CN111729073B - Application of polypeptide SE37 mutant in preparation of antitumor drugs - Google Patents

Abstract

The invention belongs to the field of polypeptide medicines, and discloses application of a polypeptide SE37 mutant in preparation of antitumor medicines, wherein the amino acid sequence of the polypeptide SE37 mutant is as follows: SETRLVLNRLFDKIRQVIRKFEKGIKEKSKRFFDGLL are provided. The anti-tumor drug is a drug for promoting tumor cell apoptosis. The polypeptide SE37 mutant has low toxicity to normal cells and high safety; and has good inhibiting and killing effects on various tumor cells including lung cancer, colorectal cancer, ovarian cancer and breast cancer.

Description

Application of polypeptide SE37 mutant in preparation of antitumor drugs

Technical Field

The invention belongs to the field of polypeptide medicines, and particularly relates to application of a polypeptide SE37 mutant in preparation of an anti-tumor medicine.

Background

Anticancer peptides (ACPs) are a series of peptide chains consisting of 10-60 amino acids whose amphiphilic structure is usually composed of a cationic face and a hydrophobic face, which are necessary to facilitate the interaction of the peptide with the target cell. The ACPs can inhibit the proliferation or migration of tumor cells or inhibit the formation of tumor thrombus, and simultaneously, the drug resistance of the tumor cells is not easily caused, so that the ACPs become the most promising candidate anticancer drugs. Compared with common anticancer drugs, the ACPs not only have more efficient antitumor activity, but also have lower toxic and side effects on normal cells and are not easy to generate drug resistance. Of course, not all ACPs can be studied as anticancer drugs, and most ACPs have the following disadvantages, such as short half-life, susceptibility to hydrolysis by proteases, strong toxicity, poor targeting, etc., and cannot meet the demand for ideal anticancer peptides.

Therefore, there is a need to develop new anticancer peptides with good antitumor effect and low toxicity to normal cells aiming at different types of tumors.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the application of the polypeptide SE37 mutant in preparing antitumor drugs, wherein the polypeptide SE37 mutant has low toxicity to normal cells, but has good killing effect on tumor cells such as lung cancer, colorectal cancer, ovarian cancer, breast cancer and the like.

The polypeptide SE37 mutant and the application of the pharmaceutically acceptable salt thereof in preparing antitumor drugs, wherein the amino acid sequence of the polypeptide SE37 mutant is as follows: SETRLVLNRLFDKIRQVIRKFEKGIKEKSKRFFDGLL are provided. The polypeptide SE37 mutant contains 37 amino acid residues, the relative molecular weight is 4509.41, the hydrophobicity is-0.33, the hydrophilicity is 0.57, and the net charge is 66.

The polypeptide SE37 mutant is a polypeptide chain independently developed and designed by the applicant, can be synthesized by a conventional chemical synthesis method, and has the characteristics of convenience and rapidness in synthesis. The polypeptide SE37 mutant is a mutant obtained by taking antibacterial peptide SE37 (the amino acid sequence of the antibacterial peptide SE37 is SETRPVLNRLFDKIRQVIRKFEKGIKEKSKRFFDGLL) as a template and replacing amino acids.

The applicant has made a series of modified peptides by performing one to several amino acid substitutions on the peptide chain in order to improve the performance of the antimicrobial peptide SE 37. Initial experiments only found that the series of modified peptides had a certain antibacterial effect. However, further research shows that part of the peptide fragments can also generate certain antitumor effect. Test results show that in a plurality of peptide chains, SE37 has higher toxicity to a plurality of tumors and normal cells, so that the safety and the patent drug property are further improved; the polypeptide SE37 mutant has slight toxicity to normal cells, generates good killing effect on a plurality of tumor cells, has outstanding comprehensive performance, can be used as a medicinal active ingredient to prepare an anti-tumor medicament, and enables the polypeptide SE37 mutant to obtain more valuable application.

Preferably, the pharmaceutically acceptable salt of the polypeptide SE37 mutant comprises acetate or citrate. In order to improve the stability or solubility of the polypeptide drug, the polypeptide SE37 mutant can be made into the form of acetate or citrate.

Preferably, the anti-tumor drug is a tumor cell apoptosis promoting drug. Experiments show that the polypeptide SE37 mutant can achieve the effect of inhibiting the growth of tumor cells by promoting the apoptosis of the tumor cells, particularly through a mitochondrial apoptosis pathway.

Preferably, the tumor is at least one of lung cancer, colorectal cancer, ovarian cancer or breast cancer.

More preferably, the tumor is lung cancer.

An antitumor drug comprises a polypeptide SE37 mutant and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable auxiliary material.

Preferably, the pharmaceutically acceptable adjuvant is at least one of a solvent, a wetting agent, an emulsifier, a thickener, an excipient, a suspending agent, a disintegrant, a filler, a lubricant or a diluent.

Preferably, the dosage form is tablet, injection, spray, freeze-dried powder injection, capsule or coated pill.

More preferably, when the pharmaceutical composition is an injection, the mass concentration of the polypeptide SE37 mutant in the injection is 1-20 mg/mL. The polypeptide SE37 mutant belongs to polypeptide drugs, is easy to degrade and difficult to pass through intestinal mucosa, and the drug effect of the polypeptide SE37 mutant can be reduced by oral administration, so that the polypeptide SE37 mutant is prepared into an injection form, and is more beneficial to absorption and utilization of drug components.

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

(1) the polypeptide SE37 mutant provided by the invention has low toxicity to normal cells and high safety.

(2) The polypeptide SE37 mutant has good inhibition and killing effects on various tumor cells including lung cancer, colorectal cancer, ovarian cancer and breast cancer, and can be used for preparing corresponding antitumor drugs.

Drawings

FIG. 1 shows the effect of SE37 on apoptosis of A549 cells;

FIG. 2 shows the effect of the SE37 mutant on A549 apoptosis;

FIG. 3 shows the effect of SE37 on mitochondrial membrane potential of A549 cells;

FIG. 4 shows the effect of the SE37 mutant on mitochondrial membrane potential of A549 cells;

FIG. 5 shows the effect of SE37 on ROS in A549 cells;

FIG. 6 shows the effect of SE37 mutant on ROS in A549 cells;

FIG. 7 shows the effect of SE37 and SE37 mutants on the change in expression of apoptosis pathway-related genes in A549 cells;

FIG. 8 shows the effect of SE37 and SE37 mutants on the expression level of the A549 cell mitochondrial apoptotic proteins Bax and Bcl-2

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Principal materials and instruments

Polypeptide synthesis: the polypeptide SE37 mutant and the polypeptide SE37 used in the experiment are synthesized by Shanghai Tanpu Biotechnology Co., Ltd, the synthesis method is a chemical solid phase synthesis method, and the purity is more than 95%.

A549 (human lung cancer cells), HCT-8 (human colorectal adenocarcinoma cells), SKVO3 (ovarian cancer cells), MCF-7 (breast cancer cells) and HK2 (human tubular epithelial cells) cell strains are purchased from Shanghai cell banks of Chinese academy of sciences, and the required experimental materials are as follows: RPMI-1640 basic medium; DMEM basal medium; f12 medium; fetal bovine serum; ampicillin and streptomycin; DMSO; annexin v-FITC double staining kit; low molecular weight protein marker; (ii) trypsin; PBS; MTT; high-efficiency RIPA tissue/cell lysate; positive polypeptide drug LL 37; PMSF; a protein quantification (BCA) test kit; an active oxygen detection kit; a mitochondrial membrane potential detection kit (JC-1); DEPC water; trizol; glycerol; chloroform.

Example 1

A mutant of polypeptide SE37, which has the amino acid sequence as follows: SETRLVLNRLFDKIRQVIRKFEKGIKEKSKRFFDGLL, can be used for preparing antitumor drugs.

Example 2: MTT method for detecting influence of SE37 mutant on proliferation of tumor cells and normal cells

Cell recovery: the frozen tubes of the A549, HCT-8, SKVO3, MCF-7 and HK2 cell strains were taken out from a liquid nitrogen tank and placed in a water bath at 37 ℃ to be shaken until being thawed. Respectively sucking the melted cell strains into a sterile centrifuge tube with the specification of 15mL in a sterile super clean bench, centrifuging for 5min at the speed of 1000r/min, and removing supernatant; 5mL of fresh RPMI-1640 complete medium was added to HCT-8 and MCF-7, respectively; adding DMEM complete medium into A549; DMEM/F12(1:1) complete medium was added to HK 2. Finally, the samples were transferred to a specification of 25cm²Sterile culture flask of (1), in 5% CO₂And cultured at 37 ℃.

Cell passage: and observing the cells by using a microscope, wherein the cell morphology is normal, and the cell passage is carried out when the growth density reaches about 90%. Firstly, taking out a culture bottle containing cells, putting the culture bottle into a super clean bench, sucking out original culture medium, washing the cells for 2 times by using PBS buffer solution, completely sucking out the PBS, adding 600 mu L of pancreatin to enable the pancreatin to completely cover the bottom of the culture bottle, screwing a bottle cover, and then placing the culture bottle into a 37 ℃ culture box for digestion. After digestion, the cells were blown down along the bottle wall with fresh complete medium, the cell suspension was then aspirated into a 15mL sterile centrifuge tube and centrifuged at 1000r/min for 5min, the supernatant was removed, the cells were resuspended in fresh complete medium and passaged at a ratio of 1: 3.

Freezing and storing cells: after the supernatant was removed by centrifugation in the above-described cell passaging procedure, a freezing medium (basal medium: fetal bovine serum: DMSO ═ 5:4:1) was added to resuspend the cells, and the suspensions were added to 1.5mL cell freezing tubes (approximately 1X 10 cells per tube of cell suspension)⁶Individual cells) are labeled and then frozen by a gradient cooling method.

When the cell density reaches about 80%, A549 cells, HCT-8 cells, SKVO3 cells, MCF-7 cells and HK2 cells are digested by trypsin and centrifuged to prepare cell suspension with the concentration of 5 × 10⁴one/mL of the cells was added to a 96-well plate in a system of 100. mu.L per well. At 37 ℃ and 5% CO₂Culturing in an incubator for 24 h.

After 24h of culture, sucking out original culture solution along the hole wall, then adding 100 mu L of SE37 drug solution and SE37 mutant drug solution which are prepared by using a basic culture medium into different cell strain experimental groups, wherein the concentrations are respectively 1 mu M, 2 mu M, 4 mu M, 6 mu M, 8 mu M and 10 mu M, 3 multiple holes are arranged in each concentration, a blank control group is 100 mu L of basic culture medium, a negative control group is a non-drug-adding hole, and a positive control group is LL37(LL37 is a C end containing 37 amino acids in hCAP-18 protein, has good anti-tumor effect besides the antibacterial effect, but has the defect of high toxicity to normal cells). At 37 ℃ with 5% CO₂The incubator continues to culture for 24 h.

And (3) preparing MTT working solution from the MTT stock solution and the basic culture medium according to the ratio of 1: 4. After 24h of drug action, the culture medium in the wells was aspirated, 100. mu.L of MTT working solution was added to each well, and 5% CO was added at 37 ℃₂And incubating for 4h in an incubator in the dark. After the incubation is finished, the supernatant of the MTT working solution is completely absorbed, 150 mu L of DMSO is added into each well, and the absorbance value of each well is detected at 490nm after shaking for 5 min. Calculating the cell survival rate by the formula: cell survival rate (%) ═ a_{Experimental group}–A_{Blank control})/(A_{Negative control}-A_{Blank control}) X 100%, and half fatality (IC50) was calculated using SPSS 21.0 analysis software; therapeutic index is IC50(HK2)/I C50 (tumor cells).

In the experiment, the MTT method is adopted to detect the killing effect, the influence of the SE37 mutant on the growth and proliferation of 4 tumor cells and normal cells HK2 is researched, and the experimental results are shown in tables 1 and 2, wherein SE37 and LL37 are known to have greater toxicity to the tumor cells, but have the defect of greater toxicity to the normal cells.

Table 1: IC50 values (unit: μ M) of the SE37 mutant on 5 cells

Note that, compared with HK2, P is < 0.01, and P is < 0.05

Table 2: therapeutic index of SE37 mutant on 4 tumor cells

As shown in Table 1, the positive drugs LL37 and SE37 both have good inhibition effects on the 4 tumor cells, but have obvious toxic and side effects on HK 2; the SE37 mutant has good inhibition effect on 4 tumor cells, and the inhibition effect on the tumor cells is similar to or better than that of the positive medicament LL 37; however, compared with the positive drug LL37, the SE37 mutant has obviously lower toxicity to normal cells and better safety. As shown in Table 2, the SE37 mutant has higher therapeutic index for 4 tumor cells in the table, and the therapeutic indexes are all higher than that of SE 37. By combining the table 1 and the table 2, the polypeptide SE37 mutant has good growth inhibition effect on tumor cells (lung cancer, colorectal cancer, breast cancer and ovarian cancer) and low toxicity on normal cells, so that the polypeptide SE37 mutant has outstanding potential for preparing antitumor drugs.

Example 3: effect of SE37 mutant on apoptosis of A549 cells

Taking the A549 with higher tumor cell therapeutic index of the SE37 mutant in example 2 as an example, the influence of the polypeptide SE37 mutant on the apoptosis of the A549 cells is researched.

Collecting A549 cells in logarithmic growth phase, preparing cell suspension respectively according to 5 × 10 per well⁵The cells were loaded into 6-well plates at 37 ℃ with 5% CO₂Culturing in an incubator for 24 h.

Discarding the original culture medium, adding SE37 and SE37 mutant drug solutions prepared from fresh basal medium at concentrations of 3 μ M, 6 μ M, 2 μ M, 4 μ M, and 8 μ M, respectively, and setting blank Control group (Control group) at 37 deg.C and 5% CO₂The incubator continues to culture for 24 h.

After 24h of drug action, the cells were collected in a centrifuge tube, 200. mu.L of EDTA-free trypsin was added to each well to digest adherent cells, the cells were gently blown with fresh medium and transferred to a sterile centrifuge tube, centrifuged at 1000r/min for 5min, and the pellet was collected. Then washing the precipitate twice with cold PBS, centrifuging at 1000r/min for 5min, discarding the supernatant. Then 1mL of 1X Binding Buffer was added to resuspend the cells, and the cells were centrifuged at 300g for 10min, and the supernatant was discarded. The cells were resuspended in 1mL of 1 × Bindin g Buffer to a cell density of 1 × 10⁶one/mL. Add 5. mu.L Annexin V-FITC and mix gently for 10min at room temperature in the dark. Then 5. mu.L of PI was added and incubated at room temperature in the dark for 5 min. Detection was performed by flow cytometry.

As shown in figure 1, A549 cells treated by SE37 with the concentration of 3 and 6 mu M for 24h show obvious early apoptosis and partial late apoptosis, and the average apoptosis rate reaches 31.19 percent and 38.73 percent respectively. This indicates that SE37 can cause significant apoptosis and necrosis in a549 cells and exhibit some concentration dependence (P < 0.01).

As shown in FIG. 2, the average apoptosis rates of A549 cells treated by SE37 mutants at the concentrations of 2. mu.M, 4. mu.M and 8. mu.M for 24h were 17.18%, 23.22% and 33.18%, respectively, and the number of cells with early apoptosis was significantly increased compared with that of a blank Control group (Control group), indicating that the SE37 mutant can cause significant apoptosis of A549 cells and is in a certain concentration dependence (P < 0.01).

Example 4: effect of the SE37 mutant on the mitochondrial Membrane potential of A549 cells

Cell suspension preparation and drug treatment As in example 3, the SE37 mutant and SE37 were administered at concentrations of 0. mu.M (Control group, i.e., blank Control group), 1. mu.M, 2. mu.M, and 4. mu.M, respectively.

After 24h of drug treatment, the culture solution is completely sucked in a centrifuge tube, PBS is used for washing for 2 times, cells are digested by trypsin without EDTA in each hole, the cells are blown and uniformly mixed by fresh culture medium and then transferred into the centrifuge tube, centrifugation is carried out for 5min at 1000r/min, and the supernatant is discarded.

Continuously adding 0.5mL of fresh culture medium to resuspend the cells, then respectively adding 0.5mL of JC-1 staining working solution, reversing for several times, uniformly mixing, and incubating for 20min at 37 ℃ in a cell incubator.

During the incubation, an appropriate amount of JC-1 staining buffer (1X) was prepared by adding 4mL of distilled water per 1mL of JC-1 staining buffer (5X) and placing in an ice bath.

After incubation at 37 ℃, centrifuging for 3min at 600g and 4 ℃, collecting the precipitate, and discarding the supernatant.

Wash twice more with JC-1 staining buffer (1X): 1mL JC-1 staining buffer (1X) was added to resuspend the cells. 600g, centrifuging at 4 ℃ for 3min, collecting the precipitate, and discarding the supernatant. Add 1mL of JC-1 staining buffer (1X) again to resuspend the cells. 600g, centrifuging at 4 ℃ for 3min, collecting the precipitate, and discarding the supernatant. The cells were resuspended in 1mL JC-1 staining buffer (1X) and then examined by flow cytometry.

As shown in FIG. 3, when the A549 cells treated with SE37 were passed through the flow cytometer at the detection concentration range (0-4. mu.M), the fluorescence collected by the FL1 channel was increased and the fluorescence collected by the FL2 channel was decreased. Compared with the blank Control group (Control group), the mitochondrial membrane potential of the A549 cells is obviously reduced (P < 0.01), but the fluorescence signal detected by the FL1 channel is not obviously increased compared with that detected by the FL1 channel at the concentration of 4 mu M (P > 0.05). This is likely to be the case at higher concentrations of SE37 drug, where cell death occurs by other mechanisms than apoptosis.

As shown in FIG. 4, when the A549 cells treated with the SE37 mutant were passed through the flow cytometer at the detection concentration range (0-4. mu.M), the fluorescence signal collected by the FL1 channel was increased, and the fluorescence signal collected by the FL2 channel was decreased, indicating that the mitochondrial membrane potential was decreased. Compared with a blank Control group (Control group), the fluorescence signal detected by the FL1 channel is enhanced, and the mitochondrial membrane potential is obviously reduced (P is less than 0.01). However, at a concentration of 4. mu.M, the fluorescence signal detected by the FL1 channel did not increase significantly (P > 0.05) compared to 2. mu.M. It was shown that the apoptotic effect of the SE37 mutant on a549 cells was achieved by a reduction in mitochondrial membrane potential.

Example 5: effect of SE37 mutant on ROS (reactive oxygen species) in A549 cells

The cell suspension preparation and dosing procedure were the same as in example 3. SE37 and SE37 mutant drug solutions were all at 1. mu.M, 2. mu.M, and 4. mu.M, and a blank Control group (Control group, drug concentration of 0. mu.M) was set. After 24h of drug treatment, the culture solution is completely sucked in a centrifuge tube, washed for 2 times by PBS, cells are digested by trypsin without EDTA in each hole, blown and uniformly mixed by fresh culture medium, transferred into the centrifuge tube, centrifuged for 5min at 1000r/min, and the supernatant is discarded. DCFH-DA (active oxygen fluorescent probe) was diluted in serum-free medium at a ratio of 1:1000 to a final concentration of 10. mu.M.

After collecting A549 cells, 1mL of diluted DCFH-DA was added for resuspension and incubation was performed in a cell incubator at 37 ℃ for 20 min. After incubation was completed, centrifugation was performed 3 times with serum-free medium washing to sufficiently remove DCFH-DA that had not entered the cells. And (4) detecting by an up-flow cytometer after resuspending by a proper amount of PBS.

As shown in FIG. 5, the ROS content in SE 37-treated A549 cells gradually increased with increasing drug concentration in the range of detection concentration (0-4 μ M), and the mean fluorescence intensity of each treatment group was 244. + -. 6, 355.333. + -. 7.095 and 360. + -. 6.557, respectively, and the fluorescence intensity was gradually increased (P < 0.01) as compared with that of the Control group (Control). It was initially shown that SE37 can exert an apoptotic effect by increasing the ROS content in a549 cells.

As shown in FIG. 6, the ROS content in A549 cells treated with the SE37 mutant gradually increased with the increase of the concentration of the drug in the range of the tested concentration (0-4. mu.M), and the mean fluorescence intensities of each treatment group were 260.667. + -. 0.577, 295.667. + -. 1.528 and 357. + -. 1.732, respectively, which were gradually increased (P < 0.01) compared with the Control group (Control). Preliminary results indicate that the SE37 mutant can promote the release of ROS in A549 cells to cause the apoptosis phenomenon.

Example 6: influence of SE37 mutant on apoptosis-related genes in A549 cells and expression quantity thereof

Total RNA extraction

(1) Collecting A549 cells in logarithmic growth phase, preparing cell suspension respectively according to 5 × 10 per well⁵The cells were plated in 6-well plates at 37 ℃ with 5% CO₂Culturing in an incubator for 24 h.

(2) The original culture medium is discarded, and a polypeptide drug solution prepared by a fresh basal culture medium is added. Setting a blank Control group (Control group without adding medicine), and a SE37 group (with the concentration of 2 mu M) and a SE37 mutant group (with the concentration of 2 mu M); at 37 ℃ with 5% CO₂The incubator continues to culture for 24 h.

(3) After the drug treatment, the cell culture plate was placed on ice, the original culture medium was aspirated, washed twice with cold PBS, 1mL RNAiso was added, and the flask was shaken horizontally on ice to distribute the lysate evenly on the cell surface and lyse the cells to shed.

(4) Transferring the lysate containing the cells into a 1.5mL centrifuge tube, repeatedly blowing and sucking by using a pipette gun until no obvious precipitate is formed in the lysate, and standing for 5min at room temperature.

(5) Adding 200 μ L chloroform into the homogenate lysate, covering the tube cover, shaking vigorously for 20s, and standing at room temperature for 5 min.

(6)12000 Xg, 4 ℃ centrifugation for 15 min.

(7) Centrifuging, collecting supernatant 400 μ L, adding 500 μ L isopropanol, mixing, and standing at room temperature for 10 min.

(8)12000 Xg, 4 ℃ centrifugation for 10min, supernatant.

(9) The pellet was washed by adding 1mL of 75% ethanol (prepared by DEPC water treatment) and the tube wall was washed by gently inverting it upside down.

(10)7500 Xg, 5min at 4 ℃.

(11) Sucking out the liquid, sucking out the residual trace ethanol by using a pipette gun, drying at room temperature for 2-3min, adding 30 mu L of EDPC water, repeatedly blowing, uniformly mixing, and fully dissolving RNA.

RNA electrophoresis and trace nucleic acid quantitative determination of concentration and OD value

(1) Using a micro nucleic acid quantitative analyzer, 2. mu.L of DEPC water was used as a blank, and 2. mu.L of a sample was used, whereby the concentration of RNA and OD value were measured.

(2) Preparing a nucleic acid electrophoresis gel: 1g of agarose powder was weighed into 100mL of 1 XTAE buffer solution and dissolved by microwave oven heating. After the solution is cooled and does not scald hands, 2 mu L of Ethidium bromide dye (EB dye) is added, and gel can be prepared after shaking and mixing.

(3) Loading: after adding electrophoresis Buffer (1 × TAE) to the electrophoresis chamber, the prepared gel was put therein to prepare for sample addition, in a Loading Buffer: the samples were mixed at a ratio of 1:2 and added to the gel wells with 3. mu.L of Marker added.

(4) Electrophoresis: 100V, 25 min.

(5) And taking a picture in a gel imaging system.

Genomic DNA was removed according to the reaction system of Table 3;

TABLE 3

X^*: the concentration of RNA can be measured by a trace nucleic acid quantitative instrument to be X ng/mL, so that the amount of X to be added can be calculated, and after the X is uniformly mixed, the mixture is placed at room temperature for 20-30min or 42 ℃ for 2 min.

Reverse transcription to form cDNA

The reaction solution was prepared on ice. In order to ensure the accuracy of the preparation of the reaction solution, Master Mix should be prepared in an amount of 2 times the reaction number, and then 10 μ L of the Master Mix should be dispensed into each reaction tube. After gentle mixing, reverse transcription was carried out immediately in the amounts shown in Table 4.

TABLE 4

After being mixed evenly, carrying out reverse transcription reaction on a PCR instrument, wherein the reaction conditions are as follows: 37 ℃, 15min, 85 ℃, 5 s.

RT-PCR was performed using the primers shown in Table 5.

TABLE 5

In order to ensure the accuracy of the preparation of the reaction solution, SYBR Premix Ex Taq is prepared according to the amount of 2 times of the reaction number in each reaction^TM(2X) + corresponding upstream and downstream primers + Rox Reference Dye II + ddH₂And O, subpackaging into each PCR tube, adding the cDNA common PCR product obtained in the step, gently mixing uniformly, and detecting on a machine (the whole process needs to be operated on ice, and the reaction system is shown in table 6).

TABLE 6

The experiment was set up with 3 replicates for the blank control group, the internal control group and the experimental group.

Reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s; circulating for 40 times at 95 ℃ for 5s and 60 ℃ for 30 s.

Analyzing the obtained melting curve, amplification curve and experimental standard curve. Obtaining C of each Gene_tValue, result, by 2^-ΔΔCtAnd (4) formula calculation. The formula is as follows:

Δ Ct1 ═ Ct (gene of interest in experimental group) -Ct (internal reference gene)

Δ Ct2 ═ Ct (blank control target gene) -Ct (internal reference gene)

- Δ Δ Ct ═ - (Δ Ct1 (gene of interest) - Δ Ct2 (blank control gene)

Subjecting cells in logarithmic growth phase to trypsinization, and processing at 5 × 10⁵Individual cells/well were added to 6-well plates at 5% CO₂After 24 hours of culture at 37 ℃ in an incubator, the medium was removed, and fresh medium (experimental group) containing 2. mu.M of each of the SE37 and SE37 mutants was added, and the culture was continued for 24 hours while using the wells without the drug as blank groups.

After cell drug treatment for 24h, collecting the supernatant by using a clean 15mL centrifuge tube for centrifugation, collecting the precipitate, and washing the precipitate for 3 times by using precooled PBS; while adherent cells in 6-well plates were washed 3 times with PBS first, the washed liquid could be centrifuged with the supernatant liquid. After washing adherent cells, 100 mu L of prepared protein lysate is obliquely added along the wall to ensure that the liquid is fully contacted with the cells, and for precipitates in a 15mL centrifuge tube, a small amount of protein lysate is used for carrying out lysis and then is transferred to a 1.5mL centrifuge tube together with the liquid in the hole, and the centrifuge tube is placed on ice for 3min for lysis (the whole process is operated on ice). After the cracking is finished, centrifuging for 30min at 12000r/min and 4 ℃, and obtaining supernatant which is the protein lysate.

Detecting protein concentration according to the protein quantitative kit instruction to obtain protein concentration, adding 5 Xprotein sample buffer solution, mixing, and placing in 100 deg.C boiled water for 5 min. Taking out the sample, placing on ice, and centrifuging for 5min at 3000r/min after the sample is cooled. At this point the next experiment can be performed or the sample can be placed in a-20 ℃ freezer.

Loading the sample after preparing the protein gel according to the kit, and adding 40 mu g of protein sample into each hole;

after sample adding, running concentrated gel at a voltage of 80V, and separating gel at an electrophoresis voltage of about 120V until bromophenol blue runs out of the gel;

cutting the PVDF membrane into gel size, soaking in methanol for 10s, adding deionized water for 10min, and balancing in electrotransformation liquid for 10 min. The porous pad, the large filter paper, the PVDF film, the protein glue, the large filter paper and the porous pad are arranged from the anode to the cathode in sequence. Transferring in ice water bath at constant current of 250mA for about 1.5 h;

blocking the PVDF membrane by 5% BSA for 1.5h, adding primary antibody, and incubating overnight at 4 ℃;

after incubation, TBST was washed 3 times for 10min each;

after washing, adding a secondary antibody (mouse antibody), and incubating for 1h at 37 ℃;

after incubation is finished, washing for 3 times by TBST, 10min each time, and detecting after washing is finished;

after mixing Millipore luminescent solution in equal volume, the mixture was mixed with PVDF membrane, imaged on an imager, and analyzed for gray value using image J.

As shown in FIG. 7, the relative expression levels of pro-apoptotic factor Bax mRNA were both up-regulated (P < 0.05) in the A549 cells treated with the SE37 and SE37 mutants after 24h treatment with the SE37 and SE37 mutants at a concentration of 2. mu.M, respectively, compared with the blank Control group (Control group); the relative expression quantity of the mRNA of the anti-apoptosis factors Bcl-2, Caspase-3 and Caspase-9 has no obvious change (P is more than 0.05). Preliminary analysis suggests that SE37 and SE37 mutants might induce apoptosis in cells by up-regulating the expression of Bax mRNA, a mitochondrial apoptosis pathway-associated gene.

FIG. 8 shows the Western Blot technique for detecting the expression of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 in A549 cells treated with the SE37 and SE37 mutants at a concentration of 2. mu.M, respectively, for 24 h. The Western Blot result is similar to the PT-PCR result, and compared with a blank Control group (Control group), the relative expression level of Bax protein of A549 cells treated by the SE37 and the SE37 mutant is up-regulated (P < 0.05), and the relative expression level of Bcl-2 protein is remarkably down-regulated (P < 0.05), thereby showing a typical mitochondrial apoptosis pathway phenomenon.

Example 7

An anti-tumor medicine is a tablet, and comprises the following raw materials in parts by weight: 10 parts of acetate of a polypeptide SE37 mutant, 60 parts of corn starch, 5 parts of magnesium stearate and 6 parts of water. Wherein corn starch is used as filler, magnesium stearate is used as lubricant, and water is used as wetting agent.

Example 8

An antitumor drug, the dosage form of which is injection, comprises polypeptide SE37 mutant with mass concentration of 10mg/mL and 0.9% sodium chloride solution as solvent.

Claims (3)

1. The polypeptide SE37 mutant and the application of the pharmaceutically acceptable salt thereof in preparing antitumor drugs are characterized in that the amino acid sequence of the polypeptide SE37 mutant is as follows: SETRLVLNRLFDKIRQVIRKFEKGIKEKSKRFFDGLL, respectively; the tumor is at least one of lung cancer, colorectal cancer, ovarian cancer or breast cancer.

2. The use of claim 1, wherein the pharmaceutically acceptable salt of the mutant polypeptide SE37 comprises acetate or citrate.

3. The use of claim 1, wherein the anti-neoplastic agent is a pro-apoptotic agent.

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