition ER α according to the method for the prior art in document, presses According to the method in embodiment 2 using pGEX-6P-1 as vector construction GST expression plasmids, using pEGFP-N2 as vector construction ER α tables Up to plasmid.Expression obtains GST-TAT- α I and GST-TAT- α I-CTM in DE3, then obtains mesh by protein expression and purification Albumen, carry out external Pull-down experiments, ER α be overexpressed in 293T cells, collect cell after 48 hours, cracking takes Clearly with after purification GST-TAT- α I fusion proteins and GST reference proteins be incubated overnight, after washing and elution, carry out Western Bloting experiments；As a result as shown in figure 1；Inventor have chosen ER α positive breast cancer cells MCF-7 and T47D has carried out external pull-down experiments, as a result as shown in fig. 1b respectively.Attached drawing 1A proves α I with 1B experimental results Directly it can act on and specifically bind with ER α.
Embodiment 4：Influences of the α I-CTM to ER α albumen in breast cancer cell
RNA extractions, reverse transcription experiment：Using OMEGA Total RNA extracts kits (purchased from Nanjing Rui Jingte biologies Science and Technology Ltd.), it is operated according to product description.RNA concentration is measured with Nanodrop 2000 after elution, carries out reverse transcription Reaction.Reverse transcription reaction system：Contain 2500ng RNA, 10 μ L5 × PrimeScript RT in the reaction volume of 50 μ L Master Mix and distilled water.It is reacted according to the program of following settings in PCR instrument：37 DEG C, 15min；85 DEG C, 5sec.
Real-time PCR experiments：Using reverse transcription product cDNA as template, PCR amplification is carried out with following primer：10μL Contain 1 μ L cDNA templates in reaction system, 0.2 μ L (10nmol) sequence is SEQ ID No：11 forward primer, 0.2 μ L (10nmol) sequence is SEQ ID No：12 reverse primer, 5 μ 2 × SYBR of L Mix, 3.6 μ L distilled waters. CFX96TMReal-Time System fluorescence quantitative PCR instruments react 40 cycles according to the program of following settings：Pre-degeneration, 95 DEG C, 30sec；Denaturation, 95 DEG C, 5sec；Annealing, 60 DEG C, 30sec.
According to the method in embodiment 2 using pEGFP-N2 as vector construction CTM-RFP, α I-CTM-RFP and α I-mCTM- RFP expression plasmids are overexpressed by plasmid and are tested, be transferred to CTM-RFP, α respectively in ER α positive breast cancer cells MCF-7 I-CTM-RFP and α I-mCTM-RFP.Cell is collected in transfection after 24 hours, extract albumen, is carried out according to the method in embodiment 3 Western Bloting experiment, be added RFP antibody (be purchased from Abcam, number ab62341,1:1000 dilution), ER Alpha antibodies (purchase From CST, number 8644,1:1000 dilutions) and β-actin antibody (purchased from Proteintech, number 60008-1-Ig, 1: 10000 dilutions)；And RNA extractions, reverse transcription experiment and Real-time PCR experiments are carried out according to the method described above and detects ER respectively The expression quantity of the mRNA of α albumen and ER α.Experimental result is respectively as shown in attached drawing 2A and attached drawing 2B.
The experimental result of Real-time PCR is as shown in table 1：
The mRNA relative expression quantities of ER α in table 1.ER α positive breast cancer cells
Group CTM-RFP αI-CTM-RFP αI-mCTM-RFP
Mrna expression amount/2- △ △ Ct 1 0.906613 0.87069
In attached drawing 2A Western Bloting test the results show that in α I-CTM-RFP groups, intracellular ER α albumen Level will be less than other groups；The experimental result of Real-time PCR is shown in attached drawing 2B, the mRNA level in-site of this group of ER α and other The no apparent difference of group, above the experimental results showed that：ER α targeting peptides α I-CTM, which can be targeted, makes intracellular ER α albumen pass through CMA approach is by lysosomal degradation.
Embodiment 5：The influence of α I-CTM cell cycles and vigor
Cell cycle is detected：The cell of logarithmic growth phase is seeded to 6 orifice plates after digestion, waits for that cell confluency rate reaches 80% or so, experiment process is carried out, cell is collected after 24 hours.After PBS is washed twice, 70% ethyl alcohol of -20 DEG C of precoolings is added It is fixed to stay overnight.Supernatant is abandoned, 300 μ L propidium iodide stains liquid (PI) is added, cell precipitation is resuspended.Room temperature is protected from light incubation dyeing 30min, stream type cell analyzer detect the cell cycle.
Cell viability detects：Single cell suspension is made with 0.25% trypsin digestion in logarithmic growth phase cell, profit It is counted with blood counting chamber, inoculating cell suspension (100 holes μ L/, 5000 cells) in 96 orifice plates, 37 DEG C, 5%CO2Cell Overnight incubation in incubator carries out experiment process.Arrive separately at for 24 hours, liquid is changed after 48h, 72h, 100 μ L are added into every hole and contain The cell culture complete medium of 10 μ L CCK-8 solution, culture plate is put into incubator and is incubated 1.5 hours.It is measured with microplate reader Light absorption value at (measuring wavelength 450).Light absorption value size reflects cell activity, according to experimental data, using the time as abscissa, Light absorption value is mapped for ordinate.According to the method in embodiment 2 using pEGFP-N2 as vector construction CTM-RFP, α I-CTM-RFP With α I-mCTM-RFP expression plasmids, α I-CTM-RFP and CTM-RFP are individually transferred in MCF-7 cells, and with CTM- RFP as a contrast, is detected its cell cycle after 24 hours, and experimental result is as shown in table 2 and attached drawing 3A；Make simultaneously With CCK8 kits, detect the influence of its cell proliferation generation according to the specification in kit, experimental result such as table 3 and Shown in attached drawing 3B：
Influences of the table 2.ER α targeting peptides α I-CTM to the MCF-7 cell cycles
Influences of the table 3.ER α targeting peptides α I-CTM to MCF-7 cell Proliferations
Cell cycle experimental result in attached drawing 3A and table 2 is shown, is transferred to the G0/G1 of the MCF-7 cells of α I-CTM-RFP Phase extends, and the S phases shorten；Cell viability testing result in attached drawing 3B and table 3 is shown, is transferred to the MCF-7 cells of α I-CTM-RFP Vigor has dropped 6.1% than be transferred to CTM-RFP, illustrates that the cell Proliferation for being transferred to α I-CTM-RFP is suppressed.The above reality Test the result shows that, α I-CTM cause intracellular ER α protein levels to reduce vigor and the period that can influence cell, and cell is caused to be lived Power declines and the cell G0/G1 phases block.
Embodiment 6：Influences of the ER α targeting peptides TAT- α I-CTM of synthesis to ER α protein expression levels in cell
According to the method chemical synthesis estrogen receptor alpha targeting peptides in embodiment 1, by the polypeptide of various dose and ER α sun Property breast cancer cell MCF-7 be incubated 24 hours, carry out Western Bloting experiments according to the method in embodiment 3, As a result as shown in fig 4；It uses 1640 complete mediums of 2ml that 200 μ g ER α targeting peptides TAT- α I-CTM are added again, adds after mixing In six orifice plates for entering positive breast cancer cells MCF-7, using blank group as control group, 200mg/ml ER α targeting peptides are added TAT- α I-CTM are experimental group.Using CCK8 kits, according to the specification detection ER α targeting peptides TAT- α I- in kit The influence that CTM cell proliferations generate, as a result as shown in attached drawing 4B and table 4.
Influences of the table 4.ER α targeting peptides TAT- α I-CTM to MCF-7 cell Proliferations
Attached drawing 4A's the results show that with ER α targeting peptides TAT- α I-CTM dosage increase, the amounts of ER α protein degradations by It is cumulative to add, the experimental results showed that the ER α targeting peptides TAT- α I-CTM of synthesis can reduce ER α protein levels in cell.Attached drawing 4B And the experimental result of table 4 is shown, MCF-7 cells are after the ER α targeting peptides TAT- α I-CTM that synthesis is added, under cell viability 24.3% has been dropped, has been proliferated by apparent inhibiting effect.
Above example shows：Small peptide α I directly can be acted on and specifically bind with ER α；ER α targeting peptides α I-CTM Can target makes intracellular ER α albumen pass through CMA approach by lysosomal degradation；To influence vigor and the period of cell, cause Cell viability declines and the cell G0/G1 phases block；Meanwhile ER α targeting peptides TAT- α I-CTM can not only reduce ER α in cell Protein level, and with the increase of targeting peptides dosage, the proliferation of MCF-7 Breast Cancer Cell is significantly inhibited.
SEQUENCE LISTING
<110>Nanjing Normal University
<120>A kind of estrogen receptor targeting peptides and its preparation and application
<130> 2017.1.9
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 29
<212> PRT
<213>It is artificial synthesized
<400> 1
Lys Phe Glu Arg Gln Lys Ile Leu Asp Gln Arg Phe Phe Glu Ser Ser
1 5 10 15
Glu Tyr Cys Phe Tyr Trp Asp Ser Ala His Cys Ser Arg
20 25
<210> 2
<211> 11
<212> PRT
<213>It is artificial synthesized
<400> 2
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
<210> 3
<211> 40
<212> PRT
<213>It is artificial synthesized
<400> 3
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Ser Glu Tyr Cys
1 5 10 15
Phe Tyr Trp Asp Ser Ala His Cys Ser Arg Lys Phe Glu Arg Gln Lys
20 25 30
Ile Leu Asp Gln Arg Phe Phe Glu
35 40
<210> 4
<211> 120
<212> DNA
<213>It is artificial synthesized
<400> 4
tatggcagga agaagcggag acagcgacga agaagcagcg agtactgctt ctactgggac 60
agcgcccact gcagccggaa gtttgagcgt cagaagattc tggaccagag gttctttgag 120
<210> 5
<211> 57
<212> DNA
<213>It is artificial synthesized
<400> 5
gatccgtatg gcaggaagaa gcggagacag cgacgaagaa agcttggtac cccgggg 57
<210> 6
<211> 57
<212> DNA
<213>It is artificial synthesized
<400> 6
aattccccgg ggtaccaagc tttcttcgtc gctgtctccg cttcttcctg ccatacg 57
<210> 7
<211> 49
<212> DNA
<213>It is artificial synthesized
<400> 7
tcgacaagtt tgagcgtcag aagattctgg accagaggtt ctttgaggc 49
<210> 8
<211> 49
<212> DNA
<213>It is artificial synthesized
<400> 8
ggccgcctca aagaacctct ggtccagaat cttctgacgc tcaaacttg 49
<210> 9
<211> 49
<212> DNA
<213>It is artificial synthesized
<400> 9
cagcagcgag tactgcttct actgggacag cgcccactgc agccggggg 49
<210> 10
<211> 57
<212> DNA
<213>It is artificial synthesized
<400> 10
aattcccccg gctgcagtgg gcgctgtccc agtagaagca gtactcgctg ctggtac 57

Claims (7)

1. a kind of estrogen receptor alpha targeting peptides, it is characterised in that：The targeting peptides have such as SEQ ID No：Amino shown in 1 Acid sequence, the nucleotide sequence such as SEQ ID No of encoding gene：In 4 34~120 shown in.
2. estrogen receptor alpha targeting peptides according to claim 1, it is characterised in that：The targeting peptides also have such as SEQ ID No：Amino acid sequence shown in 2, the nucleotide sequence such as SEQ ID No of encoding gene：In 4 1~33 shown in.
3. the estrogen receptor alpha targeting peptides described in claim 2, it is characterised in that：By SEQ ID No：Nucleotides sequence shown in 4 Row are cloned at the multiple cloning sites of expression plasmid, i.e., the amino acid sequence SEQ ID No of the described targeting peptides：2 with amino acid sequence Arrange SEQ ID No：1 is directly connected to by a peptide bond.
4. the expression vector of the encoding gene of the estrogen receptor alpha targeting peptides described in claim 1~2, it is characterised in that：It is wrapped Containing the nucleotide sequence SEQ ID No：4.
5. the host cell of the encoding gene of the estrogen receptor alpha targeting peptides described in claim 1~2, it is characterised in that：Its table Up to the estrogen receptor alpha targeting peptides.
6. the preparation method of the estrogen receptor alpha targeting peptides described in claim 2, it is characterised in that：Include the following steps：
(1) plasmid construction in first structure domain：Synthesize two mononucleotide chains in first structure domain, wherein positive sequence such as SEQ ID No：5 shown, reverse sequence such as SEQ ID No：Shown in 6, then the pET-22b loads with specific b amHI and EcoRI double digestion Body is attached reaction, obtains pET-22b-A carriers；
The plasmid construction of (2) second structural domains：Synthesize two mononucleotide chains of the second structural domain, wherein positive sequence such as SEQ ID No：7 shown, reverse sequence such as SEQ ID No：Shown in 8, then with the specific SalI and the bis- enzymes of NotI that are obtained in step (1) The pET-22b-A carriers cut are attached reaction, obtain pET-22b-A-B carriers；
(3) plasmid construction of third structural domain：Synthesize two mononucleotide chains of third structural domain, wherein positive sequence such as SEQ ID No：9 shown, reverse sequence such as SEQ ID No：Shown in 10, then it is bis- with the specific SalI and NotI that are obtained in step (2) The pET-22b-A-B carriers of digestion are attached reaction, obtain pET-22b-A-B-C plasmids；
(4) the pET-22b-A-B-C plasmids obtained in step (3) are transferred to competent E.coli DH5 α, extraction plasmid is simultaneously Sequencing；
(5) the correct plasmid of sequencing in step (4) is transferred to competent E.coli DE3, carries out protein expression and purify, obtains The estrogen receptor alpha targeting peptides.
7. the estrogen receptor alpha targeting peptides described in claim 1~2 are in preparing estrogen receptor alpha positive breast cancer drug Application.
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Application publication date: 20180803