Summary of the invention
Purpose of the present invention overcomes the deficiencies in the prior art, the polypeptide and the derived product thereof of a kind of energy treatment or preventing cancer are provided, described polypeptide or its derived product can not only be used for the treatment of separately or preventing cancer, and when itself and gene toxin coupling significantly the enhancing gene toxin optionally kill and wound the ability of cancer cells.
The inventor finds that through after the deep research a kind of polypeptide with the listed feature in the following technical proposals can achieve the above object.
The polypeptide of a kind of energy treatment or preventing cancer, its aminoacid sequence is shown in the SEQ ID NO.1.
The polypeptide of a kind of energy treatment or preventing cancer, its aminoacid sequence is shown in the SEQ ID NO.2.
The nucleotide sequence of a kind of separation and purifying, the polypeptide of its aminoacid sequence shown in SEQ ID NO.1 or the SEQ ID NO.2 of encoding.
A kind of expression vector, the encoding amino acid sequence that comprises at least one copy are the nucleotide sequence of polypeptide shown in SEQ ID NO.1 or the SEQ ID NO.2.
A kind of protokaryon or eukaryotic host cell, this host cell contains above-mentioned expression vector.
The present invention also further provide with SEQ ID NO.1 or the described polypeptide of SEQ ID NO.2 with can increase its preparation that in cell, accumulates and put together or mix the resulting product in back mutually.
The preparation that above-mentioned and described peptide is puted together is the carrier that can assist described peptide permeates cell membranes.SEQ IDNO.1 or the described peptide of SEQ ID NO.2 and the carrier that can assist the peptide permeates cell membranes, as the HIV48-57 peptide, FHV-is outer by the 35-49 peptide, HTLV-II Rex 4-16 peptide or BMV gag7-25 peptide are puted together, the compound of puting together the back generation can effectively pass through cytolemma, work in the cancer cells part, therefore have the stronger effect of killing and wounding cancer cells.
Can also be nano material, liposome or oiliness compound with the preparation of SEQ ID NO.1 or the described conjugation of polypeptides of SEQ ID NO.2.
SEQ ID NO.1 or the described polypeptide of SEQ ID NO.2 and macromolecular materials such as nano material, liposome are puted together, and the compound of puting together the back generation can make the polypeptide that the present invention relates to more stably be transported to target cell in body.The polypeptide that the present invention relates to also can mix mutually with the mixture of oiliness compound or multiple oiliness compound, and resulting mixture also can make polypeptide of the present invention more stably be transported to target cell in body.
The medicinal compositions of a kind of treatment or preventing cancer comprises: as SEQ ID NO.1 or the SEQ ID NO.2 peptide and pharmaceutical carrier, thinner and/or adjuvant composition of the medicinal significant quantity of active substance.
The present invention also provides SEQ ID NO.1 or the described polypeptide of SEQ ID NO.2 to be used for preparing the purposes of the medicine of treatment or preventing cancer.
Described cancer includes but not limited to: lung cancer, liver cancer, cancer of the stomach, colorectal carcinoma, the rectum cancer, the esophageal carcinoma, mammary cancer, leukemia, bladder cancer, cervical cancer or nasopharyngeal carcinoma etc.
The present invention also provides SEQ ID NO.1 or the described polypeptide of SEQ ID NO.2 has been used for preparing the purposes that the enhancing gene toxin optionally kills and wounds the medicine of cancer cells.
Described cancer cells includes but not limited to: cancer cells such as lung cancer, liver cancer, cancer of the stomach, colorectal carcinoma, the rectum cancer, the esophageal carcinoma, mammary cancer, leukemia, bladder cancer, cervical cancer or nasopharyngeal carcinoma.
Described gene toxin includes but not limited to: Platinol, oxaliplatin, taxol, epirubicin, Dx, pirarubicin, daunorubicin, mitomycin, Dacarbazine, endoxan, gemcitabine or capecitabine etc.
Polypeptide of the present invention can add acceptable accessories again with the gene toxin mixing or carrier obtains more effective cancer therapy drug.
Polypeptide of the present invention normally uses with the consumption that can realize intended purposes.When being used for prevention and treatment cancer, polypeptide of the present invention or its medicinal compositions are taken with the treatment significant quantity or are used.For systemic medication, can be according to experiment in vitro estimation treatment significant quantity or consumption, the common method that can also use this area estimates the intravital initial dose of polypeptide of the present invention by animal model.Certainly, the concrete significant quantity of polypeptide of the present invention will depend on severity, application method and doctor in charge's the clinical judgment of body weight, the disease of concrete treatment target, treatment target.
The inventor finds later on above-mentionedly to can be used for treating or the polypeptide of preventing cancer through deep research.This polypeptide not only has tangible lethality to cancer cells when using separately, and can share with chemotherapeutics (as Platinol) commonly used clinically, increase the susceptibility of chemotherapeutics significantly, strengthen its lethality, reduce its using dosage cancer cells to cancer cells.Polypeptide of the present invention can be to lung cancer, liver cancer, and cancer of the stomach, colorectal carcinoma, the rectum cancer, the esophageal carcinoma, mammary cancer, multiple cancer cells such as leukemia plays lethal effect.Polypeptide of the present invention has antitumor action; The effect that polypeptide of the present invention and Platinol share cancer cells significantly is better than RasGAP
317-326Peptide (RasGAP
317-326The amino acid residue sequence of peptide is: the WMWVTNLRTD) effect of share with Platinol.The polypeptide that the present invention relates to does not have tangible toxicity enhancement to normal cell, and the polypeptide that the present invention relates to can adopt the method for chemosynthesis to prepare, the purity height, and molecular weight is little, high specificity, non-immunogenicity, safe and reliable.
Polypeptide of the present invention can be used for treatment for cancer and prevention separately and good effect is arranged, polypeptide of the present invention can also and chemotherapeutics (as Platinol, taxol etc.) share, can selectivity strengthen the susceptibility of chemotherapeutics to tumour cell, obviously reduced the onset dosage of chemotherapeutics, because little, thereby the toxicity and the side effect of chemotherapeutics have been reduced to Normocellular toxic action.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Embodiment 1 RasGAP
317-326Synthesizing of peptide
1) laboratory apparatus and material:
Dimethyl formamide (DMF), piperidines, resin, methylene dichloride (DCM), Kaiser test kit, I-hydroxybenzotriazole (HOBt), tetramethyl-urea hexafluorophosphate (HBTU), diisopropylethylamine (DIEA), methyl alcohol, each seed amino acid, polypeptide solid phase synthesis pipe.
2) experimental procedure:
The weighing resin is also put in the polypeptide solid phase synthesis pipe (hereinafter to be referred as reactor), adds an amount of DMF swelling 10 minutes.Take out DMF and carry out the Fmoc protective reaction, promptly add the DMF solution that contains 20% piperidines in right amount, fully stir, take out solution after 5 minutes, the DMF solution that adds 20% piperidines again goes to protect 7 minutes.Take out and protect liquid, use DMF washing resin 4-5 time, take out DMF; with DCM washing 1-2 time, (about 5~10mg) in test tube, uses washing with alcohol 2 times for the resin that takes a morsel from reactor; the Kaiser method detects and the record color, prepares to feed intake, and enters the amino acid condensation reaction.According to RasGAP
317-326The aminoacid sequence of peptide is got corresponding amino acid, HOBt in proper order in proper container, with the DMF dissolving, adds DIEA activation 5 minutes after the dissolving fully, adds load weighted HBTU, and ice-water bath dissolving down activates 5 minutes, puts in the reactor stirring reaction.After 90 minutes, take a morsel resin from reactor in test tube, use washing with alcohol 2 times, the Kaiser method detects.Take out the liquid in the reactor,, take out DMF, obtain the peptide resin behind first amino acid condensation with DMF washing 2 times.The gained peptide resin is repeated above " Fmoc go protection---amino acid condensation " reactions steps, finish to last amino acid reaction, obtain RasGAP
317-326Peptide.Adopt aforesaid method, according to HIV
48-57The sequence of peptide is at RasGAP
317-326Continue chemosynthesis on the peptide and connect corresponding amino acid, obtain having puted together HIV
48-57RasGAP
317-326Peptide.After reaction finished, DCM washing resin 2-3 time was taken out solvent, added methyl alcohol and stirred contraction (5min+5min), took out methyl alcohol, continued to drain 15-20min.Take out the peptide resin that has synthesized in the reactor, transfer to round-bottomed flask, in moisture eliminator, drain cracking at room temperature two hours.Solution behind the resin filter is concentrated freeze-dried under vacuum.Thick peptide uses Tianjin, island LC-6AD to prepare type reversed-phase HPLC purifying, uses HPLC to detect purity>90%.Resulting pure peptide uses mass spectrum, and (MS identifies that electrospray) its molecular weight is 2813.29, and is consistent with calculated value 2813.29.
Synthesizing of the described peptide of embodiment 2 SEQ ID NO.1
1) laboratory apparatus and material:
Dimethyl formamide (DMF), piperidines, resin, methylene dichloride (DCM), Kaiser test kit, I-hydroxybenzotriazole (HOBt), tetramethyl-urea hexafluorophosphate (HBTU), diisopropylethylamine (DIEA), methyl alcohol, each seed amino acid, polypeptide solid phase synthesis pipe.
2) experimental procedure:
The weighing resin is also put in the polypeptide solid phase synthesis pipe (hereinafter to be referred as reactor), adds an amount of DMF swelling 10 minutes.Take out DMF and carry out the Fmoc protective reaction, promptly add the DMF solution that contains 20% piperidines in right amount, fully stir, take out solution after 5 minutes, the DMF solution that adds 20% piperidines again goes to protect 7 minutes.Take out and protect liquid, use DMF washing resin 4-5 time, take out DMF; with DCM washing 1-2 time, (about 5~10mg) in test tube, uses washing with alcohol 2 times for the resin that takes a morsel from reactor; the Kaiser method detects and the record color, prepares to feed intake, and enters the amino acid condensation reaction.Order according to the aminoacid sequence of the described peptide of SEQ ID NO.1 takes by weighing corresponding amino acid, HOBt in proper container, and with the DMF dissolving, dissolving back fully adds DIEA activation 5 minutes, add load weighted HBTU, ice-water bath dissolving down activates 5 minutes, puts in the reactor stirring reaction.After 90 minutes, take a morsel resin from reactor in test tube, use washing with alcohol 2 times, the Kaiser method detects.Take out the liquid in the reactor,, take out DMF, obtain the peptide resin behind first amino acid condensation with DMF washing 2 times.The gained peptide resin is repeated above " Fmoc go protection---amino acid condensation " reactions steps, finish to last amino acid reaction of SEQ ID NO.1 sequence, obtain SEQ ID NO.1 peptide.Adopt aforesaid method, according to HIV
48-57The sequence of peptide continues chemosynthesis and connects corresponding amino acid on SEQ ID NO.1 peptide, obtain having puted together HIV
48-57SEQ ID NO.1 peptide.After reaction finished, DCM washing resin 2-3 time was taken out solvent, added methyl alcohol and stirred contraction (5min+5min), took out methyl alcohol, continued to drain 15-20min.Take out the peptide resin that has synthesized in the reactor, transfer to round-bottomed flask, in moisture eliminator, drain cracking at room temperature two hours.Solution behind the resin filter is concentrated freeze-dried under vacuum.Thick peptide uses Tianjin, island LC-6AD to prepare type reversed-phase HPLC purifying, uses HPLC to detect purity>90%.Resulting pure peptide uses mass spectrum, and (MS identifies that electrospray) its molecular weight is 3411.01, and is consistent with calculated value 3412.96.
According to the method described above can be the outer quilt of FHV-
35-49Peptide, HTLV-II Rex
4-16Peptide or BMV gag
7-25Peptide and SEQ ID NO.1 peptide of the present invention are conjugated in together.
Synthesizing of the described peptide of embodiment 3 SEQ ID NO.2
1) instrument and material:
Dimethyl formamide (DMF), piperidines, resin, methylene dichloride (DCM), Kaiser test kit, I-hydroxybenzotriazole (HOBt), tetramethyl-urea hexafluorophosphate (HBTU), diisopropylethylamine (DIEA), methyl alcohol, each seed amino acid, polypeptide solid phase synthesis pipe.
2) experimental procedure:
The weighing resin is also put in the polypeptide solid phase synthesis pipe (hereinafter to be referred as reactor), adds an amount of DMF swelling 10 minutes.Take out DMF and carry out the Fmoc protective reaction, promptly add the DMF solution that contains 20% piperidines in right amount, fully stir, take out solution after 5 minutes, the DMF solution that adds 20% piperidines again goes to protect 7 minutes.Take out and protect liquid, use DMF washing resin 4-5 time, take out DMF; with DCM washing 1-2 time, (about 5~10mg) in test tube, uses washing with alcohol 2 times for the resin that takes a morsel from reactor; the Kaiser method detects and the record color, prepares to feed intake, and enters the amino acid condensation reaction.Aminoacid sequence according to the described peptide of SEQ ID NO.2 takes by weighing corresponding amino acid, HOBt in proper container, and with the DMF dissolving, dissolving back fully adds DIEA activation 5 minutes, add load weighted HBTU, ice-water bath dissolving down activates 5 minutes, puts in the reactor stirring reaction.After 90 minutes, take a morsel resin from reactor in test tube, use washing with alcohol 2 times, the Kaiser method detects.Take out the liquid in the reactor,, take out DMF, obtain the peptide resin behind first amino acid condensation with DMF washing 2 times.The gained peptide resin is repeated above " Fmoc go protection---amino acid condensation " reactions steps, finish to last amino acid reaction of SEQ ID NO.2 sequence, obtain SEQ ID NO.2 peptide.Adopt aforesaid method, according to HIV
48-57The sequence of peptide continues chemosynthesis and connects corresponding amino acid on SEQ IDNO.2 peptide, obtain having puted together HIV
48-57SEQ ID NO.2 peptide.After reaction finished, DCM washing resin 2-3 time was taken out solvent, added methyl alcohol and stirred contraction (5min+5min), took out methyl alcohol, continued to drain 15-20min.Take out the peptide resin that has synthesized in the reactor, transfer to round-bottomed flask, in moisture eliminator, drain cracking at room temperature two hours.Solution behind the resin filter is concentrated freeze-dried under vacuum.Thick peptide uses Tianjin, island LC-6AD to prepare type reversed-phase HPLC purifying, uses HPLC to detect purity>90%.Resulting pure peptide uses mass spectrum, and (MS identifies that electrospray) its molecular weight is 4660.41, meets calculated value.
According to the method described above can be the outer quilt of FHV-
35-49Peptide, HTLV-II Rex
4-16Peptide or BMV gag
7-25Peptide and SEQ ID NO.2 peptide of the present invention are conjugated in together.
Experimental example 1 mtt assay detects SEQ ID NO.1, SEQ ID NO.2 and RasGAP
317-326Peptide is tested the restraining effect of tumour cell respectively
One, laboratory sample: embodiment 1-3 institute synthetic polypeptide: RasGAP
317-326Peptide, SEQ ID NO.1 peptide, SEQ ID NO.2 peptide;
Two, experimental technique
Human cervical carcinoma cell strain Hela (purchasing in China typical culture collection center Wuhan University center) is suspended in the RPMI1640 nutrient solution that contains 10% heat-inactivated fetal bovine serum, is inoculated in 96 orifice plates with the density in 5000-10000/hole.Cell is at 37 ℃, 5%CO
2Cultivate.When cell density reaches 70%-80%, add Platinol, RasGAP
317-326Peptide, the described peptide of SEQ ID NO.1 or the described peptide of SEQ ID NO.2, wherein every kind of drug concentrations gradient is 40 μ mol/L, 30 μ mol/L, 20 μ mol/L, 10 μ mol/L, 0 μ mol/L, and each concentration is established 3 multiple holes.Continue to cultivate every hole adding MTT (5mg/ml) 20 μ l after 20 hours, inhale after 4 hours and remove nutrient solution, every hole adds dimethyl sulfoxide (DMSO) (DMSO) 150 μ l, shakes purple crystal fully to be dissolved in 10 minutes.Microplate reader 570nm measures absorbancy.
Cell inhibitory rate is calculated as follows:
Cell inhibitory rate=(blank group OD value-experimental group OD value)/blank group OD value * 100%
Data statistics is represented with mean ± standard deviation, adopts the t check.
Three, experimental result:
As seen from Figure 4, generally SEQ ID NO.1, the described polypeptide of SEQ ID NO.2 to the Hela cell inhibiting effect compare RasGAP
317-326Peptide is remarkable.When medicine at lower concentration, that is: when concentration was less than 10 μ mol/L, SEQ ID NO.1, the described peptide of SEQ ID NO.2 just had restraining effect to the Hela cell and than the Platinol or the RasGAP of same concentrations
317-326The effect of peptide is more obvious, and significant difference has statistical significance between the two.SEQ ID NO.1, the described peptide of SEQ ID NO.2 have significant inhibitory effect to the Hela cell, and the RasGAP of same concentrations
317-326Peptide does not almost have restraining effect to the Hela cell.Illustrate that polypeptide of the present invention can be used as the medicine of treatment and preventing cancer.
Experimental example 2 mtt assay detect different peptides and Platinol and share effect experiment to tumour cell
One, laboratory sample: embodiment 1-3 institute synthetic polypeptide: RasGAP
317-326Peptide, SEQ ID NO.1 peptide, SEQ ID NO.2 peptide;
Two, experimental technique
Human cervical carcinoma cell strain Hela is suspended in the RPMI1640 nutrient solution that contains 10% heat-inactivated fetal bovine serum, is inoculated in 96 orifice plates with the density in 5000-10000/hole.Cultivate after 24 hours, add Platinol and RasGAP respectively
317-326The peptide mixed solution, described peptide mixed solution of Platinol and SEQ ID NO.1 or Platinol and the described peptide mixed solution of SEQ ID NO.2.Wherein the Platinol concentration gradient is 1.1 μ mol/L, 0.37 μ mol/L, 0.12 μ mol/L, 0 μ mol/L in every kind of mixed solution, and each peptide final concentration is 20 μ mol/L, and each concentration is established 3 multiple holes.Continue to cultivate every hole adding MTT (5mg/ml) 20 μ l after 20 hours, inhale after 4 hours and remove nutrient solution, every hole adds DMSO 150 μ l, shakes purple crystal fully to be dissolved in 10 minutes.Microplate reader 570nm measures absorbancy.
Cell inhibitory rate is calculated as follows:
Cell inhibitory rate=(blank group OD value-experimental group OD value)/blank group OD value * 100%
Data statistics is represented with mean ± standard deviation, adopts the t check.
Three, experimental result:
As seen from Figure 5, when Platinol and SEQ ID NO.1 share or share with SEQ ID NO.2 peptide, perhaps with RasGAP
317-326When peptide share, they all obviously were better than independent use Platinol to the effect of Hela cell inhibiting, illustrated that polypeptide of the present invention can increase the susceptibility of Platinol to the Hela cell, strengthened it to the effect of Hela cell inhibiting.Three peptide species are different to increasing Platinol to the ability of Hela cellular sensitivity in this experiment, and wherein the described peptide of SEQ ID NO.2 is the most obvious to the sensitization of Platinol, secondly is the described peptide of SEQ ID NO.1; When the concentration of Platinol was 0 μ mol/L, the described peptide of SEQ IDNO.1, the described peptide of SEQ ID NO.2 were respectively 17.6% and 17.7% to Hela cell inhibiting rate, and RasGAP
317-326The inhibiting rate of peptide only is 2.5%, and described peptide of SEQ ID NO.1 and the described peptide of SEQ ID NO.2 are better than RasGAP to the sensitization of Platinol
317-326Peptide is to the sensitization of Platinol, and has statistical significance.
Experimental example 3 mtt assay detect different peptides and Platinol share normal cell inhibiting effect experiment
One, laboratory sample: embodiment 1-3 institute synthetic polypeptide: RasGAP
317-326Peptide, SEQ ID NO.1 peptide, SEQ ID NO.2 peptide;
Two, experimental technique
People sarcoplast (purchasing the Shanghai cell bank in the Chinese Academy of Sciences) is suspended in the RPMI1640 nutrient solution that contains 10% heat-inactivated fetal bovine serum, is inoculated in 96 orifice plates with the density in 5000-10000/hole.Cultivate after 24 hours, when cell density reaches 70%-80%, add Platinol and RasGAP respectively
317-326The mixed solution of the mixed solution of the mixed solution of peptide, Platinol and the described peptide of SEQ ID NO.1, Platinol and the described peptide of SEQ ID NO.2.Wherein the concentration gradient of Platinol is 250 μ mol/L, 200 μ mol/L, 150 μ mol/L, 100 μ mol/L, 50 μ mol/L, 0 μ mol/L, and the final concentration of polypeptide is 20 μ mol/L, and each concentration is established 3 multiple holes.Continue to cultivate every hole adding MTT (5mg/ml) 20 μ l after 20 hours, inhale after 4 hours and remove nutrient solution, every hole adds DMSO 150 μ l, shakes purple crystal fully to be dissolved in 10 minutes.Microplate reader 570nm measures absorbancy.
Cell inhibitory rate is calculated as follows:
Cell inhibitory rate=(blank group OD value-experimental group OD value)/blank group OD value * 100%
Three, experimental result:
As seen from Figure 6, when Platinol and the SEQ ID NO.1 of 50 μ mol/L share, when perhaps share, the myoblastic inhibiting rate of people is respectively 17% and 9.6%, uses same concentrations Platinol or same concentrations Platinol and RasGAP separately with the described peptide of SEQ ID NO.2
317-326Peptide share, and its inhibiting rate is respectively 25.3% and 29.1%.Progressively increase along with Platinol concentration, Platinol and SEQ IDNO.1 share or share with the described peptide of SEQ ID NO.2 the myoblastic restraining effect of people is also progressively strengthened, but compare with independent use same concentrations Platinol or with same concentrations Platinol and RasGAP
317-326Peptide share compares not notable difference.Experimental result shows when polypeptide of the present invention and Platinol share that to normal cell nontoxicity enhancement, polypeptide promptly of the present invention is little to normal cell nontoxicity or toxicity.
Experimental example 4 mtt assay detect SEQ ID NO.1, SEQ ID NO.2 and RasGAP
317-326Peptide is respectively to normal cell inhibiting effect experiment
One, laboratory sample: embodiment 1-3 institute synthetic polypeptide: RasGAP
317-326Peptide, SEQ ID NO.1 peptide, SEQ ID NO.2 peptide;
Two, experimental technique
People sarcoplast (purchasing the Shanghai cell bank in the Chinese Academy of Sciences) is suspended in the RPMI1640 nutrient solution that contains 10% heat-inactivated fetal bovine serum, is inoculated in 96 orifice plates with the density in 5000-10000/hole.Cultivate after 24 hours, when cell density reaches 70%-80%, add Platinol, RasGAP respectively
317-326Peptide, the described peptide of SEQID NO.1, the described peptide of SEQ ID NO.2, wherein every kind of drug concentrations gradient is 80 μ mol/L, 40 μ mol/L, 20 μ mol/L, 10 μ mol/L, 0 μ mol/L, and each concentration is established 3 multiple holes.Continue to cultivate every hole adding MTT (5mg/ml) 20 μ l after 20 hours, inhale after 4 hours and remove nutrient solution, every hole adds DMSO150 μ l, shakes purple crystal fully to be dissolved in 10 minutes.Microplate reader 570nm measures absorbancy.
Cell inhibitory rate is calculated as follows:
Cell inhibitory rate=(blank group OD value-experimental group OD value)/blank group OD value * 100%
Three, experimental result
Platinol has tangible lethal effect to the people sarcoplast as seen from Figure 7, and the described peptide of SEQ ID NO.1 of same concentrations, the described peptide of SEQ ID NO.2, RasGAP
317-326Peptide is less to the myoblastic lethal effect of people, and the described peptide of SEQ ID NO.1 promptly of the present invention, the described peptide of SEQ ID NO.2 do not have obvious lethal effect to normal cell.
Experimental example 5 mtt assay detection SEQ ID NO.1 peptide and Platinol share the effect to people's cancer of the stomach SGC-7901 cell
One, 2 synthetic SEQ of laboratory sample: embodiment ID NO.1 peptide;
Two, experimental technique
Human stomach cancer cell line SGC-7901 (purchasing in China typical culture collection center Wuhan University center) is suspended in the RPMI1640 nutrient solution that contains 10% heat-inactivated fetal bovine serum, is inoculated in 96 orifice plates with the density in 5000-10000/hole.Cultivate after 24 hours, control group adds Platinol, and experimental group adds Platinol and the described peptide mixed solution of SEQ ID NO.1.Wherein the Platinol concentration gradient is 90 μ mol/L, 30 μ mol/L, 10 μ mol/L, 3.3 μ mol/L, 1.1 μ mol/L, 0.37 μ mol/L in control group and the experimental group, the described peptide final concentration of SEQ ID NO.1 is 20 μ mol/L in the experimental group mixed solution, and each concentration is established 3 multiple holes.Continue to cultivate every hole adding MTT (5mg/ml) 20 μ l after 20 hours, inhale after 4 hours and remove nutrient solution, every hole adds DMSO 150 μ l, shakes purple crystal fully to be dissolved in 10 minutes.Microplate reader 570nm measures absorbancy.
Cell inhibitory rate is calculated as follows:
Cell inhibitory rate=(blank group OD value-experimental group OD value)/blank group OD value * 100%
Three, experimental result:
As seen from Figure 8, when Platinol and SEQ ID NO.1 share, effect obviously is better than independent use Platinol to the SGC-7901 cell inhibiting for they, illustrate that SEQ ID NO.1 can increase the susceptibility of Platinol to the SGC-7901 cell, strengthen it the effect of SGC-7901 cell inhibiting.
Experimental example 6 mtt assay detection SEQ ID NO.1 peptide and Platinol share the effect experiment to people's lung cancer A549 cell
One, 2 synthetic SEQ of laboratory sample: embodiment ID NO.1 peptide;
Two, experimental technique
Human lung carcinoma cell line A549 (purchasing in China typical culture collection center Wuhan University center) is suspended in the F-12K nutrient solution that contains 10% heat-inactivated fetal bovine serum, is inoculated in 96 orifice plates with the density in 5000-10000/hole.Cultivate after 24 hours, control group adds Platinol, and experimental group adds Platinol and the described peptide mixed solution of SEQ IDNO.1.Wherein the Platinol concentration gradient is 90 μ mol/L, 30 μ mol/L, 10 μ mol/L, 3.3 μ mol/L, 1.1 μ mol/L, 0.37 μ mol/L in control group and the experimental group, the described peptide final concentration of SEQ ID NO.1 is 20 μ mol/L in the experimental group mixed solution, and each concentration is established 3 multiple holes.Continue to cultivate every hole adding MTT (5mg/ml) 20 μ l after 20 hours, inhale after 4 hours and remove nutrient solution, every hole adds DMSO150 μ l, shakes purple crystal fully to be dissolved in 10 minutes.Microplate reader 570nm measures absorbancy.
Cell inhibitory rate is calculated as follows:
Cell inhibitory rate=(blank group OD value-experimental group OD value)/blank group OD value * 100%
Three, experimental result:
As seen from Figure 9, when Platinol and the described peptide of SEQ ID NO.1 share, effect obviously is better than independent use Platinol to the A549 cell inhibiting for they, illustrate that the described peptide of SEQ ID NO.1 can increase the susceptibility of Platinol to the A549 cell, strengthens it to the effect of A549 cell inhibiting.
Experimental example 7 mtt assay detection SEQ ID NO.1 peptide, SEQ ID NO.2 peptide share with Platinol respectively the effect of human colon carcinoma HCT-116 cell are tested
One, 2 synthetic SEQ of laboratory sample: embodiment ID NO.1 peptide, 3 synthetic SEQ of embodiment IDNO.2 peptide;
Two, experimental technique
Human colon cancer cell strain HCT-116 (purchasing the Shanghai cell bank in the Chinese Academy of Sciences) is suspended in the RPMI1640 nutrient solution that contains 10% heat-inactivated fetal bovine serum, is inoculated in 96 orifice plates with the density in 5000-10000/hole.Cultivate after 24 hours, control group adds Platinol, and experimental group adds Platinol and the mixed solution of the described peptide of SEQ ID NO.1 or the mixed solution of Platinol and the described peptide of SEQ ID NO.2.Wherein the Platinol concentration gradient is 90 μ mol/L, 30 μ mol/L, 10 μ mol/L, 3.3 μ mol/L, 1.1 μ mol/L, 0.37 μ mol/L in control group and the experimental group, the described peptide final concentration of described peptide of SEQ ID NO.1 or SEQ ID NO.1 is 20 μ mol/L in the experimental group mixed solution, and each concentration is established 3 multiple holes.Continue to cultivate every hole adding MTT (5mg/ml) 20 μ l after 20 hours, inhale after 4 hours and remove nutrient solution, every hole adds DMSO 150 μ l, shakes purple crystal fully to be dissolved in 10 minutes.Microplate reader 570nm measures absorbancy.
Cell inhibitory rate is calculated as follows:
Cell inhibitory rate=(blank group OD value-experimental group OD value)/blank group OD value * 100%
Three, experimental result:
As seen from Figure 10, when Platinol and SEQ ID NO.1 share, effect obviously is better than independent use Platinol to the HCT-116 cell inhibiting for they, illustrate that SEQ ID NO.1 can increase the susceptibility of Platinol to the HCT-116 cell, strengthens its restraining effect to the HCT-116 cell.
As seen from Figure 11, when Platinol and SEQ ID NO.2 share, effect obviously is better than independent use Platinol to the HCT-116 cell inhibiting for they, illustrate that SEQ ID NO.2 can increase the susceptibility of Platinol to the HCT-116 cell, strengthens its restraining effect to the HCT-116 cell.
IC
50Aspect apoptosis, can be understood as certain density certain drug-induced apoptosis of tumor cells 50%, this concentration is called 50% inhibition concentration, be apoptotic cell and the ratio of whole cell count equals 50% o'clock pairing concentration, the IC50 value can be used for weighing the ability of drug-induced apoptosis, be that inducibility is strong more, this numerical value is low more, can certainly certain cell of reverse instruction to the tolerance degree of medicine.SEQ ID NO.1 and Platinol share the result of human tumor cells effect as shown in table 1, restraining effect to tumour cell when SEQ ID NO.1 and Platinol share is obvious, and SEQ ID NO.1 peptide uses separately or and Platinol share Normocellular effect all smallerly, illustrate that peptide of the present invention can be used as the medicine of treatment and preventing cancer.
Table 1 SEQ ID NO.1 half-inhibition concentration (IC
50) observe
Medium lethal dose (LD50) experiment in the described peptide body of experimental example 8 SEQ ID NO.1
One, 2 synthetic SEQ of laboratory sample: embodiment ID NO.1 peptide;
Two, experimental technique: BALB/c kind mouse, male (the purchasing in Beijing Vital River Experimental Animals Technology Co., Ltd.) of 18-22 gram body weight used in experiment.The described peptide of SEQ ID NO.1 is established 125mg/kg, 250mg/kg, four dosage groups of 500mg/kg, 1000mg/kg, and through intraperitoneal administration once, 30 days finish experiment after the administration, weighs statistics animals survived number of elements.
Three, experimental result: the described peptide 125mg/kg of SEQ ID NO.1,250mg/kg, 500mg/kg, four dosage groups of 1000mg/kg, every group of 5 animals, through intraperitoneal administration once, when 30 days finished experiment after the administration, animal all survived.Experimental result sees Table 2, the LD50>1000mg/kg of the described peptide of experimental result proof SEQ ID NO.1.
Table 2.SEQ ID NO.1 peptide medium lethal dose (LD50) is observed
The described peptide anti-tumor in vivo of experimental example 9 SEQ ID NO.1 effect experiment
One, 2 synthetic SEQ of laboratory sample: embodiment ID NO.1 peptide;
Two, experimental technique: test BALB/c kind male mice (purchasing) in Beijing Vital River Experimental Animals Technology Co., Ltd. with the 18-22 grammes per square metre.The tumor tissues and 0.85% physiological saline of colorectal carcinoma 26 cell mice with tumor (purchasing the biotechnology research institute in the Chinese Academy of Medical Sciences) ground to form cell suspension by 1: 10, and aseptic technique is at animal right side armpit subcutaneous vaccination tumour, every animal inoculation pvaccination 0.2ml.24 hours SEQ ID of inoculated tumour NO.1 peptide is through intraperitoneal administration, every day 1 time, totally 10 times.Platinol is through intraperitoneal administration, and 1 time every other day, totally 5 times; SEQ ID NO.1 peptide and Platinol 0.85% physiological saline solution, the intraperitoneal administration amount is the 0.2ml/20g body weight.Control group (0.85% physiological saline), SEQ ID NO.1 peptide various dose group, Platinol group and SEQ ID NO.1 peptide various dose and Platinol drug combination group are established in experiment.11 days finish experiment behind the inoculated tumour, weigh, and put to death animal, strip tumour, weigh.Be calculated as follows tumor control rate, and learn by statistics and handle, judging has there was no significant difference.
Tumor control rate=heavy * 100% medication combined evaluation of effect of the average knurl of (it is heavy that average knurl is organized in the average knurl weight-treatment of control group)/control group
Two medicine association indexs (combine index, CI) CI=AB/ (A * B).AB is the T/C ratio of two medicine combined action groups, and A and B are the separately T/C ratio of effect (two medicines have synergy when CI<1 in theory) of medicine.
Three, experimental result:
SEQ ID NO.1 peptide and Platinol drug combination are to colorectal carcinoma 26 tumor tissues show dose dependent tumors restraining effect.SEQ ID NO.1 peptide dosage is respectively 25mg/kg, 50mg/kg, during 100mg/kg and Platinol dosage 1mg/kg drug combination in various degree tumor-inhibiting action is all arranged.The dosage 25mg/kg of SEQ ID NO.1 peptide, 50mg/kg, the tumour inhibiting rate of 100mg/kg is respectively 0%, 0% and 4%; The tumour inhibiting rate of the dosage 1mg/kg of Platinol is 22%.The dosage of SEQ ID NO.1 peptide is respectively 25mg/kg, 50mg/kg, and the tumour inhibiting rate of 100mg/kg and Platinol 1mg/kg drug combination is respectively 23% (P<0.05), 30% and 34% (P<0.01); Two medicine association indexs (CI) are respectively 0.99,0.90 and 0.88, show synergy.
In vivo test is the result show, SEQ ID NO.1 peptide and Platinol drug combination have the dose-dependent inhibition effect to mice transplanted tumor colorectal carcinoma 26 tumor growths, and SEQ ID NO.1 peptide and Platinol drug combination show collaborative the work
Table 3.SEQ ID NO.1 peptide and Platinol drug combination are to the effect of mouse junction cancer 26
Sequence table
<110〉the triumphant safe true tumor technology in Wuhan company limited
<120〉polypeptide or the derived product and the application thereof of treatment or preventing cancer
<160>2
<170>patentin?3.3
<210>1
<211>16
<212>PRT
<213>homo?sapiens
<400>1
Phe?Leu?Lys?Gly?Asp Met?Phe?Ile?Val?His Asn?Glu?Leu?Glu?Asp
1 5 10 15
Gly
<210>2
<211>21
<212>PRT
<213>homo?sapiens
<400>2
Phe?Leu?Lys?Gly?Asp Met?Phe?Ile?Val?His Asn?Glu?Leu?Glu?Asp
1 5 10 15
Gly?Trp?Met?Trp?Val Thr
20