HTLV-1 virus inhibition drug and application thereof

Abstract

The invention relates to an HTLV-1 virus inhibition drug and application thereof, and belongs to the technical field of virus biology. Specifically, the tegillarca granosa active peptide is extracted from the tegillarca granosa through acid pretreatment and pepsase, so that the tegillarca granosa active peptide is obtained. Experimental results show that the tegillarca granosa active peptide can significantly inhibit proliferation of HTLV-1 positive cell strains (such as ATL-2) within the concentration range of 50-400 [mu] g / mL, and meanwhile expression of Tax and Gag genes in HTLV-1 viruses is effectively reduced. In addition, the prepared tegillarca granosa active peptide has no obvious toxicity to a normal human cell strain (HEK293), which indicates that the tegillarca granosa active peptide has good safety and selectivity. Therefore, the active peptide can be used as a potential medicine for treating HTLV-1 virus related diseases (such as adult T cell leukemia / lymphoma).

Description

Technical Field

[0001] The invention belongs to the technical field of virus biology, and in particular relates to a HTLV-1 virus inhibition drug and application thereof. Background Art

[0002] Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus belonging to the lentivirus subfamily. HTLV-1 is the first retrovirus found to be associated with human disease and is mainly transmitted through mother-to-child transmission, sexual contact, blood transfusion and injection. HTLV-1 infection can cause adult T-cell leukemia / lymphoma (ATL) and a variety of diseases.

[0003] Currently, the treatment options for HTLV-1 infection and its related diseases are very limited. Although some antiretroviral drugs (such as lamivudine and zidovudine) are used clinically to inhibit the replication of HTLV-1, the efficacy is usually limited and the virus cannot be completely eliminated. In addition, long-term use of these drugs may also induce drug resistance and be accompanied by more side effects, such as bone marrow suppression, hepatotoxicity, and metabolic disorders. For the HTLV-1-related malignant disease ATL, current treatment options include chemotherapy and stem cell transplantation, but these methods have problems such as poor efficacy, high recurrence rate and high toxicity. Therefore, existing drugs have significant deficiencies in the inhibition of HTLV-1 virus. In view of the limitations of existing treatments and the serious health problems caused by HTLV-1 infection, there is an urgent need to develop new, safe and effective drugs. Summary of the invention

[0004] The purpose of the present invention is to provide a new, safe and effective HTLV-1 virus inhibitory drug.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Firstly, the present invention provides a HTLV-1 virus inhibition drug, which is composed of mud clam active peptide and physiological saline;

[0007] The concentration of the mud clam active peptide in the HTLV-1 virus inhibitory drug is 50-400 μg / mL.

[0008] Furthermore, the HTLV-1 virus inhibitory drug inhibits the HTLV-1 virus by inhibiting the infection, replication and proliferation of the HTLV-1 virus.

[0009] Furthermore, the preparation method of the active peptide of mud clam comprises the following steps:

[0010] (1) The mud clam meat tissue was crushed and suspended in a 0.1 M hydrochloric acid solution, and pretreated under acidic conditions for 12 hours;

[0011] (2) adjusting the pH of the pretreated mud clam tissue to 2.5, and adding pepsin for enzymolysis;

[0012] (3) After the enzymatic hydrolysis is completed, the enzyme is inactivated and the supernatant is collected by centrifugation;

[0013] (4) The peptides with a molecular weight of less than 3 kDa were collected by ultrafiltration and freeze-dried to obtain the active peptides of mud clam.

[0014] Furthermore, the mass volume ratio of the mud clam meat tissue to the hydrochloric acid is 1 g / 10 mL;

[0015] The pretreatment temperature is 4°C;

[0016] The enzymatic hydrolysis temperature is 37°C and the enzymatic hydrolysis time is 6h;

[0017] The enzyme inactivation temperature is 95°C and the enzyme inactivation time is 20 minutes;

[0018] The centrifugal force of the centrifugation is 8000g, and the centrifugation time is 20 minutes;

[0019] The ultrafiltration graded filtration method is to use a 10 kDa ultrafiltration membrane for the first filtration, collect the filtrate, and use a 3 kDa ultrafiltration membrane for the second filtration.

[0020] Secondly, the present invention provides an application of an active peptide in the preparation of HTLV-1 virus inhibitory drugs, wherein the active peptide is a mud clam active peptide, and the mud clam active peptide is prepared by the above-mentioned preparation method.

[0021] Furthermore, in the HTLV-1 virus inhibitory drug, the concentration range of the mud clam active peptide is 50-400 μg / mL.

[0022] Secondly, the present invention provides an application of an active peptide in the preparation of a drug for preventing or treating diseases related to HTLV-1 infection. The active peptide is a mud clam active peptide, and the mud clam active peptide is prepared by the above-mentioned preparation method.

[0023] Furthermore, the HTLV-1 infection-related disease is adult T-cell leukemia / lymphoma.

[0024] Furthermore, in the drug for preventing or treating diseases related to HTLV-1 infection, the concentration range of the active peptide from mud clam is 50-400 μg / mL.

[0025] Secondly, the present invention provides an application of an active peptide in the preparation of a drug for inhibiting the proliferation of HTLV-1 positive T cells. The active peptide is a mud clam active peptide, and the mud clam active peptide is prepared by the above-mentioned preparation method.

[0026] Furthermore, the HTLV-1 positive T cells are ATL-2 cells.

[0027] Furthermore, in the drug for inhibiting the proliferation of HTLV-1 positive T cells, the concentration range of the active peptide of mud clam is 50-400 μg / mL.

[0028] Secondly, the present invention provides an application of an active peptide in the preparation of a drug for inhibiting the expression of Tax gene and Gag gene in HTLV-1 positive T cells, wherein the active peptide is an active peptide of mud clam, and the active peptide of mud clam is prepared by the above-mentioned preparation method.

[0029] Furthermore, the HTLV-1 positive T cells are ATL-2 cells.

[0030] Furthermore, in the drug for inhibiting the expression of Tax gene and Gag gene in HTLV-1 positive T cells, the concentration range of the mud clam active peptide is 50-400 μg / mL.

[0031] The beneficial effects of the present invention are:

[0032] 1. The active peptide prepared by the present invention can significantly inhibit the proliferation ability of HTLV-1 positive cells, and the inhibitory effect increases with the increase of the active peptide concentration, providing an experimental basis for the development of dose-dependent antiviral drugs.

[0033] 2. The active peptide of mud clam prepared by the present invention effectively reduces the expression of Tax and Gag genes of HTLV-1 virus. Tax gene is a key regulatory factor for HTLV-1 virus transcription and replication, while Gag gene plays an important role in the assembly of viral particles. The active peptide of mud clam of the present invention can significantly inhibit the expression of these two key genes, preventing the replication and spread of the virus from the gene regulation level, further proving its potential as an antiviral drug.

[0034] 3. The active peptide of mud clam prepared by the present invention has no obvious toxicity to normal cells and is highly safe.

[0035] Therefore, the active peptide of the present invention not only has a significant inhibitory effect on the proliferation of HTLV-1 positive cells, but also can effectively reduce the expression of Tax and Gag genes in the HTLV-1 virus, and has potential clinical application value. At the same time, the active peptide can be used to prevent and treat HTLV-1 related diseases, such as adult T cell leukemia / lymphoma (ATL). In addition, the active peptide can also be used as a novel antiviral preparation, which can be used to assist existing antiretroviral drugs, enhance the therapeutic effect, and reduce drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the experimental result diagram of the effect of active peptides from mud clams obtained by different extraction methods on the proliferation of HTLV-1 positive cell line ATL-2;

[0037] Figure 2 This is the experimental result diagram of the effect of acidic mud clam active peptide II on the proliferation of HTLV-1 positive cell line ATL-2 at different concentrations;

[0038] Figure 3 This is the experimental result diagram of the effect of acidic mud clam active peptide II on the proliferation of human normal cell line HEK293;

[0039] Figure 4 The microscope images (a) and statistical results (b) of the inhibitory effects of different concentrations (50 μg / mL and 400 μg / mL) of acidic mud clam active peptide II on HTLV-1 positive cell clone formation;

[0040] Figure 5 This is a graph showing the inhibitory effect of acidic mud clam active peptide II on the expression of HTLV-1 virus Tax gene;

[0041] Figure 6 This is a diagram showing the inhibitory effect of acidic mud clam active peptide II on the expression of HTLV-1 virus Gag gene. DETAILED DESCRIPTION

[0042] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0043] As an important source for the development of new drugs, marine biological resources have gradually attracted widespread attention from the scientific community. A large number of studies have shown that marine organisms are rich in bioactive substances, which have multiple biological activities such as antibacterial, anti-inflammatory, antioxidant and anti-cancer. Mud clams (Tegillarca granosa), also known as mud clams, are a bivalve mollusk widely distributed in coastal areas of East Asia and Southeast Asia. Mud clams are rich in protein, minerals, vitamins and unsaturated fatty acids, and have high nutritional value. In addition, mud clams are also regarded as a marine organism with medicinal value. In traditional medicine, they are used to regulate the spleen and stomach, improve anemia and enhance immunity.

[0044] Existing studies have shown that bioactive peptides extracted from mud clams have antibacterial, antioxidant and whitening effects, but whether they can be used to inhibit HTLV-1 virus infection has not been explored. Therefore, the present invention explores whether the mud clams bioactive peptides obtained by different extraction methods can be used for HTLV-1 virus infection.

[0045] Example 1

[0046] 1. Tissue crushing:

[0047] The mud clam meat tissue was cleaned and dried, and then crushed into powder using liquid nitrogen cryo-grinding;

[0048] 2. Alkaline pretreatment:

[0049] The powder was suspended in 0.1 M NaOH solution (1 g powder: 10 mL solution) and soaked at 4 °C for 12 h to partially decompose the protein under alkaline conditions, thereby exposing more enzyme cleavage sites;

[0050] 3. Enzymatic hydrolysis:

[0051] After adjusting the pH to 9.0, alkaline protease was added (the mass ratio of enzyme to mud clam meat tissue powder was 1:100), and enzymolysis was performed at 55° C. for 6 hours to obtain an enzymolysis solution;

[0052] 4. Enzyme inactivation:

[0053] The enzymatic solution was heated to 95°C for 20 minutes to inactivate the enzyme, and then cooled to room temperature;

[0054] 5. Centrifugation

[0055] The enzymatic hydrolysate was placed in a centrifuge and centrifuged at 8000 g for 20 min, and the supernatant was collected;

[0056] 6. Molecular weight fractionation filtration

[0057] Perform the first filtration using a 10 kDa ultrafiltration membrane, collect the filtrate, and perform the second filtration using a 3 kDa ultrafiltration membrane;

[0058] The retentate of the 3 kDa ultrafiltration membrane was collected and freeze-dried to obtain the alkaline active peptide I of mud clam;

[0059] The filtrate from the 3 kDa ultrafiltration membrane was collected and freeze-dried to obtain the alkaline active peptide II from mud clam.

[0060] Example 2

[0061] 1. Tissue crushing:

[0062] The mud clam meat tissue was cleaned and dried, and then crushed into powder using liquid nitrogen cryo-grinding;

[0063] 2. Neutral pretreatment:

[0064] The powder was suspended in distilled water (1 g powder: 10 mL solution) and soaked at 4 °C for 12 h to release some of the protein;

[0065] 3. Enzymatic hydrolysis:

[0066] After adjusting the pH to 7.5, trypsin was added (the mass ratio of the enzyme to the mud clam meat tissue powder was 1:100), and enzymolysis was performed at 37° C. for 6 hours to obtain an enzymolysis solution;

[0067] 4. Enzyme inactivation:

[0068] The enzymatic solution was heated to 95°C for 20 minutes to inactivate the enzyme, and then cooled to room temperature;

[0069] 5. Centrifugation

[0070] The enzymatic hydrolysate was placed in a centrifuge and centrifuged at 8000 g for 20 min, and the supernatant was collected;

[0071] 6. Molecular weight fractionation filtration

[0072] Perform the first filtration using a 10 kDa ultrafiltration membrane, collect the filtrate, and perform the second filtration using a 3 kDa ultrafiltration membrane;

[0073] The retentate of the 3 kDa ultrafiltration membrane was collected and freeze-dried to obtain the neutral active peptide I of mud clam;

[0074] The filtrate from the 3 kDa ultrafiltration membrane was collected and freeze-dried to obtain the neutral active peptide II from mud clam.

[0075] Example 3

[0076] 1. Tissue crushing:

[0077] The mud clam meat tissue was cleaned and dried, and then crushed into powder using liquid nitrogen cryo-grinding;

[0078] 2. Neutral pretreatment:

[0079] The powder was suspended in 0.1 M hydrochloric acid solution (1 g powder: 10 mL solution) and soaked at 4 °C for 12 h to partially decompose the protein under acidic conditions, thereby exposing more enzyme cleavage sites;

[0080] 3. Enzymatic hydrolysis:

[0081] After adjusting the pH to 2.5, pepsin was added (the mass ratio of the enzyme to the mud clam meat tissue powder was 1:50), and enzymolysis was performed at 37° C. for 6 hours to obtain an enzymolysis solution;

[0082] 4. Enzyme inactivation:

[0083] The enzymatic solution was heated to 95°C for 20 minutes to inactivate the enzyme, and then cooled to room temperature;

[0084] 5. Centrifugation

[0085] The enzymatic hydrolysate was placed in a centrifuge and centrifuged at 8000 g for 20 min, and the supernatant was collected;

[0086] 6. Molecular weight fractionation filtration

[0087] Perform the first filtration using a 10 kDa ultrafiltration membrane, collect the filtrate, and perform the second filtration using a 3 kDa ultrafiltration membrane;

[0088] The retentate of the 3 kDa ultrafiltration membrane was collected and freeze-dried to obtain the acidic active peptide I of mud clams;

[0089] The filtrate from the 3 kDa ultrafiltration membrane was collected and freeze-dried to obtain the acidic active peptide II from mud clams.

[0090] Example 4

[0091] CCK-8 assay to detect the effects of active peptides extracted by different methods on the proliferation ability of HTLV-1 positive cell line ATL-2

[0092] (1) ATL-2 cells were removed from the culture flask, centrifuged, and then washed once with pre-warmed sterile PBS to remove residual culture medium and metabolites.

[0093] (2) Centrifuge again to remove PBS, resuspend the cells in RPMI 1640 complete medium for the control group, and resuspend the cells in RPMI 1640 complete medium containing different active peptides of mud clam for the treatment groups;

[0094] (3) The cell suspension was added to a 96-well plate, 100 μL per well, 5,000 cells; to ensure the repeatability of the experimental results, 3 replicate wells were set for each treatment to reduce the impact of experimental errors.

[0095] (4) Place the 96-well plate back into the cell culture incubator and continue culturing for 48 hours.

[0096] (5) After the culture was completed, 10 μL of CCK-8 reagent was added to each well, and the cell proliferation activity was detected by the reagent. After incubation with CCK-8 reagent for 4 hours, the optical density (OD) value of each well was detected at a wavelength of 450 nm using an enzyme reader. By comparing the OD values ​​of different treatment groups with the control group, the effect of active peptides on ATL-2 cell proliferation was evaluated.

[0097] The experimental results of this example are shown in Figure 1 By comparing the effects of different active peptides on the proliferation ability of HTLV-1 positive cell line ATL-2, the following conclusions can be drawn:

[0098] First, from Figure 1 The results clearly show that alkaline mud clam active peptide I, alkaline mud clam active peptide II, neutral mud clam active peptide I and neutral mud clam active peptide II failed to significantly affect the proliferation of ATL-2 cells. The experimental results showed that these active peptides had neither a promoting effect nor a significant inhibitory effect on the proliferation of HTLV-1 positive cells at the tested concentrations, and the OD450 value was not significantly different from that of the control group, indicating that these active peptides did not have the ability to inhibit the proliferation of HTLV-1 positive cell lines under the current conditions.

[0099] Acidic mud clam active peptide I showed a certain degree of inhibitory effect in the experiment. The data showed that it had a slight inhibitory effect on the proliferation of ATL-2 cells, and the P value of the result was less than 0.05, reaching the statistically significant standard. However, although this inhibitory effect reached a significant level, the overall effect was still relatively limited and could not significantly reduce the proliferation ability of HTLV-1 positive cells. Therefore, the potential of acidic mud clam active peptide I in inhibiting HTLV-1 still needs to be further optimized.

[0100] The most significant is the inhibitory effect of acidic mud clam active peptide II. The experimental results show that acidic mud clam active peptide II has a very significant inhibitory effect on the proliferation ability of ATL-2 cells. The OD450 value is significantly reduced from 0.961 in the control group to 0.443, and the inhibition rate reaches 53.90%. This inhibitory effect is statistically significant, with a P value of <0.001, indicating that acidic mud clam active peptide II has a strong inhibitory effect under the current experimental conditions. This finding indicates that acidic mud clam active peptide II extracted from mud clams may have strong anti-HTLV-1 positive cell proliferation activity and is expected to be a candidate compound for the development of new drugs to inhibit HTLV-1 infection.

[0101] The reason for this result may be that the acidic mud clam active peptides were obtained by enzymatic hydrolysis with pepsin under acidic conditions, while the alkaline and neutral mud clam active peptides were obtained by enzymatic hydrolysis with alkaline protease and trypsin under alkaline and neutral conditions, respectively. Pepsin has higher activity in a low pH environment and can cut specific peptide bonds in protein molecules to produce polypeptides with different structures and functions. The acidic environment may be more conducive to exposing some domains that have specific effects on the HTLV-1 virus or its related cellular targets, thereby generating polypeptides with antiviral activity. In contrast, the polypeptides generated under alkaline and neutral conditions may lack these specific structures and therefore do not show inhibitory effects.

[0102] At the same time, there is a significant difference in inhibitory effect between acidic mud clam active peptides I and II, which may be related to the molecular weight and length of the peptide chain. Acidic mud clam active peptide II was extracted from the filtrate below 3kDa, while acidic mud clam active peptide I was the retentate above 3kDa. This indicates that acidic mud clam active peptide II has a smaller molecular weight and may be more likely to penetrate the cell membrane and interact with target molecules in viruses or cells. Small molecule peptides generally have better bioavailability and cell penetration ability, and therefore show more significant effects in inhibiting the proliferation of HTLV-1 positive cells.

[0103] Example 5

[0104] CCK-8 assay to detect the effects of different concentrations of active peptides on the proliferation of HTLV-1 positive cell line ATL-2

[0105] (1) ATL-2 cells were removed from the culture flask, centrifuged, and then washed once with pre-warmed sterile PBS to remove residual culture medium and metabolites.

[0106] (2) The acidic mud clam active peptide II was dissolved in RPMI 1640 complete culture medium and prepared into concentrations of 50, 100, 200, and 400 μg / mL, respectively.

[0107] (3) The PBS was removed by centrifugation again, and the cells were resuspended in RPMI 1640 complete medium for the control group and in medium containing 50, 100, 200, or 400 μg / mL acidic active peptide II for the treatment group;

[0108] (4) The cell suspension was added to a 96-well plate, 100 μL per well, 5,000 cells; to ensure the repeatability of the experimental results, 3 replicate wells were set for each treatment to reduce the impact of experimental errors.

[0109] (5) Place the 96-well plate back into the cell culture incubator and continue culturing for 48 hours.

[0110] (6) After the culture was completed, 10 μL of CCK-8 reagent was added to each well, and the proliferation activity of the cells was detected by the reagent. After incubation with CCK-8 reagent for 4 hours, the optical density (OD) value of each well was detected at a wavelength of 450 nm using an enzyme marker. By comparing the OD values ​​of different treatment groups with the control group, the effect of active peptides on the proliferation of ATL-2 cells was evaluated.

[0111] The experimental results of this example are shown in Figure 2 By comparing the effects of different concentrations of acidic mud clam active peptide II on the proliferation ability of HTLV-1 positive cell line ATL-2, the following conclusions can be drawn:

[0112] At the concentrations of 50, 100, 200, and 400 μg / mL tested by the present invention, the acidic mud clam active peptide II had a significant inhibitory effect on the proliferation ability of the HTLV-1 positive cell line ATL-2. With the increase of the active peptide concentration, the proliferation ability of the HTLV-1 positive cells showed a gradually decreasing trend, which showed that the acidic mud clam active peptide II had a dose-dependent effect in inhibiting the proliferation of HTLV-1 positive cells. The OD values ​​measured at each concentration were 0.954 (control group), 0.785 (50 μg / mL), 0.640 (100 μg / mL), 0.439 (200 μg / mL), and 0.285 (400 μg / mL).

[0113] Specific analysis found that:

[0114] 50μg / mL: At a concentration of 50μg / mL, acidic mud clam active peptide II showed an obvious inhibitory effect, and the OD value decreased from 0.954 in the control group to 0.785, indicating its preliminary inhibitory effect on cell proliferation.

[0115] 100 μg / mL: When the concentration increased to 100 μg / mL, the inhibitory effect was further enhanced, and the OD value dropped to 0.640, indicating that the inhibitory effect of the active peptide became more significant as the concentration increased.

[0116] 200μg / mL: At 200μg / mL, the inhibitory effect of acidic mud clam active peptide II was more significant, and the OD value dropped to 0.439, showing a strong inhibitory ability.

[0117] 400μg / mL: At a high concentration of 400μg / mL, the acidic mud clam active peptide II achieved the strongest inhibition on the proliferation of HTLV-1 positive cells, and the OD value further dropped to 0.285, indicating that the active peptide can very effectively inhibit the proliferation of HTLV-1 positive cells at high concentrations.

[0118] In summary, acidic mud clam active peptide II showed significant inhibitory effect on the proliferation of HTLV-1 positive cell line ATL-2 at different concentrations, and the inhibitory effect increased with increasing concentration. This result provides a strong experimental basis for further developing acidic mud clam active peptide II as a potential anti-HTLV-1 drug.

[0119] Example 6

[0120] In order to exclude that the acidic active peptide II from mud clams inhibits the proliferation of HTLV-1 positive cells due to simple cytotoxicity, the present invention detected the effect of the acidic active peptide II from mud clams at the detection concentration of Example 5 on the proliferation of human normal cell line: HEK293 cells (human embryonic kidney cells).

[0121] The detection method of the experiment is exactly the same as that of Example 5, except that the cells are replaced by HEK293 cells.

[0122] The experimental results of this example are shown in Figure 3 In. From Figure 3 The results show that at different concentrations of 50, 100, 200, and 400 μg / mL, acidic mud clam active peptide II did not show a significant inhibitory effect on the proliferation of HEK293 cells. Therefore, the OD values ​​at each concentration were not significantly different from those of the control group, indicating that acidic mud clam active peptide II had no adverse effects on the normal proliferation of HEK293 cells within the above concentration range.

[0123] In summary, acidic mud clam active peptide II did not show obvious cytotoxicity to the human normal cell line HEK293 within the concentration range tested in this experiment. This indicates that its inhibitory effect on the HTLV-1 positive cell line ATL-2 is not due to nonspecific cytotoxicity, but may be achieved through a specific antiviral mechanism. This result further supports the development potential of acidic mud clam active peptide II as a HTLV-1 inhibitory drug.

[0124] Example 7

[0125] In order to further verify the results of CCK-8 detection, the present invention selected low concentration 50 μg / mL and high concentration 400 μg / mL of acidic mud clam active peptide II for cloning experiment.

[0126] (1) Remove ATL-2 cells from the culture flask, centrifuge, and wash the cells once with pre-warmed sterile PBS to remove the residual culture medium.

[0127] (2) After centrifugation to remove PBS, cell suspensions were prepared according to the following experimental groups: control group, 50 μg / mL acidic mud clam active peptide II treatment group, and 400 μg / mL acidic mud clam active peptide II treatment group.

[0128] (3) Take a 6-well plate and inoculate 2 mL of the cell suspension in each well (i.e., 2,000 cells in each well). Set up 3 replicate wells in each group and place the 6-well plate in a 37°C, 5% CO2 incubator for 14 days until clones are clearly visible. During this period, the culture medium is not changed.

[0129] (4) After the culture is completed, carefully aspirate the culture medium and gently wash the cells twice with PBS to remove residual culture medium and floating cells.

[0130] (5) Add 1 mL of methanol fixative and fix the cells for 20 min. Remove the fixative and add 1 mL of crystal violet staining solution and stain for 20 min.

[0131] (6) Remove the staining solution and gently wash the 6-well plate with tap water until there is no excess dye in the wells and colony formation is visible.

[0132] (7) The stained clones were observed under a microscope, and the clone formation of different groups was compared to evaluate the inhibitory effect of acidic mud clam active peptide II on the proliferation and clone formation of HTLV-1 positive cell lines.

[0133] The experimental results of this example are shown in Figure 4 In. From Figure 4 The results show that compared with the control group, the number of clones formed in the 50μg / mL acidic mud clam active peptide II treatment group and the 400μg / mL acidic mud clam active peptide II treatment group was significantly reduced. This result is consistent with the CCK-8 test results, further verifying that acidic mud clam active peptide II can effectively inhibit the proliferation of HTLV-1 positive cell lines.

[0134] Example 8

[0135] In order to further detect the inhibitory effect of acidic mud clam active peptide II on HTLV-1 infection, the present invention uses fluorescent quantitative PCR to detect the expression of HTLV-1 virus-related genes Tax and Gag after co-culturing HTLV-1 positive cell strains with HTLV-1 negative cell strains (Jurkat cells).

[0136] Among them, Tax (transactivator X) is an important regulatory protein of HTLV-1. It is not a viral structural protein, but plays an important role in viral replication, infected cell proliferation and tumor formation. Gag (group-specific antigen) is one of the structural proteins of HTLV-1 and is the core of viral replication and assembly.

[0137] In this example, in order to reduce the effect of inhibiting proliferation as much as possible, low concentrations of 50 μg / mL and 100 μg / mL of acidic mud clam active peptide II were selected for the experiment, and the serum concentration of RPMI 1640 complete culture medium was selected to be 3%, and the co-culture time was 24 h.

[0138] (1) After ATL-2 cells were centrifuged, they were washed once with pre-warmed sterile PBS to remove the residual culture medium.

[0139] (2) After centrifugation to remove PBS, ATL-2 cells were prepared into cell suspensions using RPMI 1640 complete medium, RPMI 1640 complete medium containing 100 μg / mL acidic mud clam active peptide II, and RPMI 1640 complete medium containing 200 μg / mL acidic mud clam active peptide II;

[0140] (3) 1000 μL ATL-2 cells were inoculated into a 6-well plate. Then, following the same steps, a Jurkat cell suspension was prepared using RPMI 1640 complete medium. 1000 μL Jurkat cells were added to the wells containing ATL-2 cells (the amount of both cells was 5 × 10 5 , after mixing, the concentration of active peptide becomes 50 μg / mL and 100 μg / mL), after mixing, shake the culture plate to mix the cells and culture medium evenly, and place in a cell culture incubator for co-culture for 24 hours;

[0141] (4) After 24 h, cells were collected by centrifugation, washed with PBS, and Trizol reagent was added to extract RNA;

[0142] (5) After the cDNA was obtained by reverse transcription using the Takara reverse transcription kit, the Takara fluorescent quantitative PCR kit was used to detect the differences in the relative expression levels of the Tax gene and the Gag gene under different treatments.

[0143] The experimental results of this embodiment are as follows Figure 5 and Figure 6 As shown, the effects of acidic mud clam active peptide II on the relative expression levels of Tax gene and Gag gene in HTLV-1 virus are respectively demonstrated.

[0144] from Figure 5 It can be seen that:

[0145] Compared with the control group, the relative expression level of Tax gene in the group treated with 50 μg / mL acidic mud clam active peptide II was 0.818±0.025, indicating that it had a certain inhibitory effect on Tax gene expression.

[0146] When the concentration of acidic mud clam active peptide II increased to 100 μg / mL, the relative expression level of Tax gene further decreased to 0.551±0.024, indicating that the inhibitory effect became more obvious with the increase of active peptide concentration.

[0147] This indicates that different concentrations of acidic mud clam active peptide II can effectively reduce the expression of Tax gene in HTLV-1, and the inhibitory effect shows a certain concentration dependence.

[0148] from Figure 6 It can be seen that:

[0149] Compared with the control group, the relative expression level of Gag gene in the group treated with 50 μg / mL acidic mud clam active peptide II was 0.859±0.024, indicating that its inhibitory effect on Gag gene was weak, but still had a certain effect.

[0150] When the concentration of acidic mud clam active peptide II increased to 100 μg / mL, the relative expression level of Gag gene significantly decreased to 0.615±0.022, showing a more obvious inhibitory effect.

[0151] This indicates that different concentrations of acidic mud clam active peptide II can also effectively reduce the expression of Gag gene in HTLV-1, and its inhibitory effect is also concentration-dependent.

[0152] The experimental results showed that acidic mud clam active peptide II had a significant inhibitory effect on the expression of Tax and Gag genes in HTLV-1 at different concentrations, and the inhibitory effect became more obvious as the concentration increased. Tax and Gag are two important genes in the infection and pathogenicity of HTLV-1. The Tax gene is responsible for activating viral replication and promoting host cell proliferation, while the Gag gene is involved in the assembly and release of viral particles. Acidic mud clam active peptide II can simultaneously inhibit the expression of these two key genes, suggesting that it has potential therapeutic value in resisting HTLV-1 infection and blocking viral replication.

[0153] These results provide a strong experimental basis for the development of acidic mud clam active peptide II as an anti-HTLV-1 virus drug, especially showing good potential in blocking viral gene expression and inhibiting viral proliferation.

[0154] Example 8

[0155] A preparation method of HTLV-1 virus inhibitory drug

[0156] (1) Prepare 0.9% sodium chloride solution using sterile distilled water and sterilize by high pressure to ensure sterility;

[0157] (2) adding the acidic mud clam active peptide II powder prepared by the method of the present invention into a 0.9% sodium chloride solution, and stirring thoroughly to ensure that the active peptide is completely dissolved;

[0158] (3) Filter the acidic mud clam active peptide II solution using a 0.22 μm sterile filter membrane to remove possible particles and microorganisms to ensure the sterility of the solution;

[0159] (4) The filtered active peptide solution was dispensed into sterile vials and stored in a refrigerator at 4°C, away from light or excessive shaking.

Claims

1. A HTLV-1 virus inhibitory drug, characterized in that: The HTLV-1 virus inhibition drug consists of active peptides from mud clams and normal saline; The concentration of the mud clam active peptide in the HTLV-1 virus inhibitory drug is 50-400 μg / mL.

2. A HTLV-1 virus inhibitory drug according to claim 1, characterized in that: The HTLV-1 virus inhibitory drug inhibits the HTLV-1 virus by inhibiting the infection, replication and proliferation of the HTLV-1 virus.

3. A HTLV-1 virus inhibition drug according to claim 2, characterized in that: The preparation method of the active peptide of mud clam comprises the following steps: (1) The mud clam meat tissue was crushed and suspended in a 0.1 M hydrochloric acid solution, and pretreated under acidic conditions for 12 hours; (2) adjusting the pH of the pretreated mud clam tissue to 2.5, and adding pepsin for enzymolysis; (3) After the enzymatic hydrolysis is completed, the enzyme is inactivated and the supernatant is collected by centrifugation; (4) The peptides with a molecular weight of less than 3 kDa were collected by ultrafiltration and freeze-dried to obtain the active peptides of mud clam.

4. Use of an active peptide in the preparation of a HTLV-1 virus inhibitory drug, characterized in that: The active peptide is a mud clam active peptide, and the mud clam active peptide is prepared by the preparation method described in claim 3.

5. Use of an active peptide in the preparation of a drug for preventing or treating HTLV-1 infection-related diseases, characterized in that: The active peptide is a mud clam active peptide, and the mud clam active peptide is prepared by the preparation method described in claim 3.

6. The use according to claim 5, characterized in that: The HTLV-1 infection-related disease is adult T-cell leukemia / lymphoma.

7. Use of an active peptide in the preparation of a drug for inhibiting the proliferation of HTLV-1 positive T cells, characterized in that: The active peptide is a mud clam active peptide, and the mud clam active peptide is prepared by the preparation method described in claim 3.

8. The use according to claim 7, characterized in that: The HTLV-1 positive T cells are ATL-2 cells.

9. Use of an active peptide in the preparation of a drug for inhibiting the expression of Tax gene and Gag gene in HTLV-1 positive T cells, characterized in that: The active peptide is a mud clam active peptide, and the mud clam active peptide is prepared by the preparation method described in claim 3.

10. The use according to claim 9, characterized in that: The HTLV-1 positive T cells are ATL-2 cells.

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