CN117695295B - Application of pyridoxal phosphate in preparation of medicines for preventing or treating silkworm nuclear polyhedrosis virus diseases - Google Patents
Application of pyridoxal phosphate in preparation of medicines for preventing or treating silkworm nuclear polyhedrosis virus diseases Download PDFInfo
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- CN117695295B CN117695295B CN202311457696.6A CN202311457696A CN117695295B CN 117695295 B CN117695295 B CN 117695295B CN 202311457696 A CN202311457696 A CN 202311457696A CN 117695295 B CN117695295 B CN 117695295B
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- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 title claims abstract description 96
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- 239000011589 pyridoxal 5'-phosphate Substances 0.000 title claims abstract description 48
- 229960001327 pyridoxal phosphate Drugs 0.000 title claims abstract description 48
- 241000255789 Bombyx mori Species 0.000 title claims abstract description 47
- 241000701366 unidentified nuclear polyhedrosis viruses Species 0.000 title claims abstract description 30
- 239000003814 drug Substances 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims description 5
- 229940079593 drug Drugs 0.000 title abstract description 4
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- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses application of pyridoxal phosphate in medicines for treating or preventing silkworm nuclear polyhedrosis virus, and researches of the invention show that small molecular pyridoxal phosphate (Pyridoxal phosphate, PL P) resisting the silkworm nuclear polyhedrosis virus (BmNPV) can effectively inhibit replication of the silkworm nuclear polyhedrosis virus (BmNPV) in cells and silkworms, thereby providing a new idea for preventing and treating BmNPV infection of silkworms and laying a foundation for preventing and treating virus infection in silkworm industry production.
Description
Technical Field
The invention belongs to the field of agricultural biology, and particularly relates to an inhibition effect of pyridoxal phosphate (Pyridoxal phosphate, PLP) on silkworm nuclear polyhedrosis virus (BmNPV) and an application thereof in preventing and treating silkworm nuclear polyhedrosis virus diseases.
Background
The silkworm has important economic value as a silk-producing insect. The silkworm nuclear polyhedrosis virus (BmNPV) is one of important diseases of silkworms, and can infect silkworms to cause nuclear polyhedrosis virus diseases, also called blood type sepsis, and symptoms such as body milky white, body trunk swelling, mania crawling and the like can appear, and finally body wall liquefaction breaks and flows out of milky white pus to die.
Silkworm nuclear polyhedrosis viruses (BmNPV) are baculoviruses, and one typical feature of them is that they produce two different types of virions during the infection cycle: inclusion body derived virions (ODV) and Budding Virions (BV), which are physically and functionally different, each play a different key role in the viral infection cycle. There are two main ways of infecting the host with baculovirus: oral infection and wound infection are mainly marked by oral infection and oral infection. After being eaten orally by a host, the inclusion body OB is split in a strong alkaline environment of the midgut of the host to release ODV, and after the ODV passes through the surrounding edible membrane, the ODV is combined with microvilli of epithelial cells of the midgut to release a nucleocapsid into cytoplasm, and as the virus replicates and the nucleocapsid is assembled in the nucleus or directly passes through, the nucleocapsid is transported to a matrix membrane and budding to form progeny virions, which are called primary infection. BV then infects other cells such as tracheal and blood cells or other tissues circulating in the haemolymph, eventually leading to systemic infection, known as secondary infection. The disease causes great economic loss to the silkworm industry every year. So far, there is no effective method for prevention and treatment.
Pyridoxal phosphate (PLP) was found to inhibit various DNA polymerase-catalyzed deoxynucleotide triphosphate polymerizations isolated from viruses and e.coli as early as 1976. It was then found that DNA polymerase activity isolated from Avian Myeloblastosis Virus (AMV), lawster's leukemia virus (RLV), hepatitis B Virus (HBV) and adenovirus was inhibited by PLP. A decrease in DNA polymerase activity results in inhibition of viral DNA replication initiation.
Disclosure of Invention
At present, no effective medicine report exists for BmNPV infection, so a method capable of rapidly and efficiently inhibiting BmNPV infection is urgently needed, and a foundation is laid for silkworm anti-BmNPV virus mechanism research and silkworm nuclear polyhedrosis virus disease prevention and treatment in silkworm production.
In view of the above, the present invention aims to provide an application of pyridoxal phosphate (Pyridoxal phosphate, PLP) in the preparation of a medicament for preventing or treating silkworm nuclear polyhedrosis virus.
In one aspect, the invention provides an application of pyridoxal phosphate in preparing a medicament for preventing silkworm from infecting nuclear polyhedrosis virus (BmN PV), wherein the pyridoxal phosphate inhibits replication of the silkworm nuclear polyhedrosis virus.
In one aspect, the invention provides the use of pyridoxal phosphate in the manufacture of a medicament for the treatment of a silkworm infected with a nuclear polyhedrosis virus (BmN PV), wherein pyridoxal phosphate inhibits replication of the silkworm nuclear polyhedrosis virus.
In the present invention, pyridoxal phosphate inhibits silkworm nuclear polyhedrosis virus BmNPV at an effective concentration of 200. Mu.M in cells and 400. Mu.M in silkworms.
In the present invention, pyridoxal phosphate was used for inhibiting silkworm nuclear polyhedrosis virus BmNPV for 24 hours before virus infection.
In the invention, the medicine also comprises pharmaceutically acceptable auxiliary materials.
In the invention, the pharmaceutical dosage form can be various pharmaceutically practical dosage forms, preferably granules and liquid preparations.
The invention discloses an application of pyridoxal phosphate in preparing medicines for inhibiting silkworm nuclear polyhedrosis virus, which can inhibit the replication of the pyridoxal phosphate in cells by reasonably utilizing small molecules of pyridoxal phosphate for resisting silkworm nuclear polyhedrosis virus, and can be mixed in the food of silkworms for eating to play a therapeutic role when infection occurs; the pyridoxal phosphate can be added 24 hours before the virus infection, so that the proliferation of the pyridoxal phosphate can be inhibited, and the effect of preventing the infection can be achieved.
Advantageous effects
The invention provides a new thought for the treatment of nuclear polyhedrosis virus infection of silkworms and lays a foundation for the prevention and treatment of virus infection in silkworm production.
The pyridoxal phosphate used in the invention is the small peptide existing in the host cell, and has the advantages of good safety, low price, safety, no toxicity and good control effect.
Drawings
FIG. 1 shows the cell viability assay after addition of pyridoxal phosphate at various concentrations
FIG. 2 shows the detection of the viral VP39 gene of pyridoxal phosphate treatment on the proliferation of BmNPV in cells
FIG. 3 shows the detection of the viral VP39 protein of pyridoxal phosphate treatment on the proliferation of BmNPV in cells
FIG. 4 shows the detection of the viral VP39 gene of pyridoxal phosphate treatment on the proliferation of BmNPV in silkworms
FIG. 5 shows the detection of the viral GP64 gene of the effect of pyridoxal phosphate treatment on BmNPV proliferation in silkworms
Detailed Description
The technical scheme of the invention is clearly and completely described below with reference to the accompanying drawings and the specific embodiments. So that those skilled in the art may better understand the present invention and be able to practice it, the examples are not intended to limit it.
Pyridoxal phosphate (Pyridoxal phosphate) of the present invention is purchased from Shanghai leaf (cat# S48206); CCK-8 is purchased from white shark (cat# BS 350A); fluorescent quantitative detection kit NovoStart SYBR qPCR SuperMix kit was purchased from an offshore protein (cat# E096); proteinase K was purchased from Beijing radix (cat# RT 403); anti-beta-Tubulin antibody, goat Ant i-Rabbit IgG antibody, goat Anti-Mouse IgG antibody were purchased from Beijing full gold (accession numbers: HC101; HS101; HS201, respectively); anti-VP39 antisera was delegated to Hangzhou Huaan.
EXAMPLE 1 inhibition of silkworm Nuclear polyhedrosis Virus replication Using Pyridoxal phosphate
When pyridoxal phosphate (Pyridoxal phosphate) is used for detecting cytotoxicity, silkworm Bm N cells with good growth state are taken, plated in advance for one day, 10mM pyridoxal phosphate storage solutions with different volumes are respectively added into different holes in the next day, the final concentrations are respectively 10, 20, 50, 100, 200 and 300 mu M, and the control is taken as a culture medium, and the culture is continued for 24 hours. Next, the activity of the cells was examined using CCK-8 assay kit, and pyridoxal phosphate had no effect on the activity of the cells within 200. Mu.M as shown in FIG. 1.
When in-vitro virus infection experiments are carried out, silkworm BmN cells with good growth state are taken and plated in advance, pyridoxal phosphate is added to a final concentration of 200 mu M in the next day, the culture medium with the same volume is used as a contrast, after the culture is continued for 24 hours, 10 mu L of virus particles BV are added, after the virus and the cells are incubated for 24 hours and 48 hours, the cells are collected for detecting the virus replication condition, and the results are shown in figures 2 and 3. The results show that: pyridoxal phosphate is capable of inhibiting the proliferation of viruses in cells compared to a control group.
In vivo virus infection experiments were carried out by taking five-year old three-day healthy silkworm larvae, orally feeding 5. Mu.L of virus particle ODV, continuously feeding 24 hours with mulberry leaves, orally feeding 400. Mu.M pyridoxal phosphate with PBS for each larva, continuously feeding 24, 48 and 72 hours with PBS for comparison, extracting genome DNA from the larvae, and detecting replication of virus genes VP39 and GP64 in silkworm bodies by fluorescent quantitative PCR, wherein the results are shown in figures 4 and 5. Pyridoxal phosphate is capable of inhibiting the proliferation of viruses in silkworms, compared with a control group.
In this example, the method for detecting replication of nuclear polyhedrosis virus by fluorescence quantitative PCR is to extract total DNA of samples respectively by using genome extraction reagents, design fluorescence quantitative primers according to nuclear polyhedrosis virus genes, and use silkworm GAPDH genes as internal references, wherein the primers used for fluorescence quantitative use are as follows:
BmNPV-VP39 Forward：5’-ACTTTTCATGATGTCACTGC-3’
BmNPV-VP39Reverse：5’-AGTACTTGCAAATCGACACG-3’
BmNPV-GP64 Forward：5’-GAAGTAGAAACGCCGCATCG-3’
BmNPV-GP64 Reverse：5’-GTGGGGTATTCAGGCAGCAGT-3’
Bm-GAPDH Forward：5’-CATTCCGCGTCCCTGTTGCTAAT-3’
Bm-GAPDH Reverse：5’-GCTGCCTCCTTGACCTTTTGC-3’
Preparing a PCR reaction system by the extracted DNA and the fluorescent quantitative primer according to a fluorescent quantitative detection kit NovoStart SYBR qPCR Super Mix kit instruction, and preheating for 30 seconds by using a fluorescent quantitative PCR instrument CFX96 Real-TIME SYSTEM (Berle) under the reaction condition of 94 ℃; denaturation at 94℃for 10 seconds, annealing at 60℃for 30 seconds and fluorescence collection were performed for 40 cycles, and the detection results were analyzed by the 2- ΔΔCT method.
In this example, the method for detecting replication of nuclear polyhedrosis virus by western blot is to separate the total protein of the cell sample by SDS-PAGE, transfer it to PVDF membrane, incubate with different antibodies, and finally color-develop and detect the expression level of the protein on the membrane.
The foregoing is a further elaboration of the present invention in connection with the detailed description, and it is not intended that the invention be limited to the specific embodiments shown, but rather that a number of simple deductions or substitutions be made by one of ordinary skill in the art without departing from the spirit of the invention, should be considered as falling within the scope of the invention as defined in the appended claims.
Claims (6)
1. The application of pyridoxal phosphate in preparing medicaments for preventing silkworm from infecting nuclear polyhedrosis virus (BmNPV) is characterized in that the pyridoxal phosphate inhibits replication of the silkworm nuclear polyhedrosis virus.
2. The use of pyridoxal phosphate for the preparation of a medicament for the treatment of a silkworm infection by a nuclear polyhedrosis virus (BmNPV), characterized in that pyridoxal phosphate inhibits replication of the silkworm nuclear polyhedrosis virus.
3. The use according to claim 1 or 2, characterized in that pyridoxal phosphate inhibits the silkworm nuclear polyhedrosis virus, bmNPV, at an effective concentration of 200 μm in cells and 400 μm in silkworms.
4. The use according to claim 1, characterized in that pyridoxal phosphate is used for inhibiting the bombyx mori nuclear polyhedrosis virus BmNPV for a period of time of 24 hours before the virus infection.
5. The use according to claim 1 or 2, wherein the medicament further comprises pharmaceutically acceptable excipients.
6. The use according to claim 1 or 2, wherein the pharmaceutical dosage form is a solid granule, a liquid formulation.
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| CN113116890A (en) * | 2021-04-25 | 2021-07-16 | 西南大学 | Application of 5-Pyridoxololactone in preparation of medicine for treating silkworm nuclear polyhedrosis virus |
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| CN113116890A (en) * | 2021-04-25 | 2021-07-16 | 西南大学 | Application of 5-Pyridoxololactone in preparation of medicine for treating silkworm nuclear polyhedrosis virus |
Non-Patent Citations (4)
| Title |
|---|
| Alan Monaghan等.Pyridoxal 5'-phosphate inhibition of adenovirus DNA polymerase.《J Biol Chem. 》.1996,第271卷(第39期),第24242-24248页. * |
| MicroRNA-6498-5pInhibits Nosemabombycis Proliferationby Downregulating BmPLPP2 in Bombyx mori;Congwu Hu等;《J.Fungi》;20211208;第7卷;第1051（1-14）页 * |
| The Antiviral Molecule 5-Pyridoxolactone Identified Post BmNPV Infection of the Silkworm, Bombyx mori;Xiaoting Hua等;《Int.J.Mol.Sci.》;20210710;第22卷;第7423（1-12）页 * |
| 家蚕核型多角体病的抗性机制研究进展;徐家萍等;《中国蚕业》;20060531;第27卷(第2期);第8-14页 * |
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