CN110664988B

CN110664988B - Application of H-Lys-Trp-Lys-OH in preparation of medicine for resisting bovine intestinal viruses

Info

Publication number: CN110664988B
Application number: CN201911078045.XA
Authority: CN
Inventors: 程凯慧; 楚会萌; 杨宏军; 任亚初; 解晓莉; 张亮; 于志君; 孙阳阳; 朱彤
Original assignee: Poultry Research Institute Shandong Academy of Agricultural Sciences; Dairy Cattle Research Center Shandong Academy of Agricultural Science
Current assignee: Poultry Research Institute Shandong Academy of Agricultural Sciences; Dairy Cattle Research Center Shandong Academy of Agricultural Science
Priority date: 2019-11-06
Filing date: 2019-11-06
Publication date: 2022-09-30
Anticipated expiration: 2039-11-06
Also published as: CN110664988A

Abstract

The invention provides application of H-Lys-Trp-Lys-OH in preparation of a medicine for resisting bovine intestinal viruses. The invention discovers for the first time that the compound H-Lys-Trp-Lys-OH can effectively inhibit the multiplication of bovine enterovirus and has low toxicity to cells, and experiments prove that the H-Lys-Trp-Lys-OH can effectively inhibit and kill the bovine enterovirus on an MDBK cell model in vitro experiments, can effectively inhibit the invasion and the replication of the bovine enterovirus, has low cytotoxicity, can be used as a novel anti-bovine enterovirus medicament, opens up a novel medicinal application for the H-Lys-Trp-Lys-OH, lays an experimental foundation for developing high-efficiency specific anti-BEV medicaments and provides a novel visual field.

Description

Application of H-Lys-Trp-Lys-OH in preparation of medicine for resisting bovine intestinal viruses

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of H-Lys-Trp-Lys-OH in preparation of a medicine for resisting bovine intestinal viruses.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Bovine Enteroviruses (BEV) are members of the Enterovirus genus of the picornaviridae family. The virus particles are spherical, icosahedral and without envelope, and the diameter of the complete virus particle is about 25-30 nm. The genome is a single-stranded positive-stranded RNA consisting of approximately 7500 nucleotides (nt), which can be used directly as mRNA, to encode and translate a polyprotein, which is then subjected to a series of degradations, yielding 4 structural proteins (VP1, VP2, VP3, and VP4) and 7 non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D). The capsid of the BEV is an icosahedron, and the virion consists of 60 asymmetric virions composed of four structural proteins (VP1, VP2, VP3, VP4) and a single-stranded positive-stranded RNA genome. Early studies separated BEV into two serotypes, BEV-1 and BEV-2; later, with the development of molecular biology, RolandZell et al performed systematic gene sequence comparison analysis of bovine enteroviruses that have been isolated at present in 2006, and further classified the bovine enteroviruses into two families A and B, BEV-A and BEV-B, which contain 2 and 3 genotypes within BEV-A and BEV-B, respectively.

After the virus is reported by Moll and Davis for the first time in 1959, the virus is generally found and prevalent in main cattle raising countries and regions all over the world, cattle farms in multiple regions of China have found that the virus can cause cattle to generate various syndromes, the syndromes are mainly transmitted through manure openings, the infection sources are mainly sick cattle and asymptomatic virus carriers, cattle infected with cattle enterovirus show mild diarrhea and mild respiratory symptoms, the appetite is reduced, severe cattle even have hematochezia, the milk yield is greatly reduced, and serious economic loss is caused to the cattle raising industry. Despite the deep understanding of their biological characteristics, the epidemiological characteristics and pathogenic mechanisms of viruses are still poorly understood. Therefore, there is an urgent need to develop specific and effective preventive and therapeutic drugs and means to prevent the harm of bovine enterovirus.

H-Lys-Trp-Lys-OH is a small molecular antibacterial peptide (CN 104072579A) reported by Ooka et al of southern medical university in China for the first time, and no report is available so far for preventing or treating bovine enterovirus.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of H-Lys-Trp-Lys-OH in preparing the medicine for resisting the bovine intestinal tract virus.

The invention aims to provide application of H-Lys-Trp-Lys-OH in preparation of a medicine for resisting bovine intestinal viruses.

The invention also aims to provide an anti-bovine enterovirus pharmaceutical composition.

In order to achieve the purpose, the invention relates to the following technical scheme:

in the first aspect of the invention, the application of H-Lys-Trp-Lys-OH in preparing the medicine for resisting bovine intestinal tract virus is provided. And therefore, H-Lys-Trp-Lys-OH is effective for the prevention and/or treatment of bovine enterovirus-associated diseases.

According to the present invention, the concept of "prevention and/or treatment" means any measure suitable for the treatment of bovine enterovirus-related diseases, or the prophylactic treatment of such manifested diseases or manifested symptoms, or the avoidance of recurrence of such diseases, such as recurrence after the end of a treatment period or treatment of symptoms of diseases that have already occurred, or the prevention or inhibition or reduction of the occurrence of such diseases or symptoms with prior intervention.

In the sense of the invention, the 'anti-bovine enterovirus medicine' represents a substance which has obvious inhibiting and killing effects on bovine enterovirus viruses, mainly acts on the early stage of the life cycle of the bovine enterovirus viruses, and has better direct killing effects on the viruses than adsorption blocking effects and replication blocking effects.

According to the present invention, not only the use of H-Lys-Trp-Lys-OH for the preparation of a medicament against bovine intestinal viruses is disclosed, but also the enhancement of this effect when H-Lys-Trp-Lys-OH is administered in combination with at least one other pharmaceutically active ingredient. As an alternative or in addition to other pharmaceutically active ingredients, H-Lys-Trp-Lys-OH may also be used in combination with other non-pharmaceutically active ingredients.

In view of the above, the second aspect of the present invention provides a pharmaceutical composition against bovine enterovirus, which comprises H-Lys-Trp-Lys-OH and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

Wherein H-Lys-Trp-Lys-OH is not less than half of Effective Concentration (EC) ₅₀ ) The drug concentration of (A), the median effective concentration of H-Lys-Trp-Lys-OH against bovine Enterovirus (EC) ₅₀ ) 60 mu M; the therapeutic index of H-Lys-Trp-Lys-OH to bovine enterovirus is greater than 1.67. Of course, when H-Lys-Trp-Lys-OH is used in combination with other drugs or active ingredients having the same application as mentioned in the summary of the invention, such as inhibiting and/or killing or assisting in inhibiting and/or killing bovine enterovirus, the drug concentration may theoretically be lower than the half effective concentration, but special exceptions are not excluded.

Other non-pharmaceutically active ingredients include, but are not limited to, pharmaceutically acceptable carriers, excipients, and/or diluents.

Compared with the prior art, the invention has the following effects and advantages:

the invention firstly discovers that the compound H-Lys-Trp-Lys-OH can effectively inhibit the multiplication of bovine enterovirus virus and has low toxicity to cells, and experiments prove that the H-Lys-Trp-Lys-OH has half Cytotoxic Concentration (CC) to MDBK cells ₅₀ ) Greater than 100. mu.M, and a median Effective Concentration (EC) against bovine enterovirus virus ₅₀ ) 60 mu M; the therapeutic index of H-Lys-Trp-Lys-OH to bovine enterovirus is larger than 1.67, which shows that the H-Lys-Trp-Lys-OH has the prospect of developing anti-bovine enterovirus drugs, opens up new drug application for the H-Lys-Trp-Lys-OH, also lays an experimental foundation for developing high-efficiency specific anti-BEV drugs and provides a new visual field.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing the effect of H-Lys-Trp-Lys-OH of the present invention on BEV-injured cells;

wherein: wherein FIG. 1A is a control group of viruses; FIG. 1B is a set of MDBK normal cells; FIG. 1C is the drug test group of infected cells (administered 100. mu. M H-Lys-Trp-Lys-OH);

FIG. 2 shows the half Cytotoxic Concentration (CC) of H-Lys-Trp-Lys-OH against MDBK cells in example 2 ₅₀ ) A drawing;

FIG. 3 is the half-Effective Concentration (EC) of H-Lys-Trp-Lys-OH against BEV in example 3 ₅₀ ) A drawing;

FIG. 4 is a graph of the effect of H-Lys-Trp-Lys-OH on BEV inhibition at various time points in example 4;

FIG. 5 is a graph showing the effect of H-Lys-Trp-Lys-OH on the direct killing of BEV in example 5.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention is further described with reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.

As described in the background art, no H-Lys-Trp-Lys-OH has been reported in the prior art for the prevention and treatment of bovine enterovirus.

In view of the above, one embodiment of the present invention provides the use of H-Lys-Trp-Lys-OH in the preparation of a medicament for treating bovine intestinal tract viruses. And therefore, H-Lys-Trp-Lys-OH is effective for the prevention and/or treatment of bovine enterovirus-associated diseases. It should be noted that the application is disclosed for the first time, and is different from the known clinical application, and the structural formula is as follows:

wherein in the anti-bovine intestinal tract virus medicine, H-Lys-Trp-Lys-OH is not less than half Effective Concentration (EC) ₅₀ ) The drug concentration of (A), the median effective concentration of H-Lys-Trp-Lys-OH against bovine Enterovirus (EC) ₅₀ ) 60 mu M;

according to the present invention, the concept "prevention and/or treatment" means any measure suitable for the treatment of a bovine enterovirus-related disease, either for prophylactic treatment of such manifested disease or manifested symptoms, or to avoid recurrence of such disease, e.g. after the end of a treatment period or treatment of symptoms of a disease that has already occurred. Or pre-interventional prevention or inhibition or reduction of the occurrence of such diseases or conditions.

In the meaning of the invention, the 'anti-bovine enterovirus medicine' represents a substance, the H-Lys-Trp-Lys-OH contained in the medicine has obvious inhibiting effect on bovine enterovirus viruses and mainly acts on the early stage of the life cycle of the bovine enterovirus viruses, and particularly, the direct killing effect on the viruses is better than the adsorption blocking effect and the replication blocking effect.

Therefore, in another embodiment of the invention, the application of H-Lys-Trp-Lys-OH in preparing the medicament for inhibiting and/or killing bovine enterovirus is provided; or, the application of H-Lys-Trp-Lys-OH in preparing the medicine for inhibiting the bovine enterovirus proliferation; specifically, the inhibition of bovine enterovirus proliferation is mainly performed by adsorption blocking effect.

In yet another embodiment of the present invention, there is provided a pharmaceutical composition against bovine enterovirus, said pharmaceutical composition consisting of H-Lys-Trp-Lys-OH and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

In still another embodiment of the present invention, H-Lys-Trp-Lys-OH is at not less than half the Effective Concentration (EC) ₅₀ ) The drug concentration of (A), the median effective concentration of H-Lys-Trp-Lys-OH against bovine Enterovirus (EC) ₅₀ ) 60 mu M;

the other pharmaceutical active ingredients include substances that inhibit and/or kill bovine enteroviruses or assist in inhibiting and/or killing bovine enteroviruses.

Other non-pharmaceutically active ingredients include pharmaceutically acceptable carriers, excipients and/or diluents. Such as pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids, pharmacologically innocuous salts such as sodium chloride, flavoring agents, vitamins such as vitamin a or vitamin E, tocopherols or provitamins, antioxidants such as ascorbic acid, and stabilizers and/or preservatives for extending the use and shelf life of the pharmaceutically active ingredient or formulation, and other common non-pharmaceutically active ingredients or adjuvants and additives known in the art, and mixtures thereof.

The pharmaceutical composition may be administered in unit dosage form. The administration form can be the form of a conventional H-Lys-Trp-Lys-OH form, or other feasible forms, such as forms suitable for H-Lys-Trp-Lys-OH can be selected by those skilled in the art from conventional forms, and is realized by adding carriers, excipients, binders, diluents and the like compatible with H-Lys-Trp-Lys-OH. Conventional dosage forms described herein such as liquid dosage forms, solid dosage forms, topical formulations, sprays, and the like, such as the following: true solutions, colloids, microparticles, emulsion, suspension, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, and liniment.

The technical scheme of the invention is further explained by combining specific embodiments. It should be noted that the bovine enterovirus used in the examples of the present application was isolated from the research center for cows of the academy of agricultural sciences of Shandong province and was identified as belonging to the BEV-A family genotype 1.

The specific separation and identification method is as follows: diluting fresh cattle feces with PBS, repeatedly freezing and thawing for 3 times, centrifuging at 5000 r/min for 5min, collecting supernatant, adding penicillin (200IU/mL) and streptomycin (100 μ g/mL), collecting 200 μ L supernatant, extracting BEV virus genome RNA according to virus genome DNA/RNA extraction kit specification, reverse transcribing into cDNA, performing PCR amplification with BEV specific primer, filtering and sterilizing the feces supernatant identified as positive by PCR with 0.22 μm filter membrane, collecting 1mL inoculated with MDBK single-layer cell, adsorbing at 37 deg.C for 1h, discarding, adding into DMEM culture medium containing 2% FBS, and culturing at 37 deg.C with 5% CO ₂ Culturing in a cell culture box, observing cytopathic condition every 12h, and continuously observing for 4 d. And (3) repeatedly freezing and thawing cells with pathological Changes (CPE) for 3 times, collecting cell virus liquid, simultaneously identifying whether the obtained cell virus liquid is BEV single infection or not, and obtaining the BEV single infection cell virus liquid by using a plaque purification method if the cell virus liquid is not the BEV single infection. This isolate was characterized by phylogenetic tree analysis of the BEV structural proteins VP1 and VP3 as belonging to the BEV-A family genotype 1.

Example 1Virus TCID ₅₀ Measurement of (2)

MDBK cells (stored in Dairy research center of Oncology institute of agriculture, Shandong province) were digested and then digested at 3X 10 per well ⁵ Cell density of one/mL was seeded into 96-well cell culture plates and placed at 37 ℃ in 5% CO ₂ In a cell culture boxCulturing to obtain monolayer cells, discarding cell growth liquid in the hole, and continuously diluting 10 times of bovine enterovirus to obtain virus diluent (10 dilution degrees respectively) ^-1 ～10 ^-10 ) Inoculating to a 96-well plate full of monolayer cells (100 μ L per well), adding 5% CO at 37 deg.C ₂ The culture was continued in the incubator, and the cells were observed day by day for CPE and the number of cytopathic wells was recorded in detail. And setting a normal cell control group and a blank control group at the same time, setting 8 repeats in each group, and judging the result when the cytopathy does not occur any more. The cell pathological pore is the corresponding cell pore to the above cell pathological change, and the virus TCID is calculated according to the Karber method ₅₀ 。

TABLE 1 TCID ₅₀ of BEV

Note: TCID ₅₀ Tissue culture infection dose, also called 50% Tissue cell infection dose; i.e., the amount of virus required to cause half of the cytopathic effect or death (CPE) in a well or tube.

As a result: morphological observation under a microscope shows that virus diluents with different concentrations all cause cytopathic effect when the virus diluents are used for 48 hours, the refractive index of cells is changed, the single-layer structure is damaged, the cells are subjected to circular shrinkage necrosis and gradually take the shape of a net and form vacuoles, some cells are cracked and fall off into fragments, cytopathic effect of each hole is not continued after 72 hours, the number of CPE holes with different concentrations is counted, the CPE ratios with different concentrations are calculated, and the TCID of the bovine enteroviruses is calculated according to a Karber method ₅₀ The value:

LgTCID ₅₀ ＝L-D(S-0.5)

(L: logarithm of highest dilution; D: difference between logarithm of dilutions; sum of S-positive well ratios)

LgTCID ₅₀ ＝L-D(S-0.5)＝-1-1×(3.25-0.5)＝-3.75

TCID ₅₀ ＝10 ^-3.75 /0.1mL

I.e. diluting the virus 10 ^3.75 Inoculation with 100. mu.L resulted in 50% of the cells being diseased.

Example 2Toxicity assay of H-Lys-Trp-Lys-OH on MDBK cells:

MDBK cells are susceptible cells to bovine enterovirus. Therefore, firstly, the cytotoxicity of H-Lys-Trp-Lys-OH on MDBK cells is detected, and the specific experimental steps are as follows:

(1) mu.L of cells (MDBK 3X 10) were seeded in 96-well plates ⁴ One/hole).

(2) After about 12h of incubation, the next dosing analysis was performed. Media was discarded and 100 μ L of 2% FBS DMEM containing different drug concentrations were added to each well, 3 replicates for each concentration. At the same time, control wells: 100 μ L of 2% FBS DMEM medium was added. And (4) zeroing holes: cells were not plated.

(3) At 37 ℃ 5% CO ₂ After culturing for 48h under the conditions, OD value at 450nm was measured with a microplate reader according to the instructions of CCK-8 kit.

(4)37℃，5％CO ₂ After further incubation for 4h under these conditions, the absorbance was measured at 450 nm. A450nm for normal growing cells was set as a 100% cell control.

(5) Data were analyzed and the half-cytotoxic concentration of H-Lys-Trp-Lys-OH (CC) was calculated using GraphPad Prism5 ₅₀ ) The value is obtained. The results are shown in FIG. 2.

As a result: H-Lys-Trp-Lys-OH has a dose-dependent relation, namely, the cytopathy is more obvious along with the increase of the concentration of the medicament. By statistical analysis, the H-Lys-Trp-Lys-OH median toxic concentration is determined to be more than 100 mu M.

Example 3Inhibition experiment of H-Lys-Trp-Lys-OH on bovine enterovirus:

(1) seeding of 3X 10 wells in 96-well plates ⁴ MDBK cells, 37 ℃, 5% CO ₂ Culturing in an incubator overnight;

(2) the medium was discarded and 100. mu.L of 100TCID was added to each well ₅₀ The bovine enterovirus diluent (virus diluent is added after 2% FBS DMEM cells are full, diluted and added according to 100 mu M initial concentration and double concentration gradient, and 5% CO is added ₂ Culturing in an incubator;

(3) after 48h, the OD at 490nm was measured with a microplate reader, following the instructions of the CCK-8 kit.

(4) The data were analyzed, and the rate of virus inhibition (%) (drug-treated D450nm value-virus control D450nm value)/(normal cell control D450nm value-virus control D450nm value) × 100%, half Effective Concentration (EC) of the compound was obtained using GraphPad Prism5 software ₅₀ ) The value is obtained. The results are shown in FIG. 3. Then according to the formula TI ═ CC ₅₀ /EC ₅₀ And calculating the corresponding therapeutic index TI value.

As a result: the CCK-8 kit is used for detecting the cell activity, so that the effective inhibition rate of the medicament on the bovine enterovirus can be calculated. From the results, it can be seen that the effective inhibition rate of H-Lys-Trp-Lys-OH in the safe concentration range is increased along with the increase of the drug concentration, and the effective inhibition rate is in a certain dose-effect relationship. Half Effective Concentration (EC) of bovine enterovirus by analytical software ₅₀ ) 60 μ M. The therapeutic index of H-Lys-Trp-Lys-OH to bovine enterovirus is greater than 1.67.

Example 4Preliminary study of mechanism of action

The test compound is added into the MDBK cells inoculated with the bovine enterovirus through different administration time, namely corresponding time points of first administration and then infection of the virus (before 0H), first infection of the virus and then administration (after 0H), and simultaneous addition of the virus and the medicine into the cells (0H), so that the action period of the H-Lys-Trp-Lys-OH is preliminarily judged. The specific experimental steps are as follows:

(1) seeding of 3X 10 wells in 96-well plates ⁴ MDBK cells, 37 ℃, 5% CO ₂ Culturing in an incubator.

(2) According to the measured pharmacodynamic evaluation result of the related medicine, the concentration of the medicine required by the experiment is determined, and the medicine is diluted to the required concentration by using a maintenance medium.

(3) After overnight incubation, the cell supernatants from the second three duplicate wells of the 96-well plate were aspirated and the cells were washed 2 times with phosphate buffer. Then 50. mu.L of the drug to be tested was added, and the time was recorded as-2 h.

(4) After 2h, the cell supernatants of the other wells were aspirated off, and the diluted bovine enterovirus diluent was added to each well of columns 2-11 at a sample addition volume of 50 μ L per well. At the same time, 50. mu.L of the corresponding analyte was added to the three duplicate wells in column 3, and this time was recorded as 0 h.

(5) And adding corresponding compounds to be detected into the three compound holes in the next row at regular intervals, and marking the corresponding time. MDBK cells from column 11 were used as virus control.

(6) After 48 hours of incubation, OD measurements were performed. The data were analyzed and concluded, the results of which are shown in fig. 4.

As a result: analysis of administration experiment results at different time points shows that the H-Lys-Trp-Lys-OH has obvious inhibition effect on viruses when the viruses infect cells for-2H, 0H and 2H, and the H-Lys-Trp-Lys-OH probably mainly acts on the early stage of the life cycle of the bovine enteroviruses.

Example 5Effect of Compound addition at different times on bovine enterovirus replication

The screened H-Lys-Trp-Lys-OH with better effect and lower toxicity is further researched for action mechanism. The in vitro antiviral inhibition test is carried out by 3 different action modes of adding medicine first and then adding virus, adding virus first and then adding medicine, and pre-acting medicine and virus.

(1) Direct killing effect of medicine on virus

Equal amount of 100TCID ₅₀ Mixing the virus solution with the medicinal diluent at different concentrations, and standing at 37 deg.C with 5% CO ₂ Pre-acting in an incubator for 4h, adding into a 96-well cell culture plate with a monolayer, allowing each liquid medicine to have a gradient of 100 μ L/well, acting in the incubator for 2h, discarding the supernatant, and adding cell maintenance liquid to continue culturing. The test simultaneously sets a normal cell control group, a virus control group and a blank control group, each concentration is set to be 3 times, cell viability detection is carried out for 48 hours, and the EC of the compound is obtained by GraphPad Prism5 software ₅₀ 。

As a result: under the administration mode of pre-action of H-Lys-Trp-Lys-OH and bovine enterovirus, cytopathic effect of the experimental medicine-adding group at each concentration is slight compared with that of a virus control group, pathological phenomena such as cell rounding, cell shedding, cell vacuolation and the like are relieved, and the action effect of the H-Lys-Trp-Lys-OH on the bovine enterovirus is shown in figure 5 through analysis software. As can be seen from the figure, under the action formula, H-Lys-Trp-Lys-OH has certain inhibiting effect on bovine enterovirus, and the effective inhibiting rate of the medicament is increased along with the increase of the concentration of the medicament within a safe concentration range, and has a certain dose-effect relationship. The result shows that H-Lys-Trp-Lys-OH has a certain direct inactivation effect on bovine enteroviruses.

(2) Blocking effect of medicine on bovine intestinal tract virus adsorption

Inoculating the digested cells into a pore plate according to the cell density of each pore, removing the supernatant after the cells grow into a monolayer, adding the drug diluents with different concentrations into a 96-pore cell culture plate with the monolayer, removing the supernatant after the drug diluents with different concentrations act for 4 hours in an incubator in advance, washing twice with PBS, adding 100TCID with the same amount ₅₀ Placing the virus liquid at 37 ℃ and 5% CO ₂ Culturing in an incubator. The test is simultaneously provided with a normal cell control group, a virus control group and a blank control group, each concentration is set for 3 times, cell activity detection is carried out after 48 hours, and the antiviral effective rate of the drugs with different concentrations under the action mode is calculated.

As a result: CPE observation results show that the degree of cytopathic effect of the drug pretreatment group is obviously changed compared with that of the virus control group. Cell viability detection results show that the effective inhibition rate of the drug on bovine enteroviruses can reach 60%, which indicates that H-Lys-Trp-Lys-OH can prevent the bovine enteroviruses from adsorbing cells.

(3) Blocking effect of medicine on bovine enterovirus replication

Inoculating the digested cells into a well plate according to the cell density of each well, removing supernatant after the cells grow into a monolayer, and adding 100TCID (100 TCID) with equal amount ₅₀ Adding virus solution into 96-well cell culture plate, and standing at 37 deg.C under 5% CO ₂ Pre-acting in an incubator for 2h, removing supernatant, washing cells for 2 times with PBS, adding drug diluents with different concentrations, each drug solution gradient being 100 μ L/well, setting a normal cell control group, a virus control group and a blank control group at the same time in the test, setting 3 repeats for each concentration, placing at 37 ℃ and 5% CO ₂ Culturing in an incubator, detecting cell viability after 48h, analyzing data and obtaining a conclusion.

As a result: CPE observations showed that there was no significant change in the extent of cytopathic effect in the virus pretreatment group compared to the virus control group. The detection result shows that the effective inhibition rate of the H-Lys-Trp-Lys-OH on the bovine enterovirus is lower than 20 percent, which indicates that the H-Lys-Trp-Lys-OH can not block the replication function in cells.

In the application example of the invention, bovine kidney cells (MDBK) are used as a carrier, and 3 different action modes of adding medicine first and then adding virus, adding medicine first and then adding medicine, and adding medicine again after virus pre-action are adopted on a cytopathogenic model to carry out in-vitro antiviral inhibition research. The novel antiviral effect of H-Lys-Trp-Lys-OH is found to have a certain inhibiting effect on bovine enteroviruses, and the direct killing effect on the viruses is better than the adsorption blocking effect and the replication blocking effect.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

The application of H-Lys-Trp-Lys-OH in preparing the medicine for resisting the bovine enterovirus is characterized in that the bovine enterovirus refers to inhibiting the adsorption of the bovine enterovirus to cells and/or killing the bovine enterovirus; the concentration of H-Lys-Trp-Lys-OH is not less than 60 mu M.
2. The use as claimed in claim 1, characterized in that a pharmaceutical composition against bovine enterovirus is constituted by H-Lys-Trp-Lys-OH and at least one other non-pharmaceutically active ingredient; the drug concentration of H-Lys-Trp-Lys-OH is not less than 60 mu M.
3. The use according to claim 2, wherein the other non-pharmaceutically active ingredients comprise pharmaceutically acceptable carriers, excipients and/or diluents.
4. The use of claim 2, wherein the pharmaceutical composition is administered in a form selected from the group consisting of a liquid form, a solid form, a topical formulation, and a spray.
5. The use of claim 2, wherein the pharmaceutical composition is administered in the form of a true solution, a colloid, a microparticle, an emulsion, a suspension, a tablet, a capsule, a drop pill, an aerosol, a pill, a powder, a solution, a suspension, an emulsion, a granule, a suppository, a lyophilized powder for injection, an inclusion compound, a landfill, a patch, or a liniment.