CN110372780A - Antineoplastic polypeptide and its application in antitumor field - Google Patents

Antineoplastic polypeptide and its application in antitumor field Download PDF

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CN110372780A
CN110372780A CN201910609086.0A CN201910609086A CN110372780A CN 110372780 A CN110372780 A CN 110372780A CN 201910609086 A CN201910609086 A CN 201910609086A CN 110372780 A CN110372780 A CN 110372780A
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马杰
焦平
陆元花
赵通鉴
宋卓瑶
魏雪晨
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Saipu Biotechnology (Changchun) Co.,Ltd.
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Jilin University
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Abstract

Application the invention discloses antineoplastic polypeptide and its in antitumor field.Be following 1) -4 the present invention provides a kind of polypeptide) in it is any: 1) include amino acid sequence be polypeptide shown in sequence 1 in sequence table；2) amino acid sequence is polypeptide shown in sequence 1 in sequence table；3) 1) or 2) N-terminal or/and the obtained fused polypeptide of C-terminal connection label；1) or 2) 4) polypeptide obtaining polypeptide shown in by the substitution and/or deletion and/or addition of one or several amino acid residues and with anti-tumor function.The present invention provides a kind of polypeptide fragments that can effectively kill cell, have very low IC50Value, while being connect with the tomour specific expression vector containing hTERT promoter, enable polypeptide fragment in tumour cell specific high-efficiency expression and killing tumor cell.

Description

Antineoplastic polypeptide and its application in antitumor field
Technical field
Application the invention belongs to gene engineering technology field more particularly to antineoplastic polypeptide and its in antitumor field.
Background technique
Antineoplastic polypeptide (Anticancer Peptides, ACPs) is that one kind is made of tens amino acid residues, is had Amphipathic (hydrophily and hydrophobicity) and positively charged small peptide, the activity with killing tumor cell.Because it has poison is secondary to make With it is small, be not likely to produce drug resistance and the low remarkable advantage of immune response, it has also become one of the hot spot of anti-tumor drug research.It is antitumor Having for collecting on polypeptide database CancerPPD (http://crdd.osdd.net/raghava/cancerppd/) is anti-swollen The polypeptide of tumor activity is 3491 kinds existing, is related to 21 kinds of tissues and 249 kinds of tumor cell lines, and part ACPs comes into clinical examination It tests.
The main mode that antineoplastic polypeptide acts on cell is lytic cell film.Natural antitumor peptide Polybia-MPI, Bombesin, synthetic peptide BTM-P1 etc. are to act on kinds of tumor cells in a manner of film cracking.It is killed in a manner of film cracking The cytosis time is short, and polypeptide does not need to enter into the cell, is not likely to produce drug resistance.Another mode of action is failure line grain Body film is to cause the Apoptosis that mitochondria pathway induces.It is broken that Eliassen et al. has found that Bovine lactoferricin LFcinB passes through Bad mitochondrial membrane inducing neural glioma cell apoptosis, and be verified in the Mice Body of heteroplastic transplantation.Leopard toxin then passes through Mitochondria is acted on, caspase3 is activated and induces epidermoid carcinoma cell apoptosis.It is sharp there are also a kind of interesting mode of action The immune response of living cells, it is dead that polypeptide LTX-315 can make cell that immunogenicity occur, and can generate a kind of lasting permanent effect It should can prevent the deterioration that same tumour occurs again.
Natural antitumor peptide is there are some apparent defects, such as hemolytic height, and tumor cell specific recognition capability is not By force, so the application of natural antitumor peptide is very restricted.The concept of gene therapy 1972 by Frideman and Roblin proposes that it is widely used in the treatment of a variety of diseases based on changing bio-genetic material at present first.
The biological targeting of gene therapy mainly includes that viral vectors targeting, the targeting of receptor mating type and cationic-liposome carry The several methods such as body.Viral vectors targeting is to assist adenovirus and retroviral vector using with amphiphatic bridging molecule Targeting transduction, can also realize changing for its gene order by the operon of encoding virus coat proteins or envelope protein Become, generates the virom crosome with the different surface protein of structure, the transfer of Lai Shixian target gene；Receptor mediated gene transfer is normal The transfer for seeing non-viral gene is to identify its corresponding ligand using receptor-specific existing for cell membrane surface, to the two In conjunction with rear by endocytosis, it would be incorporated into the target gene of ligand or carry in gene plasmid orientation importing tumour cell；Sun from Sub- liposome has good binding ability to negatively charged DNA, can binding purpose gene well, and wrap up mesh at it Gene after, can protect target gene from the degradation of lysosome etc..Due to these biological targeting carriers targeting not It is to be designed according to the characteristic of tumour cell, so not having a clear superiority in oncotherapy, and is opened using tumour-specific The targeting vector of mover design can make up this deficiency well.Telomere is to be located at eucaryote chromosome linear DNA molecule The structure of end is in expand graininess in end of chromosome, common in conjunction with multiple protein by one section of duplicate non-transcribed sequences Composition, effect are to maintain chromosome integrality and control cell cycle.The extension or shortening of telomere are adjusted by Telomerase, telomere The activity of enzyme is suppressed with cell differentiation.In recent years, effect of the Telomerase played in cell immortality and cancer occur is drawn Played extensive concern, be all presented high expression in the human tumour tissue of immortalized cell line and 85% or more, and it is most of just Expression is had no in normal body cell, so that Telomerase becomes the new target of therapy of tumor as tumour-specific markers object Point.
Summary of the invention
In order to effectively kill cell, and constructs and specifically expressing and tumour cell can be killed, to machine in tumour cell The non-viral recombinant plasmid of body safety, the present invention provides the following technical scheme that
Be following 1) -4 the present invention provides a kind of polypeptide) in it is any:
It 1) include amino acid sequence is polypeptide shown in sequence 1 in sequence table；
2) amino acid sequence is polypeptide shown in sequence 1 in sequence table；
3) 1) or 2) N-terminal or/and the obtained fused polypeptide of C-terminal connection label；
Or 2) 4) 1) polypeptide shown in is passed through to the substitution and/or deletion and/or addition of one or several amino acid residues Polypeptide obtaining and with anti-tumor function.
The nucleic acid molecules of encoding such polypeptides are also the scope of protection of the invention.
Above-mentioned nucleic acid molecules be it is any in following (a1)-(a6) shown in DNA molecular:
(a1) code area includes the DNA molecular of sequence 2 in sequence table；
(a2) code area includes the DNA molecular of sequence 3 in sequence table；
(a3) code area is the DNA molecular of sequence 2 in sequence table；
(a4) code area is the DNA molecular of sequence 3 in sequence table；
(a5) there is 75% or 75% or more identity with the nucleotide sequence of any restriction in (a1)-(a4), and encode The DNA molecular of aforementioned polypeptides；
(a6) nucleotide sequence hybridization with any restriction in (a1)-(a4), and encoding such polypeptides under strict conditions DNA molecular.
Expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing above-mentioned nucleic acid molecules are also the present invention The range of protection.
Above-mentioned recombinant vector is that above-mentioned nucleic acid molecules are inserted into expression vector, obtained carrier.
In above-mentioned recombinant vector, tomour specific promoter is contained in the expression vector；
Or the tomour specific hTERT promoter is hTERT promoter；
Or it is tomour specific promoter that the promoter of the expression of above-mentioned nucleic acid molecules is driven in the recombinant vector.
Aforementioned polypeptides or above-mentioned nucleic acid molecules, or, above-mentioned expression cassette, recombinant vector, recombinant microorganism or transgenic cell System, following 1) -6) it is at least one in application be also the scope of protection of the invention:
1) preparation treatment, inhibition and/or antitumor product；
2) preparation killing or lethal tumor cell products；
3) preparation makes tumour cell discharge LDH product；
4) tumoricidal membrane structure product is prepared；
5) preparation improves the expression quantity product of Caspase8, Caspase9, the PARP of shearing and/or Bax in tumour cell；
6) preparation reduces the expression quantity product of total PARP in tumour cell.
Another object of the present invention is to provide a kind of method for preparing treatment, inhibition and/or antitumor product.
Method provided by the invention, for individually packaging aforementioned polypeptides or above-mentioned recombinant vector.
Among the above, the tumour is liver cancer, the cancer of the esophagus, breast cancer and/or neuroblastoma.
Among the above, the product is drug or kit.
The experiment proves that having very low the present invention provides a kind of polypeptide fragment that can effectively kill cell IC50Value, while being connect with the tomour specific expression vector containing hTERT promoter, keep polypeptide fragment special in tumour cell High efficient expression and killing tumor cell, do not influence normal cell, solve many polypeptide drugs and select without cell The problem of property.Therefore the recombinant plasmid that the present invention constructs will become a kind of gene therapy medicament for potentially having much prospect.
Detailed description of the invention
Fig. 1 is the bioinformatic analysis result of polypeptide KK-63；Figure 1A is using NPS@(Network protein Sequence@nalysis) the HNN method based on neural network algorithm in system, predict alpha-helix, the beta sheet etc. of polypeptide KK-63 The content of basic second grade structure；Figure 1B is to utilize the hydrophobic of online tool ProtScale assessment polypeptide KK-63 amino acid sequence Property/hydrophily.
Fig. 2 is to handle MCF-7, TE-1, HepG2,5 hour cell metamorphosis of the cells such as HL-7702 with 3.5 μM of polypeptides.
Fig. 3 is to handle MCF-7, TE-1, HepG2, the cells such as HL-7702 24 with 0,0.5,1,2,4,6,8,10 μM of KK-63 Hour cell activity change.
Fig. 4 is that MCF-7, TE-1, HepG2 are handled with 3.5 μM of polypeptides, the cells such as HL-7702 0,0.5,1,2,4,6 hour thin Lactic dehydrogenase (LDH) relative amount of born of the same parents' release.
Fig. 5 is recombinant plasmid pcTERT-KK-63-1/KK-63-2 important feature element and its position.
Fig. 6 is to transiently transfect pc-hTERT-KK-63-1/KK-63- in MCF-7, TE1, HepG2, the cells such as HL-7702 2 plasmids 72 hours, cellular morphology variation.
Fig. 7 is to transiently transfect pc-hTERT-KK-63-1/KK-63- in MCF-7, TE1, HepG2, the cells such as HL-7702 2 plasmids 72 hours, cell activity variation.
Fig. 8 is to transiently transfect pc-hTERT-KK-63-1/KK-63- in MCF-7, TE1, HepG2, the cells such as HL-7702 2 plasmids 72 hours, the expression and activity change of apoptosis-related protein.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The screening of embodiment 1, polypeptide KK-63
1, computer simulation synthesis polypeptide and the suitable polypeptide fragment of related parameter choosing is detected
, as design considerations, to design by computer simulation the characteristics of known cell perforation peptide, optimal 8 of score are chosen Polypeptide after synthesizing the polypeptide for obtaining purifying by chemical method, is carried out bioactivity screening, i.e., is made using the hybrid peptide of various concentration For liver cancer cell lines HepG2 (HB-8065TM, ATCC, the U.S.), esophageal carcinoma cell line TE-1 (TCHu 89, China Academy of sciences's cell bank, China), breast cancer cell line MCF-7 (HTB-22DTM, American Type Culture Collection, beauty State) and hepatic cell line HL-7702 (GNHu 6, Cell Bank of Chinese Academy of Sciences, China) and cell growth state is detected, find KK- IC of 63 polypeptides for these cells50It is worth all very low.Pass through lactic dehydrogenase (LDH) detection kit (Roche, the U.S.) simultaneously LDH burst size is detected, assessment discovery is carried out to the integrality of cell membrane, the effect that KK-63 destroys cell membrane integrity is most strong.Cause This, the polypeptide KK-63 that screening obtains one section of 64 amino acid residue composition is purpose peptide, and amino acid sequence is sequence in sequence table Column 1.
2, bioinformatics technique is analyzed
By ProtParam in line computation protein physicochemical properties parameter, as relative molecular mass, isoelectric point, GRAVAY (Grand average of hydronathicity) hydrophobicity index, instability index etc. (table 1).
Table 1
As the result is shown polypeptide KK-63 relative molecular mass be 7503.14, isoelectric point 12.85, hydrophobicity index be- 0.232, illustrate that the polypeptide has hydrophily, instability index 34.19, liposoluble sex index is 120.63.
Use the HNN based on neural network algorithm in NPS@(Network protein Sequence@nalysis) system Method, prediction alpha-helix, the basic second grades structure (Fig. 1) such as beta sheet, analysis is it is found that alpha-helix rate is 73.02%, random coil The 26.98% of secondary structure conformation is accounted for, illustrates that the secondary structure of KK-63 is mainly alpha-helix.
Using hydrophobicity/hydrophily (Fig. 1) of online tool ProtScale assessment polypeptide KK-63 amino acid sequence, as a result It has been shown that, there are apparent bipolarities, i.e. both ends to be rich in hydrophily and hydrophobic amino acid respectively by KK-63, and it is anti-to meet cation The design feature of tumour peptide.
Polypeptide KK-63 is prepared by chemical synthesis.
Embodiment 2, the recombinant plasmid for constructing promoter containing hTERT and kk-63 polypeptide encoding nucleic acid
KK-63 polypeptide is obtained into amino acid sequence with the anti-DNA sequence dna kk-63-1 for translating into 192nt of source of people codon preference (sequence 2, sequence 2 is without HindIII and XbaI enzyme cutting site), and referring to Kozak sequence characteristic, in initiation codon ATG Add codon glycine GGC afterwards, obtain a 195nt DNA sequence dna kk-63-2 (sequence 3, in sequence 3 without HindIII and XbaI enzyme cutting site), HindIII restriction enzyme site is added at the end kk-63-1 and kk-63-2DNA 5 ', 3 ' ends add Behind XbaI enzyme cutting site, in Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) synthesis.
Tomour specific expression recombinant vector pcTERT contains enhancer and hTERT promoter (sequence 4；Sequence 4 is hTERT Promoter).
With restriction enzyme HindIII and XbaI (Takara, China), to carrier pcTERT, (preparation method is recorded respectively In CN201810543497.X, Publication No. 108715368A), kk-63-1, kk-63-2 sequence carry out double digestion, purify respectively After recycling, the kk-63-1 after digestion and the kk-63-2 after digestion are connected into carrier with T4DNA ligase (Takara, China) In between the HindIII and XbaI of pcTERT, pcTERT-KK-63-1 and pcTERT-KK-63-2 recombinant plasmid (Fig. 5) is obtained.
Recombinant plasmid pcTERT-KK-63-1 is that the kk-63-1DNA molecule shown in sequence 2 in sequence table replaces carrier The plasmid that DNA molecular in pcTERT between HindIII and XbaI enzyme cutting site obtains, and kk-63-1 is driven by hTERT promoter Expression；
Recombinant plasmid pcTERT-KK-63-2 is that the kk-63-2DNA molecule shown in sequence 3 in sequence table replaces carrier The plasmid that DNA molecular between the HindIII and XbaI enzyme cutting site of pcTERT obtains, and kk-63-2 is driven by hTERT promoter Expression.
Above-mentioned double digestion system and response parameter:
Enzyme disjunctor system and response parameter:
Embodiment 3, polypeptide KK-63 detect the toxic effect of cell
1, the influence that polypeptide KK-63 changes cellular morphology
(1) logarithmic growth phase MCF-7, TE-1, HepG2 and HL-7702 cell after pancreatin digestion, are trained with high sugared cell It supports base (Corning, the U.S.) HepG2, TE-1, MCF-7 are diluted to the cell suspension of 125000/ml, HL-7702 diluted At the cell suspension of 150000/ml, 6 orifice plates are inoculated in the hole 2ml/, at 37 DEG C, 5%CO2Culture 12 is small in cell incubator When, make cell adherent；
(2) polypeptide handles cell
The polypeptide KK-63 powder of synthesis: being dissolved the storing liquid for being configured to 10mM by experimental group with DMSO (Sigma, the U.S.), And 3.5 μM of concentration is diluted to culture medium, replace the original culture medium of cell.
Control group (normal condition): give the DMSO of equivalent.
After processing 5 hours, observation cellular morphology changes and takes pictures.
As a result as shown in fig. 2, it can be seen that more than KK-63 processing 4 kinds cell 5 hours, cellular morphology is substantially change, carefully Born of the same parents' structure is imperfect, many cell fragments, a large amount of cell deaths occurs.
2, polypeptide KK-63 acts on cell activity and detects
(1) logarithmic growth phase HepG2, TE-1, MCF-7 and HL-7702 cell, after pancreatin digestion, HepG2, TE-1, MCF-7 is diluted to the cell suspension of 50000/ml, and HL-7702 is diluted to the cell suspension of 80000/ml, with 100 holes μ l/ It is inoculated in 96 orifice plates, every group of 4 multiple holes, at 37 DEG C, 5%CO2It is cultivated 12 hours in cell incubator, makes cell adherent；
(2) polypeptide KK-63 is handled
Experimental group: the polypeptide KK-63 powder of synthesis is dissolved to the storing liquid for being configured to 10mM with DMSO, and dilute with culture medium 0.5 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM and 10 μM of 7 various concentrations are interpreted into, replace original training with the culture medium containing polypeptide Support base；
Control group: give the DMSO with maximum concentration group equivalent；
(3) after various concentration KK-63 is handled cell 24 hours, former culture medium is discarded, every hole, which is added, contains 20 μ l cell activity The culture medium of detection reagent (Promega, the U.S.), 37 DEG C are protected from light incubation 2 hours, and microplate reader (Biotek, the U.S.) detects 490nm Light absorption value at wavelength；
Cell survival rate: the survival rate of cell=experimental group OD value/control group OD value × 100% is calculated with following formula, And KK-63 is calculated to the IC of different cells with SPSS software50Value.
As a result as shown in figure 3, KK-63, which is acted on 24 hours, can obviously inhibit Several Kinds of Malignancy cell activity, IC50It is worth low In 6, illustrate that polypeptide KK-63 has very strong tumor cytotoxicity effect.
3, polypeptide KK-63 detects cytotoxic effect
(1) logarithmic growth phase HepG2, TE-1, MCF-7 and HL-7702 cell use cell culture medium after pancreatin digestion HepG2, TE-1, MCF-7 are diluted to the cell suspension of 50000/ml, the cell that HL-7702 is diluted to 80000/ml is outstanding Liquid is inoculated in 96 orifice plates with 100 holes μ l/, every group of 4 multiple holes, at 37 DEG C, 5%CO2It cultivates 12 hours, allows thin in cell incubator Born of the same parents are adherent；
(2) it handles
Experimental group: KK-63 is diluted to 3.5 μM with the high glucose medium containing 1% serum (Gibco, the U.S.) and handles 5 kinds Cell,
Positive controls: 5 kinds of cells are handled with the high glucose medium in X-100 containing 1%Triton (Genview, the U.S.)；
Negative control group: the DMSO for giving KK-63 equivalent handles 5 kinds of cells；
(3) it detects
100 μ l supernatants were collected at 0.5,1,2,4,6 hour respectively, detect different time points cell with LDH detection kit The content of lactic dehydrogenase in culture supernatant, after 100 μ l detection reagents are added in each sample, room temperature is protected from light incubation 30 minutes, enzyme Mark the light absorption value at instrument detection 450nm and 560nm wavelength；
KK-63 is calculated to the relative toxicity of cell with following formula: positive control light absorption value-negative control light absorption value/reality Test a group light absorption value-negative control light absorption value × 100%.
As a result as shown in Figure 4, it can be seen that KK-63 acts in 1 hour the relative toxicity of Several Kinds of Malignancy cell Just reach maximum value (100%), the release of LDH can indicate that membranolysis, KK-63 can make cell release big in a short time LDH is measured, illustrates that KK-63 can rapid damage membrane structure.
Embodiment 4, recombinant plasmid pcTERT-KK63-1 and pcTERT-KK-63-2 are in tumour cell and normal cell Active function detection
1, recombinant plasmid pcTERT-KK-63-1 and pcTERT-KK-63-2 becomes the form of tumour cell and normal cell Changing influences
(1) logarithmic growth phase MCF-7, TE-1, HepG2 and HL-7702 cell, after pancreatin digestion, HepG2, TE-1, MCF-7 is diluted to the cell suspension of 125000/ml, and HL-7702 is diluted to the cell suspension of 150000/ml, with the hole 2ml/ 6 orifice plates are inoculated in, is cultivated 12 hours in 37 DEG C, 5%CO2 cell incubator, makes cell adherent；
(2) recombinant plasmid function cells
Experimental group: recombinant plasmid pcTERT-KK-63-1 and pcTERT-KK-63-2 are used into lipofectamine respectively (Invitrogen, the U.S.) is transferred in HepG2, TE-1, MCF-7 and the HL-7702 cell of above-mentioned (1) culture in wink；
Control group: HepG2, the TE-1 that empty carrier pcTERT is cultivated with above-mentioned (1) is transferred to lipofectamine wink, In MCF-7 and HL-7702 cell；
After transfection 72 hours, observation cellular morphology variation, and take pictures.
As a result as shown in fig. 6, transfection can significantly modify for pcTERT-KK-63-1 and pcTERT-KK-63-2 plasmid 72 hours There is death in the form of the tumour cells such as HepG2, TE-1 and MCF-7, cellular morphology shrinkage, and transfection control group plasmid pair is thin Born of the same parents' form has not significant impact, it is important that transfection recombinant plasmid is to the form of normal cell HL-7702 also without obvious shadow It rings.
2, recombinant plasmid pcTERT-KK-63-1 and pcTERT-KK-63-2 presses down the activity of tumour cell and normal cell Production detection
(1) logarithmic growth phase HepG2, TE-1, MCF-7 and HL-7702 cell, after pancreatin digestion, HepG2, TE-1, MCF-7 is with 1 × 105The density in a/hole, HL-7702 is with 1.5 × 105The density in a/hole is inoculated in 12 orifice plates, with the training in the hole 1ml/ Base is supported at 37 DEG C, 5%CO2Cultivating 12 hours in cell incubator makes cell adherent；
(2) recombinant plasmid function cells
Experimental group: recombinant plasmid pcTERT-KK-63-1 and pcTERT-KK-63-2 is transferred to lipofectamine wink In HepG2, TE-1, MCF-7 and HL-7702 cell；
Control group: transfection empty carrier pcTERT is transferred to HepG2, TE-1, MCF-7 and HL- with lipofectamine wink In 7702 cells；
It after transfection 72 hours, inhales and abandons culture medium, each hole is added the 100ml containing 20 μ l cytoactive detection reagents and cultivates Base, 37 DEG C are protected from light incubation 2 hours, and microplate reader detects light absorption value at 490nm wavelength；
(3) with following formula calculating cell survival rate: the survival rate of cell=experimental group OD value/control group OD value × 100%.
As a result as shown in Figure 7, it can be seen that recombinant plasmid pcTERT-KK-63-1 and pcTERT-KK-63-2 wink turns tumour After cell 72 hours, compared with the control group, cell activity is obviously suppressed, and illustrates that kk-63 sequence can be expressed in tumour cell, And its expression product has the activity of killing tumor cell.And in normal liver cell system HL-7702, it does not detect Kk-63 is to its active effect.
Illustrate that recombinant plasmid specificity starting kk-63 sequence can express in tumour cell, and killing tumor cell.
3, shadow of the recombinant plasmid pcTERT-KK-63-1 and pcTERT-KK-63-2 to tumour cell and normal apoptosis It rings
(1) logarithmic growth phase HepG2, TE-1, MCF-7 and HL-7702 cell, after pancreatin digestion, HepG2, TE-1, MCF-7 is inoculated in 12 orifice plates with the density in 1 × 105/hole, HL-7702 with the density in 1.5 × 105/hole, with the hole 1ml/ Culture medium is cultivated 12 hours in 37 DEG C, 5%CO2 cell incubator makes cell adherent；
(2) recombinant plasmid is handled
Experimental group: by recombinant plasmid pc-hTERT-KK-63-1 and pc-hTERT-KK-63-2 with lipofectamine wink It is transferred to HepG2, TE-1, in MCF-7 and HL-7702 cell,
Control group: will transfection empty carrier pc-hTERT with being transferred to HepG2 lipofectamine wink, TE-1, MCF-7 and In HL-7702 cell.
It after transfection 72 hours, inhales and abandons culture medium, scrape cell and multigelation three times with cell pyrolysis liquid, be centrifuged, extract Albumen in supernatant；
(3) apoptosis correlation Caspase family protein Activation and PARP, Bcl2 are detected by detected by Western blot, The expression of the albumen such as Bax.
As a result as shown in figure 8, from protein immunoblot result it is found that kk-63-1 and kk-63-2 sequence is in tumour cell After expression, Caspase8,9 (Abclonal, China) are all obviously activated, total PARP (ThermoFisher Scientific, the U.S.) expression quantity downward, the PARP expression quantity rising of shearing, Bax (Cell Signaling Technologies, the U.S.) expression quantity also raise, but in normal cell HL-7702, the activation and expression of these albumen It does not substantially change.
These results explanation, tumour cell specific can start hTERT promoter and express kk-63-1 and kk-63-2 sequence Column, the polypeptide of expression can promote apoptosis in tumour cell.
The above is related embodiment of the present invention, and the description thereof is more specific and detailed, but it cannot be understood as Limitations on the scope of the patent of the present invention.It should be pointed out that for those of ordinary skill in the art, not departing from this hair Under the premise of bright design, various modifications and improvements can be made, and these are all within the scope of protection of the present invention.
SEQUENCE LISTING
<110>Jilin University
<120>antineoplastic polypeptide and its application in antitumor field
<160> 4
<170> PatentIn version 3.5
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<211> 64
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<213> Artificial sequence
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Met Lys Lys Arg Leu Leu Arg Gly Arg Leu Leu Arg Ile Lys Lys Ile
1 5 10 15
Leu Ser Leu Ile Gly Gly Leu Leu Lys Trp Lys Leu Phe Lys Lys Ile
20 25 30
Gly Ile Gly Lys Phe Leu His Ser Trp Lys Lys Phe Gly Gly Arg Trp
35 40 45
Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Lys
50 55 60
<210> 2
<211> 198
<212> DNA
<213> Artificial sequence
<400> 2
accatgaaga agagactgct gagaggcaga ttgctgagaa tcaagaagat cctgagcctg 60
atcggcggcc tgctgaagtg gaagctgttc aagaagatcg gcatcggcaa gttcctgcac 120
agctggaaga agttcggcgg cagatggggc ctgcagttcc ccgtgggcag agtgcacaga 180
ctgctgagaa agaagtaa 198
<210> 3
<211> 201
<212> DNA
<213> Artificial sequence
<400> 3
accatgggca agaagagact gctgagaggc agattgctga gaatcaagaa gatcctgagc 60
ctgatcggcg gcctgctgaa gtggaagctg ttcaagaaga tcggcatcgg caagttcctg 120
cacagctgga agaagttcgg cggcagatgg ggcctgcagt tccccgtggg cagagtgcac 180
agactgctga gaaagaagta a 201
<210> 4
<211> 378
<212> DNA
<213> Artificial sequence
<400> 4
accctgggag cgcgagcggc gcgcgggcgg ggaagcgcgg cccagacccc cgggtccgcc 60
cggagcagct gcgctgtcgg ggccaggccg ggctcccagt ggattcgcgg gcacagacgc 120
ccaggaccgc gcttcccacg tggcggaggg actggggacc cgggcacccg tcctgcccct 180
tcaccttcca gctccgcctc ctccgcgcgg accccgcccc gtcccgaccc ctcccgggtc 240
cccggcccag ccccctccgg gccctcccag cccctcccct tcctttccgc ggccccgccc 300
tctcctcgcg gcgcgagttt caggcagcgc tgcgtcctgc tgcgcacgtg ggaagccctg 360
gccccggcca cccccgcg 378

Claims (10)

  1. Be following 1) -4 1. polypeptide) in it is any:
    It 1) include amino acid sequence is polypeptide shown in sequence 1 in sequence table；
    2) amino acid sequence is polypeptide shown in sequence 1 in sequence table；
    3) 1) or 2) N-terminal or/and the obtained fused polypeptide of C-terminal connection label；
    Or 2) 4) 1) polypeptide shown in is obtained by the substitution and/or deletion and/or addition of one or several amino acid residues And with anti-tumor function polypeptide.
  2. 2. encoding the nucleic acid molecules of polypeptide described in claim 1.
  3. 3. nucleic acid molecules as claimed in claim 2, it is characterised in that: the nucleic acid molecules are any in following (a1)-(a6) DNA molecular shown in kind:
    (a1) code area includes the DNA molecular of sequence 2 in sequence table；
    (a2) code area includes the DNA molecular of sequence 3 in sequence table；
    (a3) code area is the DNA molecular of sequence 2 in sequence table；
    (a4) code area is the DNA molecular of sequence 3 in sequence table；
    (a5) there is 75% or 75% or more identity with the nucleotide sequence of any restriction in (a1)-(a4), and encode right It is required that the DNA molecular of 1 protein；
    (a6) nucleotide sequence hybridization with any restriction in (a1)-(a4) under strict conditions, and encode described in claim 1 The DNA molecular of protein.
  4. 4. expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing nucleic acid molecules described in Claims 2 or 3.
  5. 5. recombinant vector according to claim 4, it is characterised in that:
    The recombinant vector is that nucleic acid molecules described in claim 2 or 3 are inserted into expression vector, obtained carrier.
  6. 6. recombinant vector according to claim 5, it is characterised in that:
    Contain tomour specific promoter in the expression vector；
    Or the tomour specific hTERT promoter is hTERT promoter；
    Or the promoter of the expression of nucleic acid molecules described in claim 2 or 3 is driven to open in the recombinant vector for tomour specific Mover.
  7. 7. nucleic acid molecules described in polypeptide or Claims 2 or 3 described in claim 1, or, any table in claim 4-5 Up to box, recombinant vector, recombinant microorganism or transgenic cell line, following 1) -6) it is at least one in application:
    1) preparation treatment, inhibition and/or antitumor product；
    2) preparation killing or lethal tumor cell products；
    3) preparation makes tumour cell discharge LDH product；
    4) tumoricidal membrane structure product is prepared；
    5) preparation improves the expression quantity product of Caspase8, Caspase9, the PARP of shearing and/or Bax in tumour cell；
    6) preparation reduces the expression quantity product of total PARP in tumour cell.
  8. 8. a kind of method for preparing treatment, inhibition and/or antitumor product, for polypeptide or power described in individually packaging claim 1 Benefit requires any recombinant vector in 4-5.
  9. 9. it is according to claim 7 application or method described in 8, it is characterised in that: the tumour be liver cancer, the cancer of the esophagus, Breast cancer and/or neuroblastoma.
  10. 10. application according to claim 7 or method described in 8, it is characterised in that: the product is drug or reagent Box.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113583095A (en) * 2021-07-29 2021-11-02 上海卡序生物医药科技有限公司 Antitumor polypeptide and application thereof

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Publication number Priority date Publication date Assignee Title
CN103096932A (en) * 2010-06-14 2013-05-08 弗·哈夫曼-拉罗切有限公司 Cell-penetrating peptides and uses therof
WO2016111420A1 (en) * 2015-01-07 2016-07-14 서울대학교산학협력단 Cell penetration and enzymatic stabilization functions of α-helix domain in 30kc19 protein, and cargo delivery system using same
CN105829336A (en) * 2013-10-17 2016-08-03 首尔大学校产学协力团 Alpha helix cell-penetrating peptide multimer, preparation method therefor and use thereof
CN108715863A (en) * 2018-05-30 2018-10-30 吉林大学 A kind of tumor-targeting carrier pcTERT and its construction method and its application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103096932A (en) * 2010-06-14 2013-05-08 弗·哈夫曼-拉罗切有限公司 Cell-penetrating peptides and uses therof
CN105829336A (en) * 2013-10-17 2016-08-03 首尔大学校产学协力团 Alpha helix cell-penetrating peptide multimer, preparation method therefor and use thereof
WO2016111420A1 (en) * 2015-01-07 2016-07-14 서울대학교산학협력단 Cell penetration and enzymatic stabilization functions of α-helix domain in 30kc19 protein, and cargo delivery system using same
CN108715863A (en) * 2018-05-30 2018-10-30 吉林大学 A kind of tumor-targeting carrier pcTERT and its construction method and its application

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113583095A (en) * 2021-07-29 2021-11-02 上海卡序生物医药科技有限公司 Antitumor polypeptide and application thereof

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