Active polypeptides and use in the prevention and / or treatment of hpv

Abstract

The application provides an active polypeptide and application in prevention and / or treatment of HPV, and belongs to the technical field of biological medicine. The application provides a polypeptide with prevention and / or treatment activity, and the sequence of the polypeptide comprises SEQ ID No. 1. The polypeptide can directly serve as a medicine to effectively activate immunity, inhibit and eliminate human papilloma virus. The polypeptide can be used for injection alone as a medicine. The polypeptide can also induce single nucleated cells (PBMC) in vitro, culture CTL cells with specific killing of HPV, and through reinfusion of the CTL cells into a patient, directly kill tumors or stimulate immune response of the body, effectively kill HPV, make HPV in the patient turn from positive to negative, and finally achieve the effect of treatment. The polypeptide can also serve as a medicine for prevention of HPV, effectively stimulate strong immune response, and make the body produce a large number of immune cells to achieve the effect of protection and prevention.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an active polypeptide and its application in the prevention and / or treatment of HPV. Background Technology

[0002] Human papillomavirus (HPV) is a DNA virus. Based on the type and pathogenicity of HPV, it can be divided into low-risk and high-risk types. High-risk HPV is closely associated with penile cancer and common gynecological cancers such as cervical cancer.

[0003] Currently, HPV prophylactic vaccines on the domestic and international markets only provide preventative protection. For patients already infected with HPV, especially those with long-term persistent HPV infection, the therapeutic effect of HPV prophylactic vaccines alone is minimal. More importantly, postoperative recurrence of cervical disease is often caused by integrated HPV infection, and prophylactic HPV vaccines targeting L1 antigen have little effect on treating integrated HPV infection. Therefore, drugs with both preventative and / or therapeutic effects against HPV are urgently needed for development. Furthermore, how to further improve the efficacy of preventing and / or treating HPV infection, especially how to simultaneously improve specificity and immune activity, remains a technical challenge that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] This invention provides an active polypeptide and its application in the prevention and / or treatment of HPV. The polypeptide can serve as a drug for preventing and treating HPV, effectively stimulating a strong immune response and generating a large number of immune cells in the body to achieve protective and preventative effects.

[0005] The present invention provides a polypeptide having preventive and / or therapeutic activity, said polypeptide having any of the following amino acid sequences: (a) the amino acid sequence shown in SEQ ID No. 1;

[0006] (b) is an amino acid sequence that has more than 80% homology with the amino acid sequence shown in (a).

[0007] Preferably, the polypeptide can prevent and / or treat diseases associated with human papillomavirus.

[0008] Preferably, the human papillomavirus-related diseases include at least one of the following: cervical cancer, cervical and vaginal intraepithelial neoplasia (CIN I-III, VIN I-III), polyps, and warts.

[0009] The present invention also provides the use of the above-mentioned polypeptide in the preparation of medicaments for the prevention and / or treatment of diseases caused by human papillomavirus.

[0010] The present invention also provides a medicament for the prevention and / or treatment of diseases caused by human papillomavirus, wherein the active ingredient of the medicament includes the aforementioned polypeptide.

[0011] Preferably, the dosage form of the drug includes an injection.

[0012] Preferably, the administration method of the drug includes at least one of the following: subcutaneous administration, intradermal administration, intramucosal administration, submucosal administration, intramuscular administration, and intraperitoneal administration.

[0013] The present invention also provides CTL cells that specifically kill human papillomavirus-infected cells, obtained by in vitro induction of mononuclear cells based on the above-mentioned polypeptide.

[0014] The present invention also provides the use of the above-mentioned CTL cells in the preparation of drugs for the prevention and / or treatment of diseases caused by human papillomavirus.

[0015] The present invention also provides a medicament for the prevention and / or treatment of diseases caused by human papillomavirus, wherein the active ingredient of the medicament includes the above-mentioned CTL cells.

[0016] Beneficial effects: The present invention provides a polypeptide with preventive and / or therapeutic activity, which can be directly used as a drug to effectively activate immunity, inhibit and eliminate human papillomavirus; the polypeptide can be used as a drug for injection alone without the addition of any cytokines such as interleukin-2 (IL-2), interferon-α (IFN-α), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (GM-CSF).

[0017] The polypeptides described in this invention, after being artificially synthesized, can also induce PBMCs in vitro, culture CTL cells that specifically kill HPV-infected cells, and then be reinfused into the body to stimulate an immune response, effectively killing HPV-infected positive cells in the body, thereby achieving a therapeutic effect.

[0018] The polypeptide described in this invention can also be used as a drug to prevent HPV infection, effectively stimulating a strong immune response and generating a large number of immune cells in the body to achieve the effect of protection and prevention of infection progression. Attached Figure Description

[0019] Figure 1 The tumor weight (g) of mice in the experimental and control groups in the prevention model is shown below. Note: * indicates that the tumor weight of mice in the experimental group in the prevention model was significantly lower than that of mice in the control group (P<0.01).

[0020] Figure 2The tumor weight (g) of mice in the experimental and control groups in the treatment model is shown below. Note: * indicates that the tumor weight of mice in the experimental group in the treatment model was significantly lower than that of mice in the control group (P<0.01). Detailed Implementation

[0021] The present invention provides a polypeptide having preventive and / or therapeutic activity, said polypeptide having any of the following amino acid sequences: (a) the amino acid sequence shown in SEQ ID No. 1;

[0022] (b) is an amino acid sequence that has more than 80% homology with the amino acid sequence shown in (a).

[0023] The amino acid sequence of the polypeptide described in this invention includes SEQ ID No. 1: KKKKKKKALQAIELQLTLETIYNSQYSNE, and the polypeptide has activity in preventing and / or treating diseases associated with human papillomavirus (HPV). The HPV-related diseases described in this invention preferably include at least one of the following: cervical cancer, cervical and vaginal intraepithelial neoplasia (CIN I-III, VIN I-III), polyps, and warts.

[0024] The polypeptides described in this invention can be directly synthesized by chemical synthesis methods, such as the Fmoc solid-phase synthesis method used in the examples to artificially synthesize the polypeptides and perform subsequent experimental operations.

[0025] The present invention also provides the use of the above-mentioned polypeptide in the preparation of medicaments for the prevention and / or treatment of diseases caused by human papillomavirus.

[0026] The polypeptide described in this invention can be directly used as a drug to effectively activate immunity, inhibit and eliminate human papillomavirus. The polypeptide described in this invention can be used alone as a drug for injection without the addition of any cytokines such as interleukin-2 (IL-2), interferon-α (IFN-α), interferon-γ (IFN-γ), or granulocyte colony-stimulating factor (GM-CSF).

[0027] The present invention also provides a medicament for the prevention and / or treatment of diseases caused by human papillomavirus, wherein the active ingredient of the medicament includes the aforementioned polypeptide.

[0028] The drug of this invention may or may not contain cytokines. When it does not contain any cytokines, the polypeptide is used as the drug, and the dosage form of the drug preferably includes an injection. The route of administration preferably includes at least one of the following: subcutaneous administration, intradermal administration, intramucosal administration, submucosal administration, intramuscular administration, and intraperitoneal administration. The dosage of the drug of this invention preferably depends on the type and severity of the disease, as well as on individual characteristics, including general health status, age, sex, weight, and drug tolerance, etc. The dosage can be 1–100 mg / kg, or 2–20 mg / kg, for example, 2.7 mg / kg or 2.8 mg / kg.

[0029] The solvent for the injectable preparation of the present invention is preferably physiological saline. In Example 1 of the present invention, 1 mg of the polypeptide was dissolved in 2 ml of the physiological saline. In Example 2 of the present invention, experiments on C57BL / 6 mice confirmed that the polypeptide can be used alone as a drug for treatment and prevention, and the preferred administration concentration of the polypeptide as an injectable preparation is 25 μg polypeptide / 100 μL injection solution. The injection solution concentration described in Example 1 is the concentration used in the in vitro induction test, and the injection solution concentration described in Example 2 is the concentration of the polypeptide of the present invention as an injectable preparation in the in vivo test on C57BL / 6 mice.

[0030] The present invention also provides CTL cells that specifically kill human papillomavirus, obtained by in vitro induction of mononuclear cells using the above-mentioned polypeptide.

[0031] The present invention preferably utilizes the polypeptide to directly load DC cells, efficiently inducing the formation of CTL cells that specifically kill HPV, and then reinfusing them into the body to directly kill tumors or stimulate the body's immune response to effectively kill HPV-infected cells, thereby achieving a therapeutic effect.

[0032] The present invention also provides the use of the above-mentioned CTL cells in the preparation of drugs for the prevention and / or treatment of diseases caused by human papillomavirus.

[0033] The drug described in this invention can effectively stimulate a strong immune response, causing the body to produce a large number of immune cells to achieve the effects of protection and prevention.

[0034] The present invention also provides a medicament for the prevention and / or treatment of diseases caused by human papillomavirus, wherein the active ingredient of the medicament includes the above-mentioned CTL cells.

[0035] When the CTL cells are used as the active ingredient in this invention, the preferred cell quantity is (0.5~2)×10⁻⁶. 9 The sample was prepared in PBS or saline solution, with the sample size being 1 / mL.

[0036] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, describes an active polypeptide provided by the present invention and its application in the prevention and / or treatment of HPV, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] 1. The active polypeptide shown in SEQ ID No. 1 (hereinafter referred to as peptide A) was artificially synthesized using the Fmoc solid-phase synthesis method.

[0039] 2. Preparation of CTL cells loaded with peptide A:

[0040] (1) Collect 50 mL of peripheral venous whole blood from the subject using an anticoagulant syringe with added heparin sodium, and separate the subject's PBMCs by centrifugation at 2000 r / min for 10 min using lymphocyte separation fluid.

[0041] (2) After centrifugation, remove the PBMCs as cleanly as possible.

[0042] (3) Wash twice with 0.9% physiological saline, and adjust the cell concentration to 2–2.5 × 10⁻⁶ cells using serum-free lymphocyte culture medium. 6 per ml.

[0043] (4) The washed and concentrated PBMCs were seeded into culture flasks and incubated in a 37°C, 5% CO2 incubator for 2 hours. Then, adherent cells and suspension cells were collected.

[0044] (5) Collection of DC cells from the experimental and control groups: The adherent cells were placed in culture flasks for the experimental and control groups, respectively. 15 ml of serum-free lymphocyte culture medium supplemented with rhGM-CSF (1000 U / ml) and rhIL-4 (500 U / ml) was added to each flask, and the flasks were incubated at 37°C in a 5% CO2 incubator to induce DC cell growth. On day 3, rhGM-CSF (1000 U / ml) and rhIL-4 (500 U / ml) were added to each flask. On day 5, peptide A was loaded into the DC cells of the experimental group at a final concentration of 5 μg / ml, while no peptide was loaded into the control group. On day 6, TNF-α at a final concentration of 1000 U / ml and PGE2 at 1 μg / ml were added to each flask to induce DC cell maturation. On day 7, mature adherent DC cells were harvested for mixed culture.

[0045] (6) Collection of CTL cells: The suspended cells were resuspended in lymphocyte culture medium containing 5% autologous serum, and the culture flasks were coated with CD3 and CD28 monoclonal antibodies at a final concentration of 500 ng / ml 24 hours in advance to adjust the cell concentration to 2×10⁻⁶ cells / ml. 6Cells were added to a culture flask at a final concentration of 1000 U / ml using serum-free lymphocyte culture medium, and then incubated at 37°C in a 5% CO2 incubator. On day 2, IL-7, IL-15, and IL-2 were added to a final concentration of 10 ng / mL, 20 ng / mL, and 1000 U / mL, and the culture flask was incubated at 37°C in a 5% CO2 incubator. On day 4, serum-free lymphocyte culture medium was added to a final concentration of 5 ng / mL, 10 ng / mL, and 1000 U / mL, to adjust the cell concentration to 2 × 10⁻⁶ cells / ml. 6 CTL cells were cultured at a concentration of 10 cells / ml. On day 6, serum-free lymphocyte culture medium containing IL-12 (final concentration 5 ng / mL), IL-15 (final concentration 10 ng / mL), and IL-2 (final concentration 1000 U / mL) was added to adjust the cell concentration to 2 × 10⁻⁶ cells / ml. 6 CTL cells were cultured at a density of 1 cell / ml. CTL cells were harvested on day 7 for mixed culture.

[0046] (7) The above CTL cells were mixed with the experimental group and the control group DC cells at a ratio of 20:1 and cultured in a 37℃, 5% CO2 incubator.

[0047] (8) Serum-free lymphocyte culture medium containing IL-2 at a final concentration of 1000 U / ml was added every 3 days, and DC-CTL cells were obtained after 7 days of culture. The experimental group (peptide A loaded) and the control group (unloaded peptide) were obtained.

[0048] 3. CD8+ T cell detection:

[0049] The frequency of CD8+ T cells in DC-CTL cells loaded with peptide A (hereinafter referred to as peptide A-DC-CTL cells) was detected, with unloaded DC-CTL cells as a control. Cells from the experimental group (peptide A-DC-CTL cells) and the control group (DC-CTL cells without any peptide loading) were collected at the beginning (D0), middle (D13), and late (D16) stages of culture. Fluorescently labeled PE-CD8 was added according to the reagent instructions, and the cells were incubated in the dark for 30 min. After washing with PBS and resuspending, 200 μl / tube of cells was used for analysis. The average of three measurements was taken.

[0050] Table 1. Frequency of CD8+ T cells in each group of CTL cells (%)

[0051] Group D0 D13 D16 experimental group 18.9±2.9a 67.8±5.2a 92.8±5.9a control group 20.3±2.7a 39.1±2.4b 73.3±3.0b

[0052] Note: If the lowercase letters in the same column are the same, it means that under the same period conditions, P > 0.05, i.e., the difference is not significant; if the lowercase letters in the same column are different, it means that under the same period conditions, P < 0.05, i.e., the difference is significant.

[0053] The results are shown in Table 1. During the mid-stage (D13) and late-stage (D16) of DC-CTL cell culture, the proportion of CD8+ T cells in peptide A-DC-CTL cells (experimental group) was significantly higher than that in DC-CTL cells without peptide loading (control group).

[0054] 4. Detection of secretion levels of cytokines IFN-γ and TNF-α:

[0055] Cell supernatants were collected from the experimental group (peptide A loaded) and the control group (no peptide loaded) at the beginning (D0), middle (D13), and late (D16) of DC-CTL cell culture. IFN-γ and TNF-α cytokines were detected according to the ELISA kit instructions.

[0056] Table 2. IFN-γ cytokine expression levels (pg / mL) in cell supernatants of each group.

[0057]

[0058] Note: If the lowercase letters in the same column are the same, it means that under the same period conditions, P > 0.05, i.e., the difference is not significant; if the lowercase letters in the same column are different, it means that under the same period conditions, P < 0.05, i.e., the difference is significant.

[0059] Table 3. TNF-α cytokine expression levels (pg / mL) in cell supernatants of each group.

[0060]

[0061] Note: If the lowercase letters in the same column are the same, it means that under the same period conditions, P > 0.05, i.e., the difference is not significant; if the lowercase letters in the same column are different, it means that under the same period conditions, P < 0.05, i.e., the difference is significant.

[0062] The results are shown in Tables 2 and 3. During the mid-stage (D13) and late-stage (D16) of DC-CTL cell culture, the secretion levels of IFN-γ and TNF-α in the supernatant of peptide A-loaded DC-CTL cells (experimental group) were significantly higher than those in the supernatant of unloaded DC-CTL cells (control group). This result indicates that peptide A can promote the secretion of IFN-γ and TNF-α cytokines from CTL cells.

[0063] 5. In vitro killing assay of DC-CTL cells in the experimental and control groups:

[0064] The target cells used in this experiment were SIHA cells, a cervical cancer cell line infected with HPV16.

[0065] Effector cells 1: peptide A-DC-CTL cells; effector cells 2: unloaded peptide DC-CTL cells. Specifically, they were divided into two groups: the experimental group consisted of effector cells 1 and target cells, and the control group consisted of effector cells 2 and target cells (Table 4). Complete culture medium was used as a blank control group. Each group had three replicates.

[0066] Table 4. Grouping of effector cells and target cells in the experimental and control groups.

[0067]

[0068] (1) Culture of target cells: SIHA cells were cultured in RPMI-1640 medium containing 10-15% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator. The cells were passaged every 3 days. Cell morphology was observed under a microscope. Target cells in the logarithmic growth phase with a cell viability of more than 90% were used to perform MTT killing experiments with DC-CTL cells from the experimental group and the control group.

[0069] (2) Effector cell culture: The DC-CTL cells were prepared in the same way as those in the experimental and control groups in Example 1.

[0070] (3) Select target cells in the logarithmic growth phase: SIHA cells (1×10⁻⁶). 5 Add 100 μL / well to the effector cell control wells, effector cell test wells, and blank wells of a 96-well plate, and incubate overnight at 37°C in a 5% CO2 incubator.

[0071] (4) The next day, observe under a microscope whether the target cells adhere to the wall and grow normally. Remove the target cell supernatant and add 100 μL of lymphocyte culture medium to the blank well. Add 100 μL of effector cells to the effector cell test group and the effector cell control group according to the effector-target ratio of 5:1, 10:1, and 20:1. Incubate at 37°C and 5% CO2 for 4-8 hours.

[0072] (5) Observe the morphology of effector cells and target cells under a 40x inverted microscope, and detect the killing inhibition rate of effector cells on target cells using the MTT assay: Add 20 μL of 0.5% MTT reagent to all wells and incubate at 37°C and 5% CO2 for 4 h.

[0073] (6) Centrifuge, remove supernatant, add 150 μL DMSO, shake at low speed for 10 min in an ELISA reader, observe under a microscope that the formazan crystals are completely dissolved, measure the OD value at a wavelength of 490 nm, and calculate the average OD value and killing inhibition rate of each experimental group and control group.

[0074] Kill inhibition rate % = [1 - (experimental group OD - effector cell control group OD - blank group OD) / (target cell control group OD - blank group OD)] × 100%

[0075] Statistical methods:

[0076] Statistical analysis was performed using SPSS 12.0 software. Data are expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons of means among groups. The significance level was set at α = 0.05. A p-value < 0.05 was considered statistically significant.

[0077] Table 5. In vitro killing rate of CTL cells with different effector-to-target ratios in different groups

[0078]

[0079] Note: If the lowercase letters in the same column are the same, it means that under the same effect-to-target ratio, P > 0.05, i.e., the difference is not significant; if the lowercase letters in the same column are different, it means that under the same effect-to-target ratio, P < 0.05, i.e., the difference is significant.

[0080] The results are shown in Table 5. Regardless of the effector-target ratio of 5:1, 10:1 or 20:1, the effect of peptide A-DC-CTL cells (experimental group) on killing SIHA cells was significantly higher than that of unloaded peptide A-DC-CTL cells (control group) on killing SIHA cells (P<0.05).

[0081] The above data indicate that peptide A can efficiently induce the formation of specific CTL cells and effectively inhibit and target the killing of HPV16-infected cervical cancer cell lines.

[0082] Example 2

[0083] In this embodiment, 5-8 week old female C57BL / 6 mice were used. The C57BL / 6 mice were randomly divided into an experimental group and a control group to establish a prevention and treatment model of C57BL / 6 mice. Specifically, the target cells were TC-1-HPV16 cells (wherein, TC-1-HPV16 cells are C57BL / C(H-2b) mouse lung epithelial cells co-transformed with HPV16E6, E7 and ras genes). The immunizing agent injected into the experimental group mice was peptide A synthesized in Example 1; the control group mice were injected with 100 μL of sterile PBS as a control.

[0084] 1. Prevention Model:

[0085] Experimental objective: To demonstrate that peptide A of the present invention can be used as a drug for preventing HPV, effectively stimulating a strong immune response and generating a large number of immune cells in the body to achieve the effects of protection and prevention;

[0086] C57BL / 6 mice in the experimental group were intradermally injected with peptide A (25 μg / 100 μL) of the present invention on days 1, 5, 9, and 13, while C57BL / 6 mice in the control group were intradermally injected with 100 μL of sterile PBS at the same time. Subcutaneous xenograft inoculation was performed on day 3 after the last immunization. TC-1-HPV16 cells were collected, counted, and the cell concentration was adjusted to 5 × 10⁻⁶. 6 C57BL / 6 mice were subcutaneously injected with 100 μL LTC-1-HPV16 cell suspension (5 × 10⁻⁶ cells / ml) in the right groin. 5 / mouse). The growth of the transplanted tumor was observed every 2 days after inoculation. Finally, 30 days after inoculation, the mice were sacrificed, the tumor tissue was surgically removed and weighed, and the spleen cells of the mice were used for immune cell killing assay (Table 6).

[0087] Table 6. Detailed information on the C57BL / 6 mouse prevention model.

[0088]

[0089]

[0090] 2. Determination of HPV-specific immune cell activity

[0091] Single lymphocytes from the spleens of mice in the experimental and control groups were obtained as described above. The counted cells were placed in 6-well plates and cultured for 5 days in RPMI-1640 medium containing 10% fetal bovine serum (IL-2 1000 U / ml) with either peptide A or the positive stimulant PMA at 37°C in a 5% CO2 incubator. These served as effector cells for the experimental and control groups, respectively. Target cells TC-1-HPV16 were added at effector-to-target ratios of 10:1 and 20:1, respectively. The specific procedures for detecting the in vitro killing rate of immune cells were performed according to the in vitro killing assay of DC-CTL cells in the experimental and control groups described in Example 1.

[0092] Table 7. In vitro killing rate of immune cells with different effector-to-target ratios in each group.

[0093]

[0094] Note: Different lowercase letters in the same column indicate that under the same effect-to-target ratio, P < 0.05, i.e., the difference is significant.

[0095] The results are as follows Figure 1 As shown in Table 7, in the C57BL / 6 mouse prophylactic model, the tumor weight of the experimental group (injected with peptide A) was significantly lower than that of the control group. Figure 1Furthermore, the experimental group (injected peptide A) showed a significantly higher immune cell killing rate than the control group, regardless of whether the effector-to-target ratio was 10:1 or 20:1 (Table 7). These experimental results demonstrate that peptide A of the present invention can effectively prevent the growth of HPV16 cervical cancer cells.

[0096] 3. Treatment Model

[0097] Experimental objective: To demonstrate that peptide A can be used directly as a drug to effectively induce HPV-specific CTL cell immune killing of HPV and to have a therapeutic effect on HPV-related diseases.

[0098] After C57BL / 6 mice underwent adaptive growth in the experimental environment for one week, TC-1-HPV16 cells were collected, counted, and the cell concentration was adjusted to 5 × 10⁻⁶. 6 Cells / ml. On day 1, 100 μl of TC-1-HPV16 cell suspension was subcutaneously inoculated into the anterior right hind limb of mice, i.e., 5 × 10⁶ cells per mouse. 5 Mice were observed for tumor growth every two days after inoculation with TC-1-HPV16 cell suspension. Observations showed that palpable tumors formed approximately two weeks after inoculation with TC-1-HPV16 cell suspension. Therefore, mice were immunized two weeks after inoculation with TC-1-HPV16 cell suspension. Specifically, the experimental group was immunized with peptide A (25 μg / 100 μL). The control group was immunized with 100 μL of PBS. Peptide A was administered every two days for a total of six injections. Mice were sacrificed on day 30 after tumor inoculation, and tumor tissue was surgically removed, weighed, and photographed. Simultaneously, spleen cells were collected for CTL killing assays (Table 8).

[0099] Table 8. Detailed information on the treatment model of C57BL / 6 mice.

[0100] experimental group control group Mouse species 5-8 week old female C57BL / 6 mice 5-8 week old female C57BL / 6 mice quantity 10 10 Tumor transplantation time Day 1 Day 1 Initial immunization time Day 16 Day 16 Time between two immunizations Day 18 Day 18 3 immunization times Day 20 Day 20 …… …… …… Last immunization time Day 26 Day 26 Dosage of medication administered Peptide A (25 μg / 100 μL) 100μlPBS Injection method Intradermal injection Intradermal injection attacking tumor cell types TC-1-HPV16 cells TC-1-HPV16 cells Tumor cell count <![CDATA[5×10 5 [One]]> <![CDATA[5×10 5 [One]]> Immune cell detection time 30 days after tumor transplantation 30 days after tumor transplantation

[0101] 4. HPV-specific immune cell activity

[0102] Single lymphocytes from the spleens of mice in the experimental and control groups were obtained as described above. The counted cells were placed in 6-well plates and cultured for 5 days in RPMI-1640 medium containing 10% fetal bovine serum (IL-2 1000 U / ml) with either peptide A or the positive stimulant PMA at 37°C in a 5% CO2 incubator. These served as effector cells for the experimental and control groups, respectively. Target cells TC-1-HPV16 were added to the wells at effector-to-target ratios of 10:1 and 20:1, respectively. The specific procedures for detecting the in vitro killing rate of immune cells were performed according to the in vitro killing assay of DC-CTL cells in the experimental and control groups described in Example 1.

[0103] Table 9. In vitro killing rate of immune cells with different effector-to-target ratios in each group.

[0104]

[0105]

[0106] Note: Different lowercase letters in the same column indicate that under the same effect-to-target ratio, P < 0.05, i.e., the difference is significant.

[0107] The results are as follows Figure 2 As shown in Table 9, in the C57BL / 6 mouse treatment model, the tumor weight of the experimental group (injected with peptide A) was significantly lower than that of the control group. Figure 2 Furthermore, the experimental group (injected peptide A) showed a higher immune cell killing rate than the control group, regardless of whether the effector-to-target ratio was 10:1 or 20:1 (Table 9). These experimental results demonstrate that peptide A of the present invention can effectively treat HPV16 cervical cancer cells.

[0108] All experimental results from the prevention and treatment models in C57BL / 6 mice demonstrate that peptide A of the present invention can be directly injected as a drug, effectively reducing tumor weight in C57BL / 6 mice. Figures 1-2 It specifically kills TC-1-HPV16 cells (Tables 7 and 9) to achieve the effect of preventing and treating HPV infection.

[0109] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A polypeptide having preventive and / or therapeutic activity, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID No.

1.

2. The use of the polypeptide of claim 1 in the preparation of a medicament for the prevention and / or treatment of diseases caused by human papillomavirus, characterized in that, The human papillomavirus mentioned is HPV16.

3. A drug for the prevention and / or treatment of diseases caused by human papillomavirus, characterized in that, The active ingredient of the drug includes the polypeptide of claim 1, and the human papillomavirus is HPV16.

4. The drug according to claim 3, characterized in that, The dosage form of the drug includes injections.

5. The drug according to claim 4, characterized in that, The administration method of the drug includes at least one of the following: subcutaneous administration, intradermal administration, intramucosal administration, submucosal administration, intramuscular administration, and intraperitoneal administration.

6. CTL cells specifically capable of killing human papillomavirus-infected cells, obtained by in vitro induction of mononuclear cells based on the polypeptide of claim 1, characterized in that, The human papillomavirus mentioned is HPV16.

7. The use of the CTL cells of claim 6 in the preparation of medicaments for the prevention and / or treatment of diseases caused by human papillomavirus, characterized in that, The human papillomavirus mentioned is HPV16.

8. A drug for treating diseases caused by human papillomavirus, characterized in that, The active ingredient of the drug includes the CTL cells as described in claim 6, and the human papillomavirus is HPV16.

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