Application of manganese-containing compound in preparation of medicine for preventing and treating bombyx mori nuclear polyhedrosis virus

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to application of a manganese-containing compound to preparation of a medicine for preventing and treating bombyx mori nuclear polyhedrosis virus. A large number of researches find that the manganese-containing compound can be used for preventing and treating bombyx mori nuclear polyhedrosis virus (BmNPV), and meanwhile, the weight of bombyx mori and silkworm cocoons can be increased. The discovery of the invention provides a new thought for prevention and treatment of BmNPV infection of bombyx mori, and also lays a foundation for virus infection prevention and treatment in sericulture production. The compound provided by the invention is low in medication cost, energy-saving, environment-friendly and obvious in prevention and treatment effect.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and specifically, the invention relates to application of a manganese-containing compound in preparing a drug for preventing and treating Bombyx mori nuclear polyhedrosis virus. Background Art

[0002] Bombyx mori nuclear polyhedrosisvirus (BmNPV) is one of the main pathogens of silkworm breeding. The disease often breaks out in the silkworm breeding countries in the world. It has the characteristics of strong infectivity and no specific medicine, which will cause serious economic losses every year. Among the pathogens of silkworm viral diseases, baculovirus is a toxic pathogen that infects a variety of insects. BmNPV is a typical baculovirus, belonging to group I, the main envelope protein, and BmNPV causes infection through the specific interaction between the structural protein on the ODV envelope and the host midgut cell surface receptor. At present, the prevention and control of BmNPV mainly relies on "good varieties" + "good methods", that is, selecting silkworm varieties with excellent resistance and scientific and standardized feeding methods. At present, some varieties with strong resistance to BmNPV have been obtained through molecular biology assisted breeding. At the same time, attention should be paid to the disinfection of the breeding environment and raw materials when breeding silkworms, strengthening the prevention and control of mulberry garden pests and diseases, improving the quality of mulberry leaves, strengthening the management of silkworm breeding, improving the climate environment of the silkworm room, and improving the physique of silkworms. These are measures that cannot be ignored in the prevention and control of BmNPV disease. Further research is still needed to improve the disease resistance of silkworms or to discover drugs to control BmNPV in silkworms. Summary of the invention

[0003] In the course of studying the immunology of silkworms, the inventors unexpectedly discovered through extensive testing that manganese-containing compounds can be used to prevent and treat Bombyx mori nuclear polyhedrosis virus. 2+ ) can also increase the weight of silkworm cocoons themselves.

[0004] The technical solution of the present invention is as follows:

[0005] In the first aspect of the present invention, the present invention provides the use of a manganese-containing compound in the preparation of a drug for preventing and treating Bombyx mori nuclear polyhedrosis virus, wherein the manganese-containing compound is applied to silkworms, thereby enabling the silkworms to obtain disease resistance against Bombyx mori nuclear polyhedrosis virus (BmNPV). The application is to allow the silkworms to ingest the manganese-containing compound by oral feeding.

[0006] In one embodiment, the administering comprises adding the manganese-containing compound to the silkworm's food and feeding the silkworm with the food.

[0007] In one embodiment, the applying is spraying the manganese-containing compound onto mulberry leaves, which are food for the silkworms, and using the mulberry leaves as food for the silkworms.

[0008] In the present invention, the manganese-containing compound is not particularly limited, and any manganese-containing compound that is not toxic and does not affect the growth of silkworms can be used. The preferred manganese-containing compound is a compound containing divalent manganese, which can be listed as manganese compounds including manganese chloride, manganese bromide, manganese iodide, manganese sulfate, manganese nitrate, manganese perchlorate, manganese acetate, manganese carbonate, manganese borate, manganese phosphate, manganese hydrobromide, manganese tartrate, manganese fumarate, manganese maleate, manganese lactate, manganese benzenesulfonate, manganese pantothenate, manganese ascorbate, manganese phosphate, manganese carbonate and manganese hydroxide, and any combination thereof,

[0009] In the present invention, when the manganese-containing compound is used for silkworms, Mn 2+ The concentration is 50-100uM.

[0010] In the present invention, the manganese-containing compound can be administered before infection with BmNPV or within 12 hours after infection with BmNPV.

[0011] In a second aspect of the present invention, the present invention provides the use of manganese-containing compounds in the preparation of a drug for increasing the weight of silkworm cocoons. The manganese-containing compounds include manganese chloride and manganese sulfate.

[0012] In the present invention, the drug includes pharmaceutically acceptable adjuvants in addition to the manganese-containing compound. The selection and dosage of the adjuvants are based on the principle of not having a negative impact or toxicity on the growth of silkworms.

[0013] In the present invention, the dosage forms of the drug include: powder, solid granules, and liquid preparations.

[0014] Beneficial Effects

[0015] (1) The present invention finds that Mn 2+ The solution can effectively inhibit the replication of Bombyx mori nuclear polyhedrosis virus (BmNPV) in cells and silkworms, providing a new idea for the prevention and treatment of BmNPV infection in silkworms and laying a foundation for the prevention and treatment of viral infection in silkworm production.

[0016] (2) The present invention finds that Mn 2+ When the solution acts on silkworms, it can not only enhance the silkworms' immunity against BmNPV, but also increase the silkworms' own weight and the amount of cocoons.

[0017] (3) The manganese-containing compound provided by the present invention has low cost of use, is simple and convenient to use, has more environmental value than pesticides, is energy-saving and environmentally friendly, is cheap, and has obvious control effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1, feeding mulberry leaves after adding poison, the effects of different treatments on the virus in the silkworm body and the morphology of the cocoon. Among them, A is the number of viral gDNA copies in the midgut; B is the number of viral gDNA copies in the blood (hemocyte); C is the number of viral gDNA copies in the fat body (fatbody), and D is the morphology of the cocoons of different groups.

[0019] Figure 2 , 12 hours after poisoning, feeding mulberry leaves, the concentration of MnCl2 sprayed on the mulberry leaves was 100μM, and the effects of different treatments on silkworms and cocoons were analyzed. Among them, A is the weight comparison of larvae, B is the weight comparison of silkworm pupae, C is the morbidity comparison, D is the mortality comparison, E is the cocooning rate comparison, F is the weight comparison of cocoons, G is the cocoon layer rate comparison, and H is the cocoon shell weight comparison; I is the number of viral gDNA copies in the midgut; J is the number of viral gDNA copies in the blood (hemocyte); K is the number of viral gDNA copies in the fat body (fatbody).

[0020] Figure 3 , 24 hours after poisoning, the silkworms were fed mulberry leaves, and the concentration of MnCl2 sprayed on the mulberry leaves was 100μM. The effects of different treatments on silkworms and cocoons were analyzed. A is the weight comparison of larvae, B is the weight comparison of silkworm pupae, C is the morbidity comparison, D is the mortality comparison, E is the weight comparison of cocoons, F is the cocooning rate comparison, G is the cocoon layer rate comparison, and H is the cocoon shell weight comparison. I is the number of viral gDNA in the midgut; J is the number of viral gDNA in the blood (hemocyte); K is the number of viral gDNA in the fat body (fatbody).

[0021] Figure 4 , the inhibition of viral VP39 mRNA in different parts of silkworms (midgut, blood, fat body) by different treatments (C, D, E), and the amount of gDNA (F, G, H), and the effects of different treatments on the weight of silkworms themselves and the consumption of mulberry leaves (A, B).

[0022] Figure 5 , feeding mulberry leaves after adding poison, the effects of different treatments and different concentrations of MnSO4 on silkworms. A is the weight comparison of larvae, B is the morbidity comparison, C is the mortality comparison, D is the cocoon rate comparison, F is the cocoon shell weight comparison, G is the pupa weight comparison, H is the cocoon layer rate comparison, and I is the cocoon weight comparison.

[0023] Figure 6, feeding mulberry leaves after poisoning, the effects of different treatments and different concentrations of MnSO4 on the morphology of silkworms and cocoons. Among them, A is the amount of viral gDNA in the midgut; B is the amount of viral gDNA in the blood (hemocyte); C is the amount of viral gDNA in the fat body (fatbody); D is the morphology of cocoons in different groups.

[0024] Figure 7 , feeding mulberry leaves after adding poison, the effects of different groups on the virus in the silkworm body. Among them, A is the number of viral gDNA in the midgut; B is the number of viral gDNA in the blood (hemocyte); C is the number of viral gDNA in the fat body (fatbody).

[0025] Figure 8 , feeding mulberry leaves after adding poison, the effects of different treatments on the morphology of silkworms and cocoons. Among them, A is the weight comparison of larvae, B is the weight comparison of silkworm pupae, C is the morbidity comparison, D is the mortality comparison, E is the cocooning rate comparison, F is the weight comparison of cocoons, G is the weight comparison of cocoon shells, and H is the cocoon layer rate comparison; I is the morphology of silkworm cocoons under different treatments. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in the embodiments and test examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0027] Embodiment 1:

[0028] 1.1 Reagents: MnCl2, molecular weight 197.90. Dissolution and storage solubility is 200mM. Weigh 3.9858g and dissolve in 100ml of water. When using, dilute to 50uM, 75uM, and 100uM in three gradients for experimental research.

[0029] 1.2 Materials: Medicated mulberry leaves: Pick fresh mulberry leaves, weigh a fixed weight, and use a spray bottle to spray a certain amount of 50uM, 75uM, and 100uM Mn2+ on the mulberry leaves for feeding silkworms.

[0030] 1.3 Antiviral test of different concentrations of Mn2+

[0031] (1) In vivo test in silkworms: Silkworms of the fifth instar with good growth were selected and divided into five groups, namely control group (Control), polyhedra virus group (Polyhedra), PM1 group (Polyhedra + MnCl2 50μM), PM2 group (Polyhedra + MnCl2 75μM) and PM3 group (Polyhedra + MnCl2 100μM). During feeding, the control group was fed with 5ul PBS orally; the polyhedra virus group was fed with 5ul BmNPV (concentration of 10 7 ODV), PM1, PM2, and PM3 were orally fed with 5ul BmNPV (concentration of 10 7 ODV), after adding poison, spray different concentrations of Mn 2+ The reagent (50μM, 75μM, 100μM MnCl2) was sprayed on the surface of mulberry leaves in a fixed amount and used to feed silkworms. After 24, 48, and 72 hours of feeding, the blood, fat body, and midgut of the larvae were taken for genomic DNA extraction, and the replication of the viral gene VP39 in the silkworm body was detected by fluorescent quantitative PCR. At the same time, the cocoon morphology of different groups was analyzed.

[0032] Results: From Figure 1 It can be seen that using different concentrations of Mn 2+ , it was able to inhibit the proliferation of polymorphism virus in silkworm larvae at 24h, 48h, and 72h, and it was concentration-dependent. In terms of the inhibition of the virus at different concentrations, 100μM had the best inhibitory effect on BmNPV of silkworm. From the perspective of silkworm morphology, adding the virus can lead to slow growth of silkworms, reduced cocooning rate, and reduced cocoon weight, while adding high concentrations of Mn 2+ The morphology of the group was better than that of other concentration groups and virus groups.

[0033] (2) Drug-containing mulberry leaf feeding experiment: 5-year-old silkworms with good growth were divided into 5 groups, namely control group (Control), polyhedra virus group (Polyhedra), and PM group (Polyhedra + MnCl2 100 μM). During feeding, the control group was orally fed with 5ul PBS; the polyhedra virus group and PM group were orally fed with 5ul BmNPV (concentration of 10 7 ODV), feed the silkworms normally after adding the virus, feed them with medicated mulberry leaves 12 and 24 hours after virus infection to prevent BmNPV outbreaks, and continue to feed them for 24, 48, and 72 hours, extract mRNA and genomic DNA from the larvae, and detect the replication of the viral gene VP39 in the silkworm body by fluorescent quantitative PCR. At the same time, the effects of different treatments on silkworms and cocoons were observed and counted during the feeding process of silkworms.

[0034] Results: See Figure 2 and Figure 3 It can be seen that the use of 100 μM Mn within 12 h after the outbreak 2+ , can effectively inhibit the proliferation of the virus, and the larvae weight, pupa weight, cocoon weight, cocoon layer rate, cocoon shell weight and other indicators are better than the control group, and significantly better than the virus infection group, the morbidity and mortality rate are greatly reduced. However, the control effect of the drug used 24 hours after the outbreak of the virus is poor. At the same time, the results of this example show that the addition of Mn 2+ It not only has an antiviral effect, but also can improve the weight and quality of silk cocoons.

[0035] Example 2

[0036] The silkworms of the fifth instar with good growth were divided into four groups, namely the control group, MnCl2 group, polyhedra virus group, and Polyhedra + MnCl2 100 μM group. During feeding, the control group was fed with 5ul PBS orally; the polyhedra virus group and the other groups were fed with 5ul BmNPV (concentration of 10 7 ODV), and then fed with drugged mulberry leaves after poisoning. After 24, 48, and 72 hours of feeding, the larvae were taken for RNA extraction. After reverse transcription, the expression of VP39 was detected by fluorescence quantitative analysis. At the same time, the changes in the body weight of silkworms in different groups were also evaluated.

[0037] Results: See Figure 4 The invention shows that adding Mn 2+ It can inhibit the transcription of VP39 gene in the virus. At the same time, the use of Mn 2+ Increased the weight of silkworms.

[0038] Example 3

[0039] The silkworms of the fifth instar with good growth were divided into four groups, namely the control group (Control), the polyhedra virus group (Polyhedra), the PS1 group (Polyhedra + MnSO4 50μM), and the PS2 group (Polyhedra + MnSO4 100μM). During feeding, the control group was orally fed with 5ul PBS; the polyhedra virus group, PS1 and PS2 were orally fed with 5ul BmNPV (concentration of 10 7 ODV), spraying Mn 2+The larvae were fed with mulberry leaves of different concentrations (50 μM and 100 μM) and then fed for 24, 48, and 72 hours. The blood, fat body, and midgut of the larvae were taken for genomic gDNA extraction, and the replication of the viral gene VP39 in the silkworm body was detected by fluorescence quantitative PCR. At the same time, the effects of different treatments on silkworms and cocoons were observed and counted during the feeding process of silkworms.

[0040] Results: See Figure 5 , Figure 6 The results showed that the use of 50μM and 100μM MnSO4 can also inhibit BmNPV, and is superior to the control group in terms of larval weight, silkworm pupa weight, cocoon weight, cocoon layer rate, and cocoon shell weight, and is significantly superior to the virus-infected group, with a significant reduction in morbidity and mortality.

[0041] Example 4 confirmed that Mn 2+ , rather than Cl2

[0042] The 5th instar silkworms with good growth were divided into 4 groups, namely control group, polyhedra virus group, PMS group (Polyhedra+MnCl250μM), and PN group (Polyhedra+NaCl 200μM). During feeding, the control group was orally fed with 5ul PBS; the polyhedra virus group, PMS group, and PN group were orally fed with 5ul BmNP V (concentration of 10 7 ODV), and then continue to feed by spraying PBS, MnCl250μM mulberry leaves or NaCl 200μM. After 24, 48, and 72 hours of feeding, the blood, fat body, and midgut of the larvae were taken to extract mRNA and genomic DNA, and the replication of the viral gene VP39 in the silkworm body was detected by fluorescent quantitative PCR. At the same time, the effects of different treatments on silkworms and cocoons were observed and counted during the feeding process of silkworms.

[0043] Results: See Figure 7-Figure 8 The results showed that the use of 50μM MnCl2 could significantly inhibit the proliferation of the virus in silkworms, while 200μM NaCl was close to the normal control group. The same rules were observed in the larval weight, silkworm pupa weight, cocoon weight, cocoon layer rate, and cocoon shell weight, which confirmed that the Mn in the agent was the cause of the virus proliferation. 2+ It is Cl2 that plays the role, not Cl2.

[0044] In summary, the present invention has found that 50-100uM Mn 2+ The concentration can effectively inhibit the BmNPV virus. It not only has a good effect in inhibiting the virus, but also has a promoting effect on the silkworm itself, increasing Mn 2+It promotes the weight of silkworms, the weight of cocoons also increases accordingly, and the food intake also greatly increases. Through the dissection of blood, fat body, and midgut, DNA and RNA samples are extracted for testing. The virus expression amount detected by fluorescence quantitative detection has a good inhibitory effect, which is consistent with the phenotypic performance of silkworms. It can effectively inhibit the replication of Bombyx mori nuclear polyhedrosis virus (BmNPV) in cells and silkworms.

[0045] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.

Claims

1. Use of a manganese-containing compound in the preparation of a drug for preventing and treating Bombyx mori nuclear polyhedrosis virus, characterized in that: Administration of manganese-containing compounds to silkworms enables them to acquire disease resistance against Bombyx mori nuclear polyhedrosis virus (BmNPV).

2. The use according to claim 1, characterized in that: The administration is by oral administration of a manganese-containing compound to the silkworm.

3. The use according to claim 1, characterized in that: The administration is carried out by adding the manganese compound to the silkworm's food and feeding the food to the silkworm.

4. The use according to claim 3, characterized in that: The application is to spray the manganese compound onto mulberry leaves, which are food for the silkworms, and use the mulberry leaves as food for the silkworms.

5. The use according to claim 1, characterized in that: When the manganese-containing compound is used, the Mn2+ concentration is 50-100uM.

6. The use according to claim 1, characterized in that: The manganese-containing compound is administered before infection with BmNPV, or within 12 hours after infection with BmNPV.

7. Use of manganese-containing compounds in the preparation of drugs for increasing the weight of silkworm cocoons.

8. The use according to claim 1 or claim 7, characterized in that: The manganese-containing compound is a compound containing divalent manganese. Preferably, the manganese-containing compound includes manganese chloride and manganese sulfate.

9. The use according to claim 1 or claim 7, characterized in that: The medicine also contains pharmaceutically acceptable excipients.

10. The use according to claim 1 or claim 7, characterized in that: The dosage form of the medicine is powder, solid granules or liquid preparation.

Images