CN105820230A - Anti-tumor activity polypeptide and application thereof - Google Patents
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Abstract
The invention relates to anti-tumor activity polypeptide and application thereof.The polypeptide has anti-tumor activity due to the fact that the polypeptide has one of the following amino acid sequences of RMKKRYPTTFVMVV and IGLKRTPMGIV, wherein Y or the second T counting from left to right in RMKKRYPTTFVMVV is modified through phosphorylation, and T in IGLKRTPMGIV is modified through phosphorylation.The polypeptide can be used for preparing an anti-tumor drug.The sequence of the polypeptide is short, and large-scale production is easily achieved; remarkable anti-tumor activity is displayed at a low dosage.The polypeptide has no toxicity to normal tissue cells and has wide application prospects.
Description
Technical field
The present invention relates to a kind of anti-tumour active polypeptide and application thereof, belong to antitumor drug research and development and applied technical field.
Background technology
Malignant tumor is the disease of serious harm human health.Due to the impact of the factors such as environmental pollution, Exposed, food safety, life style and racial inheritance feature, the sickness rate of China's malignant tumor is notable ascendant trend.Malignant tumor including gastric cancer has become as and has a strong impact on the notable obstacle that the people are physically and mentally healthy and restriction economic society is all-round developing.Therefore, find effective antitumour medicine and method, thoroughly capture cancer, be one of common severe challenge of facing of world-wide medical interface.
The principle of Comprehensive Treatment is emphasized in the treatment of tumor, and chemotherapy is one of them important means.The research of antitumor drug in recent years achieves and develops rapidly, and occurs in that some novel antitumor drug, acts on the different links of tumor development, effectively extends the life span of tumor patient, improves quality of life.But also occur in that a lot of problem: tumor cell drug resistance constantly strengthens, and causes the using dosage of antitumor drug to be continuously increased, is greatly enhanced the toxic and side effects of body simultaneously.Now studies have found that, the therapeutic effect of chemicals and the generation of tumor cell drug resistance are all relevant with more intracellular gene expression doses, the expression controlling some gene intracellular can directly suppress the generation etc. of the growth of tumor cell, transfer, drug resistance, and it is greatly improved the therapeutic effect of other Therapeutic Method such as chemicals, reduce because of the medicine toxic and side effects to body, improve life in patients, bring glad tidings for more tumor patients.
JWA gene, i.e. ARL6IP5 gene (GenBank:AF070523.1,1998；LOCUS:AF070523, NM_006407) it is the first gene induced by all-trans-retinoic acid (ATRA) of separating clone from the primary people's trachea cultivated and bronchial epithelial cell such as inventor herein's Zhou Jianwei, encodes a kind of new cytoskeletal binding proteins.Inventor herein leads seminar member for many years all around the structure of JWA gene, function and conducting a research work with mankind's major disease relation, obtain the continuous support of state natural sciences fund committee fund project always, achieve the significant achievement in research of a series of originality, including: (1) takes the lead in proving and proposing the albumen of JWA gene code is a kind of new microtubule-associated protein (MAPs), this albumen can be combined with tubulin α and β subunit, and to environmental physics chemical factor (such as cold shock, heat shock, paclitaxel etc.) response in show the dynamic variation Tong Bu with tubulin α and β subunit (polymerization or depolymerization).(2) find that JWA albumen participates in cell mitogen process, and the dynamic stability of micro-pipe is had regulation effect.(3) JWA albumen regulates tumor cell migration and apoptosis by MAPK signal path, the depolymerization of microfilament and restructuring.(4) JWA albumen is the important molecule that suppression gastric cancer vascular generates, and in stomach organization, JWA albumen is combined with other tumor cells mark such as XRCC1, MDM2, P53, SOX2, FAK and MMP-2 etc. and can more preferably be predicted patient's prognosis；Inventor herein it has been investigated that, JWA albumen by ubiquitin-proteasome pathway negativity regulation nuclear transcription factor Sp1 and downstream gene MMP-2 express so that realize its to gastric cancer vascular generate regulation and control.
Knowable to existing JWA albumen correlational study, JWA gene expression the JWA albumen obtained has suppression tumor cell adhesion, infiltrates, shifts and suppress the functions such as tumor vascular growth, and large-scale production activity JWA albumen is imperative.But, inventor herein finds in practical studies, uses host expression system to be difficult to obtain the JWA albumen of purification at present, is carried out the poor feasibility of internal and external test by this approach with the JWA albumen of purification.
Additionally, according to known to inventor herein, matrix metalloproteinase (MMPs) is extended familys, needs the metal ions such as Ca, Zn to gain the name as cofactor because of it.MMPs almost can various protein ingredients in degradation of cell epimatrix (ECM), key effect is played in tumor cell destruction histology's barrier and Invasion and Metastasis, this makes MMPs effect in tumor invasion and metastasis be increasingly subject to pay attention to it is considered to be proteolytic enzyme main during Gai.MMPs family is separated at present identifies 26 members, and numbering is respectively MMP1～26；Wherein, the function of MMP-2 is particularly important, and MMP-2, and can be by histidine-tryptophan-Gly-Phe (HWGF) sequence institute specific recognition at stomach cancer cell surface high expressed.
Inventor herein once applied for the Chinese invention patent of entitled " polypeptide with anti-tumor activity and application thereof ", Patent No. 201310178099.X on May 14th, 2013, and Authorization Notice No. is CN103239710B.In this patent, the aminoacid sequence of polypeptide 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 aminoacid S, X, Z are respectively aminoacid or aminoacid 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-RGD、A-(G)nOne of-RGD.This polypeptide can be used for preparing antitumor drug.
After further research, from JWA protein amino acid sequence, Screening and Identification obtains another structure and is different from above-mentioned patent and has the brand-new polypeptide of anti-tumor activity inventor herein.
Summary of the invention
The technical problem to be solved is: based on prior art, proposes a kind of anti-tumour active polypeptide, and this polypeptide Screening and Identification from JWA protein amino acid sequence obtains, and its structure is different from the polypeptide invented before.Meanwhile, this polypeptide is also provided for for preparing the purposes of antitumor drug.
The major technique design of the present invention is as follows: before inventor, from JWA protein amino acid sequence, Screening and Identification obtains polypeptide FPGSDRF and the series derivatives peptide thereof with anti-tumor activity, on this basis, inventor studies discovery JWA albumen and has various biological function, and think why JWA albumen has multiple different biological function, it may be possible to the aminoacid sequence of its different fragments has what different biological this feature of activity determined；Therefore, biological function and the application prospect thereof of inquiring into different fragments JWA protein amino acid sequence have the biggest researching value, it is more likely that therefrom Screening and Identification goes out other active polypeptide.Adhering to this theory, inventor has proceeded deep experimentation, and Screening and Identification goes out invention polypeptide before structure is different from and has the JWA functional polypeptide of anti-tumor activity.
Technical scheme is as follows:
A kind of anti-tumour active polypeptide, is characterized in that, this polypeptide has anti-tumor activity because possessing one of following aminoacid sequence:
RMKKRYPTTFVMVV, wherein second phosphorylated modification of T of Y or several；
IGLKRTPMGIV, the wherein phosphorylated modification of T.
Preferably, the aminoacid sequence of described polypeptide is as shown in I, II, III or IV:
I: RMKKRYPTTFVMVV；
II: X-RMKKRYPTTFVMVV；
III: RMKKRYPTTFVMVV-Z；
IV: X-RMKKRYPTTFVMVV-Z；
Above-mentioned various in, second phosphorylated modification of T of Y or several, X, Z are respectively aminoacid or aminoacid 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 aminoacid sequence of described polypeptide is as shown in V, VI, VII or VIII:
V: IGLKRTPMGIV；
VI: X-IGLKRTPMGIV；
VII: IGLKRTPMGIV-Z；
VIII: X-IGLKRTPMGIV-Z；
Above-mentioned various in, the phosphorylated modification of T, X, Z are respectively aminoacid or aminoacid 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 span of described n is 0-10.
Preferably, the N end of described polypeptide is modified through amidatioon through acetylation modification, C end.
The present invention also proposes: anti-tumour active polypeptide described previously is for preparing the purposes of antitumor drug.
Preferably, described tumor is gastric cancer.
Peptide sequence of the present invention is shorter, it is easy to accomplish large-scale production；The most i.e. demonstrate significant anti-tumor activity.Polypeptide normal tissue cytotoxic of the present invention, has broad application prospects.
Accompanying drawing explanation
Fig. 1 is known JWA protein amino acid sequence figure.Underscore part is trans-membrane region, and shadow region is the segment portion that detailed description of the invention relates to.
The aminoacid that the most each letter represents is as follows: alanine A, arginine R, aspartic acid D, glutamine Q, glutamic acid E, histidine H, isoleucine I, glycine G, agedoite N, leucine L, lysine K, methionine M, phenylalanine F, proline P, serine S, threonine T, tryptophan W, tyrosine Y, valine V.
Fig. 2 is the one-tenth tumor comparison diagram that nonsense peptide (50mg/kg) group in the embodiment of the present invention 2, cisplatin (4mg/kg) group and each polypeptide (50mg/kg) are 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, and DDP is cisplatin.
Fig. 3 is the tumor change in volume curve chart of the embodiment of the present invention 2, and arrow represents administration time.The implication of each English word is identical with Fig. 2.In figure, by the curve point arrangement relation of the 14th day, from top to bottom it is followed successively by: 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 tumor weight weight ratio schematic diagram of the embodiment of the present invention 2.In figure, the implication of each English word is identical with Fig. 2.
Fig. 5 is the Mouse Weight change curve of the embodiment of the present invention 2.The implication of each English word is identical with Fig. 2.In figure, by the curve point arrangement relation of the 14th day, from top to bottom it is followed successively by: 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 one-tenth tumor comparison diagram that in the embodiment of the present invention 3, nonsense peptide (50mg/kg) group and each polypeptide (50mg/kg) are organized.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 tumor change in volume curve chart of the embodiment of the present invention 3.In figure, the implication of each English word is identical with Fig. 6.In figure, by the curve point arrangement relation of the 12nd day, from top to bottom it is followed successively by: 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 tumor weight weight ratio schematic diagram of the embodiment of the present invention 3, and * * represents P < 0.01.In figure, the implication of each English word is identical with Fig. 6.
Fig. 9 is the Mouse Weight change curve of the embodiment of the present invention 3.In figure, the implication of each English word is identical with Fig. 6.In figure, by the curve point arrangement relation of the 12nd day, from top to bottom it is followed successively by: polypeptide PJP3-1-HWGF, Ctrl-HWGF, polypeptide PJP5-HWGF, polypeptide PJP5-4-HWGF, polypeptide PJP3-3-HWGF, polypeptide PJP5-1-HWGF, polypeptide PJP5-5-HWGF, polypeptide PJP5-2-HWGF, polypeptide PJP5-3-HWGF.
Figure 10 is the one-tenth tumor comparison diagram that in the embodiment of the present invention 4, nonsense peptide (50mg/kg) group and each polypeptide (50mg/kg) are organized.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 tumor change in volume curve chart of the embodiment of the present invention 4.In figure, the implication of each English word is identical with Figure 10.In figure, by the curve point arrangement relation of the 12nd day, from top to bottom it is followed successively by: Ctrl-HWGF, polypeptide PJP3-3-HWGF, polypeptide A c-PJP3-3-HWGF.
Figure 12 is the tumor weight weight ratio schematic diagram of the embodiment of the present invention 4, and * represents P < 0.05.In figure, the implication of each English word is identical with Figure 10.
Figure 13 is the Mouse Weight change curve of the embodiment of the present invention 4.In figure, the implication of each English word is identical with Figure 10.In figure, by the curve point arrangement relation of the 12nd day, from top to bottom it is followed successively by: polypeptide PJP3-3-HWGF, Ctrl-HWGF, polypeptide A c-PJP3-3-HWGF.
Figure 14 is that the Westernblot of the embodiment of the present invention 4 detects the correlative protein expression level such as animal model control peptide Ctrl-HWGF group and the propagation of polypeptide A c-PJP3-3-HWGF intervention group tumor tissues, apoptosis, angiogenesis.Wherein, tubulin is internal reference albumen, and associated protein includes: MMP2, CD31, VEGFR1, PCNA, PARP1, cleavedcaspase-3.
Figure 15 is correlative protein expression level and the phosphorylation state thereof of three important branch in the Westernblot detection animal model control peptide Ctrl-HWGF group of the embodiment of the present invention 4 and the MAPK signal path of polypeptide A c-PJP3-3-HWGF intervention group tumor tissues.Wherein, tubulin is internal reference albumen, and associated protein includes: P-MEK, MEK, P-ERK, ERK, P-P38, P38, P-JNK, JNK.
Detailed description of the invention
It is described in further detail with reference to the accompanying drawings and in conjunction with the embodiments to the present invention.But the invention is not restricted to given example.
As it is shown in figure 1, JWA albumen contains 188 aminoacid.Inventor herein, for the part beyond JWA protein transmembrane region, designs a series of random polypeptide sequences fragment, and uses existing method (such as Fmoc method etc.) to synthesize, then screen and identify.Following example are to screening, the detailed description of qualification process, have chosen some representational nonactive polypeptide, not represent in research process and only relate to these polypeptide in addition to active polypeptide.
The design of embodiment 1 polypeptide and synthesis
The present embodiment design is as shown in table 1 with the polypeptide of synthesis, and the aminoacid sequence that each polypeptide is chosen is shown in the dash area in Fig. 1.
The design of table 1 embodiment 1 and the peptide sequence synthesized
| Title | Aminoacid sequence (N end-C end) | 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 aminoacid of mark underscore is the aminoacid needing phosphorylation modification.
Note 2: above peptide sequence, when synthesis, does acetylation modification at its N end, and does amidatioon modification at its C end, prevent polypeptide fast degradation.
Embodiment 2 screens the polypeptide with anti-tumor activity in the polypeptide that embodiment 1 synthesizes
The present embodiment experimental technique is as follows:
Take the logarithm people's gastric cancer BGC823 cell of trophophase, be aseptically prepared as 5 × 106The cell suspension of/200 μ l, is inoculated in each BALB/c nude mouse dorsal subcutaneous with 200 μ l respectively.With electronics vernier caliper measurement transplanted tumor major diameter and minor axis, according to formula: gross tumor volume (TV)=1/2 × major diameter × minor axis2, calculate tumor size.Treat that tumor growth arrives about 100mm3After, then by mice random packet.
Polypeptide intervention group: inject the polypeptide PJP2 of equivalent, polypeptide PJP3-1, polypeptide PJP3-2, polypeptide PJP3-3, polypeptide PJP4, polypeptide PJP5, polypeptide PJP6-1, polypeptide PJP6-2, polypeptide PJP6-3 in each group of mouse tumor respectively, the injection dosage of employing is 50mg/kg.
Negative control group: to nonsense PEPC trl-peptide (50mg/kg) of mice intratumor injection equivalent.Setting up nonsense peptide group is to observe whether each polypeptide of embodiment 1 has specificity Suppressive effect.
Positive controls: to the cisplatin DDP (4mg/kg) of mice intratumor injection equivalent.
The equal successive administration of polypeptide intervention group, negative control group and positive controls is drug withdrawal 2 days after 5 days, are administered once daily, and are administered 10 times altogether, measure Mouse Weight and tumor footpath when being administered every time.Experiment puts to death mice after terminating, and separates tumor body, measures tumor 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 demonstrate certain suppression tumor effect.
Additionally, average mice body weight no difference of science of statistics between each group, and during whole laboratory observation, have not seen that mice has drug toxicity reaction.
Gastric cancer is the tumor that a kind of grade malignancy is at a relatively high, and the present embodiment result shows, the polypeptide PJP3-1 of 50mg/kg dosage, polypeptide PJP3-3 and polypeptide PJP5 intratumor injection can suppress people's gastric cancer BGC823 cell nude mouse xenograft tumor to grow.
Embodiment 3 is screened with polypeptide PJP3-1, polypeptide PJP3-3 and polypeptide PJP5 as active center and the functional polypeptide of specific recognition MMP-2；Inquire into polypeptid acid sequence length, with or without the phosphorylation impact on antitumor action
Owing to embodiment 2 observes that the Suppressive effect of each polypeptide is the result of direct injection polypeptide in tumor, it is contemplated that be difficult to direct injection in tumor during clinical drug application, need the method using lumbar injection instead to observe each polypeptide and the most still there is good tumor killing effect.
From prior art, many malignant cell surface MMP-2 including gastric cancer express and increase, 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, filters out the functional polypeptide with function of tumor inhibition further in connecting the polypeptide having HWGF sequence with lumbar injection method.
Simultaneously, after shortening or increasing length, the most also there is similar or identical antitumor action in order to inquire into polypeptide, the present embodiment decreases 1,2,3 aminoacid on the basis of polypeptide PJP5 respectively from its N, C two ends, and increase by 2 aminoacid respectively from its N, C two ends, and observe its suppression tumor effect.
In order to inquire into the T phosphorylation impact on its antitumor action in polypeptide PJP5, the present embodiment, based on polypeptide PJP5, adds unphosphorylated polypeptide, and observes its suppression tumor effect.
The present embodiment experimental technique is as follows:
Design and synthesize the polypeptide containing HWGF sequence shown in table 2.
The table 2 polypeptide containing HWGF sequence
| Title | Aminoacid sequence (N end-C end) | 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 aminoacid of mark underscore is the aminoacid needing phosphorylation modification.
Note 2: above peptide sequence, when synthesis, does acetylation modification at its N end, and does amidatioon modification at its C end, prevent polypeptide fast degradation.
Lotus tumor model selection BALB/c nude mouse, Human gastric careinoma cells BGC823 subcutaneous lotus tumor method is with embodiment 2.Treat that transplanted tumor volume is to about 100mm3After, more random by mice group.
Polypeptide intervention group: use the mode of intraperitoneal injection, injecting the polypeptide PJP3-1-HWGF of equivalent, 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 respectively to each group of mice, injection dosage is 50mg/kg.
Negative control group: to the nonsense PEPC trl-HWGF of mouse peritoneal injection equivalent, injection dosage is 50mg/kg.Setting up nonsense peptide group is to observe whether each polypeptide has specificity Suppressive effect.
Each group is administered equal every day, injects every day 1 time, continuously injection 14 days；Measure Mouse Weight and tumor footpath simultaneously.Experiment puts to death mice after terminating, and separates tumor body, measures tumor weight.
The present embodiment experimental result is as follows:
As shown in Figures 6 to 9, (1), under the administering mode using lumbar injection, the polypeptide PJP3-3-HWGF intervention group of 50mg/kg shows and significantly suppresses tumor effect, and tumor weight weight ratio is also minimum (P < 0.01).
(2) for polypeptide PJP5, antitumor is had no significant effect by the length of its aminoacid sequence.
(3) nonsense peptide group (Ctrl-HWGF), polypeptide PJP5-5-HWGF intervention group all do not demonstrate the effect of suppression tumor.
(4) additionally, average mice body weight no difference of science of statistics between each group, and during whole experimentation, have not seen that any toxic reaction caused by polypeptide occurs in mice.
But, owing to polypeptide PJP5-HWGF itself does not demonstrates that obvious antitumor action, conclusion (2) (3) may not be applicable to polypeptide PJP3-3-HWGF.Then, on the one hand, inventor herein decreases 1,2,3 aminoacid on the basis of polypeptide PJP3-3 respectively from its N, C two ends, and increases by 2 aminoacid respectively from its N, C two ends；On the other hand, based on polypeptide PJP3-3, add unphosphorylated polypeptide.Gained sequence is as shown in table 3, then repeats above experiment..
The related polypeptide of table 3 polypeptide PJP3-3-HWGF
| Title | Aminoacid sequence (N end-C end) | 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 aminoacid of mark underscore is the aminoacid needing phosphorylation modification.
Note 2: above peptide sequence, when synthesis, does acetylation modification at its N end, and does amidatioon modification at its C end, prevent polypeptide fast degradation.
Result shows (being limited by length, unlisted specific experiment data), reduces the peptide sequence after aminoacid, and its activity is all not as polypeptide PJP3-3-HWGF, and increases the peptide sequence after aminoacid and have no significant effect antitumor action.That is, the sequence of the polypeptide PJP3-3 the shortest functional unit that is it.
Meanwhile, nonsense peptide group (Ctrl-HWGF), polypeptide PJP335-HWGF intervention group all do not demonstrate the effect of suppression tumor.From the result of polypeptide PJP335-HWGF intervention group, polypeptide site phosphorylation is the essential condition that this polypeptide possesses antitumor action, without considering further that the effect of non-phosphorylating polypeptide in follow-up study.
Add 4 G (glycine) between each polypeptide and HWGF sequence additionally, above, its objective is that the polypeptide so that having antitumor activity has certain free activity space after HWGF is attached to tumor cell surface, to strengthen the biological action of polypeptide.Inventor herein finds the most afterwards, and the quantity inserting G between polypeptide and HWGF sequence can change, and can be more than or equal to 0, but be advisable less than or equal to 10 (being limited by length, unlisted specific experiment data).
Embodiment 4 is inquired into polypeptide PJP3-3-HWGF and is modified the impact on antitumor action at its N termination Ac (acetylation)
The present embodiment experimental technique is as follows:
(1) by BGC823 cell in the operational approach of BALB/c nude mouse dorsal subcutaneous lotus tumor with embodiment 2.
Polypeptide intervention group: be respectively adopted N termination Ac (acetylation) and do not meet the polypeptide PJP3-3-HWGF of Ac (acetylation); dosage is 50mg/kg; in the way of intraperitoneal injection, identical size injection injects the BALB/c nude mouse of above-mentioned inoculation people's gastric cancer BGC823 cell.
Negative control group: set up the negative control group of 50mg/kg nonsense PEPC trl-HWGF.Setting up nonsense peptide group is to observe whether JWA polypeptide has specificity Suppressive effect.
Each group is administered equal every day, is administered once daily, successive administration 12 days；Measure Mouse Weight and tumor footpath simultaneously.Experiment puts to death mice after terminating, and separates tumor body, measures tumor weight.
(2) the correlative protein expression levels such as above-mentioned animal model control peptide Ctrl-HWGF group and the propagation of polypeptide A c-PJP3-3-HWGF group tumor tissues, apoptosis, angiogenesis are detected.
Detailed process is: take control peptide Ctrl-HWGF group and polypeptide A c-PJP3-3-HWGF group often organizes 3 tumor tissues, clip Semen Glycines size, liquid nitrogen grinding powdering, add 0.5mlRIPA (containing 0.5%PMSF) Tissue lysates and extract albumen, 12000 × g takes supernatant after being centrifuged 15min, surveying protein concentration, boiled egg is white.According to the size of institute's detection molecules amount, selecting the polyacrylamide gel of appropriate concentration to carry out protein electrophoresis, every hole adds 60 μ g albumen, and deposition condition selects 60V30min, 90V, 1-1.5h.Half-dried robin transferring film after electrophoresis so that albumen is transferred to pvdf membrane in gel.After transferring film, close 1-2h, TBST (containing 0.1%Tween20) under 5% skim milk room temperature and wash film 3 times, each 5min, hatch corresponding antibodies overnight for 4 DEG C.Next day, TBST (containing 0.1%Tween20) washes film 3 times, each 5min, the anti-1-2h of incubated at room two, and TBST (containing 0.1%Tween20) washes film 8 times, each 5min.ECL luminescent solution, exposure is dripped on film.
The present embodiment experimental result is as follows:
(1) as shown in Figure 10 to Figure 13; N termination Ac (acetylation) and do not meet the polypeptide PJP3-3-HWGF of Ac (acetylation) and all show and significantly suppress tumor effect; therefore, Ac (acetylation) modifies the suppression tumor effect on polypeptide PJP3-3-HWGF without impact.Polypeptide PJP3-3-HWGF can suppress the growth of transplanted tumor in nude mice to be the most again verified simultaneously.
Additionally, average mice body weight no difference of science of statistics between each group, and during whole experimentation, have not seen that any toxic reaction caused by polypeptide occurs in mice.
(2) WesternBlot laboratory test results finds, compared with nonsense PEPC trl-HWGF group, in Ac-PJP3-3-HWGF polypeptide group, PCNA significantly reduces, caspase3 spliced body and PARP-1 spliced body substantially increase, and angiogenesis associated protein white MMP-2, CD31, VEGFR1 significantly reduce (as shown in figure 14).
Additionally, WesternBlot also have detected P-MEK, P-ERK and P-P38 in MAPK signal path, the situation of change of tri-important branch of P-JNK.It was found that compared with nonsense PEPC trl-HWGF group, in Ac-PJP3-3-HWGF polypeptide group, P-MEK, P-ERK and P-P38 are substantially activated, and P-JNK changes inconspicuous (as shown in figure 15).
Embodiment 5 screens the functional polypeptide with polypeptide PJP3-3 as avtive spot
The purpose of the present embodiment is: on the basis of polypeptide PJP3-3, screening function polypeptide in multiple combination sequence.
The transplanted tumor mice that the present embodiment uses is same as in Example 2, and experimental technique is the most same as in Example 2, uses intratumor injection mode, and the dosage of each polypeptide is 50mg/kg, sets up nonsense peptide group simultaneously.
The sequence of each polypeptide is as shown in table 4.
The peptide sequence of table 4 multiple combination
Note 1: in each sequence, the aminoacid of mark underscore is the aminoacid needing phosphorylation modification.
Note 2: above peptide sequence, when synthesis, does acetylation modification at its N end, and does amidatioon modification at its C end, prevent polypeptide fast degradation.
Note 3:(R)9For cell-penetrating peptides CPP sequence.
Limited by length, the most unlisted specific experiment data.Obtained experimental data shows, polypeptide J1 is respectively provided with anti-tumor activity to polypeptide J57.
Embodiment 6 screens the functional polypeptide with polypeptide PJP3-1 as avtive spot
The purpose of the present embodiment is: on the basis of polypeptide PJP3-1, screening function polypeptide in multiple combination sequence.
The transplanted tumor mice that the present embodiment uses is same as in Example 2, and experimental technique is the most same as in Example 2, uses intratumor injection mode, and the dosage of each polypeptide is 50mg/kg, sets up nonsense peptide group simultaneously.
The sequence of each polypeptide is as shown in table 5.
The peptide sequence of table 5 multiple combination
Note 1: in each sequence, the aminoacid of mark underscore is the aminoacid needing phosphorylation modification.
Note 2: above peptide sequence, when synthesis, does acetylation modification at its N end, and does amidatioon modification at its C end, prevent polypeptide fast degradation.
Note 3:(R)9For cell-penetrating peptides CPP sequence.
Limited by length, the most unlisted specific experiment data.Obtained experimental data shows, polypeptide P1 is respectively provided with anti-tumor activity to polypeptide P57.
Embodiment 7 screens the functional polypeptide with polypeptide PJP5 as avtive spot
The purpose of the present embodiment is: on the basis of polypeptide PJP5, screening function polypeptide in multiple combination sequence.
The transplanted tumor mice that the present embodiment uses is same as in Example 2, and experimental technique is the most same as in Example 2, uses intratumor injection mode, and the dosage of each polypeptide is 50mg/kg, sets up nonsense peptide group simultaneously.
The sequence of each polypeptide is as shown in table 6.
The peptide sequence of table 6 multiple combination
Note 1: in each sequence, the aminoacid of mark underscore is the aminoacid needing phosphorylation modification.
Note 2: above peptide sequence, when synthesis, does acetylation modification at its N end, and does amidatioon modification at its C end, prevent polypeptide fast degradation.
Note 3:(R)9For cell-penetrating peptides CPP sequence.
Limited by length, the most unlisted specific experiment data.Obtained experimental data shows, polypeptide Q1 is respectively provided with anti-tumor activity to polypeptide Q57.
Claims (7)
1. an anti-tumour active polypeptide, is characterized in that, this polypeptide has anti-tumor activity because possessing one of following aminoacid sequence:
RMKKRYPTTFVMVV, wherein second phosphorylated modification of T of Y or several；
IGLKRTPMGIV, the wherein phosphorylated modification of T.
Anti-tumour active polypeptide the most according to claim 1, is characterized in that, the aminoacid sequence of described polypeptide is as shown in I, II, III or IV:
I: RMKKRYPTTFVMVV；
II: X-RMKKRYPTTFVMVV；
III: RMKKRYPTTFVMVV-Z；
IV: X-RMKKRYPTTFVMVV-Z；
Above-mentioned various in, several second phosphorylated modification of T, X, Z are respectively aminoacid or aminoacid 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.
Anti-tumour active polypeptide the most according to claim 1, is characterized in that, the aminoacid sequence of described polypeptide is as shown in V, VI, VII or VIII:
V: IGLKRTPMGIV；
VI: X-IGLKRTPMGIV；
VII: IGLKRTPMGIV-Z；
VIII: X-IGLKRTPMGIV-Z；
Above-mentioned various in, the phosphorylated modification of T, X, Z are respectively aminoacid or aminoacid 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.
4. according to the anti-tumour active polypeptide described in Claims 2 or 3, it is characterized in that, the span of described n is 0-10.
5. according to the anti-tumour active polypeptide described in any one of Claims 1-4, it is characterized in that, the N end of described polypeptide is modified through amidatioon through acetylation modification, C end.
6. anti-tumour active polypeptide described in any one of claim 1 to 5 is for preparing the purposes of antitumor drug.
Purposes the most according to claim 6, is characterized in that, described tumor is gastric cancer.
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| WO2021027045A1 (en) | 2019-08-12 | 2021-02-18 | 周建伟 | Anti-tumour compound based on jwa gene activation and her2 degradation, preparation method therefor, and use thereof |
| CN115414465A (en) * | 2022-10-17 | 2022-12-02 | 苏州明人医药生物科技有限公司 | Application of JWA polypeptide in preparation of antitumor drug synergist |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010008543A2 (en) * | 2008-07-15 | 2010-01-21 | Trustees Of Dartmouth College | Molecular signatures for diagnosing scleroderma |
| CN103239710A (en) * | 2013-05-14 | 2013-08-14 | 南京医科大学 | Polypeptide with anti-tumor activity and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010008543A2 (en) * | 2008-07-15 | 2010-01-21 | Trustees Of Dartmouth College | Molecular signatures for diagnosing scleroderma |
| CN103239710A (en) * | 2013-05-14 | 2013-08-14 | 南京医科大学 | Polypeptide with anti-tumor activity and application thereof |
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| KUHNAST B ET AL: "Targeting of gelatinase activity with a radiolabeled cyclic HWGF peptide", 《NUCLEAR MEDICINE AND BIOLOGY》 * |
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| WO2021027045A1 (en) | 2019-08-12 | 2021-02-18 | 周建伟 | Anti-tumour compound based on jwa gene activation and her2 degradation, preparation method therefor, and use thereof |
| CN115414465A (en) * | 2022-10-17 | 2022-12-02 | 苏州明人医药生物科技有限公司 | Application of JWA polypeptide in preparation of antitumor drug synergist |
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