CN111481541B - Application of Proline in the Prevention and Control of Bee Virus Infection - Google Patents
Application of Proline in the Prevention and Control of Bee Virus Infection Download PDFInfo
- Publication number
- CN111481541B CN111481541B CN202010255788.6A CN202010255788A CN111481541B CN 111481541 B CN111481541 B CN 111481541B CN 202010255788 A CN202010255788 A CN 202010255788A CN 111481541 B CN111481541 B CN 111481541B
- Authority
- CN
- China
- Prior art keywords
- bee
- proline
- larvae
- group
- csbv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 title claims abstract description 106
- 230000009385 viral infection Effects 0.000 title claims abstract description 23
- 230000002265 prevention Effects 0.000 title claims abstract description 13
- 239000003814 drug Substances 0.000 claims abstract description 17
- 230000035755 proliferation Effects 0.000 claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims description 47
- 239000004480 active ingredient Substances 0.000 claims description 11
- 230000002401 inhibitory effect Effects 0.000 claims description 8
- 241000030942 Sacbrood virus Species 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 241000256844 Apis mellifera Species 0.000 abstract description 139
- 241000700605 Viruses Species 0.000 abstract description 46
- 208000015181 infectious disease Diseases 0.000 abstract description 28
- 102000044503 Antimicrobial Peptides Human genes 0.000 abstract description 16
- 108700042778 Antimicrobial Peptides Proteins 0.000 abstract description 16
- 230000004083 survival effect Effects 0.000 abstract description 13
- 241000256837 Apidae Species 0.000 abstract description 4
- 238000009395 breeding Methods 0.000 abstract description 4
- 230000001488 breeding effect Effects 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 4
- 230000007124 immune defense Effects 0.000 abstract description 2
- 229960002429 proline Drugs 0.000 description 98
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 95
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 89
- 241000257303 Hymenoptera Species 0.000 description 58
- 108010002069 Defensins Proteins 0.000 description 25
- 102000000541 Defensins Human genes 0.000 description 25
- 238000001514 detection method Methods 0.000 description 25
- 108090000623 proteins and genes Proteins 0.000 description 18
- 239000006188 syrup Substances 0.000 description 16
- 235000020357 syrup Nutrition 0.000 description 16
- 108090000765 processed proteins & peptides Proteins 0.000 description 15
- 230000002829 reductive effect Effects 0.000 description 15
- 230000034994 death Effects 0.000 description 14
- 231100000517 death Toxicity 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 12
- 102000004196 processed proteins & peptides Human genes 0.000 description 12
- 241001351995 Aphomia sociella Species 0.000 description 11
- 241000256846 Apis cerana Species 0.000 description 11
- 230000000844 anti-bacterial effect Effects 0.000 description 11
- 238000011161 development Methods 0.000 description 9
- 230000018109 developmental process Effects 0.000 description 9
- 230000036039 immunity Effects 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 230000009545 invasion Effects 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 208000036142 Viral infection Diseases 0.000 description 6
- 230000007123 defense Effects 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 230000015788 innate immune response Effects 0.000 description 6
- 239000002435 venom Substances 0.000 description 6
- 231100000611 venom Toxicity 0.000 description 6
- 210000001048 venom Anatomy 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000004925 denaturation Methods 0.000 description 5
- 230000036425 denaturation Effects 0.000 description 5
- 210000000087 hemolymph Anatomy 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 238000004393 prognosis Methods 0.000 description 5
- 150000003147 proline derivatives Chemical class 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 241000317943 Acute bee paralysis virus Species 0.000 description 4
- 244000046052 Phaseolus vulgaris Species 0.000 description 4
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 235000012907 honey Nutrition 0.000 description 4
- 230000001418 larval effect Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000003053 toxin Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 230000005727 virus proliferation Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 101800003484 Apidaecin Proteins 0.000 description 3
- 241000256836 Apis Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- NUTHXVZQNRZFPR-FHDGIMILSA-N apidaecin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CN)C(C)C)C1=CC=C(O)C=C1 NUTHXVZQNRZFPR-FHDGIMILSA-N 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000008260 defense mechanism Effects 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 244000000010 microbial pathogen Species 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 101710110983 Abaecin Proteins 0.000 description 2
- 241000238876 Acari Species 0.000 description 2
- 241000358063 Apis cerana cerana Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001050320 Chronic bee paralysis virus Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 2
- 101710098413 Hymenoptaecin Proteins 0.000 description 2
- 241001163131 Israeli acute paralysis virus Species 0.000 description 2
- 229930182821 L-proline Natural products 0.000 description 2
- 241001576503 Mellea Species 0.000 description 2
- 102000015636 Oligopeptides Human genes 0.000 description 2
- 108010038807 Oligopeptides Proteins 0.000 description 2
- 241000895647 Varroa Species 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- 230000003042 antagnostic effect Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 239000003443 antiviral agent Substances 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000010138 bee pollination Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000005745 host immune response Effects 0.000 description 2
- 229960002591 hydroxyproline Drugs 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- -1 proline ester Chemical class 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 229940109850 royal jelly Drugs 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 231100000167 toxic agent Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 239000005723 virus inoculator Substances 0.000 description 2
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 241000318498 Black queen cell virus Species 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- ONIBWKKTOPOVIA-SCSAIBSYSA-N D-Proline Chemical compound OC(=O)[C@H]1CCCN1 ONIBWKKTOPOVIA-SCSAIBSYSA-N 0.000 description 1
- 229930182820 D-proline Natural products 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 241000255896 Galleria mellonella Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000258937 Hemiptera Species 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000241413 Propolis Species 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- YXWCBRDRVXHABN-JCMHNJIXSA-N [cyano-(4-fluoro-3-phenoxyphenyl)methyl] 3-[(z)-2-chloro-2-(4-chlorophenyl)ethenyl]-2,2-dimethylcyclopropane-1-carboxylate Chemical compound C=1C=C(F)C(OC=2C=CC=CC=2)=CC=1C(C#N)OC(=O)C1C(C)(C)C1\C=C(/Cl)C1=CC=C(Cl)C=C1 YXWCBRDRVXHABN-JCMHNJIXSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000000895 acaricidal effect Effects 0.000 description 1
- 239000000642 acaricide Substances 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000009341 apiculture Methods 0.000 description 1
- 229940038481 bee pollen Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- BXNANOICGRISHX-UHFFFAOYSA-N coumaphos Chemical compound CC1=C(Cl)C(=O)OC2=CC(OP(=S)(OCC)OCC)=CC=C21 BXNANOICGRISHX-UHFFFAOYSA-N 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 210000002468 fat body Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036449 good health Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000007758 mating behavior Effects 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 229940069949 propolis Drugs 0.000 description 1
- 230000019617 pupation Effects 0.000 description 1
- 229940079889 pyrrolidonecarboxylic acid Drugs 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/401—Proline; Derivatives thereof, e.g. captopril
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/30—Rearing or breeding invertebrates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/174—Vitamins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/90—Feeding-stuffs specially adapted for particular animals for insects, e.g. bees or silkworms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Physiology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Environmental Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Communicable Diseases (AREA)
- Birds (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Oncology (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Insects & Arthropods (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biochemistry (AREA)
- Botany (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及蜜蜂养殖技术领域，具体涉及脯氨酸在蜜蜂病毒感染防治中的应用。本发明发现脯氨酸能够显著提高感染中蜂囊状病毒(CSBV)的中华蜜蜂幼虫的存活率，降低其死亡率。脯氨酸能够抑制蜜蜂体内CSBV的增殖，降低病毒的拷贝数；同时，还能够诱导内源性抗菌肽的表达，提高蜜蜂的先天性免疫防御力。脯氨酸可用于制备防治CSBV感染的药物或饲料，对于中蜂囊状幼虫病的防治具有重要意义，具有良好的市场应用价值。The invention relates to the technical field of honeybee breeding, in particular to the application of proline in the prevention and control of honeybee virus infection. The present invention finds that proline can significantly improve the survival rate of Chinese honeybee larvae infected with CSBV and reduce its mortality. Proline can inhibit the proliferation of CSBV in honeybees and reduce the copy number of the virus; at the same time, it can also induce the expression of endogenous antimicrobial peptides and improve the innate immune defense of honeybees. Proline can be used to prepare medicines or feeds for preventing and controlling CSBV infection.
Description
Technical Field
The invention relates to the technical field of bee breeding, in particular to application of proline in prevention and treatment of bee virus infection.
Background
The bee is an important pollination insect in nature and has important economic value in the aspects of agricultural production and the like. About one third of the world crops need bee pollination to a certain extent, and some fruits, vegetables and the like are completely dependent on bee pollination. In addition, the bee can also produce various peak products, such as honey, royal jelly, propolis, bee pollen and the like. The quality of bee products is closely related to the health state of bees, potential safety hazards exist in the bee products produced by susceptible bees, and medicines for treating bee diseases can also be remained in the bee products to further cause the safety problems of the bee products.
The Chinese honeybee (Apis cerana) has the advantages of strong anti-mite capability, low temperature resistance and good collection of sporadic honey powder source, plays an important role in the bee-keeping industry and plays an extremely important role in maintaining the balance of a natural ecosystem. However, apis cerana are often harmed by a variety of pathogens, of which apis cerana are most seriously harmed by middle bee sacvirus (CSBV). CSBV mainly infects 1-3 day old larva, causes the larva in the bee colony to die in a large amount and can not pupate, leads to the number of worker bees newly emerged from the house to be insufficient, and then leads to the situation of the middle bee colony to be weakened, the resistance of the bee colony is reduced, and the bee colony is more easily invaded by other viruses, thereby showing the phenomenon of multi-virus common infection. CSBV can be transmitted within bee colonies by means of food mutual feeding, faecal contamination, mating behaviour and reproduction of gametes. The drone can act as a viral vector to transmit the virus to offspring through the reproductive gametes. The non-group-boundary property of the male bees can directly determine the genetic diversity of the bee colony, so that the disease resistance of the bee colony can be greatly influenced. At present, the Chinese bee sacbrood disease is mainly prevented and controlled by comprehensive prevention and control measures of changing the prince-ova, strictly disinfecting, strengthening management, preventing secondary infection by using antiviral drugs and antibiotics, but the prevention and control effect is not ideal, the harm and the spread of the virus are difficult to be effectively controlled by the common antiviral drugs, and drug residues in bee products can be caused.
When bees are infected by microorganisms or other exogenous substances, hemolymph produces a certain amount of antibacterial peptides including hymenoptera antibacterial peptide (hymenoptera antibacterial peptide), bee Defensin (Defensin), bee antibacterial peptide (Apidaecin) and bee moth antibacterial peptide (abeecin). These peptides are synthesized from fat bodies and secreted into haemolymph, which is a rapid and effective defense mechanism and can be used to rapidly kill or eliminate exogenous microorganisms. The defense mechanism is different from the immune response of animals, insects have no strict immune response mechanism, the antibacterial peptides produced by insect hemolymph can be invaded by one or more microorganisms and have no specificity, and the induction products are not only produced by specific invaded microorganisms but also have resistance to non-invaded microorganisms generally. The antibacterial peptides have different molecular weights, chemical structures and antibacterial mechanism effects, and different antibacterial peptides can resist different bacteria, fungi, viruses, tumor cells and the like, wherein the bee antibacterial peptide is the main antibacterial peptide of bees, and the hymenoptera antibacterial peptide is the most important supplementary peptide in the bee antibacterial peptide and mainly plays an inhibiting role on part of gram-negative bacteria generating resistance to the bee antibacterial peptide; the bee moth antibacterial peptide is used as a backup peptide of the bee antibacterial peptide, and only plays a role when the bactericidal abilities of the bee antibacterial peptide, the bee antibacterial peptide and the hymenoptera antibacterial peptide are lost. The bee defensin has the least expression amount and is the only antibacterial peptide for inhibiting gram-positive bacteria in bee hemolymph. The content of the bee antibacterial peptide is influenced by different factors, including bacteria, fungi, baby bugs, viruses, bee mites, medicaments and the like. The antibacterial peptide plays a role in resisting bee viruses, and researchers find that: when the bee is inoculated with Acute Bee Paralysis Virus (ABPV), the expression level of the antibacterial peptide gene is obviously increased; in the midgut epithelial tissue of the bee, the infection level of the bee residual wing virus (DWV) and the expression level of the bee antibacterial peptide gene are linearly related; after the bee is infected by the virus, the expression levels of the bee antibacterial peptide, the hymenoptera antibacterial peptide and the bee moth antibacterial peptide in the bee body are obviously reduced. Bee mites can also affect the expression of bee antimicrobial peptides. In the aspect of medicine, researchers find that the acaricide can significantly influence the expression of the bee antibacterial peptide gene, after bees contact the flumethrin vinegar, the expression level of the hymenoptera antibacterial peptide is significantly up-regulated, and the coumaphos can down-regulate the expression of the hymenoptera antibacterial peptide and the bee moth antibacterial peptide gene. In conclusion, the bee antibacterial peptide is a key part of humoral immunity of bees, and plays a role in coordination to resist invasion of various pathogenic microorganisms.
Chinese patent CN103524602A discloses the treatment of bee sacbrood by administering exogenous antibacterial peptide, but the administration of exogenous antibacterial peptide still faces the potential safety risk of antibacterial peptide drugs, the mass production of antibacterial peptide is limited, the production efficiency is low and the production cost is high (linchengde, penhongjuan, wang derived sea. the application and existing problems of antibacterial peptide, tropics medical journal 2007,1 (7): 86-90.). Therefore, the development of a high-efficiency prevention and treatment method for the Chinese sacbrood disease is of great significance to the bee breeding industry.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide the application of proline in preventing and treating bee virus infection.
Proline (Pro or P) according to the invention is a compound known in the art, known under the chemical name pyrrolidone carboxylic acid, and is a cyclic imino acid. Proline has three forms, L-proline and D-proline, and the structural formula of proline is as follows:
the invention provides the use of proline or a derivative thereof for the preparation of a medicament for the treatment or prevention of a viral infection of honey bees.
The invention provides the use of proline or a derivative thereof for the preparation of a feed for the treatment or prevention of a viral infection of bees.
The invention provides application of proline or a derivative thereof in inhibiting bee virus proliferation.
The application of the proline or the derivative thereof in inhibiting the bee virus proliferation can be used for inhibiting the virus proliferation in apparently healthy bees and can also be used for inhibiting the virus proliferation in virus disease bees.
The virus of the invention is preferably a Chinese bee sacbrood virus.
Experiments prove that the survival rate of Chinese bee larvae infected with Chinese vesicular viruses can be remarkably improved by the proline, on one hand, the proline can directly act on the viruses to reduce the copy number of the viruses, and on the other hand, the proline can also induce the expression of endogenous antibacterial peptides to enhance the innate immunity defense of organisms to resist the viruses and reduce the morbidity of infected larvae.
According to the effect of proline on bee virus infection, substances containing proline structure, capable of being metabolized in vivo to produce proline (including proline salt, proline ester, oligopeptide or polypeptide containing proline except antibacterial peptide, etc.) or other proline derivatives (such as hydroxyproline) should have the same effect.
The proline derivative comprises hydroxyproline, proline salt, proline ester, proline-containing non-antibacterial peptide oligopeptide or polypeptide.
Experiments prove that proline can induce the transcription and translation of endogenous antibacterial peptide genes, the expression level of endogenous antibacterial peptides (such as hymenoptera antibacterial peptide, bee defensin, bee antibacterial peptide and bee moth antibacterial peptide) is improved, and the immunity of an organism is further improved.
Therefore, the invention provides the application of proline or a derivative thereof in preparing a medicament for improving the immunity of bees. The invention also provides application of the proline or the proline derivative in preparing feed for improving the immunity of bees.
The improvement of the bee immunity is realized by improving the expression quantity of endogenous antibacterial peptide of the bee.
The endogenous antimicrobial peptide of the invention can be any one or more selected from hymenoptera antimicrobial peptide, bee defensin and bee antimicrobial peptide.
The active ingredient of the medicament or feed comprises one or more selected from proline and derivatives thereof.
The invention also provides a product for the treatment or prevention of a viral infection of honey bees, the active ingredient of which comprises one or more selected from proline and its derivatives; the product is a medicament, feed or feed additive.
The active ingredient of the medicament of the present invention may comprise proline or its derivatives only; may also comprise a plurality selected from proline and its derivatives; may also contain other active ingredients besides proline and its derivatives (for example, other active ingredients capable of improving the immunity or antiviral ability of bees).
The medicine of the invention can also contain auxiliary materials allowed in the field of pharmacy; the dosage form of the medicament can be any dosage form allowed in the field of pharmacy.
The feed of the present invention may comprise proline or a derivative thereof, and may also comprise a plurality selected from proline and a derivative thereof; may also contain other active ingredients besides proline and its derivatives (e.g. other active ingredients capable of improving the immunity or antiviral ability of bees); in addition to the above active ingredients, the feed according to the invention may also contain nutrients required by the bees, such as: pollen, royal jelly, fructose, glucose, sucrose, soybean flour, yeast powder, skimmed milk powder, inorganic salt, vitamins, water, etc.
The feed of the invention can be solid feed, semi-solid feed or liquid feed.
In the application of the proline provided by the invention, the dosage of the proline given to bees is preferably more than or equal to 15 ng/bee/time. More preferably 1 to 10. mu.g/unit/time. The time interval of each proline administration is preferably 22-24 h.
Specifically, for 3-day-old bees, 15 ng-1.5 mug/bee/time; 22.5 ng-2.25 mug of bees at the age of 4 days per bee; 37.5 ng-3.75 mug of bees in 5 days old per bee per time; 6-day-old bees 60 ng-6 mug/bee/time.
The invention has the beneficial effects that: the invention discovers for the first time that proline can obviously improve the survival rate of Chinese bee larvae infected with CSBV and reduce the death rate of Chinese bee larvae. On one hand, proline can inhibit the proliferation of CSBV and reduce the copy number of virus; on the other hand, proline can also induce the expression of endogenous antibacterial peptide and improve the innate immunity defense of bees. The experimental result of the invention shows that the mortality of Chinese bee larvae infected with CSBV is greatly reduced (can be reduced by 5.13 times at most) after proline intervention, and the survival larvae can pupate and emerge into adult bees; the virus copy number in the larvae of the bee which are infected with CSBV after proline intervention is greatly reduced (the maximum can be reduced by 20000 times), and the larvae show an energy-efficiency relationship, and meanwhile, the proline intervention can improve the virus infection degree of apparent healthy larvae and is beneficial to the normal growth and development of the larvae; the expression levels of hymenoptera antimicrobial peptide, bee defensin and bee antimicrobial peptide in Chinese bee larva infected with CSBV after proline intervention are all obviously up-regulated.
The new function of the proline provided by the invention on bee virus infection provides a basis for the research of bee immune defense mechanism. The proline can be applied to preparation of medicines or feeding materials for preventing and treating Chinese bee sacbrood or improving the immunity of bees, provides a new method for preventing and treating the Chinese bee sacbrood, and has good market application prospect.
Drawings
FIG. 1 is a graph showing the statistical results of the survival rate of 6-day-old larvae after proline desiccation in Experimental example 1 of the present invention; wherein CK represents the control group, 0.75 represents the low concentration group of the preparation group, 7.5 represents the medium concentration group of the preparation group, 75 represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV +0.75 represents the low concentration group of the intervention group, CSBV +7.5 represents the medium concentration group of the intervention group, and CSBV +75 represents the high concentration group of the intervention group.
FIG. 2 is a statistical result of mortality of larvae of 4-6 days old after proline intervention in Experimental example 1; wherein CK represents the control group, 0.75 represents the low concentration group of the preparation group, 7.5 represents the medium concentration group of the preparation group, 75 represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV +0.75 represents the low concentration group of the intervention group, CSBV +7.5 represents the medium concentration group of the intervention group, and CSBV +75 represents the high concentration group of the intervention group.
FIG. 3 shows the results of the detection of the viral copy number of larvae of 4-6 days old after proline intervention in Experimental example 2; wherein CK represents the control group, 0.75 represents the low concentration group of the preparation group, 7.5 represents the medium concentration group of the preparation group, 75 represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV +0.75 represents the low concentration group of the intervention group, CSBV +7.5 represents the medium concentration group of the intervention group, and CSBV +75 represents the high concentration group of the intervention group.
FIG. 4 shows the pupation of healthy larvae and post-contaminated proline-intervened larvae in Experimental example 3 of the present invention; wherein A is proline intervention group (75 μ g/ml); b is a Control (CK) group.
FIG. 5 shows the expression level of Apis mellifera antimicrobial peptide (Apidacin) for prognosis of Poison-infected and proline-dried in Experimental example 4 of the present invention; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
FIG. 6 shows the expression levels of hymenoptera antimicrobial peptide (Hymenoptaecin) for prognosis of post-infectious and proline in Experimental example 4 of the present invention; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
FIG. 7 shows the expression level of the apis cerana walker antimicrobial peptide (Abaecin) for post-infection and post-proline prognosis in Experimental example 4 of the present invention; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
FIG. 8 shows the amount of bee Defensin (Defensin) expression for post-contamination and proline block prognosis in Experimental example 4 of the present invention; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Statistical analysis of the data in the following examples was performed using SPSS 22.0 (SPSS corporation, chicago, usa). The effect of proline on apis cerana larvae is expressed as "Mean ± SE". And comparing the change of the virus copy number infected with CSBV in the Chinese bee larva by adopting an analysis of variance and an LSD multiple analysis of variance method.
Example 1 intervention of healthy Apis cerana larvae and Poison larvae with proline
1. Sample supply book
The Apis cerana Fabricius is from Apis cerana Fabricius research institute of China academy of agricultural sciences. In order to obtain 2-day-old larvae, after queen bees are closed on the son spleens to lay eggs for 96 hours, the son spleens containing the 2-day-old larvae are taken out from the bee colony and put into a 24-well plate, and the plate is placed in a constant temperature and humidity incubator with the temperature of 32 +/-1 ℃ and the relative humidity of 75 +/-5% RH for pre-culture.
The presence or absence of CSBV and other viral infections (including BQCV, DWV, IAPV, KBV, ABPV, CBPV) in the larvae was determined by RT-PCR, and healthy larvae without viral infections were used as samples in this example.
The primer sequences provided in the above-mentioned detection primer references for various viruses are as follows: CSBV detection primers were referenced to Grabenstein, E., W.Ritter, M.J.Carter, S.Davison, H.Pechhalker, J.Kolodziejek, O.Boeckking, I.Derakshifar, R.Moosbeckhofer, E.Licek, and N.Nowotny.Sacbrood virus of the honeybee (Apis mellea): rapid identification and phylogenetic analysis conversion-PCR.clin.Diagen. Lab.Immunol.2001,8: 93-104; BQCV detection primer references M, Benjeddou, N, Leat, M, Allsop, S, Davison.detection of acid bean analysis virus and black queen cell virus from the host genes by reverse transcription transcriptional pcr.applied and environmental microbiology.2001,67(5): 2384-; DWV detection primer refer to Tentcheva, D., L.Gauthier, S.Jouvee, L.Canaday-Rochelle, B.Dainat, F.Counters, M.E.Colin, B.V.Ball, and M.Berginin.Polymeraschen reaction detection of formed with virus (DWV) in Apis melifera and Varroa detector. idiology.2004, 35: 431-; IAPV detection primers are referred to Eyal, Maori, Shai, Lavi, Rita, Mozes-Koch, Yulia, Gantman, Yuv al, Peretz, Orit, Edelbaum, Edna, Tanne, Ilan, Sela. isolation and characterization of analysis resources, a direct infection of genes in an array, identification for direction product to intra-and inter-site recognition, the Journal of general vision, 2007,88(Pt 12): 3428-38; KBV detection primers were Stoltz, D., X.R.Shen, C.Boggis, and G.Sisson.molecular diagnostics of Kashmir bean virus infection.J.apic.Res.1995,34: 153-; ABPV detection primer refer to Bakonyi, T., R.Farkas, A.Szendroi, M.Dobos-Kovacs, and M.Rusvai.detection of acid bean analysis virus by RT-PCR in the gene bean and Varroa derivative field samples, screening of sensing human diagnosis experiments.Apidiologue.2002, 33: 63-74; CBPV detection primers are referred to Ribie're, M., C.Triboultot, L.Mathieu, C.Aurie ' res, J.P.Faucon, and M.Pe ' pin.molecular diagnostic of molecular be medical science in Apidiologie, 2002,33: 339-.
The detection method specifically comprises the following steps: RNA was extracted using RNeasy mini kit (edley) according to the product instructions (the method includes a step of removal of DNA, thus ensuring that only RNA is present in the final extract). Total RNA was eluted in 30. mu.L of elution buffer and used directly for RT-PCR. First strand cDNA was generated immediately using the extracted RNA using the Quantitec reverse transcription kit (Takara) (according to the product instructions) and the cDNA was stored at-20 ℃. The PCR amplification reaction is a 50 mu L system; PCR reaction conditions included pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 15 seconds, and 35 cycles; extension for 5min at 72 ℃. The results showed that no CSBV and other viruses were detected.
2. Preparation of syrup
Preparation and feeding of the syrups are carried out according to the Standard for feeding Apis mellifera in 2016 and adjusted on this basis (Karl Crailsheim, Robert Brodschneider, Pierrick Aupinel, Dieter Behrens, Elke Genersch, Jutta Vollmann & Ulrike Riessberger-Gall. Standard methods for identifying the real area re-addressing of Apis mellea. journal of Apimulturral Research,2013,52(1): 1-15.). The formula, ratio and dosage of the syrup fed to the larvae of different ages of days are shown in table 1.
TABLE 1 syrup formulation, ratio and dosage to feed
3. Preparation of virus liquid
Chinese bee sacbrood with typical sacbrood morphology is selected from another Chinese bee farm of the bee institute of Chinese agricultural academy of sciences, and the existence of CSBV in the Chinese bee body is confirmed by RT-PCR. To obtain CSBV, 210 larvae infected with CSBV were taken, placed in a sterile triturator and 2100 μ L sterile Phosphate Buffered Saline (PBS) was added and triturated. Grinding, centrifuging at 8000rpm at 4 deg.C for 30min, repeating for 2 times, collecting supernatant, performing RT-PCR detection with cDNA reverse transcribed after RNA extraction as template, and storing the supernatant at-80 deg.C as original venom without other viruses.
Virus detection Using the Absolute quantitative PCR method (K.M.Hong, H.Najjar, M.Hawley, R.D.Press.quantitative real-time PCR with automatic sample preparation for diagnosis and monitoring of genomic infection in bone matrix genomic infection. clinical chemistry.2004,50(5):846 856; HU Zhi Gang, CHEN Ke Ping, YAO Qin, GAO Gui Tian, XU Jia-Ping, CHEN Hui-Qing.cloning and Characterification of genomic movement PP-BP, Gene Induced by Viral infection. acta.acta.acta.2005, En.48: 875)
PCR reaction in BIOER LineGene9600 real-time PCR lineThe method is carried out in a unified way, and the upstream primer and the downstream primer are respectively as follows: 5'-ccttggagtttgctatttacg-3', and 5'-cctacatccttgggtcag-3'. The real-time fluorescent quantitative PCR reaction system is 15 mu L, comprises 7.5 mu L of reaction solution of 0.3 mu L, SYBR of each forward primer and reverse primer, 1 mu L of template and water till 15 mu L. The qPCR reaction conditions adopt a two-step method: the first step comprises a constant temperature section (denaturation at 95 ℃ for 3min) and a circulation section (denaturation at 95 ℃ for 5 s; annealing at 60 ℃ for 30s, 40 cycles); the second step is a melting section (denaturation at 95 ℃ for 15 s; annealing at 60 ℃ for 5 s; denaturation at 95 ℃ for 15 s.). The concentration of the original venom was 6.74X 10 by qPCR detection4copies/. mu.L, this original venom was used for subsequent vaccination experiments.
Preparing the toxic syrup: IC based on Pre-test Virus infection50And (3) detecting results, selecting the feed venom according to the proportion of the original venom: syrup (syrup composition see table 1) ═ 1: 3, preparing a mixture of 5 μ L virus stock solution and 15 μ L syrup, i.e. CSBV of 1.685 × 104copies/. mu.L of toxic syrup. The toxicant-containing syrup was fed to 3-day-old test larvae.
4. Prolinamic acid formulations
Purchasing L-proline standard (source leaf product), preparing 1mg/mL mother liquor with high-purity water, filtering, and sterilizing for later use. In the test, the syrup of each day age in Table 1 was diluted in multiple proportions as required to obtain a proline preparation, and a low concentration group (0.75. mu.g/mL), a medium concentration group (7.5. mu.g/mL) and a high concentration group (75. mu.g/mL) were provided, respectively.
Preparation of the toxin-containing preparation: the original venom was mixed with a proline preparation (syrup composition see table 1) at a ratio of 1: 3 to obtain a mixture of 5. mu.L of virus stock and 15. mu.L of proline preparation, i.e. CSBV of 1.685X 104copies/. mu.L of the toxin-containing preparation. The toxicant-containing formulation was fed to 3-day-old test larvae.
5. Proline intervention test
The larvae after preculture for 1 day (3 days old) were taken out of the incubator, and the larvae with good health status were placed in 48-well plates, and randomly divided into a control group, an infected group, a preparation group and an intervention group, each group consisting of 24 samples, and the steps were repeated three times. Grouping and feeding are shown in table 2, and the composition of the syrup in table 2 is shown in table 1 (control day age).
TABLE 2 proline intervention test grouping
The optimal time for bee larvae to infect CSBV is 2-3 days old, so that CSBV infection and proline intervention treatment are carried out when the larvae are 3 days old, and the prevention and treatment effects of proline on the CSBV infection of bees are analyzed. Feeding the infection group with the syrup containing toxin (preparation method is described in the above 3), and feeding the intervention group with the preparation containing toxin (preparation method is described in the above 4); and respectively feeding syrup or proline preparations to the larvae of 4-6 days old according to groups, and taking the larvae fed with syrup as CK control (the feeding formulas and feeding amounts of the larvae of different days old refer to table 1). The above fed food is placed on one side of the bottom of the culture plate to avoid contacting with larvae. Feeding was done every 24h and feeding and mortality of larvae were observed and recorded.
Forceps were sterilized with 75% ethanol (prepared with DEPC-treated water) for 5min and rinsed three times with DEPC-treated water, 5 each of the differently treated live larvae were removed each day, immediately treated with liquid nitrogen for freezing, and then stored in a-80 ℃ freezer for subsequent viral copy number detection.
Experimental example 1 survival and mortality analysis after proline intervention in healthy and infected larvae
1. Survival rate
Survival rate statistics is carried out on each group of bee larvae in example 1, the results are shown in fig. 1, and the results show that the survival rate of larvae of CK group and preparation group fed conventionally can reach over 86%, which indicates that proline intervention does not cause abnormal death of healthy larvae and does not affect growth and development of healthy larvae.
The larvae of the infected group die in a large amount, the survival rate is only 43.05 percent, the survival rate of the larvae of the proline intervention group is greatly improved to more than 77 percent, the survival rate of the larvae of the high-concentration group (75 mu g/mL) in the 6-day-old infected larvae is the highest and reaches 88.89 percent, and the result shows that about 50 percent of the infected larvae can be prevented from dying due to illness by adopting the proline intervention, so that the infected bee colony is prevented from collapsing, and the proline intervention plays an important role in recovering and breeding the bee colony.
2. Mortality rate
Mortality statistics were performed on each group of bee larvae in example 1, and the mortality of the larvae was determined as follows: the death numbers of the larvae in the pre-culture (2 days old) and the virus inoculation day (3 days old) are not counted and analyzed to eliminate the influence of mechanical death of the larvae in the experimental process, the death numbers of the larvae are counted at regular time every day from the next day (4 days old) of virus inoculation, the death larvae are removed, and the steps are repeated until the 6 days old is finished.
Larval mortality per day-number of larvae dead per day/number of larvae alive per day x 100%;
overall larval mortality rate-number of larval deaths/total number of samples x 100%.
The statistical results of the mortality of all groups are shown in fig. 2, and the results show that the mortality of 4-6-day-old larvae in CK groups fed conventionally is lower than 3% every day, and the mortality of 4-6-day-old larvae after being dried by proline at various concentrations is not greatly increased every day, which indicates that the growth and development of healthy larvae are not influenced by proline intervention, and the feed can be used for auxiliary feeding of larvae.
CSBV is a typical induction source, the death rate of larvae at the next day (4 days old) after 3 days old larvae are infected with virus is greatly increased and reaches 25%, the death rate at 5 days old and 6 days old is reduced, but the death rate per day is more than 20%, and only 43% of experimental larvae survive by 6 days old (figure 1). The study of Liang Qin et al (Liang Qin, Chendafu, bee protection science [ M ]. China agricultural Press, 2009.2-3.) found that CSBV is most susceptible to 2-3 days old larvae, the infected larvae cannot pupate and die in large quantities in the later stage of the larvae, which is consistent with the experimental results of the present invention.
Compared with the infected group, the death rate of the larvae of each day age in the proline intervention group is greatly reduced, particularly the death rate of the larvae of 4 days age is less than 10%, and the survival rate of the larvae of 6 days age is higher than 77% (shown in figure 1).
Experimental example 2 detection of viral copy number following proline intervention in healthy and infected larvae
CSBV test was performed on the apparently healthy 4-6 day old Apis cerana larvae (CK group) in example 1, and the number of Virus copies per day old larvae was found to be 66-70, which indicated that apparently healthy larvae were also infected with Virus, but showed no obvious symptoms, indicating that the Virus was universally present in bee colonies and that the larvae could live with Virus in a certain concentration, which is consistent with the results of Liu san (Shan, L., Liuhao, W., Jun, G., Yujie, T., Yanping, C., Jie, W., Jilian, L., Chinese Sacbroud infestations in Asian Honey Bees (Apis cerana cerana cerana cerana) and Host response to the Virus Infect, Journal of the ecology Patch.2017, and htx.20125/3509. The virus copy number of the internal bodies (preparation groups) of the larvae of the 4-6-day-old Apis cerana Fabricius with the apparent healthy proline intervention prognosis is reduced to below 14, which indicates that the virus infection degree of the apparent healthy larvae can be improved by proline intervention, and the normal growth and development of the larvae are facilitated.
The virus copy number in the larvae of the infected group grows in an explosive manner, and the virus copy number in the larvae of 4 days old is as high as 12.1 multiplied by 104Is 1700 times of CK group, so the multiplication of a large amount of virus leads to the death of infected larvae, the death rate is up to 25 percent, the virus copy number in bodies of 5 days old and 6 days old is reduced along with the growth and development, but the virus copy number is still maintained at 2.3 multiplied by 104Above, it was shown that the single-day larval mortality rate was still above 20% (fig. 3). The virus copy number in the larvae of 4 days old in each proline intervention group with different concentrations is reduced to less than 22, the virus copy number in the larvae of 5 days old and 6 days old is reduced to about 10, and no CSBV detection can be detected, which shows that strong antagonistic virus reaction is generated in the infected larvae after proline feeding, the infection degree of the virus to the larvae is obviously reduced, and the fatality rate of the infected larvae is reduced.
EXAMPLE 3 morphological analysis of proline after intervention in healthy and infected larvae
On the 18 th day (22 th day from the egg stage) of the experiment of example 1, the states of the larvae of each group in example 1 were observed with a stereomicroscope and recorded by photographing.
Morphological observation results are shown in FIG. 4, in which the larvae in the virus-infected group did not pupate even though they did not die, but the larvae in the CK group and post-infection proline intervention group (75. mu.g/ml) pupate and eclose into adult bees, and the life activities were very active. The CSBV infection is characterized in that 1-3 day old larvae are infected, so that the larvae in bee colonies die in large quantity and cannot pupate, healthy larvae pupate and emerge into adult bees, which is the normal development process of honey, and infected CSBV larvae can pupate and emerge into adult bees, which shows that proline intervention plays a role in antagonizing viruses, so that infected larvae can develop normally.
Experimental example 4 influence of CSBV infection and proline intervention on the expression level of genes involved in larva immunity
To analyze the effect of CSBV Infection and proline intervention on the Immune system of bee larvae, the expression levels of the antimicrobial peptide genes in each group of bee larvae in example 1 were examined by real-time fluorescent quantitative PCR using β -actin as an internal reference gene (Shan, L., Liuhao, W., Jun, G., Yujie, T., Yanping, C., Jie, W., Jilian, L., Chinese Sacbrood Virus Infection in Asian Honey Bees (Apis cerana) and Host Immune Responses to the Virus Infection, Journal of Invertebrate Pathology.2017, doi: http:// dx. doi.org/10.1016/j.jj.2017.09.006). The detection result is specifically as follows:
1. expression detection of bee antibacterial peptide (Apidaecin) gene
The bee antibacterial peptide (Apidaecin) is the main antibacterial peptide of bees, the detection result is shown in fig. 5, the expression quantity of the bee antibacterial peptide of the larvae of the control group (CK) is only 1.38-1.74, the expression quantity of the bee antibacterial peptide is increased in the larvae after the larvae are infected with CSBV for 24 hours and reaches 30, but the relative expression quantity of the bee antibacterial peptide of the larvae of 5-6 days old falls back and is maintained at about 20.
The proline intervention (preparation group) on healthy larvae can cause the up-regulation of the expression quantity of the bee antibacterial peptide, and the maximum relative expression quantity is up-regulated to about 46; and (3) applying proline intervention (intervention group) to the infected larvae, so that the expression quantity of the bee antibacterial peptide is greatly increased, the expression quantity of the bee antibacterial peptide of the larvae of 5 days old and 6 days old is mainly increased, and the highest single-day expression quantity can reach about 112.
The result shows that the exogenous approach intervenes by proline to strongly induce the expression capacity of endogenous bee antibacterial peptide, enhance the innate immunity defense of the larva to resist CSBV invasion and reduce the fatality rate. The above results indicate that proline has the ability to strongly induce the expression of endogenous honey bee antimicrobial peptide from honey larvae to antagonize the invasion of CSBV to the larvae.
2. Hymenoptera antibacterial peptide (Hymenoptaecin)
Hymenoptera antimicrobial peptides are the most important supplementary peptides in bee antimicrobial peptides. The detection result of the hymenoptera antibacterial peptide is shown in fig. 6, the relative expression quantity of the hymenoptera antibacterial peptide of the larvae in the Control (CK) group is only 1.31-1.56, the expression quantity of the hymenoptera antibacterial peptide in the larvae is increased to about 22 after the larvae are infected with CSBV for 24 hours, the expression quantity of the larvae aged 4-6 days is not obviously changed, and the expression quantity is maintained at 20.47-21.86.
The proline intervention (preparation group) on healthy larvae can cause the expression level of the hymenoptera antibacterial peptide to be obviously increased, the maximum increase range of the expression level of 5-day-old larvae can be up to about 370, the expression level of the hymenoptera antibacterial peptide is increased along with the increase of the proline administration amount, and a good dose-effect relationship is shown; and (3) applying proline intervention (intervention group) to the infected larvae, so that the expression quantity of the hymenoptera antibacterial peptide is greatly increased, the expression quantity of 5-day-old larvae is increased to the maximum, and the maximum expression quantity can reach about 450.
The result shows that the exogenous approach intervenes by proline to strongly induce the expression capacity of endogenous antibacterial peptide hymenoptera antibacterial peptide, enhance the innate immunity defense of larvae to resist CSBV invasion and reduce the fatality rate.
3. Bee moth antibacterial peptide (Abaecin)
The bee moth antibacterial peptide is used as a backup peptide of the bee antibacterial peptide, and only plays a role when the bactericidal capability of the bee antibacterial peptide and the hymenoptera antibacterial peptide is lost.
The detection result of the bee moth antibacterial peptide is shown in figure 7, the relative expression quantity of the bee moth antibacterial peptide of the larvae of the control group (CK) is only 1.52-3.68, after the larvae are infected with CSBV for 24 hours, the expression quantity of the bee moth antibacterial peptide in the larvae is obviously increased to 73.25, but the expression quantity of 5-6 days old falls back to 25.4-26.97.
The proline intervention (preparation group) on the healthy larvae can cause the expression level of the bee moth antibacterial peptide to be up-regulated to about 25; proline intervention (intervention group) on the infected larvae can greatly reduce the bee moth antibacterial peptide expression quantity which is increased by the infection.
4. Bee Defensin (Defensin)
The detection result of the bee defensin is shown in figure 8, the relative expression quantity of the bee defensin of the larva in the Control (CK) group is only 1.32-1.62, the expression quantity of the bee defensin in the larva is increased after the larva is infected with CSBV for 24 hours, the maximum expression quantity of the bee defensin reaches 46.26, the expression quantity of the bee defensin in 5 days is 52.59, and the bee defensin in 6 days falls back to 22.28.
The proline intervention (preparation group) is given to healthy larvae to cause the up-regulation of the bee defensin expression quantity, and the bee defensin expression quantity of the larvae of 6 days old is the highest and can be up-regulated to about 77; and (3) applying proline intervention (intervention group) to the infected larvae greatly up-regulates the expression quantity of the bee defensin, and the maximum expression quantity of the bee defensin can reach about 130 by using 6-day-old larvae.
The bee defensin is the only antibacterial peptide in bee hemolymph for inhibiting gram-positive bacteria, but the expression amount is the least. According to the invention, the expression capacity of endogenous antimicrobial peptide bee defensins is strongly induced through proline intervention, the innate immunity defense of larvae is enhanced to resist CSBV invasion, and the fatality rate is reduced. The above results indicate that proline has the ability to strongly induce the expression of bee defensin, an endogenous antimicrobial peptide of infected larvae.
According to the invention, the dynamic change of the expression of the Immune related genes in larvae of 4-6 days old of Chinese Bees infected with CSBV is analyzed, and the expression level of the antibacterial peptide genes in the infected larvae is higher than that of healthy larvae on the whole, which indicates that the infected CSBV can promote the expression of the Immune related genes in the larvae of the Bees, and the result is matched with the research result of Liu-san (Shan, L., Liuhao, W., Jun, G., Yujie, T., Yanping, C., Jie, W., Jiian, L., Chinese Sacbrood Virus Infection in Asian Home Bees (Apis cerana cerana) and Host Immune Responses to the Virus Infection, Journal of Inverteby Pathology.2017, and phi: http:// dx.doi.org/10.1016/j.j.006).
The invention takes Chinese bee infected with CSBV and healthy Chinese bee as research objects, and utilizes a fluorescent quantitative PCR method to quantitatively detect and compare the expression conditions of 4 antibacterial peptide genes in the disease-sensitive and healthy bee larva development stages, thereby defining the immune response condition of the Chinese bee after CSBV infection; and quantitatively detecting and comparing the expression conditions of 4 antibacterial peptide genes after proline is given to sick and healthy bee larvae: the proline intervention strongly induces the expression capacity of endogenous antibacterial peptides of infected larvae, namely hymenoptera antibacterial peptide (Hymenoptacin), bee Defensin (Defensin) and bee antibacterial peptide (Apidacin). The high expression of the three antibacterial peptides for one day or more resists the invasion of CSBV to larvae, reduces the fatality rate of the larvae, promotes the larvae to pupate and emerge into adult bees, and is an effective method for enhancing the immunity of the larvae of the Chinese bees and resisting CSBV infection; meanwhile, the proline intervention can also up-regulate the expression level of the healthy Chinese bee larva antibacterial peptide to form a quick and effective defense mechanism for quickly killing or eliminating exogenous pathogenic microorganisms and protecting the growth and development of the larva.
In conclusion, proline can inhibit the proliferation of CSBV and reduce the copy number of CSBV in bees; can also induce the expression of the endogenous antibacterial peptide of the bee, improve the innate immunity defense of the bee, resist the invasion of the larva by the CSBV in vivo and prevent the further infection of the CSBV and other pathogenic microorganisms, thus being used for preventing and treating the sacbrood disease of the bee.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> bee institute of Chinese academy of agricultural sciences
Application of <120> proline in prevention and treatment of bee virus infection
<130> KHP201110402.1
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ccttggagtt tgctatttac g 21
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cctacatcct tgggtcag 18
Claims (6)
1. Use of proline in the manufacture of a medicament for the treatment or prevention of a Chinese bee-saclike virus infection.
2. The use according to claim 1, wherein the active ingredient of the medicament comprises proline.
3. Use of proline in the preparation of a feed for the treatment or prevention of a Chinese bee-saclike virus infection.
4. Use according to claim 3, wherein the active ingredient of the feed comprises proline.
5. Use of proline in the preparation of a medicament or feed for inhibiting the proliferation of Chinese bee sacbrood virus.
6. Use according to claim 5, wherein the active ingredient of the medicament or feed comprises proline.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010255788.6A CN111481541B (en) | 2020-04-02 | 2020-04-02 | Application of Proline in the Prevention and Control of Bee Virus Infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010255788.6A CN111481541B (en) | 2020-04-02 | 2020-04-02 | Application of Proline in the Prevention and Control of Bee Virus Infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111481541A CN111481541A (en) | 2020-08-04 |
| CN111481541B true CN111481541B (en) | 2021-04-13 |
Family
ID=71790109
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010255788.6A Active CN111481541B (en) | 2020-04-02 | 2020-04-02 | Application of Proline in the Prevention and Control of Bee Virus Infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111481541B (en) |
-
2020
- 2020-04-02 CN CN202010255788.6A patent/CN111481541B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| Imidacloprid, Deformed wing virus, proline, and immune defense in apis mellifera;Mammone;《Honors Theses》;20151231 * |
| 维生素 C 与表观遗传调控;郭琳等;《科学通报》;20141231;第59卷(第28-29期);第2833-2839页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111481541A (en) | 2020-08-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Goblirsch | Nosema ceranae disease of the honey bee (Apis mellifera) | |
| Yoshiyama et al. | Inhibition of Paenibacillus larvae by lactic acid bacteria isolated from fermented materials | |
| Defer et al. | Screening for antibacterial and antiviral activities in three bivalve and two gastropod marine molluscs | |
| US9609875B2 (en) | Methods for the prevention and control of pathogenic infections in bees and relative composition | |
| Maruščáková et al. | Effect of application of probiotic pollen suspension on immune response and gut microbiota of honey bees (Apis mellifera) | |
| Royan | Mechanisms of probiotic action in the honeybee | |
| Huang et al. | A potential probiotic Leuconostoc mesenteroides TBE-8 for honey bee | |
| Pluta et al. | Changes in the expression of antimicrobial peptide genes in honey bees (Apis mellifera) under the influence of various pathogens | |
| Usta et al. | Investigation of deformed wing virus, black queen cell virus, and acute bee paralysis virus infections in honey bees using reverse transcriptase-polymerase chain reaction (RT-PCR) method | |
| da Luz et al. | Cuticle melanization and the expression of immune-related genes in the honeybee Apis mellifera (Hymenoptera: Apidae) adult workers | |
| CN111297884B (en) | Application of Adenosine in Prevention and Control of Honeybee Virus Infection | |
| Lietze et al. | Transmission of MdSGHV among adult house flies, Musca domestica (Diptera: Muscidae), occurs via oral secretions and excreta | |
| Yang et al. | Immune responses in the haemolymph and antimicrobial peptide expression in the abdomen of Apis mellifera challenged with Spiroplasma melliferum CH-1 | |
| CN111481541B (en) | Application of Proline in the Prevention and Control of Bee Virus Infection | |
| Li et al. | Heat stress affects the expression of antimicrobial peptide genes in adult honeybee (Apis cerana and Apis mellifera) | |
| CN111297851B (en) | Application of Coumarin in Antiviral Infection of Honeybees | |
| Bahreini et al. | A study of the efficacy of formic acid in controlling Varroa destructor and its correlation with temperature in Iran | |
| Zhang et al. | Toll receptor ligand Spätzle 4 responses to the highly pathogenic Enterococcus faecalis from Varroa mites in honeybees | |
| Fedoruk et al. | The Effect of Probiotic Microorganisms on Catalase Activity, Fractional Composition of Soluble Proteins, and Intestinal Microbiota of Honey Bee | |
| Vung et al. | Controlling sacbrood virus disease in Apis cerana colonies with biological methods in Korea | |
| CN111265596B (en) | Traditional Chinese medicine composition for preventing and treating middle capsule disease | |
| CN103524602B (en) | Application of antibacterial peptide in pharmacy | |
| Tejerina et al. | Effect of Lactic Bacteria on Ascosphaera apis and A. atra | |
| Prayugo | Israeli acute paralysis virus (IAPV) and deformed wing virus (DWV) infection progression in honey bees | |
| Durand et al. | Co-infection with two iflaviruses (deformed wing virus and sacbrood virus) affects viral and immune dynamics and synergistically increases honey bee mortality |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |