CN108129560B

CN108129560B - A kind of mutant melittin MEL-pep and its application

Info

Publication number: CN108129560B
Application number: CN201711354495.8A
Authority: CN
Inventors: 柯梦云; 吴荣谦; 吕毅
Original assignee: First Affiliated Hospital of Xian Jiaotong University
Current assignee: Chonghao Technology Co ltd
Priority date: 2017-12-15
Filing date: 2017-12-15
Publication date: 2020-04-28
Anticipated expiration: 2037-12-15
Also published as: CN108129560A

Abstract

The invention discloses a mutant melittin MEL-pep and application thereof, wherein the amino acid sequence of the polypeptide is shown as SEQ ID: shown in NO. 1. Experimental results show that MEL-pep has obvious effect of inhibiting growth of 5-fluorouracil (5-FU) -resistant human hepatoma cell BEL-7402/5-FU, remarkably reduces P-gp on the cell surface of BEL-7402/5-FU, and can inhibit expression of BEL-7402/5-FU cell drug-resistant protein P-gp protein and genes; MEL-pep can obviously inhibit the growth of BEL-7402/5-FU cell tumor-bearing mouse tumor in vivo, is dose-dependent, and has obvious MEL-pep in vivo anti-BEL-7402/5-FU cell proliferation activity.

Description

Mutant melittin MEL-pep and application thereof

Technical Field

The invention belongs to the technical field of polypeptide medicines in biochemistry, and relates to mutant melittin MEL-pep and application thereof.

Background

The antibiotic peptide is a kind of natural small molecular polypeptide existing widely in various organisms, is a component of natural immune system, and has the functions of resisting exogenous microbe, eliminating pathological change cell in vivo, etc. In addition to their antimicrobial action, antimicrobial peptides are of particular interest for their unique, rapid antitumor activity. Most of the antibacterial peptide has positive charges, is rich in hydrophobic bases and can form an amphiphilic structure. The antibacterial peptide has a special anti-tumor mechanism and has a quick action; compared with common antitumor drugs, the compound can selectively act on tumor cells; the antibacterial peptide has low toxicity to normal cells of mammals and is not easy to generate drug resistance, and the advantages make the antibacterial peptide become one of hot spots for developing the antitumor drugs.

Bee venom is a light yellow transparent venom with aromatic odor secreted by worker bee venom glands and accessory glands, and has a complex mixture with various pharmacological and biological activities. Melittin (MEL) is the major component of bee venom, accounting for about 50% of the dry weight of bee venom, and is a linear polypeptide consisting of 26 amino acid residues, with a relative molecular weight of 2847, an isoelectric point of about 12.02, and the complete sequence: glycine-isoleucine-glycine-alanine-valine-leucine-lysine-valine-leucine-threonine-glycine-leucine-proline-alanine-leucine-isoleucine-serine-tryptophan-isoleucine-lysine-arginine-glutamic acid. The melittin can inhibit the growth of various tumor cells, and is an anti-tumor natural medicine with a good application prospect.

Disclosure of Invention

The invention aims to provide mutant melittin MEL-pep and application thereof, and the mutant melittin MEL-pep synthesizes novel polypeptide based on an amino acid sequence of the melittin and has the effect of resisting liver cancer.

The invention is realized by the following technical scheme:

a mutant melittin MEL-pep, the amino acid sequence of which is shown in SEQ ID: shown in NO. 1.

The application of the mutant melittin MEL-pep in preparing the medicine for treating and/or preventing tumor diseases.

The application of the mutant melittin MEL-pep in preparing a medicament for treating liver cancer.

The application of the mutant melittin MEL-pep in preparing a medicine for treating liver cancer resistant to 5-FU.

The application of the mutant melittin MEL-pep in preparing the medicine which can act on 5-FU drug-resistant liver cancer cell membrane.

The application of the mutant melittin MEL-pep in preparing a medicine for reversing 5-FU drug-resistant liver cancer.

The application of the mutant melittin MEL-pep in preparing a medicine for inhibiting the growth inhibition of 5-FU-resistant liver cancer cells in vivo.

Compared with the prior art, the invention has the following beneficial technical effects:

the mutant melittin MEL-pep provided by the invention takes an α -helical wheel model as a theoretical model, improves the helical degree of polypeptide, enhances the positive charge of the polypeptide, changes valine at the eighth site and proline at the tenth site of the MEL into lysine to obtain the novel polypeptide MEL-pep, which not only improves the helical degree, but also increases the net charge number from 5 to 7 so as to have stronger antitumor activity.

The mutant melittin MEL-pep provided by the invention has more remarkable effect of inhibiting cell growth than polypeptide MEL, and the half inhibitory concentration IC of the polypeptide MEL₅₀A value of 11.09. mu.M, polypeptide MEL-pep IC₅₀The value was 4.44. mu.M, which is a decrease of 6.85. mu.M compared to MEL (about MEL IC)₅₀59.96% of value); moreover, the polypeptide MEL-pep can obviously inhibit BEL-7402/5-FU cell growth in a time, dose dependent relationship.

The cell membrane of the cell treated by the mutant melittin MEL-pep provided by the invention has obvious holes, and the polypeptide MEL-pep has obvious damage effect on the cell membrane of BEL-7402/5-FU; MEL-pep was dose-dependent on LDH release from BEL-7402/5-FU cells and had statistical differences (^**P<0.01)。

The mutant melittin MEL-pep provided by the invention can obviously reduce the cell surface P-gp of BEL-7402/5-FU, and proves that the MEL-pep can inhibit the expression of the P-gp protein and gene of BEL-7402/5-FU cell drug-resistant protein; MEL-pep can obviously inhibit the growth of BEL-7402/5-FU cell tumor-bearing mouse tumor in vivo, is dose-dependent, and has obvious MEL-pep in vivo anti-BEL-7402/5-FU cell proliferation activity.

Drawings

FIG. 1 shows the in vitro growth inhibitory effect of the polypeptide MEL-pep on different cells;

FIG. 2 is a comparison of the growth inhibitory activities of polypeptide MEL and MEL-pep on 5-FU-resistant hepatoma carcinoma cell BEL-7402/5-FU;

FIG. 3 shows the effect of polypeptide MEL-pep on the growth of 5-FU-resistant hepatoma cell BEL-7402/5-FU;

FIG. 4 shows the binding effect of the polypeptide FITC-labeled-MEL-pep and 5-FU-resistant hepatoma cell BEL-7402/5-FU;

FIG. 5 shows the effect of polypeptide MEL-pep on the cell membrane structure of 5-FU-resistant hepatoma cell BEL-7402/5-FU;

FIG. 6 shows the effect of polypeptide MEL-pep on the release of 5-FU-resistant hepatoma cell BEL-7402/5-FU LDH;

FIG. 7 shows the effect of polypeptide MEL-pep on 5-FU resistance of hepatoma cell BEL-7402/5-FU reversing 5-FU resistance;

FIG. 8 shows the effect of polypeptide MEL-pep on the expression of 5-FU-resistant hepatoma cell BEL-7402/5-FU MDR1 mRNA;

FIG. 9 shows the effect of polypeptide MEL-pep on the expression of cell membrane P-gp of 5-FU-resistant hepatoma cell BEL-7402/5-FU;

FIG. 10 shows the effect of polypeptide MEL-pep on accumulation of 5-FU-resistant hepatoma cell BEL-7402/5-FU Rhodamine 123;

FIG. 11 shows the effect of polypeptide MEL-pep on tumor growth of 5-FU-resistant hepatoma cell BEL-7402/5-FU in vivo in tumor-bearing mice;

FIG. 12 shows the effect of polypeptide MEL-pep on tumor weight of 5-FU-resistant hepatoma cell BEL-7402/5-FU in vivo;

FIG. 13 shows the effect of polypeptide MEL-pep on the body weight of 5-FU-resistant hepatoma cell BEL-7402/5-FU tumor-bearing mice in vivo;

FIG. 14 is a picture of HE staining of tumor tissues of mice in each experimental group.

Detailed Description

The present invention is described in further detail below with reference to the attached drawings.

The amino acid sequence of the MEL is shown as SEQ ID No.1, the whole amino acid sequence is glycine-isoleucine-glycine-alanine-valine-leucine-lysine-threonine-glycine-leucine-lysine-alanine-leucine-isoleucine-serine-tryptophan-isoleucine-lysine-arginine-glutamine, the molecular weight is 2907.5, the isoelectric point is 12.04, the helix degree and the charge number of the polypeptide are closely related to the activity of the polypeptide, and compared with the melittin MEL, the polypeptide MEL-pep not only improves the helix degree, but also improves the net charge number from 5 to 7, so that the polypeptide has stronger anti-tumor activity.

The invention adopts a solid phase chemical synthesis method to obtain the anti-tumor polypeptide MEL-pep with higher anti-liver cancer activity, and carries out in-vivo and in-vitro pharmacodynamic experiments, and the results show that the MEL-pep has obvious effect of inhibiting the growth of 5-fluorouracil (5-FU) resistant human liver cancer cell BEL-7402/5-FU, and can be used for preparing the anti-liver cancer treatment medicine.

The following are some of the pharmacodynamic experiments and results of the present invention

In vitro growth inhibition of different cells by polypeptide MEL-pep

Rat primary bone marrow mesenchymal stem cells (BMSC), human normal liver cells L02 in logarithmic growth phase, human liver cancer cells BEL-7402 and human 5-FU drug-resistant liver cancer cells BEL-7402/5-FU are taken. Among them, DMEM-F12 medium containing 10% FBS was used for BMSC cells, RPMI1640 medium containing 10% FBS was used for L02 cells and BEL-7402 cells, and RPMI1640 medium containing 10% FBS and 20. mu.g/mL 5-FU was used for BEL-7402/5-FU cells.

Each cell was adjusted to 5X 10 cell concentration with the respective medium⁴Perml, 100. mu.l of cell suspension was added to a 96-well plate. After overnight incubation, 20. mu.l/well of MEL-pep diluted with culture medium at various concentrations (final concentrations: 0, 2.5, 5, 7.5, 10, 20, 40, 80. mu.M, respectively) was added, the culture medium was zeroed, after incubation at 37 ℃ for 24, 48, 72 hours with 5% CO2, the supernatant was aspirated, 90. mu.l of fresh culture medium was added, 10. mu.l CCK-8 was added to each well, incubation was continued for 1.5-2.5 hours, absorbance was measured at 490nm using a microplate reader, and the cell viability was calculated according to the formula:

as shown in FIG. 1, at 48 hours, the polypeptide MEL-pep had no inhibitory effect on the growth of rat primary BMSC, human normal hepatocyte L02, but on tumor cells in a concentration range of 10. mu.M: BEL-7402 cell and BEL-7402/5-FU cell have obvious inhibitory effect, obvious dose-effect relationship and statistical difference compared with blank control group of each cell ()^**P<0.01). The IC50 values for the polypeptide MEL-pep for each cell line are shown in Table 1:

TABLE 1 IC of polypeptide MEL-pep on individual cell lines₅₀(μM)

Secondly, comparing the growth inhibition activity of polypeptide MEL and polypeptide MEL-pep on 5-FU-resistant hepatoma carcinoma cell BEL-7402/5-FU

The human 5-FU-resistant hepatoma cell BEL-7402/5-FU (RPMI 1640 medium containing 10% FBS and 20. mu.g/mL 5-FU) in logarithmic growth phase was taken and the cell concentration was adjusted to 5X 10 by using the medium⁴and/mL, adding 100 μ l of cell suspension into a 96-well culture plate, adding 20 μ l/well of MEL and MEL-pep with different concentrations (final concentrations are respectively: 0, 1.25, 2.5, 5, 10, 20 and 40 μ M) diluted by culture solution, adjusting the culture solution to zero, absorbing supernatant after culturing for 48 hours at 37 ℃ and 5% CO2, adding 90 μ l of fresh culture solution, adding 10 μ l CCK-8 into each well, continuing culturing for 1.5-2.5 hours, measuring absorbance at 490nm by using a microplate reader, and calculating the cell activity rate according to the formula:

as shown in FIG. 2, both MEL and MEL-pep polypeptides inhibited the growth of BEL-7402/5-FU cells. Compared with polypeptide MEL, MEL-pep has more significant effect of inhibiting cell growth, and half inhibitory concentration IC of polypeptide MEL₅₀A value of 11.09. mu.M, polypeptide MEL-pep IC₅₀The value was 4.44. mu.M, which is a decrease of 6.85. mu.M compared to MEL (about MEL IC)₅₀59.96% of the value).

Thirdly, the time and dosage dependence relationship of the polypeptide MEL-pep on the in vitro growth inhibition of the 5-FU drug-resistant hepatoma cell BEL-7402/5-FU

The logarithmic growth phase of BEL-7402/5-FU cells were cultured in RPMI1640 containing 10% FBS and 20. mu.g/mL 5-FU to adjust the cell concentration to 5X 10⁴and/mL. Add 150. mu.l of medium to the wells of the E-Plate L8 Plate and place the E-Plate L8 on the iCelligence system, which will automatically scan. The E-Plate L8 was removed and 300. mu.l of a well-mixed cell suspension of BEL-7402/5-FU was added to the wells to give a cell number of 15,000cells per well. E-Plate L8 was placed in a clean bench at room temperature for 30min, the plates were then placed on an iCelligence in an incubator, the machine was started and the cell proliferation curve was examined. After overnight incubation, 50. mu.L of different concentrations of MEL-pep (final concentrations of 0, 2, 4, 6, 8, 10. mu.M) were added and the assay was performed every 15minCell proliferation curves.

As shown in FIG. 3, the polypeptide MEL-pep can obviously inhibit the growth of BEL-7402/5-FU cells and is in a time-dependent and dose-dependent relationship.

Fourth, the combination effect of the polypeptide MEL-pep and 5-FU drug-resistant liver cancer cell BEL-7402/5-FU cell

Taking BEL-7402/5-FU cells in logarithmic growth phase, adjusting cell concentration to 3 × 10 with PBS⁵mL, 500. mu.L/sample. To each sample was added 50. mu.L of FITC-labeled polypeptide MEL-pep (FITC-labeled MEL-pep) diluted in PBS to a final concentration of: 0.1, 2, 4. mu.M, incubating at 4 ℃ for 30min in the dark. Subsequently, the cells were collected, centrifuged at 1000rpm for 5min, the supernatant was discarded, 5mL of precooled PBS was added, the cell pellet was slowly blown down and homogenized, centrifuged at 1000rpm for 5min, and the supernatant was discarded. PBS washing was repeated 2 times. Cells were resuspended in 500. mu.l PBS and FITC mean fluorescence intensity was measured by flow cytometry.

The results are shown in figure 4, compared with the control group, the fluorescence intensity of the cells of the FITC-labeled-MEL-pep and BEL-7402/5-FU cell incubation group is obviously enhanced, mainly as the peak shape is obviously shifted to the right, and the polypeptide MEL-pep can be combined with BEL-7402/5-FU cells and is in metering dependence.

Fifthly, the influence of polypeptide MEL-pep on the cell membrane structure of 5-FU drug-resistant hepatoma carcinoma cell BEL-7402/5-FU

BEL-7402/5-FU cells were collected in the logarithmic growth phase and plated on 10cm cell culture dishes. When the cells grew to 60% confluence, MEL-pep was added to the medium (

final concentrations

0, 4. mu.M, respectively). Culturing in a 5% CO2 cell culture box at 37 deg.C for 24 hr, collecting cells, centrifuging at 1000rpm for 5min, and discarding supernatant. Washing was repeated 2 times with 5mL of PBS, centrifugation was performed at 1000rpm for 5min, and the supernatant was discarded. Adding 500. mu.L of 2.5% glutaraldehyde for fixation, and observing the cell membrane structure by a scanning electron microscope.

The result is shown in figure 5, the cell membrane structure of the control group cell is complete, and the cell membrane of the cell treated by the polypeptide MEL-pep has obvious holes, so the polypeptide MEL-pep has obvious destructive effect on the cell membrane of BEL-7402/5-FU.

Sixth, Effect of polypeptide MEL-pep on LDH Release of 5-FU-resistant hepatoma cell BEL-7402/5-FU

Collecting BEL-7402/5-FU cells in logarithmic growth phase, subjecting to trypsinization, centrifuging, collecting cells, culturing in RPMI1640 containing 10% FBS and 20 μ g/mL5-FU, adjusting cell concentration to 5 × 10⁴mL, cells were seeded into 96-well plates at 100 μ l per well. Placing at 37 ℃ and 5% CO₂And (5) continuously culturing in the cell culture box, and after 24 hours, adhering the cells to the wall. Mu.l of polypeptide MEL-pep was added to each well at various concentrations (final concentrations of 0, 2, 4, 6, 8, 10. mu.M) and stimulated for 24 h. The cell culture supernatant was collected.

When measuring, the test wells are divided into 4 groups of blank wells, standard wells, measurement wells and control wells. The procedure was carried out as follows.

The results are shown in FIG. 6, where at 24h, compared to the blank control group at the same time point, MEL-pep was dose-dependent on LDH release from BEL-7402/5-FU cells and was statistically different (see (R) (R))^**P<0.01). The experiment further verifies that the polypeptide MEL-pep kills BEL-7402/5-FU through membrane rupture.

Seventhly, the drug resistance effect of the polypeptide MEL-pep reversal 5-FU drug resistance liver cancer cell BEL-7402/5-FU

Collecting BEL-7402/5-FU cells in logarithmic growth phase, subjecting to trypsinization, centrifuging, collecting cells, culturing in RPMI1640 containing 10% FBS and 20 μ g/mL5-FU, adjusting cell concentration to 5 × 10⁴mL, cells were seeded into 96-well plates at 100 μ l per well. Mu.l/well of different concentrations MEL-pep (final concentration: 0, 2, 4. mu.M) diluted with the culture medium were added, the culture medium was zeroed, cultured at 37 ℃ with 5% CO2 for 2 hours, and then 20. mu.l/well of different concentrations 5-FU (final concentration: 0, 1, 10, 100, 1000. mu.g/mL) was added. After culturing at 37 ℃ and 5% CO2 for 24h, the supernatant was aspirated, 90. mu.l of fresh culture medium was added, 10. mu.l of CCK-8 was added to each well, the culture was continued for 1.5 to 2.5 hours, the absorbance at 490nm was measured with a microplate reader, and the cell viability was calculated according to the formula:

the results are shown in FIG. 7, MEL-pep in IC, compared to the 5-FU-treated group alone₅₀(4. mu.M) and one-half IC₅₀(2. mu.M) IC of 5-FU after respective combination with 5-FU₅₀The temperature is remarkably reduced from 1298.97 mu g/mL to 519.76 and 295.67 mu g/mL. Thus, MEL-pep was able to reverse the resistance of BEL-7402/5-FU cells to 5-FU.

Eighthly, polypeptide MEL-pep inhibits the expression of the drug-resistant gene MDR1mRNA of 5-FU-resistant hepatoma carcinoma cell BEL-7402/5-FU

Collecting BEL-7402/5-FU cells in logarithmic growth phase, subjecting to trypsinization, centrifuging, collecting cells, culturing in RPMI1640 containing 10% FBS and 20 μ g/mL5-FU, adjusting cell concentration to 2 × 10⁵Cells were seeded into 6-well plates at 1.5mL per well. After overnight incubation, 500. mu.l/well of different concentrations of MEL-pep (final concentrations: 0, 1, 2, 4. mu.M) diluted with culture medium was added and incubation was continued for 24h at 37 ℃ with 5% CO 2. After 3mL of pre-cooled PBS was added to the cell culture wells and washed 3 times, 500 μ Ltrizol was added. Extracting cell RNA, and performing fluorescent quantitative PCR detection.

As shown in FIG. 8, the expression of MDR1mRNA of the BEL-7402/5-FU cell resistance gene was reduced after the MEL-pep dry prognosis and was significantly dose-dependent as compared to the control group.

Ninth, the expression of polypeptide MEL-pep for inhibiting 5-FU drug-resistant hepatoma cell BEL-7402/5-FU P-glycoprotein (P-gp)

Collecting BEL-7402/5-FU cells in logarithmic growth phase, subjecting to trypsinization, centrifuging, collecting cells, culturing in RPMI1640 containing 10% FBS and 20 μ g/mL5-FU, adjusting cell concentration to 2 × 10⁵Cells were seeded into 6-well plates at 1.5mL per well. After overnight incubation, 500. mu.l/well of different concentrations of MEL-pep (final concentrations: 0, 1, 2, 4. mu.M) diluted with culture medium was added and incubation was continued for 24h at 37 ℃ with 5% CO 2. The cells were collected, centrifuged and the supernatant discarded. Washing was repeated 2 times with 5mL of PBS, centrifugation was performed at 1000rpm for 5min, and the supernatant was discarded. Add 100 u L PBS heavy suspension, add 5 u L anti-P-glycoprotein-PE labeled antibody, room temperature incubation for 30 min. Cells were collected by centrifugation and the supernatant discarded by centrifugation. Washing was repeated 2 times with 5mL of PBS, centrifugation was performed at 1000rpm for 5min, and the supernatant was discarded. Adding 500. mu.L PBS for resuspension and detecting by flow cytometryAnd (6) measuring.

As shown in the attached figure 9, similar to the result of the fluorescent quantitative PCR detection, MEL-pep significantly reduces the P-gp on the cell surface of BEL-7402/5-FU, and the MEL-pep is proved to be capable of inhibiting the expression of the P-gp protein and gene of the BEL-7402/5-FU cell drug-resistant protein.

Ninthly, polypeptide MEL-pep inhibits 5-FU drug resistant liver cancer cell BEL-7402/5-FU Rhodamine123 intracellular accumulation

Collecting BEL-7402/5-FU cells in logarithmic growth phase, subjecting to trypsinization, centrifuging, collecting cells, culturing in RPMI1640 containing 10% FBS and 20 μ g/mL5-FU, adjusting cell concentration to 2 × 10⁵Cells were seeded into 6-well plates at 1.5mL per well. After overnight incubation, 500. mu.l/well of different concentrations of MEL-pep (final concentrations: 0, 1, 2, 4. mu.M) diluted with culture medium was added and incubation was continued for 24h at 37 ℃ with 5% CO 2. The cells were collected, centrifuged and the supernatant discarded. Washing was repeated 2 times with 5mL of PBS, centrifugation was performed at 1000rpm for 5min, and the supernatant was discarded. Add 500. mu.L PBS for resuspension, add Rhodamine123 (final concentration 5. mu.g/mL)

Incubating at 37 deg.C in dark for 60min, and adding equal volume of culture solution into blank control group. After incubation, the cells were washed 2 times with cold PBS and the mean intracellular fluorescence intensity was measured by flow cytometry.

As a result, it is shown in FIG. 10 that Rhodamine123 is a specific P-gp fluorogenic substrate, and intracellular accumulation of Rhodamine123 reflects the efficiency of inhibition of P-gp activity. After MEL-pep action, intracellular Rhodamine123 accumulation in BEL-7402/5-FU was significantly reduced, confirming that MEL-pep was able to inhibit P-gp function.

Tenth, in vivo growth inhibition effect of Cathelicidin-BF on 5-FU drug-resistant liver cancer cell BEL-7402/5-FU

Diluting BEL-7402/5-FU cells in logarithmic growth phase with PBS, staining with trypan blue, counting with a blood counting plate under an inverted microscope to obtain viable cells with a count of 98% or more, and adjusting cell concentration to 5 × 10⁸and/mL. 24 nude mice of 3 weeks old were used for the experiment, and 0.1mL of cells were subcutaneously inoculated to the right underarm of the mice. The tumor volume of the mouse is up to 100-³Then, randomly divided into 4 groups: tumor model groups, MEL-pep low, medium, high dose treatment groups (1, 1.5, 2mg/kg), 6 per group. The administration mode is intratumoral injectionInjections were administered three times a week for a total of three weeks. The antitumor effect of the test object is dynamically observed by using a method for measuring the tumor size. Tumor diameter was measured 1 time every 3 days. After 21 days of dosing, the mice were sacrificed and the tumor mass was surgically removed and weighed.

The formula for Tumor Volume (TV) is:

TV＝1/2×a×b²wherein a and b represent length and width, respectively.

The tumor inhibition rate calculation formula is as follows:

as shown in FIGS. 11-12, MEL-pep significantly inhibited the growth of BEL-7402/5-FU cells in tumor-bearing mice in vivo, and was dose-dependent. Wherein the tumor inhibition rates of three MEL-pep1, 1.5 and 2mg/kg dose groups are respectively as follows: 50.69%, 61.46%, and 72.92%. FIG. 13 shows that the weight curve of MEL-pep administered mice did not significantly decrease compared to the model group. FIG. 14 shows that HE staining results of tumor tissues of mice in each group of experiments show that the tumor tissues of the mice in the model group have obvious proliferation and division, and the tumor tissues of the mice in the administration group have obvious necrosis areas. Therefore, MEL-pep was significantly active in vivo against BEL-7402/5-FU cell proliferation.

Sequence listing

<110> first subsidiary Hospital of medical college of Western-Ann transportation university

<120> mutant melittin MEL-pep and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>3

<211>26

<212>PRT

<213> melittin MEL-pep (2 Ambystoma latex x Ambystoma jeffersonia)

<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

1. a mutant melittin MEL-pep, is characterized in that, the aminoacid sequence of this polypeptide is as shown in SEQ ID NO.1.

2. The application of the mutant melittin MEL-pep according to claim 1 in the preparation of a medicine for the treatment of liver cancer.

3. application as claimed in claim 2 is characterized in that, the application of described mutant melittin MEL-pep in the preparation of the medicine for the treatment of 5-FU resistant liver cancer.

4. The application according to claim 2, characterized in that, the application of the mutant melittin MEL-pep in the preparation of a medicine for reversing 5-FU drug-resistant liver cancer.

5. The application of claim 3, wherein the mutant melittin MEL-pep is used in the preparation of a medicine for inhibiting the growth of 5-FU drug-resistant liver cancer cells in vivo.