CN105820230B - A kind of anti-tumour active polypeptide and application thereof - Google Patents

A kind of anti-tumour active polypeptide and application thereof Download PDF

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CN105820230B
CN105820230B CN201610164491.2A CN201610164491A CN105820230B CN 105820230 B CN105820230 B CN 105820230B CN 201610164491 A CN201610164491 A CN 201610164491A CN 105820230 B CN105820230 B CN 105820230B
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周建伟
陈敏娟
李爱萍
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Suzhou Mingren Pharmaceutical Biotechnology Co.,Ltd.
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周建伟
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Abstract

The present invention relates to a kind of anti-tumour active polypeptides and application thereof.The polypeptide has anti-tumor activity because having one of following amino acid sequence: RMKKRYPTTFVMVV, wherein Y or from left to right second phosphorylated modification of T of number；IGLKRTPMGIV, the wherein phosphorylated modification of T.The polypeptide can be used for preparing anti-tumor drug.Polypeptide sequence of the present invention is shorter, it is easy to accomplish large-scale production；Significant anti-tumor activity is shown at lower doses.Polypeptide normal tissue cytotoxic of the present invention, has broad application prospects.

Description

A kind of anti-tumour active polypeptide and application thereof
Technical field
The present invention relates to a kind of anti-tumour active polypeptides and application thereof, belong to anti-tumor drug research and development and lead with application technology Domain.
Background technique
Malignant tumour is to seriously endanger the disease of human health.Due to environmental pollution, Exposed, food safety, life The influence of the factors such as mode and racial inheritance feature, the disease incidence of China's malignant tumour are in significant ascendant trend.Including gastric cancer Malignant tumour inside has become the notable obstacle for seriously affecting people's physical and mental health and restricting economic society development in an all-round way.Cause This, finds effective antitumour drug and method, thoroughly captures cancer, be the common severe challenge faced of world-wide medical interface it One.
The treatment of tumour emphasizes that the principle of complex treatment, chemotherapy are one of important means.Anti-tumor drug in recent years Research achieve rapid development, there are some novel anti-tumor drugs, act on the different links of tumor development, It effectively extends the life span of tumor patient, improve quality of life.But also occur many problems: tumour cell simultaneously Drug resistance constantly enhances, and the dosage of anti-tumor drug is caused to be continuously increased, and greatly enhances to the toxic side effect of body.It is existing The study found that chemicals therapeutic effect and tumor cell drug resistance generation with intracellular some gene expression doses Related, the expression for controlling intracellular certain genes can directly inhibit growth, transfer, the generation of drug resistance of tumour cell etc., and The therapeutic effect of other treatment methods such as chemicals is greatly improved, reduces because drug is to the toxic side effect of body, improves patient Life quality brings glad tidings for more tumor patients.
JWA gene, i.e. ARL6IP5 gene (GenBank:AF070523.1,1998；LOCUS:AF070523, NM_ 006407) be that inventor Zhou Jianwei is equal separates clone first from the primary popularity pipe and bronchial epithelial cell of culture The gene induced by all-trans retinoic acid (ATRA), encode a kind of new cytoskeletal binding proteins.Inventor with Neck project group membership conducts a research work all around the structure of JWA gene, function and its with mankind's major disease relationship for many years Make, obtains the continuous support of state natural sciences fund committee fund project always, achieve that a series of originality are significant to grind Study carefully achievement, comprising: (1) albumen for taking the lead in proving and proposing JWA gene coding is a kind of new microtubule associated protein (MAPs), should Albumen can be in conjunction with tubulin α and β subunit, and to environmental physics chemical factor (such as cold shock, heat shock, taxol Deng) response in show the dynamic variation synchronous with tubulin α and β subunit (polymerization or depolymerization).(2) JWA is found Albumen participates in cell mitogen process, and has adjustment effect to the dynamic stability of micro-pipe.(3) JWA albumen passes through MAPK Signal path, the depolymerization of microfilament and recombination adjust tumor cell migration and apoptosis.(4) JWA albumen is to inhibit gastric cancer vascular raw At important molecule, in stomach organization JWA albumen and other tumor cells marker such as XRCC1, MDM2, P53, SOX2, FAK and The joints such as MMP-2 can more preferably predict patient's prognosis；Inventor it has been investigated that, JWA albumen passes through Ubiquitin-proteasome Approach negativity adjusts nuclear transcription factor Sp1 and its downstream gene MMP-2 expresses and then realize its regulation generated to gastric cancer vascular.
Inhibit tumour cell from existing JWA albumen correlative study it is found that being had by the JWA albumen that JWA gene expression obtains Functions, the large-scale production activity JWA albumen such as adherency, infiltration, transfer and inhibition tumor vascular growth are imperative.However, this Invention inventor has found in practical studies, is difficult to obtain the JWA albumen of purifying using host expression system at present, passes through the way Diameter carries out the poor feasibility of internal and external test with the JWA albumen purified.
In addition, matrix metalloproteinase (MMPs) is a large family according to known to inventor, because its need Ca, The metal ions such as Zn are gained the name as confactor.MMPs can almost degrade the various protein ingredients in extracellular matrix (ECM), Play the role of key in tumor cell destruction histology barrier and invasion transfer, this makes MMPs in tumor invasion and metastasis Effect be paid more and more attention, it is considered to be should during main proteolytic enzyme.MMPs family is separated at present identifies 26 members, number are respectively MMP1~26；Wherein, the function of MMP-2 is particularly important, and MMP-2 is on stomach cancer cell surface Height expression, and can be by histidine-tryptophan-Gly-Phe (HWGF) sequence institute specific recognition.
Inventor had once applied for entitled " polypeptide with anti-tumor activity and its use on May 14th, 2013 Chinese invention patent on the way ", Patent No. 201310178099.X, Authorization Notice No. CN103239710B.It is more in the patent The amino acid sequence of peptide is as shown in I, II, III or IV: I: FPGSDRF；II: X-FPGSDRF；III: FPGSDRF-Z；IV: X- FPGSDRF-Z；Wherein, the phosphorylated modification of amino acid S, X, Z are respectively amino acid or amino acid sequence；X is selected from F, (R)9、 (R)9- F, 6-aminocaprolc acid, 6-aminocaprolc acid-F, 6-aminocaprolc acid-(R)9, 6-aminocaprolc acid-(R)9One of-F；Z be selected from A, (G)n-RGD、A-(G)nOne of-RGD.The polypeptide can be used for preparing anti-tumor drug.
After further research, screening and identification obtains another knot to inventor from JWA protein amino acid sequence Structure is different from above-mentioned patent and completely new polypeptide with anti-tumor activity.
Summary of the invention
The technical problems to be solved by the present invention are: being based on the prior art, a kind of anti-tumour active polypeptide is proposed, the polypeptide It is that screening and identification obtains from JWA protein amino acid sequence, the polypeptide that structure is invented before being different from.Meanwhile it also providing The polypeptide is used to prepare the purposes of anti-tumor drug.
Major technique design of the invention is as follows: inventor's screening and identification from JWA protein amino acid sequence before Polypeptide FPGSDRF and its series derivatives peptide with anti-tumor activity are obtained, on this basis, inventor studies discovery JWA Albumen has multiple biological function, and thinks JWA albumen why there are many different biological function, it may be possible to its different piece There is the amino acid sequence of section this active feature of different biological to determine；Therefore, different fragments JWA Argine Monohydrochloride is inquired into The biological function and its application prospect of sequence have very big researching value, it is more likely that therefrom screening and identification goes out other living Property polypeptide.Adhere to this theory, inventor has continued deep experimental study, and screening and identification goes out before structure is different from Invention polypeptide and JWA functional polypeptide with anti-tumor activity.
Technical scheme is as follows:
A kind of anti-tumour active polypeptide, characterized in that the polypeptide has anti-swollen because having one of following amino acid sequence Tumor activity:
RMKKRYPTTFVMVV, wherein Y or from left to right second phosphorylated modification of T of number；
IGLKRTPMGIV, the wherein phosphorylated modification of T.
Preferably, the amino acid sequence of the polypeptide is as shown in I, II, III or IV:
I: RMKKRYPTTFVMVV；
II: X-RMKKRYPTTFVMVV；
III: RMKKRYPTTFVMVV-Z；
IV: X-RMKKRYPTTFVMVV-Z；
In the above formulas, Y or from left to right second phosphorylated modification of T of number, X, Z are respectively amino acid or amino acid sequence Column；
X is selected from F, (R)9、(R)9- F, 6-aminocaprolc acid, 6-aminocaprolc acid-F, 6-aminocaprolc acid-(R)9, 6-aminocaprolc acid- (R)9One of-F；
Z is selected from A, (G)n-HWGF、A-(G)nOne of-HWGF, n are the integer more than or equal to 0.
Preferably, the amino acid sequence of the polypeptide is as shown in V, VI, VII or VIII:
V: IGLKRTPMGIV；
VI: X-IGLKRTPMGIV；
VII: IGLKRTPMGIV-Z；
VIII: X-IGLKRTPMGIV-Z；
In the above formulas, the phosphorylated modification of T, X, Z are respectively amino acid or amino acid sequence；
X is selected from F, (R)9、(R)9- F, 6-aminocaprolc acid, 6-aminocaprolc acid-F, 6-aminocaprolc acid-(R)9, 6-aminocaprolc acid- (R)9One of-F；
Z is selected from A, (G)n-HWGF、A-(G)nOne of-HWGF, n are the integer more than or equal to 0.
Preferably, the value range of the n is 0-10.
Preferably, the N-terminal of the polypeptide is modified through acetylation modification, C-terminal through amidation.
The present invention also proposes: anti-tumour active polypeptide described previously is used to prepare the purposes of anti-tumor drug.
Preferably, the tumour is gastric cancer.
Polypeptide sequence of the present invention is shorter, it is easy to accomplish large-scale production；It shows at lower doses significant anti-swollen Tumor activity.Polypeptide normal tissue cytotoxic of the present invention, has broad application prospects.
Detailed description of the invention
Fig. 1 is known JWA protein amino acid sequence figure.Underscore part is trans-membrane region, and shadow region is specific implementation The segment portion that mode is related to.
The amino acid that wherein each letter represents is as follows: alanine A, arginine R, aspartic acid D, glutamine Q, glutamic acid E, histidine H, isoleucine I, glycine G, asparagine N, leucine L, lysine K, methionine M, phenylalanine F, dried meat Propylhomoserin P, serine S, threonine T, tryptophan W, tyrosine Y, valine V.
Fig. 2 is nonsense peptide (50mg/kg) group, cis-platinum (4mg/kg) group and each polypeptide (50mg/ in the embodiment of the present invention 2 Kg) the tumor formation comparison diagram organized.Wherein, each polypeptide is respectively as follows: polypeptide PJP2, polypeptide PJP3-1, polypeptide PJP3-2, polypeptide PJP3- 3, polypeptide PJP4, polypeptide PJP5, polypeptide PJP6-1, polypeptide PJP6-2 and polypeptide PJP6-3.In figure, ctrl-peptide is nonsense Peptide, DDP are cis-platinum.
Fig. 3 is the knurl product change curve of the embodiment of the present invention 2, and arrow indicates administration time.Each English word contains Justice is identical as Fig. 2.In figure, by the 14th day curve point arrangement relationship, from top to bottom successively are as follows: polypeptide PJP6-3, polypeptide PJP6- 2, polypeptide PJP6-1, ctrl-peptide, polypeptide PJP3-2, polypeptide PJP4, polypeptide PJP3-1, polypeptide PJP2, polypeptide PJP3-3, Polypeptide PJP5, DDP.
Fig. 4 is the knurl weight weight ratio schematic diagram of the embodiment of the present invention 2.The meaning of each English word is identical as Fig. 2 in figure.
Fig. 5 is the mouse weight change curve of the embodiment of the present invention 2.The meaning of each English word is identical as Fig. 2.Figure In, by the 14th day curve point arrange relationship, from top to bottom successively are as follows: polypeptide PJP6-2, polypeptide PJP4, ctrl-peptide, Polypeptide PJP3-1, polypeptide PJP3-3, polypeptide PJP6-1, polypeptide PJP3-2, polypeptide PJP6-3, polypeptide PJP5, polypeptide PJP2, DDP.
Fig. 6 is the tumor formation comparison of nonsense peptide (50mg/kg) group and each polypeptide (50mg/kg) group in the embodiment of the present invention 3 Figure.Wherein, each polypeptide is respectively as follows: polypeptide PJP3-1-HWGF, polypeptide PJP3-3-HWGF, polypeptide PJP5-HWGF, polypeptide PJP5- 1-HWGF, polypeptide PJP5-2-HWGF, polypeptide PJP5-3-HWGF, polypeptide PJP5-4-HWGF and polypeptide PJP5-5-HWGF.In figure, Ctrl-HWGF is nonsense peptide.
Fig. 7 is the knurl product change curve of the embodiment of the present invention 3.The meaning of each English word is identical as Fig. 6 in figure.Figure In, by the 12nd day curve point arrangement relationship, from top to bottom successively are as follows: Ctrl-HWGF, polypeptide PJP5-HWGF, polypeptide PJP5- 1-HWGF, polypeptide PJP5-3-HWGF, polypeptide PJP5-5-HWGF, polypeptide PJP3-1-HWGF, polypeptide PJP5-4-HWGF, polypeptide PJP5-2-HWGF, polypeptide PJP3-3-HWGF.
Fig. 8 is the knurl weight weight ratio schematic diagram of the embodiment of the present invention 3, and * * indicates P < 0.01.Each English word contains in figure Justice is identical as Fig. 6.
Fig. 9 is the mouse weight change curve of the embodiment of the present invention 3.The meaning of each English word is identical as Fig. 6 in figure. In figure, by the 12nd day curve point arrangement relationship, from top to bottom successively are as follows: polypeptide PJP3-1-HWGF, Ctrl-HWGF, polypeptide It is PJP5-HWGF, polypeptide PJP5-4-HWGF, polypeptide PJP3-3-HWGF, polypeptide PJP5-1-HWGF, polypeptide PJP5-5-HWGF, more Peptide PJP5-2-HWGF, polypeptide PJP5-3-HWGF.
Figure 10 is the tumor formation comparison of nonsense peptide (50mg/kg) group and each polypeptide (50mg/kg) group in the embodiment of the present invention 4 Figure.Wherein, each polypeptide is respectively as follows: polypeptide PJP3-3-HWGF, polypeptide A c-PJP3-3-HWGF.In figure, Ctrl-HWGF is nonsense Peptide.
Figure 11 is the knurl product change curve of the embodiment of the present invention 4.The meaning of each English word is identical as Figure 10 in figure. In figure, by the 12nd day curve point arrangement relationship, from top to bottom successively are as follows: Ctrl-HWGF, polypeptide PJP3-3-HWGF, polypeptide Ac-PJP3-3-HWGF。
Figure 12 is the knurl weight weight ratio schematic diagram of the embodiment of the present invention 4, and * indicates P < 0.05.Each English word contains in figure Justice is identical as Figure 10.
Figure 13 is the mouse weight change curve of the embodiment of the present invention 4.The meaning of each English word and Figure 10 phase in figure Together.In figure, arrange relationship by the 12nd day curve point, from top to bottom successively are as follows: polypeptide PJP3-3-HWGF, Ctrl-HWGF, more Peptide Ac-PJP3-3-HWGF.
Figure 14 is that the Western blot of the embodiment of the present invention 4 detects animal model control peptide Ctrl-HWGF group and polypeptide The correlative protein expressions such as proliferation, apoptosis, the angiogenesis of Ac-PJP3-3-HWGF intervention group tumor tissues are horizontal.Wherein, Tubulin is internal reference albumen, and GAP-associated protein GAP includes: MMP2, CD31, VEGFR1, PCNA, PARP1, cleaved caspase-3.
Figure 15 is that the Western blot of the embodiment of the present invention 4 detects animal model control peptide Ctrl-HWGF group and polypeptide In the MAPK signal path of Ac-PJP3-3-HWGF intervention group tumor tissues the correlative protein expression of three important branch it is horizontal and Its phosphorylation state.Wherein, tubulin be internal reference albumen, GAP-associated protein GAP include: P-MEK, MEK, P-ERK, ERK, P-P38, P38、P-JNK、JNK。
Specific embodiment
Present invention is further described in detail with reference to the accompanying drawings and in conjunction with the embodiments.But the present invention is not limited to be given Example out.
As shown in Figure 1, JWA albumen contains 188 amino acid.Inventor is directed to other than JWA protein transmembrane region Part, design a series of random polypeptide sequences segments, and synthesize using existing method (such as Fmoc method), then screened And identification.Following embodiment is that some representatives are had chosen in addition to active polypeptide to the detailed description of screening, qualification process Property nonactive polypeptide, not indicate research process in only relate to these polypeptides.
The design and synthesis of 1 polypeptide of embodiment
The present embodiment design and the polypeptide of synthesis are as shown in table 1, and the amino acid sequence that each polypeptide is chosen is shown in the shade in Fig. 1 Part.
The polypeptide sequence of 1 embodiment 1 of table design and synthesis
Title Amino acid sequence (N-terminal-C-terminal) Phosphorylation site SEQ ID No:
PJP2 Ac-6- aminocaproic acid-DFRDISKWNNR-NH2 S phosphorylation 1
PJP3-1 Ac-6- aminocaproic acid-RMKKRYPTTFVMVV-NH2 Y phosphorylation 2
PJP3-2 Ac-6- aminocaproic acid-RMKKRYPTTFVMVV-NH2 T phosphorylation 2
PJP3-3 Ac-6- aminocaproic acid-RMKKRYPTTFVMVV-NH2 T phosphorylation 2
PJP4 Ac-6- aminocaproic acid-MFIHASLRLRN-NH2 S phosphorylation 3
PJP5 Ac-6- aminocaproic acid-IGLKRTPMGIV-NH2 T phosphorylation 4
PJP6-1 Ac-6- aminocaproic acid-NRLTDYISKVKE-NH2 T phosphorylation 5
PJP6-2 Ac-6- aminocaproic acid-NRLTDYISKVKE-NH2 Y phosphorylation 5
PJP6-3 Ac-6- aminocaproic acid-NRLTDYISKVKE-NH2 S phosphorylation 5
Ctrl-peptide Ac-6- aminocaproic acid-EEMQRR-NH2 Non- phosphorylation 6
Note 1: in each sequence, the amino acid for marking underscore is the amino acid for needing phosphorylation modification.
Note 2: the above polypeptide sequence does acetylation modification in its N-terminal, and do amidation modification in its C-terminal in synthesis, prevents Only polypeptide fast degradation.
Embodiment 2 screens polypeptide with anti-tumor activity in the polypeptide that embodiment 1 synthesizes
The present embodiment experimental method is as follows:
The human gastric cancer BGC823 cell of logarithmic growth phase, is aseptically prepared into 5 × 106The cell of/200 μ l is outstanding Liquid is inoculated in each BALB/c nude mouse dorsal subcutaneous respectively with 200 μ l.With electronics vernier caliper measurement transplantable tumor major diameter and short Diameter, according to formula: gross tumor volume (TV)=1/2 × major diameter × minor axis2, calculate tumor size.To tumour growth to about 100mm3 Afterwards, then by mouse it is grouped at random.
Polypeptide intervention group: the polypeptide PJP2, polypeptide PJP3-1, polypeptide PJP3- of equivalent are injected respectively into each group mouse tumor 2, polypeptide PJP3-3, polypeptide PJP4, polypeptide PJP5, polypeptide PJP6-1, polypeptide PJP6-2, polypeptide PJP6-3, the injection of use Amount is 50mg/kg.
Negative control group: to the nonsense Peptide C trl-peptide (50mg/kg) of mouse intratumor injection equivalent.Set up nonsense peptide Group is in order to which whether each polypeptide for observing embodiment 1 has specific Suppressive effect.
Positive controls: to the cis-platinum DDP (4mg/kg) of mouse intratumor injection equivalent.
Polypeptide intervention group, negative control group and the equal successive administration of positive controls are discontinued 2 days after 5 days, are administered once daily, It is administered 10 times altogether, every time measurement mouse weight and knurl footpath when administration.Mouse is put to death in experiment after terminating, separate knurl, measures knurl weight.
The present embodiment experimental result is as follows:
As shown in Figures 2 to 5, compared with negative control group, polypeptide PJP3-1, polypeptide PJP3-3, polypeptide PJP5 intervention group All show certain inhibition tumor effect.
In addition, average mice body weight no difference of science of statistics between each group, and during entire Germicidal efficacy, have not seen that mouse has Drug toxicity reaction.
Gastric cancer is a kind of quite high tumour of grade malignancy, and the present embodiment the result shows that, the polypeptide of 50mg/kg dosage It is raw that PJP3-1, polypeptide PJP3-3 and polypeptide PJP5 intratumor injection are able to suppress human gastric cancer BGC823 cell nude mouse xenograft tumor It is long.
Embodiment 3 is screened using polypeptide PJP3-1, polypeptide PJP3-3 and polypeptide PJP5 as activated centre and specific recognition The functional polypeptide of MMP-2；Inquire into polypeptid acid sequence length, the influence whether there is or not phosphorylation to antitumor action
Due to embodiment 2 observe each polypeptide Suppressive effect be in tumor direct injection polypeptide as a result, in view of drug Direct injection in tumor is difficult to when clinical application, whether the method for needing to use instead intraperitoneal injection observes each polypeptide still with good Good tumor killing effect.
By the prior art it is found that many malignant cell surface MMP-2 expression including gastric cancer is increased, and HWGF Sequence can be with specific recognition MMP-2, therefore the present embodiment is on the basis of polypeptide PJP3-1, polypeptide PJP3-3 and polypeptide PJP5 On, further filtering out in the polypeptide for being connected with HWGF sequence in intraperitoneal injection method has the function of that function of tumor inhibition is more Peptide.
Meanwhile whether also there is similar or identical antitumor action to inquire into polypeptide after shortening or increasing length, The present embodiment reduces 1,2,3 amino acid from its both ends N, C on the basis of polypeptide PJP5 respectively, and from its both ends N, C 2 amino acid are increased separately, and observes it and inhibits tumor effect.
In order to inquire into influence of the T phosphorylation to its antitumor action in polypeptide PJP5, the present embodiment is using polypeptide PJP5 as base Plinth adds unphosphorylated polypeptide, and observes it and inhibit tumor effect.
The present embodiment experimental method is as follows:
Design and synthesize the polypeptide containing HWGF sequence shown in table 2.
Polypeptide of the table 2 containing HWGF sequence
Title Amino acid sequence (N-terminal-C-terminal) Phosphorylation site
PJP3-1-HWGF Ac-6- aminocaproic acid-RMKKRYPTTFVMVV-GGGG-HWGF-NH2 Y phosphorylation
PJP3-3-HWGF Ac-6- aminocaproic acid-RMKKRYPTTFVMVV-GGGG-HWGF-NH2 T phosphorylation
PJP5-HWGF Ac-6- aminocaproic acid-IGLKRTPMGIV-GGGG-HWGF-NH2 T phosphorylation
PJP5-1-HWGF Ac-6- aminocaproic acid-GLKRTPMGI-GGGG-HWGF-NH2 T phosphorylation
PJP5-2-HWGF Ac-6- aminocaproic acid-LKRTPMG-GGGG-HWGF-NH2 T phosphorylation
PJP5-3-HWGF Ac-6- aminocaproic acid-KRTPM-GGGG-HWGF-NH2 T phosphorylation
PJP5-4-HWGF Ac-6- aminocaproic acid-EGIGLKRTPMGIVLD-GGGG-HWGF-NH2 T phosphorylation
PJP5-5-HWGF Ac-6- aminocaproic acid-IGLKRTPMGIV-GGGG-HWGF-NH2 Non- phosphorylation
Ctrl-HWGF Ac-6- aminocaproic acid-EEMQRR-GGGG-HWGF-NH2 Non- phosphorylation
Note 1: in each sequence, the amino acid for marking underscore is the amino acid for needing phosphorylation modification.
Note 2: the above polypeptide sequence does acetylation modification in its N-terminal, and do amidation modification in its C-terminal in synthesis, prevents Only polypeptide fast degradation.
Lotus knurl model selection BALB/c nude mouse, the subcutaneous lotus knurl method of Human gastric careinoma cells BGC823 is the same as embodiment 2.Wait transplant Knurl is long-pending to about 100mm3Afterwards, then at random by mice group.
Polypeptide intervention group: by the way of intraperitoneal injection, the polypeptide PJP3-1- of equivalent is injected respectively to each group mouse HWGF, polypeptide PJP3-3-HWGF, polypeptide PJP5-HWGF, polypeptide PJP5-1-HWGF, polypeptide PJP5-2-HWGF, polypeptide PJP5- 3-HWGF, polypeptide PJP5-4-HWGF, polypeptide PJP5-5-HWGF, injection dosage are 50mg/kg.
Negative control group: to nonsense the Peptide C trl-HWGF, injection dosage 50mg/kg of mouse peritoneal injection equivalent.It sets up Nonsense peptide group is to observe whether each polypeptide has specific Suppressive effect.
The equal daily administration of each group, daily injection 1 time, continuous injection 14 days；Mouse weight and knurl footpath are measured simultaneously.Experiment is eventually Mouse is put to death after only, separates knurl, measures knurl weight.
The present embodiment experimental result is as follows:
As shown in Figures 6 to 9, (1) using intraperitoneal injection administration mode under, the polypeptide PJP3-3-HWGF of 50mg/kg Intervention group shows significantly to inhibit tumor effect, and knurl weight weight ratio is also minimum (P < 0.01).
(2) for polypeptide PJP5, the length of amino acid sequence has no significant effect to antitumor.
(3) nonsense peptide group (Ctrl-HWGF), polypeptide PJP5-5-HWGF intervention group do not show the effect for inhibiting tumour Fruit.
(4) it in addition, average mice body weight no difference of science of statistics between each group, and during whole experiment process, has not seen There is any toxic reaction as caused by polypeptide in mouse.
However, conclusion (2) (3) may not be suitable for since polypeptide PJP5-HWGF itself does not show obvious antitumor action Polypeptide PJP3-3-HWGF.Then, on the one hand, inventor subtracts on the basis of polypeptide PJP3-3 from its both ends N, C respectively Lack 1,2,3 amino acid, and increases separately 2 amino acid from its both ends N, C；On the other hand, using polypeptide PJP3-3 as base Plinth adds unphosphorylated polypeptide.Gained sequence is as shown in table 3, then repeats the above experiment.
The related polypeptide of 3 polypeptide PJP3-3-HWGF of table
Title Amino acid sequence (N-terminal-C-terminal) Phosphorylation site
PJP3-3-HWGF Ac-6- aminocaproic acid-RMKKRYPTTFVMVV-GGGG-HWGF-NH2 T phosphorylation
PJP331-HWGF Ac-6- aminocaproic acid-MKKRYPTTFVMV-GGGG-HWGF-NH2 T phosphorylation
PJP332-HWGF Ac-6- aminocaproic acid-KKRYPTTFVM-GGGG-HWGF-NH2 T phosphorylation
PJP333-HWGF Ac-6- aminocaproic acid-KRYPTTFV-GGGG-HWGF-NH2 T phosphorylation
PJP334-HWGF Ac-6- aminocaproic acid-EGRMKKRYPTTFVMVVLD-GGGG-HWGF-NH2 T phosphorylation
PJP335-HWGF Ac-6- aminocaproic acid-RMKKRYPTTFVMVV-GGGG-HWGF-NH2 Non- phosphorylation
Ctrl-HWGF Ac-6- aminocaproic acid-EEMQRR-GGGG-HWGF-NH2 Non- phosphorylation
Note 1: in each sequence, the amino acid for marking underscore is the amino acid for needing phosphorylation modification.
Note 2: the above polypeptide sequence does acetylation modification in its N-terminal, and do amidation modification in its C-terminal in synthesis, prevents Only polypeptide fast degradation.
The result shows that (being limited by length, unlisted specific experiment data), the polypeptide sequence after reducing amino acid, activity It is not so good as polypeptide PJP3-3-HWGF, and increases the polypeptide sequence after amino acid and antitumor action is had no significant effect.That is, more The sequence of peptide PJP3-3 is its most short functional unit.
Meanwhile nonsense peptide group (Ctrl-HWGF), polypeptide PJP335-HWGF intervention group do not show and inhibit tumour Effect.By the result of polypeptide PJP335-HWGF intervention group it is found that polypeptide site phosphorylation is that the polypeptide has antitumor action Necessary condition, without considering further that the effect of non-phosphorylated polypeptide in follow-up study.
In addition, increasing 4 G (glycine) between above each polypeptide and HWGF sequence, the purpose is to make with tumor suppression Active polypeptide have after HWGF is integrated to tumor cell surface it is certain move freely space, made with enhancing the biology of polypeptide With.Inventor has found that the quantity that G is inserted between polypeptide and HWGF sequence can change, Ke Yi great after studying In or be equal to 0, but 10 be advisable and (limited by length, unlisted specific experiment data) to be less than or equal to.
Embodiment 4 inquires into polypeptide PJP3-3-HWGF and connects influence of Ac (acetylation) modification to antitumor action in its N-terminal
The present embodiment experimental method is as follows:
(1) by BGC823 cell in BALB/c nude mouse dorsal subcutaneous lotus knurl operating method with embodiment 2.
Polypeptide intervention group: being respectively adopted the polypeptide PJP3-3-HWGF that N-terminal meets Ac (acetylation) and do not meet Ac (acetylation), Dosage is 50mg/kg, and in a manner of intraperitoneal injection, equivalent is injected into above-mentioned inoculation human gastric cancer BGC823 cell BALB/c nude mouse.
Negative control group: the negative control group of 50mg/kg nonsense Peptide C trl-HWGF is set up.Set up nonsense peptide group and be in order to Whether observation JWA polypeptide has specific Suppressive effect.
The equal daily administration of each group, is administered once daily, and successive administration 12 days；Mouse weight and knurl footpath are measured simultaneously.Experiment is eventually Mouse is put to death after only, separates knurl, measures knurl weight.
(2) above-mentioned animal model control peptide Ctrl-HWGF group and polypeptide A c-PJP3-3-HWGF group tumor tissues are detected The correlative protein expressions such as proliferation, apoptosis, angiogenesis are horizontal.
Detailed process are as follows: every group of 3 tumor tissues of control peptide Ctrl-HWGF group and polypeptide A c-PJP3-3-HWGF group are taken, Clip soya bean size, liquid nitrogen grinding powdering are added 0.5mlRIPA (containing 0.5%PMSF) Tissue lysates and extract albumen, Supernatant is taken after 12000 × g centrifugation 15min, surveys protein concentration, boiled egg is white.According to the size of institute's detection molecules amount, select appropriate dense The polyacrylamide gel of degree carries out protein electrophoresis, and 60 μ g albumen are added in every hole, and deposition condition selects 60V30min, 90V, 1- 1.5h.Half-dried robin transferring film after electrophoresis, so that albumen is transferred on pvdf membrane out of gel.After transferring film, 5% degreasing 1-2h is closed under milk room temperature, TBST (containing 0.1%Tween20) washes film 3 times, each 5min, 4 DEG C incubation corresponding antibodies and stays overnight. Next day, TBST (containing 0.1%Tween20) are washed film 3 times, each 5min, are incubated at room temperature secondary antibody 1-2h, TBST (contains 0.1% Tween20 film 8 times) are washed, each 5min.ECL luminescent solution, exposure are added dropwise on film.
The present embodiment experimental result is as follows:
(1) as shown in Figure 10 to Figure 13, N-terminal meets Ac (acetylation) and does not meet the polypeptide PJP3-3-HWGF of Ac (acetylation) It shows significantly to inhibit tumor effect, therefore, Ac (acetylation) modifies the inhibition tumor effect to polypeptide PJP3-3-HWGF Without influence.The growth that polypeptide PJP3-3-HWGF is able to suppress transplanted tumor in nude mice simultaneously is verified again.
In addition, average mice body weight no difference of science of statistics between each group, and during whole experiment process, have not seen mouse There is any toxic reaction as caused by polypeptide.
(2) Western Blot laboratory test results are found, compared with nonsense Peptide C trl-HWGF group, Ac-PJP3-3- In HWGF polypeptide group, PCNA is significantly reduced, and caspase3 spliced body and PARP-1 spliced body obviously increase, angiogenesis associated protein White MMP-2, CD31, VEGFR1 significantly reduce (as shown in figure 14).
In addition, Western Blot also has detected tri- P-MEK, P-ERK and P-P38 in MAPK signal path, P-JNK weights Want the situation of change of branch.As a result, it has been found that compared with nonsense Peptide C trl-HWGF group, in Ac-PJP3-3-HWGF polypeptide group, P- MEK, P-ERK and P-P38 are obviously activated, and P-JNK variation is unobvious (as shown in figure 15).
Embodiment 5 is screened using polypeptide PJP3-3 as the functional polypeptide of active site
The purpose of the present embodiment is: on the basis of polypeptide PJP3-3, the screening function polypeptide in multiple combinations sequence.
The transplantable tumor mouse that the present embodiment uses is same as Example 2, and experimental method is also same as Example 2, using tumor Interior injection system, the dosage of each polypeptide are 50mg/kg, while setting up nonsense peptide group.
The sequence of each polypeptide is as shown in table 4.
The polypeptide sequence of 4 multiple combinations of table
Note 1: in each sequence, the amino acid for marking underscore is the amino acid for needing phosphorylation modification.
Note 2: the above polypeptide sequence does acetylation modification in its N-terminal, and do amidation modification in its C-terminal in synthesis, prevents Only polypeptide fast degradation.
Infuse 3:(R)9For cell-penetrating peptides CPP sequence.
It is limited by length, herein unlisted specific experiment data.Obtained experimental data shows that polypeptide J1 is equal to polypeptide J57 With anti-tumor activity.
Embodiment 6 is screened using polypeptide PJP3-1 as the functional polypeptide of active site
The purpose of the present embodiment is: on the basis of polypeptide PJP3-1, the screening function polypeptide in multiple combinations sequence.
The transplantable tumor mouse that the present embodiment uses is same as Example 2, and experimental method is also same as Example 2, using tumor Interior injection system, the dosage of each polypeptide are 50mg/kg, while setting up nonsense peptide group.
The sequence of each polypeptide is as shown in table 5.
The polypeptide sequence of 5 multiple combinations of table
Note 1: in each sequence, the amino acid for marking underscore is the amino acid for needing phosphorylation modification.
Note 2: the above polypeptide sequence does acetylation modification in its N-terminal, and do amidation modification in its C-terminal in synthesis, prevents Only polypeptide fast degradation.
Infuse 3:(R)9For cell-penetrating peptides CPP sequence.
It is limited by length, herein unlisted specific experiment data.Obtained experimental data shows that polypeptide P1 is equal to polypeptide P57 With anti-tumor activity.
Embodiment 7 is screened using polypeptide PJP5 as the functional polypeptide of active site
The purpose of the present embodiment is: on the basis of polypeptide PJP5, the screening function polypeptide in multiple combinations sequence.
The transplantable tumor mouse that the present embodiment uses is same as Example 2, and experimental method is also same as Example 2, using tumor Interior injection system, the dosage of each polypeptide are 50mg/kg, while setting up nonsense peptide group.
The sequence of each polypeptide is as shown in table 6.
The polypeptide sequence of 6 multiple combinations of table
Note 1: in each sequence, the amino acid for marking underscore is the amino acid for needing phosphorylation modification.
Note 2: the above polypeptide sequence does acetylation modification in its N-terminal, and do amidation modification in its C-terminal in synthesis, prevents Only polypeptide fast degradation.
Infuse 3:(R)9For cell-penetrating peptides CPP sequence.
It is limited by length, herein unlisted specific experiment data.Obtained experimental data shows that polypeptide Q1 is equal to polypeptide Q57 With anti-tumor activity.

Claims (4)

1. a kind of anti-tumour active polypeptide, characterized in that the amino acid sequence of the polypeptide is as shown in I, II, III or IV:
I: RMKKRYPTTFVMVV；
II: X-RMKKRYPTTFVMVV；
III: RMKKRYPTTFVMVV-Z；
IV: X-RMKKRYPTTFVMVV-Z；
In the above formulas, Y or second phosphorylated modification of T of number, X, Z are respectively amino acid or amino acid sequence from left to right；
X is selected from F, (R)9、(R)9- F, 6-aminocaprolc acid, 6-aminocaprolc acid-F, 6-aminocaprolc acid-(R)9, 6-aminocaprolc acid-(R)9- One of F；
Z is selected from A, (G)n-HWGF、A-(G)nOne of-HWGF, n are the integer more than or equal to 0.
2. anti-tumour active polypeptide according to claim 1, characterized in that the value range of the n is 0-10.
3. anti-tumour active polypeptide according to claim 1 or 2, characterized in that the N-terminal of the polypeptide is repaired through acetylation Decorations, C-terminal are modified through amidation.
4. anti-tumour active polypeptide as claimed in claim 1 or 2 is used to prepare the purposes of anti-tumor drug, the tumour is gastric cancer.
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