CN107056887A

CN107056887A - A kind of polypeptide and its application in the medicine for treating and preventing tumour is prepared

Info

Publication number: CN107056887A
Application number: CN201610921217.5A
Authority: CN
Inventors: 胡卓伟; 花芳; 李珂
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-10-22
Filing date: 2016-10-21
Publication date: 2017-08-18
Anticipated expiration: 2036-10-21
Also published as: CN107056887B; WO2017067510A1

Abstract

Application the invention discloses the polypeptide of specific binding TRB3 a kind of and its in the medicine for treating and preventing tumour is prepared.Two or more amino acid substitutions in amino acid sequence of the amino acid sequence of the polypeptide as shown in by sequence table SEQ ID No.8 are shown in the alpha-non-natural amino acid for making other side chains connected.The polypeptide can be combined specifically with TRB3, so that the interaction of TRB3 and P62 albumen is blocked, therefore in the preparation applied to the medicine for treating and preventing tumour.Prepared medicine is in treatment tumor disease, and with evident in efficacy, toxic side effect is few, uses the advantage of safety.

Description

A kind of polypeptide and its application in the medicine for treating and preventing tumour is prepared

Technical field

The invention belongs to biological technical field, and in particular to a kind of polypeptide and its medicine in preparation treatment and prevention tumour In application.

Background technology

TRB3 (Tribbles Homologue 3) is one of Tribbles homologous protein family members, participates in regulation development During propagation, migration and the form of cell formed.TRB3 is as false kinase protein family member, the work(with adaptor protein sample Can, participate in the assembling of multiple protein complex.Multinomial research thinks, TRB3 can be with a variety of transcription factors, ubiquitin ligase, thin II types bmp receptor and MAPK, PI3K signal path member protein interact on after birth, participate in glycolipid metabolism, fat Cell differentiation, apoptosis and the regulation and control that stress be waited.Recently, a variety of evidences show, TRB3 is in kinds of tumor cells system and human tumour group Knit it is middle high expression is presented, and play in the evolution of tumour important facilitation.Research find, TRB3 by with from Bite lorry albumen p62 to interact, suppress the autophagy activity of cell, promote propagation and the transfer of tumour cell.Thus may be used See, the interaction between targeting TRB3 and p62 is a potential target spot for treating tumour.Therefore, research and development is blocked TRB3 and P62 protein-interactings material, with the good patent medicine prospect for suppressing tumour occurrence and development.

Protein-protein interaction (PPIs) plays important role, the increasing of such as cell in many bioprocess Grow, grow, breaking up and programmed death.Many potential therapeutic targets are mainly protein-protein interaction in human diseases. During protein-protein interaction, α spirals and β-pleated sheet secondary structure are the principal contact surfaces units for participating in PPIs.Closely Nian Lai, high activity is obtained with chemical synthesis mode, the synthesis polypeptide class medicine of high selectivity has become new study hotspot.So And, the binding ability of polypeptide and action protein is very weak, and common linear polypeptide can not be through cell membrane and easily by protease water Solution.Therefore, polypeptide target drug also has many not enough at present.As from the foregoing, it would be highly desirable to obtain the phase between targeting TRB3 and p62 Interaction, high activity, the synthesis polypeptide class medicine of high selectivity.

The content of the invention

The technical problems to be solved by the invention are to lack the interaction targetted between TRB3 and p62 for current, High activity, the present situation of the synthesis polypeptide class medicine of high selectivity are there is provided the polypeptide of specific binding TRB3 a kind of and its are preparing Treat and prevent the application in the medicine of tumour.

Experiment of the present inventor by in-depth study and repeatedly is found, targets TRB3 and p62 albumen phase interactions The specific binding capacity and biological stability of polypeptide A 2 (amino acid sequence is referring to sequence table SEQ ID No.8) and TRB3 All than relatively low.And it can not stablize that to form the required alpha helical conformation of activity directly related with polypeptide A 2 in the solution.Thus, send out A person of good sense is targetedly studied and tested, and has been found that the amino acid residue of ad-hoc location in polypeptide A 2 replacing with side chain Two grades of the alpha-non-natural amino acid that can be connected, such as S- amylenes alanine (S5), then α spiral of the improved polypeptide with stabilization Structure, makes improved polypeptide have high affinity, antienzyme Numerical solution and cell-penetrating, that is, improves its α spiral Stability, TRB3 binding abilities and metabolic stability, suppress kinds of tumor cells propagation and shift.Experiments verify that, after transformation Polypeptide can be applied to prepare treat and prevent tumour medicine in.Research work based on inventor, under the present invention is provided The technical scheme stated.

One of technical scheme that the present invention is provided is：A kind of specific binding TRB3 polypeptide, the amino acid of the polypeptide Two or more amino acid substitutions in amino acid sequence of the sequence as shown in by sequence table SEQ ID No.8 are side chain Shown in connectable alpha-non-natural amino acid.

Described makes the connected alpha-non-natural amino acid of other side chains be the conventional alpha-non-natural amino acid in this area, is preferably S- amylenes alanine (S5).

Preferably, in described polypeptide, the number of the amino acid of the replacement is two and the amino acid of the replacement Position is respectively i-th bit and the i-th+3, or, it is i-th bit and the i-th+4, wherein 1≤i≤7, i is positive integer.

More preferably, the amino acid sequence of described polypeptide is such as by sequence table SEQ ID No.1, SEQ ID No.2, SEQ ID Shown in any one of No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7.

Wherein, suitably amino acid can be carried out in the amino acid sequence shown in above-mentioned SEQ ID No.1-SEQ ID No.7 Replace, lack or add, as long as making improved amino acid sequence remain able to that transformation is specifically bound and kept with TRB3 Preceding activity.

The two of the technical scheme that the present invention is provided are：A kind of specific binding TRB3 polypeptide is in preparation treatment and/or in advance Application in the medicine of preventing tumor.

Described tumour is the conventional tumour in this area.Preferably liver cancer, lung cancer, breast cancer, intestinal cancer or leukaemia.Its In, the liver cancer is the conventional liver cancer in this area, preferably primary carcinoma of liver or secondary carcinoma of liver.The lung cancer is this area Conventional lung cancer, preferably ED-SCLC or non-small cell lung cancer.The breast cancer is the conventional breast cancer in this area, compared with It is good for non-infiltration breast cancer, early stage breast cancer, wellability Special Types of Breast Cancer or wellability no special type Breast cancer.The intestinal cancer is the conventional intestinal cancer in this area, preferably colon cancer or the carcinoma of the rectum.The leukaemia is that this area is normal The leukaemia of rule, preferably leukemia or non-lymphocyte type leukaemia.

The prevention is the conventional prevention in this area, is preferably referred to when there is possible tumor diathesis, prevented after use or Reduce the generation of tumour.The treatment is the conventional treatment in this area, preferably refers to the degree for mitigating tumour, or cure tumour Normalization is allowed to, or slows down the process of tumour.

The three of the technical scheme that the present invention is provided are：A kind of antitumor medicine composition, it contains described specificity Active component is used as with reference to TRB3 polypeptide.

Described active component refers to the compound for preventing or treating tumour function.In described pharmaceutical composition, The polypeptide of the specific binding TRB3 can have the compound one of antitumor activity separately as active component or with other Rise and be used as active component.

The method of administration of pharmaceutical composition of the present invention is preferably drug administration by injection or oral administration.It is described to be administered to Medicine preferably includes the approach such as intravenous injection, intramuscular injection, intraperitoneal injection, intracutaneous injection or hypodermic injection.Described medicine group Compound is the conventional various formulations in this area, preferably solid, the form of semi-solid or liquid, can be the aqueous solution, non-aqueous Solution or suspension, more preferably tablet, capsule, granule, injection are transfused agent etc..

It is preferred that pharmaceutical composition of the present invention also includes one or more pharmaceutical carriers.Described pharmaceutical carrier For this area conventional pharmaceutical carrier, described pharmaceutical carrier can be any appropriate physiology or pharmaceutically acceptable medicine Auxiliary material.Described excipient substance is the conventional excipient substance in this area, preferably including pharmaceutically acceptable excipient, filling Agent or diluent etc..More preferably, described pharmaceutical composition includes 0.01-99.99% above-mentioned protein and 0.01- 99.99% pharmaceutical carrier, the percentage is the mass percent for accounting for described pharmaceutical composition.

It is preferred that the amount of application of described pharmaceutical composition is effective dose, the effective dose is that can alleviate or postpone disease The amount of disease, degenerative or damaging disease progression.The effective dose can be determined with individual primary, and will be based partially on and be waited to control Treat the consideration of symptom and sought result.

On the basis of common sense in the field is met, above-mentioned each optimum condition can be combined, and produce each preferable reality of the present invention Example.

Agents useful for same and raw material of the present invention are commercially available.

The positive effect of the present invention is：The polypeptide of the present invention can be combined specifically with TRB3, block TRB3 With the interaction of P62 albumen so that applied to treat and prevent tumour medicine preparation in.Prepared medicine is in treatment In tumor disease, with evident in efficacy, toxic side effect is few, uses the advantage of safety.

Brief description of the drawings

Fig. 1 is the knot of surface plasma resonance method validation polypeptide A 2, S1, S2, S3, S4, S5, S6 and S7 and triblles 3 Conjunction ability.Wherein Fig. 1 (A) is the kinetic curve of polypeptide A 2 and triblles 3；Fig. 1 (B) is that polypeptide S1 is combined with triblles 3 Kinetic curve；Fig. 1 (C) is polypeptide S2 and triblles 3 binding kineticses curve；Fig. 1 (D) is polypeptide S3 and triblles 3 knot Close kinetic curve；Fig. 1 (E) is polypeptide S4 and triblles 3 binding kineticses curve；Fig. 1 (F) is polypeptide S5 and triblles 3 Binding kineticses curve；Fig. 1 (G) is polypeptide S6 and triblles 3 binding kineticses curve；Fig. 1 (H) is polypeptide S7 and TRB3 eggs White binding kineticses curve.Abscissa in Fig. 1 is the reaction time, and unit is the second.Ordinate is reaction chip surface and polypeptide Response intensity, unit is RU.

Fig. 2 is polypeptide A 2, S1, S2, S3, S4, S5, S6 and S7 interference TRB3 and p62 protein-interacting collection of illustrative plates.Wherein A Show that A2, S1, S2 and S3 disturb the interaction of TRB3 and P62 albumen；Wherein B show S4, S5, S6 and S7 interference TRB3 with The interaction of P62 albumen." one " represents input, i.e., the protein content of triblles 3 and P62 albumen in cell pyrolysis liquid；" two " table Show output, i.e., the protein content of contained triblles 3 and P62 albumen after P62 antibody precipitation.

Fig. 3 is that polypeptide S1, S2, S3, S4, S5, S6 and S7 suppress Growth of Human Hepatoma Cell Line HepG 2 result figure.Abscissa be to Medicine time, unit is day.Ordinate is cell quantity, and unit is ten thousand.Compare as polypeptide A 2.

Fig. 4 is that polypeptide S1, S2, S3, S4, S5, S6 and S7 suppress lung cell A549 growth result figure.Abscissa is administration Time, unit is day.Ordinate is cell quantity, and unit is ten thousand.Compare as polypeptide A 2.

Fig. 5 is that polypeptide S1, S2, S3, S4, S5, S6 and S7 suppress breast cancer cell MDA-MB-231 growth result figures.It is horizontal to sit Administration time is designated as, unit is day.Ordinate is cell quantity, and unit is ten thousand.Compare as polypeptide A 2.

Fig. 6 is that polypeptide S1, S2, S3, S4, S5, S6 and S7 suppress colon-cancer cell HCT-8 growth result figures.Abscissa be to Medicine time, unit is day.Ordinate is cell quantity, and unit is ten thousand.Compare as polypeptide A 2.

Fig. 7 is that polypeptide S1, S2, S3, S4, S5, S6 and S7 suppress K562 Leukaemia growth result figure.Abscissa be to Medicine time, unit is day.Ordinate is cell quantity, and unit is ten thousand.Compare as polypeptide A 2.

Fig. 8 is polypeptide A 2, S1, S2, S3, S4, S5, S6 and S7 suppression hepatocellular carcinoma H22 migration results figure.Ordinate is Repaired area ratio after cell cut, unit is percentage.Compare as polypeptide A 2.* * represent p<0.001.

Fig. 9 is polypeptide A 2, S1, S2, S3, S4, S5, S6 and S7 suppression lung cell A549 migration results figure.Ordinate is Repaired area ratio after cell cut, unit is percentage.Compare as polypeptide A 2.* * represent p<0.001.

Figure 10 is polypeptide A 2, S1, S2, S3, S4, S5, S6 and S7 suppression breast cancer cell MDA-MB-231 migration results figures. Ordinate is repaired area ratio after cell cut, and unit is percentage.Compare as polypeptide A 2.* * represent p<0.001.

Figure 11 is polypeptide A 2, S1, S2, S3, S4, S5, S6 and S7 suppression colon-cancer cell HCT-8 migration results figures.Ordinate For repaired area ratio after cell cut, unit is percentage.Compare as polypeptide A 2.* * represent p<0.001.

Figure 12 is polypeptide A 2, S1, S2, S3, S4, S5, S6 and S7 suppression K562 Leukaemia Clone formation result figure.It is vertical Coordinate is Clone formation number, and unit is individual.Compare as polypeptide A 2.* * represent p<0.001.

Embodiment

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to described reality Apply among a scope.The experimental method of unreceipted actual conditions in the following example, conventionally and condition, or according to business Product specification is selected.

Unless otherwise noted, PBS solution used in embodiment, it is the phosphate-buffered that 0.1M, pH value are 7.2 to refer to concentration Liquid.

Room temperature in embodiment is the conventional room temperature in this area, preferably 15-30 DEG C.

Experimental result is represented with mean value ± standard error, through parameter or nonparametric variance test, through comparing p<0.05 thinks There are significant difference, p<0.01 thinks to have extremely significant difference.

The synthesis of the polypeptide of embodiment 1

The amino acid sequence of polypeptide A 2 is referring to sequence table SEQ ID No.8.Polypeptide A 2 is had by Beijing SBS Genetech gene technology Limit company synthesizes and purified.

Introduce two alpha-non-natural amino acid S- amylenes alanine (S5) and carry out solid-phase polypeptide chain synthesis.Solid-phase polypeptide chain is synthesized After the completion of using ruthenium as catalyst carry out olefin metathesis reaction (RCM) cyclisation produce target polypeptides.Finally by target polypeptides Cut down and purified from resin.The step of above-mentioned solid phase polypeptide chain is synthesized and purified is by company of Chinese Peptide Co., Ltd. Complete.Wherein, two S- amylene alanine be inserted in the amino acid sequence of polypeptide A 2 i-th, i+3 or i, i+4, thus Not homotactic improved polypeptide (amino acid sequence is referring to sequence table SEQ ID No.1-SEQ ID No.7) is obtained, it has Body insertion point is as follows：

S1：S5-Gly-Trp-S5-Thr-Arg-Leu-Leu-Gln-Thr-Lys；Insertion point i-th, i+3, i=1；

S2：Gly-S5-Trp-Leu-Thr-S5-Leu-Leu-Gln-Thr-Lys；Insertion point i-th, i+4, i=2；

S3：Gly-Gly-S5-Leu-Thr-Arg-S5-Leu-Gln-Thr-Lys；Insertion point i-th, i+4, i=3；

S4：Gly-Gly-Trp-S5-Thr-Arg-Leu-S5-Gln-Thr-Lys；Insertion point i-th, i+4, i=4；

S5：Gly-Gly-Trp-Leu-S5-Arg-Leu-Leu-S5-Thr-Lys；Insertion point i-th, i+4, i=5；

S6：Gly-Gly-Trp-Leu-Thr-S5-Leu-Leu-Gln-S5-Lys；Insertion point i-th, i+4, i=6；

S7：Gly-Gly-Trp-Leu-Thr-Arg-S5-Leu-Gln-Thr-S5；Insertion point i-th, i+4, i=7.

The method detection polypeptide and the binding ability of triblles 3 of the surface plasma resonance of embodiment 2

Surface plasma resonance experiment is carried out in surface plasma resonance instrument Biacore T200, operating procedure according to etc. Ion resonance instrument Biacore T200 specification is carried out.Comprise the following steps that：

1. the triblles 3 (being purchased from RD companies) of purifying is pressed by (being purchased from GE companies) in amino coupled to CM5 chips 10 μ L/min flow velocity elution removes uncombined albumen, and balances chip surface 2 hours.Wherein, amino coupled, elution and The specific steps of balance referring to GE companies CM5 chips instructions book.

2. the institute of embodiment 1 of the μ L various concentrations (800,400,200,50,12.5,6.25 and 3.125nM) of auto injection 250 S1-S7 the and A2 polypeptide fragments of preparation, the experiment of whole surface plasma resonance is in 25 DEG C of progress.Used buffer solution is HBS- EP buffer solutions [0.01M HEPES, 0.15M NaCl, 3mM EDTA and 0.005% (w/w) surfactant].Use Biacore T200 carries analysis software simulation various concentrations polypeptide and TRB3 binding curve, as a result as shown in Fig. 1 (A)-(H) and table 1.Figure 1 (A)-(H) and table 1 illustrate, the affinity of peptide fragment S1, S2, S3, S4, S5, S6 and S7 and triblles 3 apparently higher than polypeptide A 2 with The affinity of triblles 3.

The affinity test of the polypeptide S1-S7 and A2 of table 1 and triblles 3

The circular dichroism detector of embodiment 3 detects the α spiral rates of polypeptide

With the α spiral rates of circular dichroism spectrometer (being purchased from Jasco companies of Japan) detection polypeptide.Will be many prepared by embodiment 1 Peptide A2, S1, S2, S3, S4, S5, S6 and S7 are dissolved into PBS solution, and the upper machine concentration of circular dichroism spectrometer is adjusted into 1mg/mL, As a result it is as shown in table 2.Table 2 illustrates that polypeptide S1, S2, S3, S4, S5, S6 and S7 α spirals rate are apparently higher than polypeptide A 2, due to many The α helix secondary structure direct polypeptides of peptide are combined with triblles 3, therefore, the raising of polypeptide S1-S7 α spiral rates with itself and The increase of triblles 3 binding ability, and the propagation of kinds of tumor cells are relevant with transfer.Wherein, α spirals rate refers to holding two The peptide segment number of level structure α spirals accounts for the percentage of total peptide segment number.

The circular dichroism detector of table 2 determines polypeptide α spiral rates

Polypeptide title	A2	S1	S2	S3	S4	S5	S6	S7
									α spiral rates	0.87%	42.1%	39.6%	50.2%	47.8%	46.0%	41.6%	45.0%

The method validation polypeptide of the co-immunoprecipitation of embodiment 4 suppresses albumen p62 and TRB3 combination in cellular level

Wherein, the relevant reagent of co-immunoprecipitation is as follows：

Lysate A liquid：0.6057g Tris alkali, 1.7532g NaCl, 0.1017g MgCl₂·6H₂O、0.0742g EDTA, 10mL glycerine and 10mL 10% (v/v) NP40, plus deionized water are adjusted pH value to 7.6, are settled to 150mL with HCl 191mL, is fully mixed, 0.45 μm of membrane filtration, 4 DEG C of storages.

Lysate B liquid：200 μ L 2M β-phosphoglycerol, 4mL 2.5M NaF, 2mL 100mM PMSF, 200 μ L 1M DTT With 1mg/mL Leu, Pep and Apr each 200 μ L, cumulative volume 9mL, stored in -20 DEG C.Wherein, 2M β-phosphoglycerol, 2.5M NaF, 100mM PMSF, 1M DTT and 1mg/mL Leu, Pep and Apr are stored with stock solutions, except PMSF mother liquor is with first Alcohol is that remaining is using water as solvent beyond solvent is prepared.In use, lysate B lyolysis is frozen, by lysate B liquid:Lysate A Liquid 1:Lysate B liquid is added in lysate A liquid and mixed by 100 volume ratios.

Co-immunoprecipitation washing lotion：1% 7.510% (v/v) glycerine of (v/v) NP40,150mM NaCl, 20mM Hepes, pH With 1mM EDTA.

Protein A/G Plus-Agarose are purchased from Santacruz companies of the U.S..

Concrete operation step is as follows：

1. by hepatoma Hep G 2 cells (being purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) paving 90mm²Culture dish, treats thin Polypeptide A 1, S1, S2, S3, S4, S5, S6 or S7 made from 1mg/mL embodiment 1 are separately added into after born of the same parents are adherent, in 37 DEG C of incubations Cell is collected after being incubated 12 hours in case.

2. with 100:Lysate A liquid and lysate B liquid are configured to 10mL lysates, plus 550 μ l lysates by 1 volume ratio Total protein in the cell that cleavage step 1 is collected, harvesting, each histone is adjusted to same concentrations.200 are taken per histone μ g, are used as control.

3. taking each histone obtained by 200 μ g steps 2 respectively, 2 μ g P62 antibody (being purchased from Sigma companies) are added, simultaneously Add 10 μ L Protein A/G Plus-Agarose (being purchased from Santa Cruze companies) to be fully resuspended, 4 DEG C of slow rotations are shaken It is dynamic to stay overnight.4 DEG C, 3000rpm centrifugation 5min, carefully absorb supernatant.0.5mL co-immunoprecipitation washing lotions are added, are mixed, ice bath is stood 4 DEG C after 1min, 3000rpm centrifuge 30 seconds, carefully absorb supernatant.Repeated washing 5 times, last time stands 5min before centrifuging.It is small The heart absorbs supernatant, adds 30 μ L 2 × sds gel sample loading buffers, mixes, 95 DEG C of denaturation 3min, is transferred quickly to ice bath cold But.12000rpm room temperatures centrifuge 2min, and supernatant is the protein sample of precipitation, take and partly or entirely carry out SDS- polyacrylamides Gel electrophoresis.As a result it is as shown in Figure 2.Fig. 2 result explanation, polypeptide S1, S2, S3, S4, S5, S6 and S7 interference TRB3/p62 eggs White interaction ability is apparently higher than polypeptide A 2.

Embodiment 5 cell count experimental verification polypeptide S1, S2, S3, S4, S5, S6 and S7 can suppress the life of tumour cell It is long

Concrete operation step is as follows：

1. collect hepatocellular carcinoma H22 (being purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), the lung of exponential phase Cancer cell A549 (being purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), colon cancer cell HCT-8 (are purchased from China Medical Science Institute of Basic Medical Sciences of institute), breast cancer cell MDA-MB-231 (be purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) and in vain Blood disease cell K562 (being purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), adjusts cell concentration, the thin of 150,000/mL is made Born of the same parents' suspension.

2. by obtained by 1mL steps 1 cell suspension add 12 orifice plates cultivated (wherein HepG2, A549, HCT-8 and MDA-MB-231 cells used medium is DMEM culture mediums, and K562 cells used medium is 1640 culture mediums, is purchased from Invitrogen companies；Cultivation temperature is 37 DEG C, and culture volume is 1mL), change new culture medium after 12 hours into, and add Enter polypeptide S1, S2, S3, S4, S5, S6 and S7 obtained by 1 μ g/mL embodiments 1.Every other day passed on once, and carried out Count.Culture draws out growth curve after 12 days.Experimental result is shown in Fig. 3-7.Fig. 3-7 illustrates polypeptide S1, S2, S3, S4, S5, S6 It can more suppress the growth of tumour cell compared to A2 with S7.Wherein, growths of the polypeptide S1 relative to polypeptide A 2 to lung carcinoma cell Suppression level be up to 4 times, polypeptide S2 is up to 3 relative to polypeptide A 2 to the suppression level of the growth of liver cancer and colon cancer cell Times, polypeptide S3 is up to 3 times to the suppression level of the growth of lung carcinoma cell relative to polypeptide A 2, and polypeptide S4 is relative to 2 pairs of breasts of polypeptide A The suppression level of the growth of adenocarcinoma cell is up to 3 times, and polypeptide S5 is relative to suppression level of the polypeptide A 2 to the growth of lung carcinoma cell Up to 3 times, polypeptide S6 is up to 3 times to the suppression level of the growth of leukaemia relative to polypeptide A 2, and polypeptide S7 is relative to more Peptide A2 is up to 3 times to the suppression level of the growth of breast cancer cell.

The cell scratch experiment of embodiment 6 checking polypeptide A 1, S1, S2, S3, S4, S5, S6 and S7 suppress after tumour cell cut Healing

Concrete operation step is as follows：

1. first compared with ruler in 6 orifice plates behind with marking pen and draw horizontal line, cross via.

2. it is separately added into 5 × 10 in each hole⁵Individual tumour cell, 37 DEG C of incubator overnight incubations in DMEM culture mediums Cell attachment afterwards.The tumour cell is hepatocellular carcinoma H22, lung cell A549, the colon cancer cell HCT-8 of exponential phase With breast cancer cell MDA-MB-231.

3. second day compares ruler with pipette tips, the horizontal line perpendicular to behind carries out cut as far as possible, and pipette tips are vertical.

4. washing cell with PBS 3 times, the cell under drawing is removed, new culture medium is added, while adding 1 μ g/mL embodiments 1 Prepared polypeptide A 1, S1, S2, S3, S4, S5, S6 and S7.

5. it is then placed in 37 DEG C of 5% (v/v) CO₂Incubator culture, sampling is taken pictures after 24 hours.Experimental result is shown in Fig. 8-11 With table 3-6, wherein ratio of the injury repair area than referring to the damaged area after repairing and preprosthetic damaged area, unit is hundred Fraction.Table 3-6 result explanation, injury repair area is stronger than the transfer ability of bigger explanation tumour cell, after cell cut Healing ability is stronger.Therefore polypeptide S1, S2, S3, S4, S5, S6 and S7 can reduce the healing ability after tumour cell cut.

Table 3 polypeptide S1-S7 and A2 suppress hepatocellular carcinoma H22 and migrated

Polypeptide title	Injury repair area ratio
		A2 (control)	79.4±1.05
S1	22.6±0.33
		S2	18.7±0.51
S3	19.8±0.69
		S4	12.1±0.47
S5	13.3±0.31
		S6	29.1±0.41
S7	20.0±0.35

Table 4 polypeptide S1-S7 and A2 suppress lung cell A549 and migrated

Table 5 polypeptide S1-S7 and A2 suppress colon cancer cell HCT-8 and migrated

Polypeptide title	Injury repair area ratio
		A2 (control)	67.7±2.33
S1	24.5±1.97
		S2	18.2±2.30
S3	12.7±2.37
		S4	12.6±2.29
S5	12.5±2.60
		S6	12.1±1.92
S7	11.2±1.01

Table 6 polypeptide S1-S7 and A2 suppress breast cancer cell MDA-MB-231 and migrated

Polypeptide title	Injury repair area ratio
		A2 (control)	91.3±3.22
S1	36.7±2.91
		S2	20.3±2.11
S3	10.5±1.92
		S4	21.7±3.35
S5	20.4±4.47
		S6	10.4±1.96
S7	17.9±1.05

The colony formation of embodiment 7 checking polypeptide A 1, S1, S2, S3, S4, S5, S6 and S7 suppress gram of leukaemia It is grand to be formed

Its operating procedure is as follows：

1. spread bottom agar：5% (w/w) agar boiling water bath is cooled to 50 DEG C to complete thawing, plus 9 times of 37 DEG C of volumes are in advance The 1640 culture medium (being purchased from Invitrogen companies) of temperature, mixes, adds 24 orifice plates, per hole 0.8mL, room temperature solidification is standby.

2. spread top-layer agar：50 DEG C of 5% (w/w) agar of 0.6mL is added in 9.4mL cell suspensions, is mixed, addition has been spread 24 orifice plates of good bottom agar, per hole 0.8mL.Room temperature solidifies.Every hole cell number 100.Wherein, the preparation method of cell suspension For：K562 Leukaemia is diluted with 1640 culture medium, adjustment concentration is 132 cell/mL.

3. the cell 1640 culture medium obtained by step 2 is incubated into culture 3 weeks in 37 DEG C of incubators, Clone formation is calculated Quantity.

Its result is as shown in Figure 12 and table 7.The result explanation of table 7, polypeptide S1-S7 is relative to polypeptide A 2 to leukaemia The suppression level of Clone formation is significantly improved.

Table 7 polypeptide S1-S7 and A2 suppress the Clone formation of leukaemia

Polypeptide title	Clone formation number (individual)
		A2 (control)	110±2.94
S1	53±5.7
		S2	47±2.3
S3	27±5.5
		S4	30±5.4
S5	34±6.1
		S6	29±5.3
S7	19±2.1

The tumour subcutaneous growth experimental verification polypeptide A 2 of embodiment 8, S1, S2, S3, S4, S5, S6 and S7 suppress tumour cell and existed Growth in Mice Body

Operating procedure is as follows：

1. test consumptive material and reagent：Sterilize EP pipes 1.5mL, 15mL centrifuge tube, pipette tips, filter screen (100 mesh), rayon balls, Tweezers number handle, cotton ball soaked in alcohol, sterile 1mL syringes, 500mL beakers (sterilizing, with preceding according to ultraviolet), PBS (filtering), pancreatin, blood Clearly.

2. experimental animal and packet：4-6 week old male nude mouse 80 is (purchased from the limited public affairs of Beijing magnificent experimental animal of dimension tonneau Department), it is randomly divided into 8 groups：A2, S1, S2, S3, S4, S5, S6 and S7 group, every group 10.

3. it is prepared by cell：The tumour cell of adhere-wall culture is digested with pancreatin, (now cell is reached after pancreatin digestion time State should be unicellular and just adherent not fall), sop up pancreatin.Terminated with the PBS containing 1% serum by 2mL/ wares, by cell Blow down, move in 15mL centrifuge tubes, 1200 leave heart 5min.Supernatant is abandoned, PBS is resuspended, and crosses 100 mesh filter screens once；Cell count, Final concentration of cells is adjusted to 2.5 × 10⁷/mL.The tumour cell is hepatocellular carcinoma H22, the lung carcinoma cell of exponential phase A549, colon cancer cell HCT-8 and breast cancer cell MDA-MB-231.The K562 Leukaemia of suspension is directly collected to 15mL Centrifuge tube, 1200 leave heart 5min.Supernatant is abandoned, PBS is resuspended, and crosses 100 mesh filter screens once；Cell count, adjusts final concentration of cells To 2.5 × 10⁷/mL。

4. tumor cell inoculation：Inoculation 5 × 10⁶Individual tumour cell (μ l of cell suspension 200) is in the nearly armpit of nude mice left upper abdomen It is lower subcutaneous.

5. tumour growth is observed：Subcutaneous injection of tumor cells one week after is treated (5mg/kg body weight, weekly two with polypeptide It is secondary), slide measure record tumor size.

Its result is as shown in table 8- tables 12, and polypeptide S1-S7 is respectively less than 0.001 with the p value that control group A 2 is compared in each table.It is swollen Knurl volume shows that more greatly tumour growth is faster, therefore polypeptide S1, S2, S3, S4, S5, S6 and S7 can suppress tumour cell small Mouse tumor growth.

Table 8 polypeptide S1-S7 and A2 suppress hepatocellular carcinoma H22 in mouse tumor growth

Table 9 polypeptide S1-S7 and A2 suppress colon cancer cell HCT-8 in mouse tumor growth

Polypeptide title	Gross tumor volume (mm³)
		A2 (control)	1754±107.9
S1	583.4±68.2
		S2	501.8±81.9
S3	488.8±62.4
		S4	496.5±81.7
S5	606.4±74.3
		S6	511.1±54.6
S7	619.8±75.5

Table 10 polypeptide S1-S7 and A2 suppress breast cancer cell MDA-MB-231 in mouse tumor growth

Polypeptide title	Gross tumor volume (mm³)
		A2 (control)	2379.4±165.4
S1	666.1±74.3
		S2	718.7±67.5
S3	570.8±69.4
		S4	730.1±84.7
S5	737.3±83.1
		S6	629.5±54.1
S7	598.8±75.9

Table 11 polypeptide S1-S7 and A2 suppress lung cell A549 in mouse tumor growth

The polypeptide S1 of table 12~S7 and A2 suppresses K562 Leukaemia in mouse tumor growth

Polypeptide title	Gross tumor volume (mm3)
		A2 (control)	1996.4±1.05
S1	572.6±63.9
		S2	638.9±75.1
S3	699.8±69.0
		S4	722.1±94.7
S5	703.3±73.6
		S6	693.1±54.8
S7	589.9±75.2

Above-described embodiment result shows that polypeptide of the invention has significant antitumor action, can be used as active ingredient In preparing anti-tumor drug.

It should be understood that after the above of the present invention has been read, those skilled in the art can make various to the present invention Change or change, these equivalent form of values equally fall within the application appended claims limited range.

Claims

1. a kind of specific binding TRB3 polypeptide, it is characterised in that the amino acid sequence of the polypeptide is such as by sequence table SEQ Two or more amino acid substitutions in amino acid sequence shown in ID No.8 are the connectable non-natural amino of side chain Shown in sour.

2. TRB3 polypeptide is specifically bound as claimed in claim 1, it is characterised in that described makes what other side chains were connected Alpha-non-natural amino acid is S- amylene alanine.

3. TRB3 polypeptide is specifically bound as claimed in claim 2, it is characterised in that the number of the amino acid of the replacement It is respectively i-th bit and the i-th+3 for the position of two and the amino acid of the replacement, or, it is i-th bit and the i-th+4, wherein 1≤i≤7。

4. TRB3 polypeptide is specifically bound as claimed in claim 3, it is characterised in that the amino acid sequence of described polypeptide Such as sequence table SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID Shown in any one of No.6 and SEQ ID No.7.

5. the polypeptide of the specific binding TRB3 as any one of claim 1-4 is preparing treatment and/or pre- preventing tumor Medicine in application.

6. application as claimed in claim 5, it is characterised in that described tumour is liver cancer, lung cancer, breast cancer, intestinal cancer or white Blood disease.

7. application as claimed in claim 6, it is characterised in that described liver cancer is primary carcinoma of liver or secondary carcinoma of liver；Institute Lung cancer is stated for ED-SCLC or non-small cell lung cancer；The breast cancer be non-infiltration breast cancer, early stage breast cancer, Wellability Special Types of Breast Cancer or wellability no special type of mammary cancer；The intestinal cancer is colon cancer or the carcinoma of the rectum；It is described white Blood disease is leukemia or non-lymphocyte type leukaemia.

8. a kind of antitumor medicine composition, it is characterised in that it contains special as any one of claim 1-4 Property combination TRB3 polypeptide.

9. pharmaceutical composition as claimed in claim 8, it is characterised in that it also includes one or more pharmaceutical carriers.

10. pharmaceutical composition as claimed in claim 8, it is characterised in that it contains as any one of claim 1-4 Specific binding TRB3 polypeptide be used as active component；Or, it contains special as any one of claim 1-4 Property combination TRB3 polypeptide and other there is the compound of antitumor activity together as active component.