Application of glutamic acid in preparation of medicine for treating pseudorabies virus infection
By using drugs prepared by glutamic acid, the problem of insufficient effectiveness of existing antiviral drugs on pseudorabies virus has been solved, effective inhibition of pseudorabies virus and control of viral transmission has been achieved, and a safe and economical treatment plan has been provided.
Patent Information
- Application Number
- CN202510404688.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-01
- Publication Date
- 2025-07-25
Smart Images
Figure CN120360980A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of glutamic acid in the preparation of a drug for treating pseudorabies virus infection. Background Art
[0002] Porcine pseudorabies (PR) is an acute, high fever and highly lethal infectious disease caused by pseudorabies virus (PRV), which can infect livestock and various animals, and pigs are the only natural host of PRV. The clinical manifestations of this disease vary depending on the age of the pigs. After pregnant sows are infected, abortion, stillbirth and mummification of fetuses can occur; after newborn piglets are infected, severe neurological symptoms such as ataxia, paralysis and exhaustion death will appear, and the mortality rate is as high as 100%; most adult pigs show latent infection, but may present respiratory symptoms. Although vaccines are currently the main means of controlling porcine pseudorabies, live vaccines and inactivated vaccines have some inherent defects, such as the restoration of virulence, the risk of virus transmission and the insufficient protection ability against new strains. In addition, with the continuous mutation of the virus, porcine pseudorabies has still been reported to be prevalent in some areas.
[0003] In this context, the development of highly efficient and safe antiviral drugs has become an important supplementary means to control PRV infection. In recent years, the research and development of drugs for porcine pseudorabies has gradually become a research hotspot. However, currently, the drugs used for treating PRV infection clinically are relatively limited. For the currently visible effective antiviral treatment regimens, broad-spectrum antiviral drugs are mostly used for antiviral treatment, such as acyclovir, ribavirin, interferon, etc. However, the effects of most broad-spectrum antiviral drugs on PRV are relatively limited because they are usually not specifically targeted at specific targets of PRV. In addition, some broad-spectrum drugs (such as acyclovir) may cause some side effects, such as renal toxicity, embryotoxic effects, etc., and long-term use may have an adverse impact on animal health. Long-term use of these drugs may lead to the generation of drug resistance in the virus, resulting in a decline in curative effect, especially for pseudorabies virus variants, this problem is particularly serious. Therefore, it is necessary to propose a new solution to solve the problems such as the restoration of virulence, virus transmission and insufficient defense against new variants existing in the existing vaccines and drugs when dealing with pseudorabies virus infection. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide the use of glutamic acid in the preparation of a drug for treating pseudorabies virus infection.
[0005] To solve the technical problem, the solution of the present invention is:
[0006] Provide an application of glutamic acid in the preparation of a drug for treating pseudorabies virus infection.
[0007] As a preferred embodiment of the present invention, the drug comprises an effective amount of glutamic acid and a pharmaceutically acceptable excipient.
[0008] The present invention also provides a drug for treating pseudorabies virus infection, which uses glutamic acid as the active ingredient.
[0009] As a preferred embodiment of the present invention, the drug further comprises an excipient; glutamic acid and the excipient are made into a clinically acceptable preparation through a preparation process.
[0010] As a preferred embodiment of the present invention, the excipient is any one or a combination of two or more of the following: dextrin, lactose, gelatin, microcrystalline cellulose.
[0011] As a preferred embodiment of the present invention, the preparation is any one of the following forms: aqueous solution, capsule, granule, injection, spray.
[0012] As a preferred embodiment of the present invention, the drug is an environmental disinfectant used in a spraying manner, and the drug is diluted with water into a solution with a glutamic acid concentration of 2.5 - 10 mM before use.
[0013] As a preferred embodiment of the present invention, the drug is an oral drug used in an oral manner, or an injection used in an injection manner, with a concentration of 10 mM during use; the dosage of the oral drug or injection is 12 - 36 mg / Kg body weight.
[0014] Compared with the prior art, the technical effects of the present invention are as follows:
[0015] 1. The present invention first proves that glutamic acid can significantly inhibit the replication of PRV in PK-15 cells. Combining in vivo (C57BL / 6 mouse model) and in vitro (PK-15 cell model) experiments, it verifies the effectiveness of glutamic acid in inhibiting virus replication and improving infection symptoms, as well as the significant effect in improving the survival rate and body weight change of infected mice, providing a new scientific basis and practical support for the prevention and control of PRV infection.
[0016] 2. The present invention proves that glutamic acid can inhibit the replication of pseudorabies virus in cells, reduce the viral load in the brain and trigeminal nerve tissues; significantly reduce the mortality rate of infected mice and inhibit weight loss. The virus positive rate of the brain tissue and trigeminal nerve of mice treated with glutamic acid is significantly reduced, especially showing the most significant antiviral effect in the medium-dose group (24 mg / kg). The survival rate of mice in the glutamic acid treatment group is significantly higher than that of the control group, and shows a dose-dependent trend.
[0017] 2. As a natural amino acid, glutamic acid has the characteristics of high safety, low toxicity and side effects, and at the same time is low in price and easy to obtain, which is suitable for popularization and use in farms.
[0018] 3. The present invention provides a brand-new anti-pseudorabies virus drug option, offering a new treatment method for dealing with the prevalence of porcine pseudorabies, and is expected to prevent virus transmission in pig farms and reduce economic losses. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Absorbance changes of cell lysate at OD 450nm after adding different concentrations of glutamate.
[0020] Figure 2 Changes in cell viability after adding different concentrations of glutamate.
[0021] Figure 3 Detection results of virus titers after adding different concentrations of glutamate.
[0022] Figure 4 qPCR detection results after adding different concentrations of glutamate.
[0023] Figure 5 Inhibitory effect of glutamate on PRV titer. Detection results of virus titer (log 10 TCID 50 / mL) of PRV-infected PK-15 cells treated with glutamate under the condition of MOI = 0.01.
[0024] Figure 6 Results of detecting the nucleic acid copy number of PRV genome by qPCR, showing the effect of glutamate on the nucleic acid copy number of PRV in PK-15 cells at different time points (36 hours and 48 hours).
[0025] Figure 7 Results of detecting the expression of PRV gB protein by Western Blot, showing the protein expression differences between the glutamate treatment group and the control group at 12 hours and 24 hours after infection.
[0026] Figure 8 Results of gray scale analysis of PRV gB protein in the glutamate treatment group and the control group at 12 hours and 24 hours after infection.
[0027] Figure 9 Median lethal dose (LD 50 ) of PRV to C57BL / 6 mice in the present invention. Survival curves were used to evaluate the virulence level of PRV with different infection doses and calculate LD 50 .
[0028] Figure 10 Effect of glutamate treatment on the body weight change of PRV-infected mice, showing the body weight change records of each group of mice after infection (n = 19).
[0029] Figure 11 Effect of different doses of glutamate on the survival rate of PRV-infected mice (n = 19).
[0030] Figure 12 Detection results of the positive sample rate of virus in brain tissue after PRV infection, showing the effect of different doses of glutamate on the viral load in brain tissue.
[0031] Figure 13 Detection results of the positive sample rate of virus in trigeminal nerve tissue after PRV infection, showing the effect of different doses of glutamate on the viral load in trigeminal nerve. Detailed implementation mode
[0032] The present invention discloses the use of glutamate in the preparation of a drug for treating pseudorabies virus infection. This scheme verified the antiviral effect of glutamate through in vivo and in vitro experiments. In vitro experiments used PK-15 porcine kidney cells as a model, and multiple detection methods (virus titer, qPCR and Western Blot) were used to prove the inhibitory effect of glutamate on the replication of pseudorabies virus (PRV). Under the infection condition of MOI = 0.01, treatment with glutamate can significantly reduce the virus titer and nucleic acid copy number. In vivo experiments used C57BL / 6 mice as an animal model. Each mouse was infected with PRV by intraperitoneal injection, and the first dose was given 5 hours after infection, and then the drug was given by intraperitoneal injection every 24 hours. The survival rate, body weight change and improvement of clinical symptoms of the mice were observed, and the nucleic acid copy number and positive rate of PRV were detected in brain tissue and trigeminal nerve respectively.
[0033] In in vivo experiments, mice were treated with doses of 12 mg / kg, 24 mg / kg, and 36 mg / kg. The experimental results showed that glutamate reduced the viral load and positive sample rate of infected mice in a dose-dependent manner. As a preferred scheme, glutamate inhibited PRV replication in vitro at a concentration of 10 mM and could significantly reduce the virus titer 36 hours and 48 hours after infection.
[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below.
[0035] The following are the detection methods used in the embodiments of the present invention, specifically including:
[0036] CCK-8 assay: PK-15 cells were seeded into 96-well plates at a density of 9000 cells per well and cultured in an incubator with 5% CO2 at 37°C for about 24 h. The cell culture medium was discarded and the cells were washed 3 times with PBS. Then, the cells were treated with medium containing 2% serum and different concentrations of glutamate, and the culture plates were incubated in the incubator for 48 h. A 10% CCK-8 solution was prepared and 100 μl of the CCK-8 solution was added to each well of the plate. After incubation for 30 min, the absorbance value at OD 450 nm was measured using a microplate reader. Six replicates were set for each group.
[0037] Western Blot assay: Cell lysate was added to the cell plate to collect cell samples. The collected samples were centrifuged at 13000×g for 10 min at 4°C, and the supernatant was taken. 5× protein loading buffer was added and incubated at 96°C for 10 min to denature the proteins. Equal amounts of whole cell proteins were electrophoresed on a 10% separating gel. The electrophoresis conditions were: 80 V was set when the sample passed through the stacking gel, and 120 V was set when it passed through the separating gel. Transfer: A PVDF membrane was prepared during electrophoresis. A PVDF membrane with a suitable size of 0.45 μm was cut and activated in methanol. After electrophoresis, the protein was cut according to the expected size with reference to the protein marker. The transfer membrane device was assembled in the order of filter mesh, filter paper, PVDF membrane, protein gel, filter paper, and filter mesh. Transfer buffer was added, and transfer was carried out at a constant current of 200 mA for 35 min. Blocking: After transfer, the PVDF membrane was taken out, placed in the blocking solution, and incubated on a horizontal shaker at 40 r / min at room temperature for 1.5 h. The blocking solution was removed, and TBST buffer was added. It was washed 3 times at 90 r / min at room temperature for 5 min each time. The primary antibody working solution was prepared according to the antibody instruction manual and added to the membrane, and incubated at 40 r / min at 4°C overnight. The primary antibody working solution was removed, and it was washed 3 times with TBST buffer. The secondary antibody working solution was prepared according to the antibody instruction manual and added to the membrane, and incubated at 40 r / min at room temperature for 2 h. The secondary antibody working solution was removed, and it was washed 3 times with TBST buffer. Exposure: According to the instruction manual, the A and B solutions of the ECL developing solution were mixed at a ratio of 1:1 to prepare the ECL developing solution, which was evenly dripped on the PVDF membrane and developed in a protein gel imaging system.
[0038] TCID 50 assay: The virus titer was determined according to the Reed-Muench method. PK-15 cells were seeded into 96-well plates at a density of 9000 cells per well and cultured in an incubator with 5% CO2 at 37°C for about 24 h. The virus solution to be tested was serially diluted 10-fold until a dilution of 10 -8 dilution. After dilution, it was inoculated into 96-well plates, and each dilution was repeated 6 times. The inoculated cells were placed in an incubator at 37°C. After 72 h of infection, the cytopathic effect (CPE) of the cells was observed under a microscope, and the TCID of the sample to be tested was calculated.50 。
[0039] qPCR detection: Extract the viral genomic DNA according to the Tiangen Virus Genomic DNA / RNA Rapid Extraction Kit. Perform qPCR detection on the extracted viral genomic DNA. Configure the reaction system according to the 2×ChamQ Universal SYBR qPCR Master Mix Kit, and the fluorescence quantitative PCR system is shown in Table 1. The reaction conditions are pre-denaturation at 95°C for 60 s, denaturation at 95°C for 10 s, and annealing at 62°C for 20 s for a total of 45 cycles. Among them, the primer sequences are: qPCR-gB-F: CGCAACAACCACAAGGTGA, qPCR-gB-R: TGGACAGGGCGAAGGAG.
[0040] Table 1 Fluorescence quantitative PCR system
[0041]
[0042] Example 1: Identification of the cytotoxicity of glutamic acid to PK-15 cells
[0043] Seed 9000 PK-15 cells per well into a 96-well plate and culture them in an incubator at 5% CO2 and 37°C for about 24 h. Discard the cell culture medium and wash 3 times with PBS. Add medium containing 2% serum with different concentrations of glutamic acid to the wells to treat the cells. Incubate the culture plate in the incubator for 48 h. Prepare a 10% CCK-8 solution and add 100 μl of the CCK-8 solution to each well of the plate. After incubating for 30 min, measure the absorbance value at OD 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and set 6 replicates for each group.
[0044] In the present invention, the results of the cytotoxicity assay of glutamic acid on PK-15 cells are as Figure 1 、 2 shown. Among them, Figure 1 is the change in the absorbance of the cell lysate at OD 450 nm after adding different concentrations of glutamic acid, Figure 2 is the change in the cell viability after adding different concentrations of glutamic acid.
[0045] Glutamic acid has no promoting or toxic effect on cell growth at concentrations of 0.625 mM and 1.25 mM. Glutamic acid has a promoting effect on cell growth at concentrations of 2.5 mM, 5 mM, and 10 mM, but is toxic to cells at concentrations exceeding 20 mM. Subsequent virus efficacy experiments verified that the optimal drug concentration can be selected as the maximum concentration of 10 mM.
[0046] Example 2: Effect of different glutamic acids on the replication of PRV in PK-15 cells
[0047] Inoculate PK-15 cells at a cell density of 6×10 5 cells per well into a 6-well plate; after culturing at 37°C for 15 h, infect the cells with PRV (MOI = 0.01). After the virus has infected the cells for 1 h, discard the supernatant and add DMEM containing 2% FBS with different concentrations of glutamate. The control group is added with DMEM containing 2% FBS. Collect the cell supernatant 36 h after infection for virus titer detection and qPCR detection.
[0048] TCID 50 The results of the TCID Figure 3 、 4 assay and qPCR assay are shown in
[0049] Figures
[0050] and
[0051] show the effects of different concentrations of glutamate on PRV replication. From the titer results, 5 mM and 10 mM glutamate can significantly downregulate the titer of pseudorabies virus, indicating that glutamate has an obvious inhibitory effect on PRV replication in PK-15 cells. It can also be seen from the fluorescence quantitative PCR results that 5 mM and 10 mM glutamate significantly reduce the DNA copy number of pseudorabies virus. These results indicate that glutamate has an inhibitory effect on PRV replication in PK-15 cells, and 10 mM glutamate has the best inhibitory effect on PRV. 5 Inoculate PK-15 cells at a cell density of 6×10
[0052] cells per well into a 6-well plate; after culturing at 37°C for 15 h, infect the cells with PRV (MOI = 0.01). After the virus has infected the cells for 1 h, discard the supernatant and add DMEM containing 2% FBS with 10 mM glutamate. The control group is added with DMEM containing 2% FBS. Collect the cell supernatant at different time points after infection for virus titer detection and qPCR detection. 50 The results of the TCID Figure 5 、 6 assay and qPCR assay are shown in
[0053] Figures
[0054] Inoculate PK-15 cells at a cell density of 6×10 5 cells per well into a 6-well plate; after culturing at 37°C for 15 h, infect the cells with pseudorabies virus (MOI = 0.01). After 1 h of virus infection of the cells, discard the supernatant and add DMEM containing 2% FBS with 10 mM glutamate. Add DMEM containing 2% FBS to the control group. Collect samples at different time points after infection. Discard the culture medium and wash once with PBS. Add lysis buffer according to the ratio of 100 μl of lysis buffer per well of the 6-well plate. Pipette several times to ensure sufficient contact between the lysis buffer and the cells. Perform Western blot analysis on the collected cell protein samples.
[0055] The results of the Western blot analysis are shown in Figure 7 , 8 . The results of the Western blot analysis indicate that the expression level of the gB protein of pseudorabies virus significantly decreases under the action of 10 mM glutamate. Combining with the previous experimental results, it shows that glutamate inhibits the proliferation of PRV in vitro.
[0056] Example 5: Median lethal dose (LD 50 )
[0057] Evaluate the lethality of different doses of PRV in C57BL / 6 mice (n = 6). The mice were intraperitoneally injected with 101, 10 2 , 10 3 , and 10 4 TCID 50 of PRV virus, and observe the survival situation within 10 days. The results are shown in Figure 9 . Use SPSS software for Probit analysis to calculate the LD 50 of PRV to be 108 TCID 50 . This result serves as the basis for selecting the challenge doses of 400 TCID 50 and 800 TCID 50 in this study to ensure that the severity of infection is sufficient to evaluate the antiviral effect of glutamate.
[0058] Example 6: Effect of treatment with different doses of glutamate on the survival rate of PRV-infected mice
[0059] Using a C57BL / 6 mouse model, each mouse was infected with 400 TCID 50PRV. The number of mice in each group was n = 19. The first dose was administered 5 hours after infection, and then once every 24 hours. The groups were the control group (Control), the 12 mg / kg group, the 24 mg / kg group, and the 36 mg / kg group. The body weight changes of the mice within 10 days after infection were observed and recorded, and the data were presented in the form of percentage of weight change (Weight change percentage %). The survival curve recorded the survival of the mice 10 days after infection, and Kaplan-Meier survival analysis was used for statistics.
[0060] The body weight changes and survival curves were recorded as Figure 10 , 11 , and the results showed that glutamate could be used after PRV infection. The body weight results showed that in the early stage of infection, the degree of weight loss of the mice in the glutamate treatment group was similar to that of the control group, but the mice in the glutamate treatment group could recover to the initial body weight earlier than the control group; glutamate treatment could significantly improve the survival rate after PRV infection; and the clinical symptoms of the mice in the glutamate treatment group were better than those of the control group after infection. The above results indicated that glutamate had a protective effect on mice infected with PRV.
[0061] Example 7
[0062] Using the C57BL / 6 mouse model, it was divided into 4 groups with 6 mice in each group. The groups were the control group (Control), the 12 mg / kg group, the 24 mg / kg group, and the 36 mg / kg group. Each mouse was infected with 800 TCID 50 of PRV virus, and the first dose was administered 5 hours after infection, and then once every 24 hours. After obvious morbidity symptoms were observed, samples of the mouse brain and trigeminal nerve tissues were collected, and qPCR was performed to detect the nucleic acid copy number of the PRV genome, and the positive sample rate was calculated. Three biological replicate experiments were carried out.
[0063] The statistical results were as Figure 12 , 13 shown, indicating that after PRV infection, drug treatment could significantly reduce the PRV positive sample rate in the brain and trigeminal nerve tissues. Among them, the 24 mg / kg dose group showed the best antiviral effect, showing good dose-dependence and treatment potential.
[0064] Based on the content described in the above examples, it can be confirmed that glutamate can be used to prepare drugs for the treatment of pseudorabies virus infection and can stably exert antiviral effects.
[0065] Based on the common knowledge of those skilled in the art, the drug comprises an effective amount of glutamic acid and a pharmaceutically acceptable excipient. The drug uses glutamic acid as the active ingredient and includes the excipient, and glutamic acid and the excipient are made into a clinically acceptable preparation through a formulation process. The excipient can be optionally any one or a combination of two or more of the following: dextrin, lactose, gelatin, microcrystalline cellulose. The preparation can be optionally any one of the following forms: aqueous solution, capsule, granule, injection, spray.
[0066] When using the drug, environmental disinfection treatment or treatment of diseased individuals can be selected according to the actual situation. The former refers to using the drug as an environmental disinfectant in the form of spraying, and diluting the drug with water into a solution with a glutamic acid concentration of 2.5 - 10 mM before use. The latter refers to an oral drug used in an oral manner or an injection used in an injection manner, and the concentration during use is 10 mM; the dosage of the oral drug or injection is 12 - 36 mg / Kg body weight.
[0067] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.
Claims
1. Use of glutamic acid in the preparation of a drug for treating pseudorabies virus infection.
2. The application according to claim 1, wherein The drug comprises an effective amount of glutamic acid and a pharmaceutically acceptable excipient.
3. A drug for treating pseudorabies virus infection, characterized in that, The drug uses glutamic acid as the active ingredient.
4. The drug according to claim 3, characterized in that, The drug further comprises an excipient; glutamic acid and the excipient are made into a clinically acceptable preparation through a formulation process.
5. The drug according to claim 3, characterized in that, The excipient is any one or a combination of two or more of the following: dextrin, lactose, gelatin, microcrystalline cellulose.
6. The drug according to claim 3, characterized in that, The preparation is any one of the following forms: aqueous solution, capsule, granule, injection, spray.
7. The drug according to claim 3, characterized in that, The drug is an environmental disinfectant used by spraying. Before use, the drug is diluted with water into a solution with a glutamic acid concentration of 2.5 - 10 mM.
8. The drug according to claim 3, characterized in that, The drug is an oral drug used by oral administration or an injection used by injection, with a concentration of 10 mM during use; the dosage of the oral drug or injection is 12 - 36 mg / Kg body weight.