CN108203458A - A kind of small peptide and its application - Google Patents
A kind of small peptide and its application Download PDFInfo
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- CN108203458A CN108203458A CN201611190218.3A CN201611190218A CN108203458A CN 108203458 A CN108203458 A CN 108203458A CN 201611190218 A CN201611190218 A CN 201611190218A CN 108203458 A CN108203458 A CN 108203458A
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- albumen
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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
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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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 invention discloses a kind of peptide and its applications.A kind of small peptide provided by the invention, amino acid sequence areIt is described
Description
Technical field
The present invention relates to a kind of small peptide and its applications.
Background technology
Radiotherapy is the clinical classical antitumour treatments for generally employing a nearly century.Why ray can answer extensively
It is because ionising radiation can effectively killing tumor cell for oncotherapy.But in the radiotherapy of tumour, many tumours
It is not high to radiation sensitive, and the serious genomic instability damage such as chromosome double-strand DNA cleavage has frequently been lived through in canceration
The tumour cell of wound has obtained the tolerance to ionization radiation injury, and curative effect is caused to decline, and therefore, improves tumour cell to putting
The problem of sensibility and Mechanism of enhancement sensitivity for the treatment of are always interesting in tumor radiotherapy field.
Radiation is that the G2/M of cell cycle is caused to block to the effect of the eukaryocyte most feature of growth, the tune of G2/M phases
Control is also mostly concerned to the sensibility of radiation with eukaryocyte.On the one hand, the length of G2/M residence times is directly related to tumour
For cell to the sensibility of radiation, this may be because after cell is by actinism, G2/M retardances occur first, when G2/M blocks
Time lengthening makes cell have the sufficient time to carry out DNA reparations, avoids mitotic failure, reduce withering for cell
It dies, therefore cell declines the sensibility of radiation.On the other hand, there is suitable one in radiogenic G2/M retardances signal path
Point gene be both cell cycle checkpoint and with the relevant gene of radiosusceptibility.Such as cell-cycle checkpoint kinase -2
(checkpoint kinase 2, CHK2), the main function in ionization radiation injury access is under damage signal is conducted to
Effector is swum, G2/M is caused to block, meanwhile, overexpression can also increase repellence of the tumour cell to radiation.It is clinical
Statistical result showed, the expression of CHK2 albumen is related with radiotherapy resistance in tumor tissues.Therefore, these cell cycle regulations G2/M
The checkpoint of retardance is often used as the novel targets of tumor radiotherapy enhanced sensitivity.
The cell cycle G2/M retardances of caused by ionizing radiation are a very complicated processes, are related to multiple checkpoints, more
The regulation and control of approach, many levels, wherein most important approach be by two kinase c HK1 of ATM/ATR kinase activators and
CHK2.Damage signal is passed to the effector in downstream by CHK1 and CHK2, leads to cell-cycle arrest in the G2 phases.The effect of the mankind
The factor is answered to include 3 kinds of phosphates, respectively cdc25a, cdc25b, cdc25c, wherein cdc25c turns in phase cell cycle G2-M
It plays a major role during changing.CHK1 and CHK2 can make cdc25c phosphorylations, generate the binding site with 14-3-3 albumen.
Cdc25c is with after 14-3-3 protein bindings, being trapped in cytoplasm, it is impossible to enter nucleus and lose activity.cdc25c
After Ser216 phosphorylations, phosphate esterase active is suppressed, the 14th 's of protein kinase cdc2 that then cell cycle relies on
Threonine (Thr14) and the tyrosine (Tyr15) of the 15th are unable to dephosphorylation, the cdc2/ as G2/M checkpoints master switch
The activity of cyclinB1 compounds will be suppressed, and master switch is in "Off" state, and cell is then arrested in the G2 phases, it is impossible to carry out
Mitosis.In this process, the activity of cdc25c is to ensure that G2/M phase master switch activity is in correct horizontal, can be correct
Carry out mitotic prerequisite.
Invention content
The object of the present invention is to provide a kind of small peptide and its applications.
The present invention provides a kind of small peptide, amino acid sequence is W/FH χ ψIt is describedFor arbitrary amino acid；
The χ is acidic amino acid or basic amino acid.
If χ is acidic amino acid, ψ is arbitrary amino acid.
If χ is basic amino acid, ψ is acidic amino acid.
The amino acid sequence of the small peptide concretely WHDTCFRCAKC.
The amino acid sequence of the small peptide concretely WHEACFRCAKC.
The amino acid sequence of the small peptide concretely WHKDCFTCSNC.
The amino acid sequence of the small peptide concretely WHDKCFTCSNC.
The amino acid sequence of the small peptide concretely WHKDCFVCVTC.
The amino acid sequence of the small peptide concretely WHENCFSCARC.
The amino acid sequence of the small peptide concretely WHEDCLKCACC.
The amino acid sequence of the small peptide concretely WHDYCFHCKKC.
The present invention also application of the protection any description above small peptide in product is prepared.
The purposes of the product is at least one of following (b1)-(b8)：
(b1) increase sensibility of the tumour cell to radiation；
(b2) promote lethal effect of the radiation treatment to tumour cell；
(b3) promote apoptosis-promoting effect of the radiation treatment to tumour cell；
(b4) increase sensibility of the cancerous tissue to radiotherapy；
(b5) promote inhibiting effect of the radiotherapy to cancerous tissue；
(b6) promote lethal effect of the radiotherapy to cancerous tissue；
(b7) radiation treatment is promoted to block the M phases of tumour cell；
(b8) treating cancer.
The purposes of the product is at least one of following (c1)-(c3)：
(c1) with reference to CHK2 albumen or CDC25C albumen；
(c2) CHK2 albumen or CDC25C albumen are detected；
(c3) CHK2 albumen or CDC25C albumen are purified.
The purposes of the product is at least one of following (d1)-(d3)：
(d1) phosphorylation level of CDC25C albumen and/or the phosphorylation level of CDC2 albumen in tumour cell are reduced；
(d2) inhibit the combination of CDC25C albumen and CHK2 albumen in tumour cell；
(d3) inhibit the combination of CDC25C albumen and 14-3-3 albumen in tumour cell.
The present invention also protects a kind of product, and active constituent is the small peptide of any description above.
The purposes of the product is at least one of following (e1)-(e14)：
(e1) increase sensibility of the tumour cell to radiation；
(e2) promote lethal effect of the radiation treatment to tumour cell；
(e3) promote apoptosis-promoting effect of the radiation treatment to tumour cell；
(e4) increase sensibility of the cancerous tissue to radiotherapy；
(e5) promote inhibiting effect of the radiotherapy to cancerous tissue；
(e6) promote lethal effect of the radiotherapy to cancerous tissue；
(e7) treating cancer；
(e8) radiation treatment is promoted to block the M phases of tumour cell；
(e9) with reference to CHK2 albumen or CDC25C albumen；
(e10) CHK2 albumen or CDC25C albumen are detected；
(e11) CHK2 albumen or CDC25C albumen are purified；
(e12) phosphorylation level of CDC25C albumen and/or the phosphorylation level of CDC2 albumen in tumour cell are reduced；
(e13) inhibit the combination of CDC25C albumen and CHK2 albumen in tumour cell；
(e14) inhibit the combination of CDC25C albumen and 14-3-3 albumen in tumour cell.
The present invention also protects a kind of product, the small peptide including irradiation devices and any description above.
The purposes of the product the following is (f1) and/or (f2)：
(f1) killing tumor cell；
(f2) treating cancer.
The irradiation devices can be arbitrary for treating cancer or to cancerous tissue progress radiotherapy or killing tumor cell
Radiotherapy unit.
The present invention also protects the application of any description above small peptide, at least one of following (g1)-(g14)：
(g1) increase sensibility of the tumour cell to radiation；
(g2) promote lethal effect of the radiation treatment to tumour cell；
(g3) promote apoptosis-promoting effect of the radiation treatment to tumour cell；
(g4) increase sensibility of the cancerous tissue to radiotherapy；
(g5) promote inhibiting effect of the radiotherapy to cancerous tissue；
(g6) promote lethal effect of the radiotherapy to cancerous tissue；
(g7) treating cancer；
(g8) radiation treatment is promoted to block the M phases of tumour cell；
(g9) with reference to CHK2 albumen or CDC25C albumen；
(g10) CHK2 albumen or CDC25C albumen are detected；
(g11) CHK2 albumen or CDC25C albumen are purified；
(g12) phosphorylation level of CDC25C albumen and/or the phosphorylation level of CDC2 albumen in tumour cell are reduced；
(g13) inhibit the combination of CDC25C albumen and CHK2 albumen in tumour cell；
(g14) inhibit the combination of CDC25C albumen and 14-3-3 albumen in tumour cell.
The present invention also protects a kind of method of killing tumor cell, includes the following steps：To carrying out the tumour of radiation treatment
Cell uses the small peptide of any description above.
In the method, a concentration of 50nM of use of the small peptide.
Any description above cancerous tissue concretely cervical carcinoma cancerous tissue.
Any description above cancer in particular can be cervical carcinoma.
Any description above tumour cell concretely cervical cancer cell, more specifically can be Hela cells.
Concretely cobalt-60 gamma-radiations are handled any description above radiation treatment.
The dosage of the radiation treatment concretely 8Gy.
The dosage of the radiation treatment concretely 10cGy.
The dosage rate of the radiation treatment concretely 101.8cGy min-1。
There is the shortcomings that specificity is not high, and toxicity is larger in current radiosensitizer.Small peptide radiation provided by the invention
The radiosensitizer that sensitizer will be expected to become new type of safe, and will be the molecule machine that deep excavation tumour cell radiation is resisted
Reason provides new thinking.
Description of the drawings
Fig. 1 is the magnetic bead western blot qualification results of the magnetic bead and combination GST albumen with reference to GST-FHL1 albumen.
Fig. 2 is embodiment 2eLIM small peptides and the combination situation testing result of CHK2 albumen.
Fig. 3 is each magnetic bead western blot qualification results of 3 step 9 of embodiment.
Fig. 4 is 3 step 14eLIM small peptides of embodiment and the combination situation testing result of cell cycle key protein.
Fig. 5 is 4 step 1 western blot testing results of embodiment.
Fig. 6 is 4 step 2 western blot testing results of embodiment.
Fig. 7 is 4 step 3 fluorescence microscope result of embodiment.
Fig. 8 is 4 step 4 flow cytomery result of embodiment.
Fig. 9 is 4 step 4 western blot testing results of embodiment.
Figure 10 is 4 step 5 cell survival rate statistical result of embodiment.
Figure 11 carries out cell division obstacle using immunofluorescence technique for 4 step 6 of embodiment and multinuclear multilevel cases are analyzed
As a result.
Figure 12 is 4 step 7 Apoptosis situation statistical result of embodiment.
Figure 13 takes pictures and volume statistical result for 4 step 8 tumor mass of embodiment.
Figure 14 is 4 step 8 tumor mass western bolt testing results of embodiment.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is conventional method unless otherwise specified.Test material used in following embodiments is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Escherichia coli Rossate：Novagen companies.
PGEX-KG carriers：Amersham companies.
PXJ-40-myc carriers：Biovectors companies.
Hela cells：ATCC, article No.：CCL-2TM。
Sepharose 4B sepharose 4Bs：Pharmacia companies.
C-Myc antibody (article No.s：Sc-40), Cdc25C antibody (article No.：Sc-13138), p-Cdc25C antibody (article No.：sc-
12354), Cdc2 antibody (article No.：Sc-137035), p-Cdc2 antibody (article No.：Sc-7989), Chk2 antibody (article No.：sc-
17747), p-Chk2 antibody (article No.：sc-16297)：Santa Cruz Biotechnology.
GAPDH antibody：Sigma-Aldrich, article No.：G9295.
- 3 antibody of histone (H3Ser10)：Cell Signaling Technology.
α-tubulin antibody, γ-tubulin antibody：Proteintech companies.
FHL1 antibody：Proteintech companies, article No.：10991-1-AP.
Balb/c nude mices：Beijing Vital River Experimental Animals Technology Co., Ltd..
IP buffer solutions：20mM Tris-HCL pH 8.0,0.25MNaCl, 0.5M NP-40,5mM EDTA, when use, add
Enter 1/1000 protease inhibitors and 1M DTT.
CHK2i：Calbiochem companies, No. CAS：516480-79-8.
αR-cdc25C：Santa Cruz Biotechnology, article No.：sc-13138.
14-3-3 antibody：Santa Cruz Biotechnology, article No.：sc-292984.
The recombinant plasmid using carrying target gene in following embodiment prepares the sepharose 4B of binding purpose albumen
Preparation method includes the following steps：
(1) by recombinant plasmid transformed Escherichia coli Rossate, recombinant bacterium is obtained.
(2) recombinant bacterium that step (1) obtains is inoculated in LB fluid nutrient mediums, 37 DEG C, 200rpm shaken cultivations stay overnight,
Obtain seed liquor；Seed liquor is forwarded in fresh LB fluid nutrient mediums by 10% inoculum concentration, 30 DEG C, 200rpm oscillation trainings
It supports to A600nmAddition IPTG (a concentration of 0.1mmol/Ls of the IPTG in cultivating system) during=0.4-0.6, then 20 DEG C,
200rpm shaken cultivations 16-20h.
(3) after completing step (2), by cultivating system, thalline were collected by centrifugation, adds in protein lysate, carries out ultrasound on ice
Broken, then supernatant is collected by centrifugation in 12 000rpm.Add in 100 μ L Sepharose 4B sepharose 4Bs into 20mL supernatants, 4 DEG C
3000r/min low-speed centrifugals collect sepharose 4B after rotation combines 4h, and IP buffer solutions fully elute unbinding protein, and centrifugation is received
Collection precipitation, the sepharose 4B of binding purpose albumen is obtained after precipitation is resuspended using IP buffer solutions.
The method of destination protein supernatant is prepared in following embodiment using the recombinant plasmid for carrying target gene including as follows
Step：By Transfected Recombinant Plasmid Hela cells, 37 DEG C of quiescent cultures collect cell afterwards for 24 hours, after cell is resuspended using IP buffer solutions
30min is placed on ice, and then 4 DEG C, 12000rpm centrifugation 10min collect supernatant, as destination protein supernatant.
Specifically using cobalt-60 gamma-radiations, dosage rate is 101.8cGy min for 8Gy radiation in following embodiment-1。
The discovery of embodiment 1, small peptide
By carrying out sequence analysis and functional analysis to a variety of LIM albumen, a kind of and tumour cell radiosusceptibility is found
Relevant small peptide, is named as eLIM, and block is(Represent any amino acid；χ is acidic amino acid
Or basic amino acid, if χ is an acidic amino acid, ψ can be the amino acid of any property, if χ is a basic amine group
Acid, then ψ can only be acidic amino acid).
The combination detection of embodiment 2, eLIM small peptides and CHK2 albumen
1st, artificial synthesized following 12 small peptides：
Small peptide I：WHDTCFRCAKC；
Small peptide II：WHTDCFRCAKC；
Small peptide III：WHKACFRCAKC；
Small peptide IV：WHEACFRCAKC；
Small peptide V：WHKDCFTCSNC；
Small peptide VI：WHDKCFTCSNC；
Small peptide VII：WHADCFVCVTC；
Small peptide VIII：WHKDCFVCVTC；
Small peptide IX：WHHNCFSCARC；
Small 1-9Nac MBP：WHKNCFSCARC；
Small peptide XI：WHENCFSCARC；
Small peptide XII：WHEDCLKCACC.
The 2nd, small fragment between EcoR I and Xho the I restriction enzyme sites of pGEX-KG carriers is replaced with to the sequence 1 of sequence table
Shown DNA molecular obtains recombinant plasmid pGEX-KG-FHL1.
3rd, the agarose with reference to GST-FHL1 albumen is prepared in the recombinant plasmid pGEX-KG-FHL1 obtained using step 2
Pearl.
4th, recombinant plasmid pGEX-KG-FHL1 is substituted using pGEX-KG carriers, is operated according to step 3, with reference to
The sepharose 4B of GST albumen.
The magnetic bead western blot qualification results that step 3 and step 4 obtain are shown in Fig. 1 (one of western blot uses
Resist for GST antibody).
5th, the small fragment between HindIII the and Kpn I restriction enzyme sites of pXJ-40-myc carriers is replaced with into sequence table
DNA molecular shown in sequence 2 obtains recombinant plasmid pXJ-40-myc-CHK2.
6th, Myc-CHK2 albumen supernatants are prepared in the recombinant plasmid pXJ-40-myc-CHK2 obtained using step 5.
7th, following packet transaction is carried out：
(1) 4 DEG C of incubation 4h of small peptide I that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains,
Then the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h are added in.
(2) II4 DEG C of incubation 4h of small peptide that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains,
Then the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h are added in.
(3) III4 DEG C of incubation of small peptide that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains
Then 4h adds in the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h.
(4) IV4 DEG C of incubation 4h of small peptide that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains,
Then the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h are added in.
(5) V4 DEG C of incubation 4h of small peptide that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains,
Then the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h are added in.
(6) VI4 DEG C of incubation 4h of small peptide that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains,
Then the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h are added in.
(7) VII4 DEG C of incubation of small peptide that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains
Then 4h adds in the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h.
(8) VIII4 DEG C of incubation of small peptide that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains
Then 4h adds in the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h.
(9) IX4 DEG C of incubation 4h of small peptide that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains,
Then the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h are added in.
(10) X4 DEG C of incubation 4h of small peptide that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains,
Then the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h are added in.
(11) XI4 DEG C of incubation of small peptide that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains
Then 4h adds in the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h.
(12) XII4 DEG C of incubation of small peptide that the Myc-CHK2 albumen supernatant and 2 μ g steps 1 obtained 500 μ L steps 6 obtains
Then 4h adds in the sepharose 4B of combination GST-FHL1 albumen that 2 μ g steps 3 obtain and 4 DEG C of incubation 4h.
(13) fine jade of combination GST albumen that the Myc-CHK2 albumen supernatant and 2 μ g steps 4 obtained 500 μ L steps 6 obtains
4 DEG C of incubation 4h of lipolysaccharide pearl.
(14) the combination GST-FHL1 eggs that the Myc-CHK2 albumen supernatant and 2 μ g steps 3 obtained 500 μ L steps 6 obtains
White 4 DEG C of incubation 4h of sepharose 4B.
After completing above-mentioned packet transaction, the magnetic bead of each group is collected respectively, carries out western bolt detections (detection primary antibody
For：C-Myc antibody).The Myc-CHK2 albumen supernatants that step 6 is obtained are as positive control.
Shown in result figure 2.In Fig. 2, swimming lane 1-14 is corresponding in turn to the testing result of the magnetic bead of sample (1)-(14) collection, swimming
Road 15 is positive control testing result.
The result shows that swimming lane 1,4,5,6,8,11,12 does not show the Myc positives, corresponding small peptide and CHK2 albumen are represented
With reference to so as to the result of Reverse transcriptase CHK2 albumen and GST albumen.
The eLIM that small peptide I, small peptide IV, small peptide V, small peptide VI, small peptide VIII, small peptide XI, small peptide XII meet embodiment 1 is small
The block component law of peptide, can be combined with CHK2；Remaining small peptide is unsatisfactory for the block composition rule of the eLIM small peptides of embodiment 1
Rule, it is impossible to be combined with CHK2.
Embodiment 3, eLIM small peptides and the Binding experiment of cell cycle key protein
The 1st, small fragment between BamH I and the HindIII restriction enzyme sites of pGEX-KG carriers is replaced with to the sequence of sequence table
DNA molecular shown in row 3 obtains recombinant plasmid pGEX-KG-A.
The 2nd, small fragment between BamH I and the HindIII restriction enzyme sites of pGEX-KG carriers is replaced with to the sequence of sequence table
DNA molecular shown in row 4 obtains recombinant plasmid pGEX-KG-B.
The 3rd, small fragment between BamH I and the HindIII restriction enzyme sites of pGEX-KG carriers is replaced with to the sequence of sequence table
DNA molecular shown in row 5 obtains recombinant plasmid pGEX-KG-C.
The 4th, small fragment between BamH I and the HindIII restriction enzyme sites of pGEX-KG carriers is replaced with to the sequence of sequence table
DNA molecular shown in row 6 obtains recombinant plasmid pGEX-KG-D.
The 5th, small fragment between BamH I and the HindIII restriction enzyme sites of pGEX-KG carriers is replaced with to the sequence of sequence table
DNA molecular shown in row 7 obtains recombinant plasmid pGEX-KG-E.
The 6th, small fragment between BamH I and the HindIII restriction enzyme sites of pGEX-KG carriers is replaced with to the sequence of sequence table
DNA molecular shown in row 8 obtains recombinant plasmid pGEX-KG-F.
The 7th, small fragment between BamH I and the HindIII restriction enzyme sites of pGEX-KG carriers is replaced with to the sequence of sequence table
DNA molecular shown in row 9 obtains recombinant plasmid pGEX-KG-G.
The 8th, small fragment between BamH I and the HindIII restriction enzyme sites of pGEX-KG carriers is replaced with to the sequence of sequence table
DNA molecular shown in row 10 obtains recombinant plasmid pGEX-KG-H.
DNA molecular shown in sequence 3-10 encodes following small peptide successively：
Small peptide A：WHDTCFRCAKC；
Ala-Ile-Ala-Cys-Cys-Arg-Arg：WHKDCFTCSNC；
Small peptide C：WHADCFVCVTC；
Small peptide D：WHDYCFHCKKC；
Small peptide E：WHNDCFNCKKC；
Small peptide F：WHHNCFSCARC；
Small peptide G：YHLDCFACQLC；
Small peptide H：FHKSCFLCMVC.
9th, the magnetic bead with reference to GST- small peptide A fusion proteins, knot is prepared in the recombinant plasmid that step 1-8 preparations are respectively adopted
Close GST- Ala-Ile-Ala-Cys-Cys-Arg-Arg fusion proteins magnetic bead, with reference to the magnetic bead of GST- small peptide C fusion proteins, with reference to GST- small peptide D fusion proteins
Magnetic bead, the magnetic bead with reference to GST- small peptide F fusion proteins, melts with reference to GST- small peptides G the magnetic bead with reference to GST- small peptide E fusion proteins
The magnetic bead of hop protein, the magnetic bead with reference to GST- small peptide H fusion proteins carry out western blot detections, and (western blot are used
Primary antibody be GST antibody).
The western blot testing results of step 9 are shown in Fig. 3.In Fig. 3, swimming lane 1 is the GST eggs that 2 step 4 of embodiment obtains
The white testing result for combining sepharose 4B, swimming lane 2-9 are corresponding in turn to the fine jade of combination GST- small peptide A fusion proteins that step 9 obtains
Lipolysaccharide pearl, the sepharose 4B with reference to GST- small peptide C fusion proteins, combines the sepharose 4B with reference to GST- Ala-Ile-Ala-Cys-Cys-Arg-Arg fusion proteins
The sepharose 4B of GST- small peptide D fusion proteins is merged with reference to the sepharose 4B of GST- small peptide E fusion proteins, with reference to GST- small peptides F
The sepharose 4B of albumen, the sepharose 4B with reference to GST- small peptide G fusion proteins, the agarose with reference to GST- small peptide H fusion proteins
The testing result of pearl.
10th, the small fragment between BamH I and the HindIII restriction enzyme sites of pXJ-40-myc carriers is replaced with into sequence table
Sequence 11 shown in DNA molecular, obtain recombinant plasmid pXJ-40-myc-cdc25C.
11st, the small fragment between BamH I and the HindIII restriction enzyme sites of pXJ-40-myc carriers is replaced with into sequence table
Sequence 12 shown in DNA molecular, obtain recombinant plasmid pXJ-40-myc-14-3-3 ε.
12nd, the small fragment between BamH I and the HindIII restriction enzyme sites of pXJ-40-myc carriers is replaced with into sequence table
Sequence 13 shown in DNA molecular, obtain recombinant plasmid pXJ-40-myc-cdc2.
13rd, the recombinant plasmid that step 10-12 is obtained is respectively adopted, Myc-cdc25C albumen supernatant, Myc-14- is prepared
3-3 3-3 yupsilon protein supernatant Myc-cdc2 albumen supernatants.
14th, the Myc-cdc25C that the Myc-CHK2 albumen supernatant, step 13 that 2 step 6 of embodiment obtains obtain is respectively adopted
Albumen supernatant, Myc-14-3-3 3-3 yupsilon protein supernatant Myc-cdc2 albumen supernatant carry out following packet transaction：
(1) 4 DEG C of incubations of the magnetic bead for the combination GST- small peptide A fusion proteins for obtaining 500 μ L albumen supernatants and 2 μ g steps 9
4h。
(2) 4 DEG C of incubations of the magnetic bead for the combination GST- Ala-Ile-Ala-Cys-Cys-Arg-Arg fusion proteins for obtaining 500 μ L albumen supernatants and 2 μ g steps 9
4h。
(3) 4 DEG C of incubations of the magnetic bead for the combination GST- small peptide C fusion proteins for obtaining 500 μ L albumen supernatants and 2 μ g steps 9
4h。
(4) 4 DEG C of incubations of the magnetic bead for the combination GST- small peptide D fusion proteins for obtaining 500 μ L albumen supernatants and 2 μ g steps 9
4h。
(5) 4 DEG C of incubations of the magnetic bead for the combination GST- small peptide E fusion proteins for obtaining 500 μ L albumen supernatants and 2 μ g steps 9
4h。
(6) 4 DEG C of incubations of the magnetic bead for the combination GST- small peptide F fusion proteins for obtaining 500 μ L albumen supernatants and 2 μ g steps 9
4h。
(7) 4 DEG C of incubations of the magnetic bead for the combination GST- small peptide G fusion proteins for obtaining 500 μ L albumen supernatants and 2 μ g steps 9
4h。
(8) 4 DEG C of incubations of the magnetic bead for the combination GST- small peptide H fusion proteins for obtaining 500 μ L albumen supernatants and 2 μ g steps 9
4h。
(9) 4 DEG C of incubation 4h of the magnetic bead for the combination GST albumen for obtaining 500 μ L albumen supernatants and 2 μ g embodiments, 2 step 2.
After completing above-mentioned packet transaction, the magnetic bead of each group is collected respectively, carries out western bolt detections (detection primary antibody
For：C-Myc antibody).Each albumen supernatant is set as positive control.
The results are shown in Figure 4.In Fig. 4, the testing result of the magnetic bead of 1-9 correspondence (1)-(9) collection of swimming lane.Swimming lane 10 is
The testing result of positive control.Swimming lane 1,2,4 shows positive findings, and representing corresponding small peptide can be combined with destination protein.
The result shows that small peptide A, Ala-Ile-Ala-Cys-Cys-Arg-Arg, small peptide D meet the block component law of the eLIM small peptides of embodiment 1, therefore energy
It is enough to be combined with CHK2 and CDC25C；Remaining small peptide is unsatisfactory for the block component law of the eLIM small peptides of embodiment 1, therefore can not
It is combined with CHK2 and CDC25C.
Show that the small peptide of the block component law for the eLIM small peptides for meeting embodiment 1 can be with CHK2 based on the above results
It is specifically bound with CDC25C, the small peptide I that step 1 synthesizes is named as eLIM-1, small peptide XI is named as eLIM-2, small peptide VII
It is named as eLIM-C；ELIM-1 and eLIM-2 meets the block component law of the eLIM small peptides of embodiment 1, and eLIM-C is discontented with full
Apply the block component law of the eLIM small peptides of example 1.
Embodiment 4, the property of eLIM small peptides and application
First, eLIM small peptides reduce the phosphorylation level of CDC2 and CDC25C
1st, by Hela cell inoculations to 6cm2Ware is (per hole 106A cell), using DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-) 37 DEG C of quiescent cultures until cell length to 80%.
2nd, after completing step 1, the 6cm is taken2Ware carries out following packet transaction：
Experimental group 1：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% pair
It is anti-), 37 DEG C stand 12 hours；
Experimental group 2：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% pair
It is anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 3：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 5nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 37 DEG C stand 12 hours；
Experimental group 4：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 5nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 5：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 37 DEG C stand 12 hours；
Experimental group 6：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 7：Culture solution supernatant is abandoned in suction, add in the eLIM-2 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 37 DEG C stand 12 hours；
Experimental group 8：Culture solution supernatant is abandoned in suction, add in the eLIM-2 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 9：Culture solution supernatant is abandoned in suction, add in containing 50nMeLIM-C DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 37 DEG C stand 12 hours；
Experimental group 10：Culture solution supernatant is abandoned in suction, add in containing 50nMeLIM-C DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 8Gy radiation is given, 37 DEG C stand 12 hours.
3rd, it after completing step 2, collects cell and carries out western-blot experiments.
The results are shown in Figure 5.Swimming lane 1-10 is followed successively by the experimental result of experimental group 1-10.The result shows that eLIM-1 and
ELIM-2 reduces the phosphorylation level of CDC25C and CDC2 in Hela cells, and eLIM-C cannot reduce CDC25C in Hela cells
With the phosphorylation level of CDC2.
2nd, eLIM small peptides weaken the combination of CDC25C and CHK2, CDC25C and 14-3-3
1st, by Hela cell inoculations to 10cm2Ware is (per hole 107A cell), using DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 37 DEG C of quiescent cultures are up to cell length to 80%.
2nd, after completing step 1, the 10cm is taken2Ware carries out following packet transaction：
Group 1：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% is dual anti-), 37
DEG C stand 12 hours；
Group 2：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% is dual anti-), is given
8Gy is given to radiate, 37 DEG C stand 12 hours；
Group 3：Culture solution supernatant is abandoned in suction, adds in DMEM culture mediums (10% fetal calf serum+0.1% pair of the eLIM-1 containing 50nM
It is anti-), 37 DEG C stand 12 hours；
Group 4：Culture solution supernatant is abandoned in suction, adds in DMEM culture mediums (10% fetal calf serum+0.1% pair of the eLIM-1 containing 50nM
It is anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Group 5：Culture solution supernatant is abandoned in suction, adds in DMEM culture mediums (10% fetal calf serum+0.1% pair containing 50nMeLIM-C
It is anti-), 37 DEG C stand 12 hours；
Group 6：Culture solution supernatant is abandoned in suction, adds in DMEM culture mediums (10% fetal calf serum+0.1% pair containing 50nMeLIM-C
It is anti-), 8Gy radiation is given, 37 DEG C stand 12 hours.
3rd, after completing step 2, cell precipitation is collected by centrifugation, proceeds as follows：
(1) cell is resuspended using 0.5ml IP buffer solutions, 12000rpm centrifuges 10min after ice bath 30min ultrasonications, inhales
Take supernatant.
(2) supernatant that 420 μ L steps (1) obtain is taken to add in 50 μ L protein G closing 1h, 3000rpm centrifugations 2min to take
Supernatant.
(3) by 4 DEG C of incubation 4h of the supernatant that step (2) is collected and α R-cdc25C (20 μ L) or Normal IgG (1 μ L), add
Enter 50 μ L of protein G, 4 DEG C of incubation 1h, 3000rpm centrifugation 2min, abandon supernatant and collect precipitation.
(4) precipitation that step (3) obtains is washed 4 times using IP buffer solutions, 3000rpm centrifugation 2min abandon supernatant, use IP
Buffer solution carries out Western-blot detections after being resuspended.As a result as shown in Figure 6A.
4th, after completing step 2, cell is collected, carries out Western-blot detections.As a result as shown in Figure 6B.
The result shows that eLIM-1 can weaken the combination of CDC25C and CHK2, CDC25C and 14-3-3, and eLIM-C is not
Energy.
3rd, the Cell permeable of eLIM small peptides
1st, the eLIM-C of eLIM-1 and the FITC label of artificial synthesized FITC labels.
2nd, by Hela cell inoculations to six orifice plates (per hole 105A cell), using DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-) 37 DEG C of quiescent cultures until cell length to 80%.
3rd, after completing step 2, six orifice plate is taken, carries out following packet transaction：
Experimental group 1：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 37 DEG C stand 2 hours；
Experimental group 2：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 37 DEG C stand 12 hours；
Experimental group 3：Culture solution supernatant is abandoned in suction, add in containing 50nMeLIM-C DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 37 DEG C stand 2 hours；
Experimental group 4：Culture solution supernatant is abandoned in suction, add in containing 50nMeLIM-C DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 37 DEG C stand 12 hours.
4th, after completing step 3, nucleus is dyed with DAPI, is observed under inverted fluorescence microscope.
The results are shown in Figure 7.The result shows that eLIM-1 and eLIM-C are respectively provided with cell-penetrating power.
4th, the M phases that eLIM small peptides increase after tumour cell irradiation block
1st, by Hela cell inoculations to 6cm2Ware is (per hole 106A cell), using DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-) 37 DEG C of quiescent cultures until cell length to 80%.
2nd, after completing step 1, the 6cm is taken2Ware carries out following packet transaction：
Experimental group 1：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% pair
It is anti-), 37 DEG C stand 12 hours；
Experimental group 2：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% pair
It is anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 3：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 37 DEG C stand 12 hours；
Experimental group 4：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 5：Culture solution supernatant is abandoned in suction, add in containing 50nMeLIM-C DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 6：Culture solution supernatant is abandoned in suction, adds in CHK2i containing 100nM (CAS 516480-79-8-Calbiochem)
DMEM culture mediums (10% fetal calf serum+0.1% is dual anti-), give 8Gy radiation, 37 DEG C stand 12 hours.
3rd, after completing step 2, cell is collected, M phases cell is carried out using -3 antibody of histone (H3Ser10) of phosphorylation
Dyeing, fluidic cell are detected.The results are shown in Figure 8.In Fig. 8,1-6 is followed successively by the corresponding testing results of experimental group 1-6.
4th, after completing step 2, cell is collected, carries out Western-blot detections.
The results are shown in Figure 9.Swimming lane 1-6 is followed successively by the corresponding testing results of experimental group 1-6.
The result shows that after eLIM-1 and CHK2 inhibitor can significantly increase irradiation M phases of tumour cell block, and
ELIM-C unobvious.
5th, eLIM small peptides reduce the survival rate after tumour cell irradiation
1. by Hela cell inoculations to six orifice plates (per 2000, hole cell), using DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 37 DEG C of quiescent cultures are until cell is adherent.
2. after completing step 1, six orifice plate is taken, carries out following packet transaction：
Experimental group 1：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% pair
It is anti-)；
Experimental group 2：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% pair
It is anti-), give 8Gy radiation；
Experimental group 3：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-)；
Experimental group 4：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), give 8Gy radiation；
Experimental group 5：Culture solution supernatant is abandoned in suction, add in containing 50nMeLIM-C DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), give 8Gy radiation；
Experimental group 6：Culture solution supernatant is abandoned in suction, add in the CHK2i containing 100nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), give 8Gy radiation.
3rd, after completing step 2, continue culture 2-3 weeks, cell is collected, using Crystal Violet Dye to the cell shape in six orifice plates
Into clone dyed, every group of clone is counted respectively.
The results are shown in Figure 10.In Figure 10,1-6 is followed successively by the corresponding testing results of experimental group 1-6.The result shows that eLIM-
1 and CHK2 inhibitor can significantly reduce the survival of tumour cell after irradiation, and eLIM-C unobvious.
6th, eLIM small peptides increase cell division obstacle and multinuclear multilevel cell quantity after tumour cell irradiation
1st, by Hela cell inoculations to 6 orifice plates (per hole 105A cell), using DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-) 37 DEG C of quiescent cultures until cell length to 60%.
2nd, after completing step 1,6 orifice plate is taken, carries out following packet transaction：
Experimental group 1：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% pair
It is anti-), 37 DEG C stand 12 hours；
Experimental group 2：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% pair
It is anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 3：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 4：Culture solution supernatant is abandoned in suction, add in containing 50nMeLIM-C DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 5：Culture solution supernatant is abandoned in suction, adds in CHK2i containing 100nM (CAS 516480-79-8-Calbiochem)
DMEM culture mediums (10% fetal calf serum+0.1% is dual anti-), give 8Gy radiation, 37 DEG C stand 12 hours.
3rd, it after completing step 2, carries out cell division obstacle using immunofluorescence technique and multinuclear multilevel cases is analyzed.Primary antibody
Using α-tubulin antibody (green) and γ-tubulin antibody (red), nucleus is dyed (blue) with DAPI.
As a result as shown in figure 11.Arrow in the first row figure represents multistage spindle, and the arrow in the second row figure represents more
Core.
The result shows that eLIM-1 and CHK2 inhibitor can significantly increase irradiation after tumour cell division obstacle and multinuclear it is more
The quantity of grade cell, and eLIM-C unobvious.
7th, eLIM small peptides increase the Apoptosis after tumour cell irradiation
1st, by Hela cell inoculations to 6 orifice plates (per hole 105A cell), using DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-) 37 DEG C of quiescent cultures until cell length to 80%.
2nd, after completing step 1,6 orifice plate is taken, carries out following packet transaction：
Experimental group 1：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% pair
It is anti-), 37 DEG C stand 12 hours；
Experimental group 2：Culture solution supernatant is abandoned in suction, is added in containing fresh DMEM culture mediums (10% fetal calf serum+0.1% pair
It is anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 3：Culture solution supernatant is abandoned in suction, add in the eLIM-1 containing 50nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 4：Culture solution supernatant is abandoned in suction, add in containing 50nMeLIM-C DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 8Gy radiation is given, 37 DEG C stand 12 hours；
Experimental group 5：Culture solution supernatant is abandoned in suction, add in the CHK2i containing 100nM DMEM culture mediums (10% fetal calf serum+
0.1% is dual anti-), 8Gy radiation is given, 37 DEG C stand 12 hours.
3rd, after completing step 2, cell, flow cytometry analysis Apoptosis situation are collected.
As a result as shown in figure 12.In Figure 12,1-5 is followed successively by the corresponding testing results of experimental group 1-5.The result shows that eLIM-
1 and CHK2 inhibitor can significantly increase the apoptosis of HeLa cells after irradiation, and eLIM-2 unobvious.
8th, zoopery
Experimental animal is：Balb/c nude mices (female, 6 week old)
1st, to 5,000,000 Hela cells of every experimental animal right hind intramuscular injection.
2nd, treat tumour length to 150mm3Experimental animal is randomly divided into five groups (every group 10) by size, respectively the 0th day,
It is handled as follows within 7th day, the 14th day and the 21st day：
Group I (control group)：Without any processing.
Group II：Carry out tumor by local radiotherapy in the treatment.
Group III：200 μ g/kg eLIM-1 of intratumor injection (eLIM-1 uses physiological saline solution) carry out tumor by local and put afterwards
Treat treatment.
Group IV：200 μ g/kgeLIM-C of intratumor injection (eLIM-C uses physiological saline solution) carry out tumor by local radiotherapy afterwards
Treatment.
Group V：100 μ g/kgCHK2i of intratumor injection (CHK2i uses physiological saline solution) carry out tumor by local radiotherapy and control afterwards
It treats.
Radiotherapy in the treatment method is cobalt-60 gamma-radiations, and dosage rate is 101.8cGy min-1, dosage 10cGy.
Measure gross tumor volume at the 0th day, the 7th day, the 14th day and the 21st day, the 28th day respectively, the 28th day when is taken off at neck
Dead mouse removes tumor mass.Tumor mass is taken pictures as shown in figure 13 with volume statistical result.
Tumour tumor mass is chosen from every group at random, carries out Western-blot analyses.As a result as shown in figure 14.
The result shows that after intratumor injection eLIM-1 and CHK2 inhibitor carry out tumor by local radiotherapy, compared with the control group, swell
Knurl size is obviously reduced, and illustrates that eLIM-1 and CHK2 inhibitor can enhance sensibility of the tumour to radiotherapy, and eLIM-C effects
Unobvious.Carry out Western-blot analyses to tumor mass, pCDC25C the and pCDC2 levels of eLIM-1 or CHK2i groups will less than pair
According to group, and eLIM-C groups change unobvious.
<110>Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A
<120>A kind of small peptide and its application
<130> GNCYXMN162207
<160> 13
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<220>
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atggcggaga agtttgactg ccactactgc agggatccct tgcaggggaa gaagtatgtg 60
caaaaggatg gccaccactg ctgcctgaaa tgctttgaca agttctgtgc caacacctgt 120
gtggaatgcc gcaagcccat cggtgcggac tccaaggagg tgcactataa gaaccgcttc 180
tggcatgaca cctgcttccg ctgtgccaag tgccttcacc ccttggccaa tgagaccttt 240
gtggccaagg acaacaagat cctgtgcaac aagtgcacca ctcgggagga ctcccccaag 300
tgcaaggggt gcttcaaggc cattgtggca ggagatcaaa acgtggagta caaggggacc 360
gtctggcaca aagactgctt cacctgtagt aactgcaagc aagtcatcgg gactggaagc 420
ttcttcccta aaggggagga cttctactgc gtgacttgcc atgagaccaa gtttgccaag 480
cattgcgtga agtgcaacaa ggccatcaca tctggaggaa tcacttacca ggatcagccc 540
tggcatgccg attgctttgt gtgtgttacc tgctctaaga agctggctgg gcagcgtttc 600
accgctgtgg aggaccagta ttactgcgtg gattgctaca agaactttgt ggccaagaag 660
tgtgctggat gcaagaaccc catcactggg tttggtaaag gctccagtgt ggtggcctat 720
gaaggacaat cctggcacga ctactgcttc cactgcaaaa aatgctccgt gaatctggcc 780
aacaagcgct ttgttttcca ccaggagcaa gtgtattgtc ccgactgtgc caaaaagctg 840
taa 843
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atgtctcggg agtcggatgt tgaggctcag cagtctcatg gcagcagtgc ctgttcacag 60
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tcctctacca gcacgatgcc aaactccagc cagtcctctc actccagctc tgggacactg 180
agctccttag agacagtgtc cactcaggaa ctctattcta ttcctgagga ccaagaacct 240
gaggaccaag aacctgagga gcctacccct gccccctggg ctcgattatg ggcccttcag 300
gatggatttg ccaatcttga atgtgtgaat gacaactact ggtttgggag ggacaaaagc 360
tgtgaatatt gctttgatga accactgctg aaaagaacag ataaataccg aacatacagc 420
aagaaacact ttcggatttt cagggaagtg ggtcctaaaa actcttacat tgcatacata 480
gaagatcaca gtggcaatgg aacctttgta aatacagagc ttgtagggaa aggaaaacgc 540
cgtcctttga ataacaattc tgaaattgca ctgtcactaa gcagaaataa agtttttgtc 600
ttttttgatc tgactgtaga tgatcagtca gtttatccta aggcattaag agatgaatac 660
atcatgtcaa aaactcttgg aagtggtgcc tgtggagagg taaagctggc tttcgagagg 720
aaaacatgta agaaagtagc cataaagatc atcagcaaaa ggaagtttgc tattggttca 780
gcaagagagg cagacccagc tctcaatgtt gaaacagaaa tagaaatttt gaaaaagcta 840
aatcatcctt gcatcatcaa gattaaaaac ttttttgatg cagaagatta ttatattgtt 900
ttggaattga tggaaggggg agagctgttt gacaaagtgg tggggaataa acgcctgaaa 960
gaagctacct gcaagctcta tttttaccag atgctcttgg ctgtgcagta ccttcatgaa 1020
aacggtatta tacaccgtga cttaaagcca gagaatgttt tactgtcatc tcaagaagag 1080
gactgtctta taaagattac tgattttggg cactccaaga ttttgggaga gacctctctc 1140
atgagaacct tatgtggaac ccccacctac ttggcgcctg aagttcttgt ttctgttggg 1200
actgctgggt ataaccgtgc tgtggactgc tggagtttag gagttattct ttttatctgc 1260
cttagtgggt atccaccttt ctctgagcat aggactcaag tgtcactgaa ggatcagatc 1320
accagtggaa aatacaactt cattcctgaa gtctgggcag aagtctcaga gaaagctctg 1380
gaccttgtca agaagttgtt ggtagtggat ccaaaggcac gttttacgac agaagaagcc 1440
ttaagacacc cgtggcttca ggatgaagac atgaagagaa agtttcaaga tcttctgtct 1500
gaggaaaatg aatccacagc tctaccccag gttctagccc agccttctac tagtcgaaag 1560
cggccccgtg aaggggaagc cgagggtgcc gagaccacaa agcgcccagc tgtgtgtgct 1620
gctgtgttgt ga 1632
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<213>Artificial sequence
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<400> 3
tggcatgaca cctgcttccg ctgtgccaag tgc 33
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<213>Artificial sequence
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<400> 4
tggcacaaag actgcttcac ctgtagtaac tgc 33
<210> 5
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<213>Artificial sequence
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tggcatgccg attgctttgt gtgtgttacc tgc 33
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tggcacgact actgcttcca ctgcaaaaaa tgc 33
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tggcaccaca actgcttctc ctgcgcccgc tgc 33
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tatcacctcg actgcttcgc ctgccagctc tgc 33
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ttccataaat cctgcttcct gtgcatggtc tgc 33
<210> 11
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<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 11
atgtctacgg aactcttctc atccacaaga gaggaaggaa gctctggctc aggacccagt 60
tttaggtcta atcaaaggaa aatgttaaac ctgctcctgg agagagacac ttcctttacc 120
gtctgtccag atgtccctag aactccagtg ggcaaatttc ttggtgattc tgcaaaccta 180
agcattttgt ctggaggaac cccaaaacgt tgcctcgatc tttcgaatct tagcagtggg 240
gagataactg ccactcagct taccacttct gcagaccttg atgaaactgg tcacctggat 300
tcttcaggac ttcaggaagt gcatttagct gggatgaatc atgaccagca cctaatgaaa 360
tgtagcccag cacagcttct ttgtagcact ccgaatggtt tggaccgtgg ccatagaaag 420
agagatgcaa tgtgtagttc atctgcaaat aaagaaaatg acaatggaaa cttggtggac 480
agtgaaatga aatatttggg cagtcccatt actactgttc caaaattgga taaaaatcca 540
aacctaggag aagaccaggc agaagagatt tcagatgaat taatggagtt ttccctgaaa 600
gatcaagaag caaaggtgag cagaagtggc ctatatcgct ccccgtcgat gccagagaac 660
ttgaacaggc caagactgaa gcaggtggaa aaattcaagg acaacacaat accagataaa 720
gttaaaaaaa agtatttttc tggccaagga aagctcagga agggcttatg tttaaagaag 780
acagtctctc tgtgtgacat tactatcact cagatgctgg aggaagattc taaccagggg 840
cacctgattg gtgatttttc caaggtatgt gcgctgccaa ccgtgtcagg gaaacaccaa 900
gatctgaagt atgtcaaccc agaaacagtg gctgccttac tgtcggggaa gttccagggt 960
ctgattgaga agttttatgt cattgattgt cgctatccat atgagtatct gggaggacac 1020
atccagggag ccttaaactt atatagtcag gaagaactgt ttaacttctt tctgaagaag 1080
cccatcgtcc ctttggacac ccagaagaga ataatcatcg tgttccactg tgaattctcc 1140
tcagagaggg gcccccgaat gtgccgctgt ctgcgtgaag aggacaggtc tctgaaccag 1200
tatcctgcat tgtactaccc agagctatat atccttaaag gcggctacag agacttcttt 1260
ccagaatata tggaactgtg tgaaccacag agctactgcc ctatgcatca tcaggaccac 1320
aagactgagt tgctgaggtg tcgaagccag agcaaagtgc aggaagggga gcggcagctg 1380
cgggagcaga ttgcccttct ggtgaaggac atgagcccat ga 1422
<210> 12
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<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 12
atggatgatc gagaggatct ggtgtaccag gcgaagctgg ccgagcaggc tgagcgatac 60
gacgaaatgg tggagtcaat gaagaaagta gcagggatgg atgtggagct gacagttgaa 120
gaaagaaacc tcctatctgt tgcatataag aatgtgattg gagctagaag agcctcctgg 180
agaataatca gcagcattga acagaaagaa gaaaacaagg gaggagaaga caagctaaaa 240
atgattcggg aatatcggca aatggttgag actgagctaa agttaatctg ttgtgacatt 300
ctggatgtac tggacaaaca cctcattcca gcagctaaca ctggcgagtc caaggttttc 360
tattataaaa tgaaagggga ctaccacagg tatctggcag aatttgccac aggaaacgac 420
aggaaggagg ctgcggagaa cagcctagtg gcttataaag ctgctagtga tattgcaatg 480
acagaacttc caccaacgca tcctattcgc ttaggtcttg ctctcaattt ttccgtattc 540
tactacgaaa ttcttaattc ccctgaccgt gcctgcaggt tggcaaaagc agcttttgat 600
gatgcaattg cagaactgga tacgctgagt gaagaaagct ataaggactc tacacttatc 660
atgcagttgt tacgtgataa tctgacacta tggacttcag acatgcaggg tgacggtgaa 720
gagcagaata aagaagcgct gcaggacgtg gaagacgaaa atcagtga 768
<210> 13
<211> 894
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 13
atggaagatt ataccaaaat agagaaaatt ggagaaggta cctatggagt tgtgtataag 60
ggtagacaca aaactacagg tcaagtggta gccatgaaaa aaatcagact agaaagtgaa 120
gaggaagggg ttcctagtac tgcaattcgg gaaatttctc tattaaagga acttcgtcat 180
ccaaatatag tcagtcttca ggatgtgctt atgcaggatt ccaggttata tctcatcttt 240
gagtttcttt ccatggatct gaagaaatac ttggattcta tccctcctgg tcagtacatg 300
gattcttcac ttgttaagag ttatttatac caaatcctac aggggattgt gttttgtcac 360
tctagaagag ttcttcacag agacttaaaa cctcaaaatc tcttgattga tgacaaagga 420
acaattaaac tggctgattt tggccttgcc agagcttttg gaatacctat cagagtatat 480
acacatgagg tagtaacact ctggtacaga tctccagaag tattgctggg gtcagctcgt 540
tactcaactc cagttgacat ttggagtata ggcaccatat ttgctgaact agcaactaag 600
aaaccacttt tccatgggga ttcagaaatt gatcaactct tcaggatttt cagagctttg 660
ggcactccca ataatgaagt gtggccagaa gtggaatctt tacaggacta taagaataca 720
tttcccaaat ggaaaccagg aagcctagca tcccatgtca aaaacttgga tgaaaatggc 780
ttggatttgc tctcgaaaat gttaatctat gatccagcca aacgaatttc tggcaaaatg 840
gcactgaatc atccatattt taatgatttg gacaatcaga ttaagaagat gtag 894
Claims (10)
1. a kind of small peptide, amino acid sequence are
It is describedFor arbitrary amino acid；
The χ is acidic amino acid or basic amino acid；
If χ is acidic amino acid, ψ is arbitrary amino acid；
If χ is basic amino acid, ψ is acidic amino acid.
2. small peptide as described in claim 1, it is characterised in that：The amino acid sequence of the small peptide is in following (a1) (a8)
It is any：
(a1)WHDTCFRCAKC；
(a2)WHEACFRCAKC；
(a3)WHKDCFTCSNC；
(a4)WHDKCFTCSNC；
(a5)WHKDCFVCVTC；
(a6)WHENCFSCARC；
(a7)WHEDCLKCACC；
(a8)WHDYCFHCKKC。
3. application of the small peptide described in claims 1 or 2 in product is prepared；The purposes of the product is following (b1)-(b8)
At least one of：
(b1) increase sensibility of the tumour cell to radiation；
(b2) promote lethal effect of the radiation treatment to tumour cell；
(b3) promote apoptosis-promoting effect of the radiation treatment to tumour cell；
(b4) increase sensibility of the cancerous tissue to radiotherapy；
(b5) promote inhibiting effect of the radiotherapy to cancerous tissue；
(b6) promote lethal effect of the radiotherapy to cancerous tissue；
(b7) radiation treatment is promoted to block the M phases of tumour cell；
(b8) treating cancer.
4. application of the small peptide described in claims 1 or 2 in product is prepared；The purposes of the product is following (c1)-(c3)
At least one of：
(c1) with reference to CHK2 albumen or CDC25C albumen；
(c2) CHK2 albumen or CDC25C albumen are detected；
(c3) CHK2 albumen or CDC25C albumen are purified.
5. application of the small peptide described in claims 1 or 2 in product is prepared；The purposes of the product is following (d1)-(d3)
At least one of：
(d1) phosphorylation level of CDC25C albumen and/or the phosphorylation level of CDC2 albumen in tumour cell are reduced；
(d2) inhibit the combination of CDC25C albumen and CHK2 albumen in tumour cell；
(d3) inhibit the combination of CDC25C albumen and 14-3-3 albumen in tumour cell.
6. a kind of product, active constituent is the small peptide described in claims 1 or 2；The purposes of the product be following (e1)-
At least one of (e14)：
(e1) increase sensibility of the tumour cell to radiation；
(e2) promote lethal effect of the radiation treatment to tumour cell；
(e3) promote apoptosis-promoting effect of the radiation treatment to tumour cell；
(e4) increase sensibility of the cancerous tissue to radiotherapy；
(e5) promote inhibiting effect of the radiotherapy to cancerous tissue；
(e6) promote lethal effect of the radiotherapy to cancerous tissue；
(e7) treating cancer；
(e8) radiation treatment is promoted to block the M phases of tumour cell；
(e9) with reference to CHK2 albumen or CDC25C albumen；
(e10) CHK2 albumen or CDC25C albumen are detected；
(e11) CHK2 albumen or CDC25C albumen are purified；
(e12) phosphorylation level of CDC25C albumen and/or the phosphorylation level of CDC2 albumen in tumour cell are reduced；
(e13) inhibit the combination of CDC25C albumen and CHK2 albumen in tumour cell；
(e14) inhibit the combination of CDC25C albumen and 14-3-3 albumen in tumour cell.
7. a kind of product, including the small peptide described in irradiation devices and claims 1 or 2；The purposes of the product the following is (f1)
And/or (f2)：
(f1) killing tumor cell；
(f2) treating cancer.
8. the application of the small peptide described in claims 1 or 2, at least one of following (g1)-(g14)：
(g1) increase sensibility of the tumour cell to radiation；
(g2) promote lethal effect of the radiation treatment to tumour cell；
(g3) promote apoptosis-promoting effect of the radiation treatment to tumour cell；
(g4) increase sensibility of the cancerous tissue to radiotherapy；
(g5) promote radiotherapy to cancerous tissue to the inhibiting effect of tumour；
(g6) promote radiotherapy to cancerous tissue to the lethal effect of tumour；
(g7) treating cancer；
(g8) radiation treatment is promoted to block the M phases of tumour cell；
(g9) with reference to CHK2 albumen or CDC25C albumen；
(g10) CHK2 albumen or CDC25C albumen are detected；
(g11) CHK2 albumen or CDC25C albumen are purified；
(g12) phosphorylation level of CDC25C albumen and/or the phosphorylation level of CDC2 albumen in tumour cell are reduced；
(g13) inhibit the combination of CDC25C albumen and CHK2 albumen in tumour cell；
(g14) inhibit the combination of CDC25C albumen and 14-3-3 albumen in tumour cell.
9. the product as described in application or, claim 4 or 5 as described in claim 3 or 6, it is characterised in that：The tumour
Cell is cervical cancer cell.
10. a kind of method of killing tumor cell, includes the following steps：It will using right to the tumour cell for carrying out radiation treatment
Seek the small peptide described in 1 or 2.
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| CN201611190218.3A CN108203458B (en) | 2016-12-20 | 2016-12-20 | Small peptide and application thereof |
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| CN201611190218.3A CN108203458B (en) | 2016-12-20 | 2016-12-20 | Small peptide and application thereof |
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| US6656705B1 (en) * | 1998-03-26 | 2003-12-02 | The General Hospital Corporation | Sciellin and uses thereof |
| US6756355B1 (en) * | 1996-07-12 | 2004-06-29 | University Of Medicine And Dentistry Of New Jersey | HMGI proteins in cancer and obesity |
| CN101248177A (en) * | 2005-06-24 | 2008-08-20 | 沃尔特及伊莱萨霍尔医学研究院 | Therapeutic pro-apoptotic BH3-like molecules and methods for generating and/or selecting the same |
| CN101683518A (en) * | 2008-09-25 | 2010-03-31 | 中国人民解放军军事医学科学院生物工程研究所 | Application of FHL 1 in preparing medicament for treating tumour |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6756355B1 (en) * | 1996-07-12 | 2004-06-29 | University Of Medicine And Dentistry Of New Jersey | HMGI proteins in cancer and obesity |
| US6656705B1 (en) * | 1998-03-26 | 2003-12-02 | The General Hospital Corporation | Sciellin and uses thereof |
| CN101248177A (en) * | 2005-06-24 | 2008-08-20 | 沃尔特及伊莱萨霍尔医学研究院 | Therapeutic pro-apoptotic BH3-like molecules and methods for generating and/or selecting the same |
| CN101683518A (en) * | 2008-09-25 | 2010-03-31 | 中国人民解放军军事医学科学院生物工程研究所 | Application of FHL 1 in preparing medicament for treating tumour |
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