阅读说明：本技术 一种抗菌肽在制备抗草鱼呼肠孤病毒药物中的应用 (Application of antibacterial peptide in preparation of anti-grass carp reovirus medicine ) 是由 黄贞胜 庄淑芳 谢晓露 林斌 于 2021-10-21 设计创作，主要内容包括：本发明涉及拟穴青蟹阴离子抗菌肽的技术领域,公开了一种抗菌肽在制备抗草鱼呼肠孤病毒药物中的应用,所述抗菌肽为专利CN201110250662.0所述的拟穴青蟹阴离子抗菌肽Scygonadin,穴青蟹阴离子抗菌肽Scygonadin能够明显的抑制草鱼呼肠孤病毒的活性。(The invention relates to the technical field of scylla paramamosain anionic antibacterial peptides, and discloses application of antibacterial peptides in preparation of a medicine for resisting grass carp reovirus, wherein the antibacterial peptides are Scygonadin of scylla paramamosain described in patent CN201110250662.0, and the Scygonadin can obviously inhibit the activity of grass carp reovirus.)

1. An application of an antibacterial peptide in preparation of an anti-grass carp reovirus medicine is characterized in that the antibacterial peptide is Scygonadin which is an anionic antibacterial peptide of scylla paramamosain and is described in patent CN 201110250662.0.

2. The use of the antimicrobial peptide of claim 1 as an antiviral active ingredient in the manufacture of a medicament against reovirus in grass carp at a dose of 1ug/g to 30 ug/g.

3. The use of the antimicrobial peptide of claim 2 in the preparation of an antiviral medicament, wherein the antimicrobial peptide is used at a dose of from 10ug/g to 20 ug/g.

4. The use of the antimicrobial peptide of claim 1 in the preparation of an antiviral medicament, wherein said anti-grass carp reovirus medicament further comprises a pharmaceutically acceptable adjuvant.

5. The use of the antimicrobial peptide according to claim 1, in the preparation of an antiviral medicament, wherein the dosage form of the anti-grass carp reovirus medicament comprises a tablet, a powder, a granule, a capsule or a sustained release agent.

6. The use of the antimicrobial peptide of claim 1 for the preparation of an antiviral medicament, wherein said anti-grass carp reovirus medicament is administered by a gavage feeding regimen.

Technical Field

The invention relates to the technical field of scylla paramamosain anionic antibacterial peptides, in particular to application of antibacterial peptides in preparation of an anti-grass carp reovirus medicament.

Background

At present, the culture amount of grass carps in China is about 20 percent of the total culture amount, and the yield reduction caused by diseases accounts for more than 30 percent of the total culture amount of the annual grass carps. Among Grass carp pathogens, Grass Carp Reovirus (GCRV) has the biggest harm, and Grass carp hemorrhagic disease caused by the infection of Grass carp reovirus has high infectivity and high lethality, so that large-scale yield reduction is caused, and the Grass carp reovirus is always regarded as one of the most prominent problems in freshwater aquaculture in China. The grass carp reovirus particles are in icosahedral symmetry, have double-layer capsids and no cyst membrane, the genome consists of 11 double-stranded RNAs, and the grass carp reovirus infection can cause hyperemia and hemorrhage phenomena of various organs and tissues of grass carps to different degrees, and the morbidity and mortality rate is more than 80%. At present, no ideal treatment method for grass carp reovirus infection exists, and the prevention work is mainly taken as the main treatment.

The antibacterial peptide is considered to be one of main components of defense systems of fishes, shrimps, crabs, shellfish and the like, and is an important component of an organism innate immune system. The antibacterial peptide is expected to be an anti-drug-resistance bacterial medicament and feed immune additive with wide application prospect in the prevention and treatment of diseases of marine culture fishes, crabs, shellfish and the like. The research, development and application of the antibacterial peptide have important scientific significance and practical application value for solving the problems of bacterial drug resistance in mariculture production, aquatic product drug residue caused by abuse of antibiotics in fish feed, water environment pollution and the like, and can obtain remarkable economic benefit.

Disclosure of Invention

Therefore, the application of the antibacterial peptide in preparing the anti-grass carp reovirus medicine is needed, and the new application of the Scygonadin in resisting the grass carp reovirus is developed.

In order to achieve the purpose, the invention provides an application of an antibacterial peptide in preparation of an anti-grass carp reovirus drug, wherein the antibacterial peptide is Scygonadin, an anionic antibacterial peptide of scylla paramamosain, and the anti-grass carp reovirus drug is described in patent CN 201110250662.0.

Furthermore, the antibacterial peptide is used as an antiviral active ingredient for preparing an anti-grass carp reovirus medicament, and the application dose of the antibacterial peptide is 1ug/g-30 ug/g.

Further, the application dosage of the antibacterial peptide is 10ug/g-20 ug/g.

Further, the anti-grass carp reovirus medicine also comprises pharmaceutically acceptable auxiliary materials.

Furthermore, the dosage form of the anti-grass carp reovirus medicine comprises tablets, powder, granules, capsules or sustained-release agents.

Furthermore, the administration mode of the anti-grass carp reovirus medicine is mixed feeding.

The technical scheme has the following beneficial effects:

according to the invention, the Scygonadin is innovatively applied to the anti-grass carp reovirus medicine, the grass carp reovirus infection can be effectively inhibited, the inhibiting effect is strong, in addition, the administration mode of the antibacterial peptide is mixed feeding, the administration is convenient, and the market application of the antibacterial peptide can be greatly facilitated.

Detailed Description

In order to explain technical contents, structural features, and objects and effects of the technical means in detail, the following detailed description is given with reference to specific embodiments.

The following experiment was performed using Scygonadin, an anionic antibacterial peptide of Scygonadin described in patent CN 201110250662.0.

Example 1 safe concentration assay of antimicrobial peptides on CIK cells

Subculturing CIK cells by conventional method, adjusting cell number to 2 × 10⁵Perml, 0.1mL of cell suspension was added to each well of a 96-well microplate and incubated overnight in an incubator at 28 ℃. Cells containing fetal bovine serum were cultured. Preparing antibacterial peptide solution by using cell culture medium, setting the concentration of test groups to be 1ug/g, 5ug/g, 10ug/g, 15ug/g, 20ug/g, 25ug/g and 30ug/g respectively, adding 0.1ml into each hole, setting a blank control group and a normal cell group, culturing at 28 ℃ for 96h, and determining the cell survival rate of CIK by using MTT colorimetric method. The results are shown in Table 1.

The cell viability rate was determined to be (test group D490nm value-blank control group D490 nm)/(cell normal group D490nm value-blank control group D490nm), and the highest concentration of the antimicrobial peptide solution at which the cell viability rate was 95% or more was determined to be the upper limit of the safe concentration.

TABLE 1 safe concentration test of antimicrobial peptides on CIK cells.

Concentration of antimicrobial peptide

1ug/g

5ug/g

10ug/g

15ug/g

20ug/g

25ug/g

30ug/g

Cell survival Rate%

99.8％

99.1％

98.3％

96.1％

95.7％

94.3％

91.7％

As can be seen from Table 1, the antibacterial peptide has a small influence on the activity of CIK cells at a concentration of 20ug/g or less, and has a large influence on the cell viability at a concentration of 25ug/g or more, and the cell viability is less than 95%.

Example 2 ex vivo antiviral effect of antimicrobial peptides.

Transferring the CIK cells to a 24-well plate, culturing in an incubator at 28 ℃ for 12h, adding GCRV virus, incubating for 48h, removing supernatant, adding 1ug/g, 5umg/g, 10ug/g, 15ug/g and 20ug/g of antibacterial peptide cell culture medium solution, and adding cell culture medium into a blank control group. We determined the virus titer at each concentration by fluorescent quantitative PCR 24h and 48h after detection of infection, the results are shown in table 2.

TABLE 2 in vitro antiviral Effect of antibacterial peptides

As can be seen from Table 2, the antimicrobial peptides have effective inhibitory effect on GCRV virus in a safe concentration range, and the inhibitory effect is enhanced with the increase of the dosage.

Example 3 effect of antimicrobial peptide-mixed feed on anti-GCRV virus.

Experimental materials: 300 tails of grass carps are used for the test, the body length is 8 +/-1 cm, the body weight is 6 +/-1 g, the grass carps are averagely divided into 6 groups, and 50 tails are respectively used for each group. The test was performed after one week of acclimation of the test cell. The feeding and test conditions are that the water temperature is 26-28 ℃, and the feed is fed twice a day.

Healthy control group: the fish were not treated and fed with normal commercial fish feed.

PBS control group: 100uL of PBS solution is injected into the abdominal cavity, and common commercial fish feed is fed.

Control group for challenge: 100uL of GCRV virus is injected into the abdominal cavity, and common commercial fish feed is fed.

10ug/g antibacterial peptide group: 100uL of GCRV virus is injected into the abdominal cavity, and the fish feed mixed with 10ug/g of antibacterial peptide is fed.

15ug/g antibacterial peptide group: 100uL of GCRV virus is injected into the abdominal cavity, and fish feed mixed with 15ug/g of antibacterial peptide is fed.

20ug/g antibacterial peptide group: 100uL of GCRV virus is injected into the abdominal cavity, and the fish feed mixed with 20ug/g of antibacterial peptide is fed.

The number of surviving individuals in each group was counted until 7 consecutive days of the experiment revealed no dead fish, and the statistical results are shown in table 3.

Table 3 survival rate effect of antimicrobial peptides on GCRV virus infected grass carp.

Item	Number of dead individuals	Number of surviving individuals	Survival rate
				Healthy control group	0	50	100％
PBS control group	1	49	98％
				Control group for counteracting toxic pathogen	43	7	14％
10ug/g antibacterial peptide group	9	41	82％
				15ug/g antibacterial peptide group	7	43	86％
20ug/g antibacterial peptide group	6	44	88％

As can be seen from table 3, when the scylla paramamosain anionic antimicrobial peptide is applied to fish feed for feeding, the effect of obviously improving the survival rate of the GCRV infected grass carp is achieved, which indicates that the antimicrobial peptide has a significant anti-GCRV virus effect in vivo, and the survival rate of the GCRV virus infected grass carp is higher with the increase of the concentration of the antimicrobial peptide.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

Although the embodiments have been described, once the basic inventive concept is obtained, other variations and modifications of these embodiments can be made by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all the modifications of the equivalent structure or equivalent flow path using the present specification, or the direct or indirect application to other related fields are included in the scope of the present invention.