CN108129560A - A kind of mutation melittin MEL-pep and its application - Google Patents
A kind of mutation melittin MEL-pep and its application Download PDFInfo
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- CN108129560A CN108129560A CN201711354495.8A CN201711354495A CN108129560A CN 108129560 A CN108129560 A CN 108129560A CN 201711354495 A CN201711354495 A CN 201711354495A CN 108129560 A CN108129560 A CN 108129560A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses a kind of mutation melittin MEL pep and its application, the amino acid sequence such as SEQ ID of the polypeptide：Shown in NO.1.The experimental results showed that MEL pep have the function of significantly to inhibit drug resistant 7402/5 FU of the human liver cancer cell BEL growths of 5 fluorouracils (5 FU), 7402/5 FU cell surface P gp of BEL are significantly lowered, the expression of 7402/5 FU cells resistance albumen P gp albumen of BEL and gene can be inhibited；MEL pep can significantly inhibit 7402/5 FU cell mice with tumor tumor growth in vivo of BEL, and in dose dependent, and anti-7402/5 FU cell-proliferation activities of BEL are notable in MEL pep bodies.
Description
Technical field
The invention belongs to the polypeptide drugs technical field in biochemistry, be related to a kind of mutation melittin MEL-pep and its
Using.
Background technology
Antibacterial peptide is a kind of natural small molecule polypeptide being widely present in various organisms, is the group of natural immune system
Into part, have the effects that resist inoculating microbe and remove internal sick cell.Other than antibacterial action, antibacterial peptide is only with it
Special, rapid antitumor activity, is concerned.Antibacterial peptide is largely positively charged, rich in hydrophobic bases, can form amphipathic knot
Structure.Antibacterial peptide has special antitumor mechanism, and acts on rapid；It, being capable of selectively acting compared with general antitumor drug
In tumour cell；It is relatively low to the normal cell toxicity of mammal, drug resistance is not easy to produce, these advantages become antibacterial peptide
One of hot spot of antitumor drug research and development.
Bee venom is the pale yellow transparent venom with aromatic odor that worker bee poison gland and accessory gland secret out of, and has a variety of pharmacology
Learn the complex mixture with biological activity.Melittin (Melittin, MEL) is the chief component of bee venom, accounts for about bee venom
The 50% of dry weight, is a kind of linear polypeptide being made of 26 amino acid residues, relative molecular weight 2847, and isoelectric point is about
12.02 total order is classified as：Glycine-Isoleucine-glycine-alanine-valine-leucine-lysine-valine-bright ammonia
Acid-thr-thr-Gly-Leu-Pro-Ala-Leu-Ile-serine-tryptophan-different
Leucine-Lys-Arg-Lys-Arg-Glu-Glu.Melittin can inhibit multiple types tumour thin
The growth of born of the same parents is an antitumor natural drug very with application prospect.
Invention content
The content that the present invention solves is to provide a kind of mutation melittin MEL-pep and its application, with the amino of melittin
Acid sequence is basic synthesizing new polypeptide, with anti-liver cancer efficacy.
The present invention is to be achieved through the following technical solutions：
A kind of mutation melittin MEL-pep, the amino acid sequence such as SEQ ID of the polypeptide：Shown in NO.1.
Applications of the mutation melittin MEL-pep in the drug for preparing treatment and/or prevention tumor disease.
Applications of the mutation melittin MEL-pep in the drug for preparing treatment liver cancer.
The mutation melittin MEL-pep is preparing application of the treatment to 5-FU in the drug of drug resistant liver cancer.
The mutation melittin MEL-pep can act in preparation in the drug of the drug resistant hepatoma cell membranes of 5-FU
Using.
Applications of the mutation melittin MEL-pep in the drug for reversing the drug resistant liver cancer of 5-FU is prepared.
The mutation melittin MEL-pep inhibits the drug of the drug resistant liver cancer cells tumor growth inhibition of 5-FU preparing
In application.
Compared with prior art, the present invention has technique effect beneficial below：
Mutation melittin MEL-pep provided by the invention, using alpha-helix wheel model as theoretical model, improves the spiral of polypeptide
The valine of MEL the 8th and the proline of the 14th are changed to lysine by degree, and enhance the positive charge of polypeptide,
Obtain novel polypeptide MEL-pep.Polypeptide MEL-pep not only increases helix degree, and its net charges is promoted to 7 by 5,
Make it have stronger antitumor activity.Significantly inhibit 5 FU 5 fluorouracil (5-FU) resistance to the experimental results showed that MEL-pep has
The effect of the human liver cancer cell BEL-7402/5-FU growths of medicine can be used to prepare the drug of anti-liver cancer and anti-treatment.
Mutation melittin MEL-pep provided by the invention, cytostatic effect is more more notable than polypeptide MEL,
The half-inhibition concentration IC of polypeptide MEL50It is 11.09 μM to be worth, polypeptide MEL-pep IC50It is 4.44 μM to be worth, and is declined compared with MEL
6.85 μM of (about MEL IC50The 59.96% of value)；And polypeptide MEL-pep can significantly inhibit BEL-7402/5-FU cells
Growth, and in the time, dose-dependence.
There is apparent hole, polypeptide in mutation melittin MEL-pep provided by the invention, treated cell, cell membrane
MEL-pep has apparent destruction to BEL-7402/5-FU cell membranes；MEL-pep is to BEL-7402/5-FU cells
LDH release actions be in dose-dependence, and with significant difference (**P<0.01)。
Mutation melittin MEL-pep provided by the invention significantly lowers BEL-7402/5-FU cell surfaces P-gp, it was demonstrated that
MEL-pep can inhibit the expression of BEL-7402/5-FU cells resistances albumen P-gp albumen and gene；MEL-pep can be significantly
Inhibit BEL-7402/5-FU cell mice with tumor tumor growth in vivo, and in dose dependent, and anti-BEL- in MEL-pep bodies
7402/5-FU cell-proliferation activities are notable.
Description of the drawings
Fig. 1 is growth in vitro inhibitory action of the polypeptide MEL-pep to different cells；
Fig. 2 for polypeptide MEL, MEL-pep to 5-FU drug resistant liver cancer cells BEL-7402/5-FU growth inhibitory activities ratio
Compared with；
Fig. 3 is the influence of polypeptide MEL-pep drug resistant liver cancer cells BEL-7402/5-FU growths to 5-FU；
Fig. 4 is the combination of the drug resistant liver cancer cells BEL-7402/5-FU of polypeptide FITC-labeled-MEL-pep and 5-FU
Effect；
Fig. 5 is the influence of polypeptide MEL-pep drug resistant liver cancer cells BEL-7402/5-FU membrane structures to 5-FU；
Fig. 6 is the influence of polypeptide MEL-pep drug resistant liver cancer cells BEL-7402/5-FU LDH releases to 5-FU；
Fig. 7 is that polypeptide MEL-pep reverses drug-resistant effects of the drug resistant liver cancer cells BEL-7402/5-FU of 5-FU to 5-FU；
Fig. 8 is the influence of polypeptide MEL-pep drug resistant liver cancer cells BEL-7402/5-FU MDR1mRNA expression to 5-FU；
Fig. 9 is the shadow of polypeptide MEL-pep drug resistant liver cancer cells BEL-7402/5-FU cell membranes P-gp expression to 5-FU
It rings；
For polypeptide MEL-pep, to 5-FU, drug resistant liver cancer cells BEL-7402/5-FU Rhodamine 123 accumulate Figure 10
Influence；
Figure 11 is liver cancer cells BEL-7402/5-FU mice with tumor tumour growths drug resistant to 5-FU in polypeptide MEL-pep bodies
Influence；
Figure 12 is the shadow of liver cancer cells BEL-7402/5-FU mice with tumor knurl weights drug resistant to 5-FU in polypeptide MEL-pep bodies
It rings；
Figure 13 is the shadow of liver cancer cells BEL-7402/5-FU mice with tumor weight drug resistant to 5-FU in polypeptide MEL-pep bodies
It rings；
Figure 14 dyes picture for experiment each group mouse tumor tissue HE.
Specific embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings.
The present invention is based on the amino acid sequence of melittin MEL, using alpha-helix wheel model as theoretical model, improves polypeptide
Helix degree, and enhance the positive charge of polypeptide.The valine of MEL the 8th and the proline of the 14th are changed to rely
Propylhomoserin obtains novel polypeptide MEL-pep.The base acid sequence of polypeptide MEL-pep such as SEQ ID：Shown in No.1, amino acid total order
It is classified as：Glycine-Isoleucine-glycine-alanine-valine-leucine-lysine-lysine-leucine-threonine-
Threonine-Gly-Leu-lysine-Ala-Leu-isoleucine-serine-tryptophan-isoleucine-rely
Propylhomoserin-arg-lys-arg-glutamine-glutamine, molecular weight 2907.5, isoelectric point 12.04.It is more
The helix degree and charge number of peptide and its activity are closely related, and compared with melittin MEL, polypeptide MEL-pep is not only increased
Helix degree, and its net charges is promoted to 7 by 5, make it have stronger antitumor activity.
The present invention is obtained using solid-state chemical reaction method method has higher resisting liver cancer activity antineoplastic polypeptide MEL-pep, and its
Carry out in vivo and Pharmacodynamics in vitro is tested, the results showed that MEL-pep has apparent inhibition 5 FU 5 fluorouracil (5-FU) drug resistance
Human liver cancer cell BEL-7402/5-FU growths effect, can be used to prepare the drug of anti-liver cancer and anti-treatment.
Here is the part pharmacodynamic experiment and result of the present invention
First, polypeptide MEL-pep is to the growth in vitro inhibitory action of different cells
Take rat primary mesenchymal stem cell (BMSC), the human normal liver cell L 02 of exponential phase, human liver cancer thin
The drug resistant liver cancer cells BEL-7402/5-FU of born of the same parents BEL-7402, people 5-FU.Wherein, BMSC cells are with containing 10%FBS's
1640 culture mediums of RPMI of DMEM-F12 culture mediums, L02 cells and BEL-7402 cells containing 10%FBS, BEL-
RPMI 1640 culture medium of the 7402/5-FU cells containing 10%FBS and 20 μ g/mL 5-FU.
It is 5 × 10 that the respective culture medium of each cell, which adjusts cell concentration,4/ mL, addition cell hangs in 96 well culture plates
100 μ l of liquid.After overnight incubation, add in that (final concentration is respectively with the diluted 20 μ l/ holes of various concentration MEL-pep of culture solution：0、
2.5th, 5,7.5,10,20,40,80 μM), culture solution zeroing, 37 DEG C, 5%CO2 cultivate 24,48,72 hours after, suck supernatant, add
Enter 90 μ l fresh mediums, 10 μ l CCK-8 are then added in per hole, continue culture 1.5-2.5 hours, enzyme is used at 490nm wavelength
It marks instrument and surveys light absorption value, Cell viability is calculated by formula：
The results are shown in Figure 1, and polypeptide MEL-pep is to rat primary cell BMSC, people in 48 hours, 10 μM of concentration ranges
The no inhibiting effect of growth of normal liver cell L02, but to tumour cell：BEL-7402 cells and BEL-7402/5-FU
Cell has apparent inhibiting effect, and agent effect relationship is apparent, and has significant difference compared with the blank control group of each cell
(**P<0.01).Polypeptide MEL-pep is as shown in table 1 to the IC50 values of each cell strain：
1 polypeptide MEL-pep of table is to the IC of each cell strain50(μM)
2nd, polypeptide MEL and polypeptide MEL-pep to 5-FU drug resistant liver cancer cells BEL-7402/5-FU growth inhibitory activities
Comparison
Take the logarithm growth period the drug resistant liver cancer cells BEL-7402/5-FU of people 5-FU (containing 10%FBS and 20 μ g/
1640 culture mediums of RPMI of mL 5-FU), it is 5 × 10 with its culture medium adjustment cell concentration4/ mL adds in 96 well culture plates
Enter 100 μ l of cell suspension, and add in that (final concentration is respectively with the diluted 20 μ l/ holes of various concentration MEL, MEL-pep of culture solution：
0th, 1.25,2.5,5,10,20,40 μM), culture solution zeroing, 37 DEG C, after 5%CO2 cultivates 48 hours, suck supernatant, add in 90 μ l
Then fresh medium adds in 10 μ l CCK-8 per hole, continue culture 1.5-2.5 hours, surveyed at 490nm wavelength with microplate reader
Light absorption value calculates Cell viability by formula：
The results are shown in Figure 2, and polypeptide MEL and MEL-pep can inhibit the growth of BEL-7402/5-FU cells.With polypeptide
MEL is compared, and the cytostatic effect of MEL-pep is more notable, the half-inhibition concentration IC of polypeptide MEL50Be worth is 11.09
μM, polypeptide MEL-pep IC50It is 4.44 μM to be worth, and 6.85 μM of (about MEL IC are had dropped compared with MEL50The 59.96% of value).
3rd, the polypeptide MEL-pep times that drug resistant liver cancer cells BEL-7402/5-FU growth in vitro inhibits to 5-FU and agent
Measure dependence
It takes the logarithm the BEL-7402/5-FU cells in growth period, with the RPMI containing 10%FBS and 20 μ g/mL5-FU
1640 culture adjustment cell concentrations are 5 × 104/mL.150 μ l culture mediums are added in the hole of E-Plate L8 culture plates, by E-
Plate L8 are put into iCelligence systems, and system can be scanned automatically.E-Plate L8 are taken out, 300 are added in hole
The BEL-7402/5-FU cell suspensions that μ l are uniformly mixed, it is 15,000cells to make cell number in every hole.E-Plate L8 are put
30min is placed at room temperature in super-clean bench, then culture plate is put on the iCelligence in incubator, starts machine, detection
Cell Proliferation curve.After being incubated overnight, the MEL-pep (final concentration of 0,2,4,6,8,10 μM) of 50 μ L various concentrations is added in, often
15min detects a cell proliferation curve.
The results are shown in Figure 3, and polypeptide MEL-pep can significantly inhibit the growth of BEL-7402/5-FU cells, and in when
Between, dose-dependence.
4th, the combination of polypeptide MEL-pep and the drug resistant liver cancer cells BEL-7402/5-FU cells of 5-FU
It takes the logarithm the BEL-7402/5-FU cells in growth period, is 3 × 10 with PBS adjustment cell concentrations5/ mL, 500 μ L/
Sample.The polypeptide MEL-pep (FITC-labeled-MEL-pep) of the diluted FITC labels of 50 μ L PBS is added in into each sample,
It is final concentration of：0th, 1,2,4 μM, 4 DEG C are protected from light incubation 30min.Cell is then collected, 1000rpm centrifugation 5min are discarded supernatant, added
Enter the PBS 5mL of precooling, cell precipitation is slowly blown even, and 1000rpm centrifugation 5min are discarded supernatant.PBS is washed repeatedly 2 times.Cell
It is resuspended in 500 μ l PBS, flow cytomery FITC average fluorescent strengths.
As a result see attached drawing 4, compared with the control group, FITC-labeled-MEL-pep and BEL-7402/5-FU cell incubations
The fluorescence intensity of group cell is remarkably reinforced, and being mainly shown as peak shape figure, displacement is apparent to the right, it was demonstrated that polypeptide MEL-pep can be with
BEL-7402/5-FU cell combinations, and in metering dependence.
5th, the influence of polypeptide MEL-pep drug resistant liver cancer cells BEL-7402/5-FU membrane structures to 5-FU
It takes the logarithm the BEL-7402/5-FU cells in growth period, is spread into the Tissue Culture Dish of 10cm.Treat cell grow to
During 60% fusion, MEL-pep is added in into culture medium (final concentration is respectively 0,4 μM).It is placed in 37 DEG C, 5%CO2 cell incubators
In continue to cultivate, be incubated for 24 hours after, collect cell, 1000rpm centrifugation 5min, discard supernatant.Add in 5mL PBS repeated washings 2
Secondary, 1000rpm centrifugation 5min are discarded supernatant.It adds in 500 μ L, 2.5% glutaraldehydes to fix, scanning electric mirror observing cell membrane structure.
As a result see attached drawing 5, the membrane structure of cellular control unit is complete, and polypeptide MEL-pep treated cell, cells
There is apparent hole in film, and therefore, polypeptide MEL-pep has apparent destruction to BEL-7402/5-FU cell membranes.
6th, the influence of the LDH releases of polypeptide MEL-pep drug resistant liver cancer cells BEL-7402/5-FU to 5-FU
Take the logarithm growth period BEL-7402/5-FU cell, cell be collected by centrifugation after pancreatin digestion, with containing 10%FBS with
And 20 μ g/mL 5-FU RPMI 1640 culture adjustment cell concentration be 5 × 104/ mL, by cell inoculation to 96 orifice plates, often
100 μ l of hole.It is placed in 37 DEG C, 5%CO2Continue to cultivate in cell incubator, for 24 hours after, cell is adherent.It is dense that 20 μ l differences are added in per hole
Polypeptide MEL-pep (final concentration of 0,2,4,6,8,10 μM) is spent, stimulation is for 24 hours.Collect cell culture supernatant.
During measure, it is divided into blank well, gauge orifice measures 4 groups of hole and control wells.Operation is carried out according to the following table.
As a result see attached drawing 6, for 24 hours, compared with the blank control group at same time point, MEL-pep is to BEL-7402/5-FU
The LDH release actions of cell be in dose-dependence, and with significant difference (**P<0.01).This experiment is further verified
Polypeptide MEL-pep killings BEL-7402/5-FU is the conclusion acted on by rupture of membranes.
7th, polypeptide MEL-pep reverses the drug-resistant effect of the drug resistant liver cancer cells BEL-7402/5-FU of 5-FU
Take the logarithm growth period BEL-7402/5-FU cell, cell be collected by centrifugation after pancreatin digestion, with containing 10%FBS with
And 20 μ g/mL 5-FU RPMI 1640 culture adjustment cell concentration be 5 × 104/ mL, by cell inoculation to 96 orifice plates, often
100 μ l of hole.The diluted 20 μ l/ holes of various concentration MEL-pep of addition culture solution are (final concentration of：0th, 2,4 μM), culture solution tune
Zero, 37 DEG C, to add in various concentration 5-FU 20 μ l/ holes after 5%CO2 cultures 2h (final concentration of：0、1、10、100、1000μg/
mL).37 DEG C, after 5%CO2 cultures for 24 hours, suck supernatant, add in 90 μ l fresh mediums, 10 μ l CCK-8 are then added in per hole,
Continue culture 1.5-2.5 hours, light absorption value is surveyed with microplate reader at 490nm wavelength, Cell viability is calculated by formula：
The results are shown in Figure 7, and compared with 5-FU individually processing group, MEL-pep is in IC50(4 μM) and half IC50(2μ
M after) being combined respectively with 5-FU, the IC of 5-FU50It is significantly reduced by 1298.97 μ g/mL to 519.76,295.67 μ g/mL.Therefore,
MEL-pep can reverse drug resistance of the BEL-7402/5-FU cells to 5-FU.
8th, polypeptide MEL-pep inhibits the drug resistant liver cancer cells BEL-7402/5-FU drug resistant genes MDR1mRNA expression of 5-FU
Take the logarithm growth period BEL-7402/5-FU cell, cell be collected by centrifugation after pancreatin digestion, with containing 10%FBS with
And 20 μ g/mL 5-FU RPMI 1640 culture adjustment cell concentration be 2 × 105/ mL, by cell inoculation to 6 orifice plates, per hole
1.5mL.After overnight incubation, the diluted 500 μ l/ holes of various concentration MEL-pep of addition culture solution are (final concentration of：0、1、2、4μ
M), 37 DEG C, 5%CO2 continue culture for 24 hours.After the PBS of addition 3mL precoolings is washed 3 times into cell culture well, 500 μ L are added in
trizol.Cell RNA is extracted, carries out fluorescence quantitative PCR detection.
As a result as shown in Figure 8, compared with the control group, after MEL-pep intervenes, BEL-7402/5-FU cells resistance genes
MDR1mRNA expression reduces, and in significant dose dependent.
9th, polypeptide MEL-pep inhibits the table of the drug resistant liver cancer cells BEL-7402/5-FU P- glycoprotein (P-gp) of 5-FU
It reaches
Take the logarithm growth period BEL-7402/5-FU cell, cell be collected by centrifugation after pancreatin digestion, with containing 10%FBS with
And 20 μ g/mL 5-FU RPMI 1640 culture adjustment cell concentration be 2 × 105/ mL, by cell inoculation to 6 orifice plates, per hole
1.5mL.After overnight incubation, the diluted 500 μ l/ holes of various concentration MEL-pep of addition culture solution are (final concentration of：0、1、2、4μ
M), 37 DEG C, 5%CO2 continue culture for 24 hours.Cell is collected, supernatant is abandoned in centrifugation.It adds in 5mL PBS to wash repeatedly 2 times, 1000rpm
5min is centrifuged, is discarded supernatant.100 μ L PBS resuspensions are added in, add in 5 μ L anti-P-glycoprotein-PE labelled antibodies, room
Temperature is incubated 30min.Cell is collected by centrifugation, supernatant is abandoned in centrifugation.It adds in 5mL PBS to wash repeatedly 2 times, 1000rpm centrifugation 5min are abandoned
Remove supernatant.Add in 500 μ L PBS resuspensions, flow cytomery.
As a result as shown in Figure 9, similar to fluorescence quantitative PCR detection result, MEL-pep significantly lowers BEL-7402/5-
FU cell surfaces P-gp, it was demonstrated that MEL-pep can inhibit BEL-7402/5-FU cells resistances albumen P-gp albumen and gene
Expression.
9th, polypeptide MEL-pep inhibits the drug resistant liver cancer cells BEL-7402/5-FU Rhodamine123 intracellulars of 5-FU to store
Product
Take the logarithm growth period BEL-7402/5-FU cell, cell be collected by centrifugation after pancreatin digestion, with containing 10%FBS with
And 20 μ g/mL 5-FU RPMI 1640 culture adjustment cell concentration be 2 × 105/ mL, by cell inoculation to 6 orifice plates, per hole
1.5mL.After overnight incubation, the diluted 500 μ l/ holes of various concentration MEL-pep of addition culture solution are (final concentration of：0、1、2、4μ
M), 37 DEG C, 5%CO2 continue culture for 24 hours.Cell is collected, supernatant is abandoned in centrifugation.It adds in 5mL PBS to wash repeatedly 2 times, 1000rpm
5min is centrifuged, is discarded supernatant.500 μ L PBS resuspensions are added in, add in Rhodamine 123 (5 μ g/mL of final concentration)
It is protected from light under the conditions of 37 DEG C and is incubated 60min, blank control group adds in isometric culture solution.After incubation, cold PBS
Washing 2 times, the intracellular average fluorescent strength of flow cytomery.
The results are shown in Figure 10, and Rhodamine 123 is a species specific P-gp fluorogenic substrates, into the cell
The accumulation of Rhodamine 123 can reflect the efficiency for inhibiting P-gp activity.After MEL-pep effects, BEL-7402/5-FU cells
The interior accumulations of Rhodamine 123 significantly reduce, it was demonstrated that MEL-pep can inhibit the function of P-gp.
Tenth, Cathelicidin-BF to 5-FU drug resistant liver cancer cells BEL-7402/5-FU tumor growth inhibiting effect
It takes the logarithm the BEL-7402/5-FU cells in growth period, PBS dilutions are being fallen with blood counting chamber after Trypan Blue
It puts and is counted under formula microscope, viable count is more than 98%, adjusts cell concentration 5 × 108/mL.Experiment is using 3 week old nude mouses
24, mouse oxter right side inoculates 0.1mL cells.Treating mouse tumor, the bulk grows to 100-300mm3When, it is randomly divided into 4 groups：
Tumor model group, the basic, normal, high dosage treatment groups of MEL-pep (1,1.5,2mg/kg), every group 6.Administering mode is notes in knurl
It penetrates, weekly administration three times, is administered three weeks altogether.Use the method for measuring knurl footpath, the antitumor effect of dynamic observation subject.It is swollen
The pendulous frequency of knurl diameter was surveyed 1 time for every 3 days.After administration 21 days, mouse is put to death, and operation strips tumor mass and weighs.
The calculation formula of gross tumor volume (tumor volume, TV) is：
TV=1/2 × a × b2, wherein a, b represent length and width respectively.
Tumour tumour inhibiting rate calculation formula is as follows：
As a result as shown in attached drawing 11-12, MEL-pep can be significantly inhibited in BEL-7402/5-FU cell mice with tumor tumour bodies
Growth, and in dose dependent.Wherein MEL-pep1,1.5, the tumour inhibiting rates of tri- dosage groups of 2mg/kg are respectively：50.69%th,
61.46%, and 72.92%.13 weight curve of attached drawing shows the weight curve of MEL-pep administration group mouse and model group phase
Than having no and being remarkably decreased.Attached drawing 14 is tested each group mouse tumor tissue HE coloration results and is shown, model group mouse tumor tissue increases
It is apparent to grow division, and administration group mouse tumor tissue necrosis region is apparent.Therefore, anti-BEL-7402/5-FU is thin in MEL-pep bodies
Born of the same parents' proliferation activity is notable.
Sequence table
<110>No.1 Hospital Attached to Medical College, Xi'an Communication Univ
<120>A kind of mutation melittin MEL-pep and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 3
<211> 26
<212> PRT
<213>Melittin MEL-pep (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
Gly Ile Gly Ala Val Leu Lys Lys Leu Thr Thr Gly Leu Lys Ala Leu
1 5 10 15
Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln
20 25
Claims (7)
- A kind of 1. mutation melittin MEL-pep, which is characterized in that the amino acid sequence of the polypeptide such as SEQID：Shown in NO.1.
- 2. mutation melittin MEL-pep described in claim 1 answering in the drug for preparing treatment and/or prevention tumor disease With.
- 3. application as described in claim 1, which is characterized in that the mutation melittin MEL-pep is preparing treatment liver cancer Drug in application.
- 4. application as claimed in claim 3, which is characterized in that the mutation melittin MEL-pep is preparing treatment to 5- Application in the drug of the drug resistant liver cancer of FU.
- 5. application as claimed in claim 4, which is characterized in that the mutation melittin MEL-pep is being prepared and can acted on Application in the drug of the drug resistant hepatoma cell membranes of 5-FU.
- 6. application as claimed in claim 2, which is characterized in that the mutation melittin MEL-pep is preparing reverse 5-FU Application in the drug of drug resistant liver cancer.
- 7. application as claimed in claim 3, which is characterized in that the mutation melittin MEL-pep is preparing inhibition 5-FU Application in the drug that drug resistant liver cancer cells tumor growth inhibits.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110498848A (en) * | 2019-09-10 | 2019-11-26 | 中国医学科学院基础医学研究所 | A kind of novel melittin variant and its application |
| CN110551191A (en) * | 2019-09-10 | 2019-12-10 | 中国医学科学院基础医学研究所 | Improved melittin with low hemolytic activity and application thereof |
| CN113150074A (en) * | 2021-03-16 | 2021-07-23 | 西安交通大学医学院第一附属医院 | Antibacterial peptides GK-19 and FK-20 as well as preparation method and application thereof |
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| CN113150074A (en) * | 2021-03-16 | 2021-07-23 | 西安交通大学医学院第一附属医院 | Antibacterial peptides GK-19 and FK-20 as well as preparation method and application thereof |
| CN113150074B (en) * | 2021-03-16 | 2023-03-21 | 西安交通大学医学院第一附属医院 | Antibacterial peptides GK-19 and FK-20 as well as preparation method and application thereof |
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| CN108129560B (en) | 2020-04-28 |
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