CN107446041A - A kind of antiserum of anti-apple necrosis mosaic virus and preparation method thereof - Google Patents
A kind of antiserum of anti-apple necrosis mosaic virus and preparation method thereof Download PDFInfo
- Publication number
- CN107446041A CN107446041A CN201710790136.0A CN201710790136A CN107446041A CN 107446041 A CN107446041 A CN 107446041A CN 201710790136 A CN201710790136 A CN 201710790136A CN 107446041 A CN107446041 A CN 107446041A
- Authority
- CN
- China
- Prior art keywords
- apple
- mosaic virus
- necrosis mosaic
- protein
- necrosis
- 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.)
- Granted
Links
- 241000700605 Viruses Species 0.000 title claims abstract description 71
- 230000017074 necrotic cell death Effects 0.000 title claims abstract description 58
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 39
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 34
- 101710132601 Capsid protein Proteins 0.000 claims abstract description 30
- 101710094648 Coat protein Proteins 0.000 claims abstract description 30
- 101710125418 Major capsid protein Proteins 0.000 claims abstract description 30
- 101710141454 Nucleoprotein Proteins 0.000 claims abstract description 30
- 101710083689 Probable capsid protein Proteins 0.000 claims abstract description 30
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 claims abstract description 28
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 23
- 241000283977 Oryctolagus Species 0.000 claims abstract description 9
- 201000010099 disease Diseases 0.000 claims abstract description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 7
- 241000588724 Escherichia coli Species 0.000 claims abstract description 5
- 241001465754 Metazoa Species 0.000 claims abstract description 5
- 230000002163 immunogen Effects 0.000 claims abstract description 3
- 108020004414 DNA Proteins 0.000 claims description 20
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 13
- 230000009465 prokaryotic expression Effects 0.000 claims description 11
- 239000002574 poison Substances 0.000 claims description 6
- 231100000614 poison Toxicity 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 5
- 102000037865 fusion proteins Human genes 0.000 claims description 3
- 108020001507 fusion proteins Proteins 0.000 claims description 3
- 102000053602 DNA Human genes 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 125000000539 amino acid group Chemical group 0.000 claims description 2
- 210000004899 c-terminal region Anatomy 0.000 claims description 2
- 238000010367 cloning Methods 0.000 claims description 2
- 230000004069 differentiation Effects 0.000 claims description 2
- 239000000835 fiber Substances 0.000 claims description 2
- 238000006467 substitution reaction Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 238000009396 hybridization Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 21
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000002265 prevention Effects 0.000 abstract description 3
- 230000003612 virological effect Effects 0.000 abstract description 3
- 244000141359 Malus pumila Species 0.000 description 44
- 235000011430 Malus pumila Nutrition 0.000 description 43
- 235000015103 Malus silvestris Nutrition 0.000 description 43
- 239000000523 sample Substances 0.000 description 16
- 230000014509 gene expression Effects 0.000 description 14
- 238000002965 ELISA Methods 0.000 description 12
- 239000000499 gel Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 241000196324 Embryophyta Species 0.000 description 9
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 9
- 238000001262 western blot Methods 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 230000029087 digestion Effects 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 239000013256 coordination polymer Substances 0.000 description 5
- 235000013399 edible fruits Nutrition 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000012134 supernatant fraction Substances 0.000 description 4
- 241000724277 Ilarvirus Species 0.000 description 3
- 240000000220 Panda oleosa Species 0.000 description 3
- 235000016496 Panda oleosa Nutrition 0.000 description 3
- 101100191561 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PRP3 gene Proteins 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 239000010977 jade Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- 230000000087 stabilizing effect Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 241000459419 Apple necrotic mosaic virus Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001533462 Bromoviridae Species 0.000 description 2
- 101150083464 CP gene Proteins 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- -1 Polysaccharide polyphenol Chemical class 0.000 description 2
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 108010087924 alanylproline Proteins 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000010241 blood sampling Methods 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 150000002460 imidazoles Chemical class 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 108010078274 isoleucylvaline Proteins 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 229940005654 nitrite ion Drugs 0.000 description 2
- 235000013824 polyphenols Nutrition 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- SGIWPAGWLKAFCF-FLKCQLHMSA-N (2s)-2-[[(2s)-2-[[(2s)-4-amino-2-[[(2s)-2-[[(2s)-2-amino-3-methylbutanoyl]amino]-3-methylbutanoyl]amino]-4-oxobutanoyl]amino]-3-carboxypropanoyl]amino]-4-methylpentanoic acid Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)C(C)C SGIWPAGWLKAFCF-FLKCQLHMSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- SBGXWWCLHIOABR-UHFFFAOYSA-N Ala Ala Gly Ala Chemical compound CC(N)C(=O)NC(C)C(=O)NCC(=O)NC(C)C(O)=O SBGXWWCLHIOABR-UHFFFAOYSA-N 0.000 description 1
- CSAHOYQKNHGDHX-ACZMJKKPSA-N Ala-Gln-Asn Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CSAHOYQKNHGDHX-ACZMJKKPSA-N 0.000 description 1
- BLIMFWGRQKRCGT-YUMQZZPRSA-N Ala-Gly-Lys Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN BLIMFWGRQKRCGT-YUMQZZPRSA-N 0.000 description 1
- SIGTYDNEPYEXGK-ZANVPECISA-N Ala-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)[C@@H](N)C)C(O)=O)=CNC2=C1 SIGTYDNEPYEXGK-ZANVPECISA-N 0.000 description 1
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 1
- AUFACLFHBAGZEN-ZLUOBGJFSA-N Ala-Ser-Cys Chemical compound N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O AUFACLFHBAGZEN-ZLUOBGJFSA-N 0.000 description 1
- KTXKIYXZQFWJKB-VZFHVOOUSA-N Ala-Thr-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O KTXKIYXZQFWJKB-VZFHVOOUSA-N 0.000 description 1
- SGYSTDWPNPKJPP-GUBZILKMSA-N Arg-Ala-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SGYSTDWPNPKJPP-GUBZILKMSA-N 0.000 description 1
- RWCLSUOSKWTXLA-FXQIFTODSA-N Arg-Asp-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O RWCLSUOSKWTXLA-FXQIFTODSA-N 0.000 description 1
- PTVGLOCPAVYPFG-CIUDSAMLSA-N Arg-Gln-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O PTVGLOCPAVYPFG-CIUDSAMLSA-N 0.000 description 1
- HPSVTWMFWCHKFN-GARJFASQSA-N Arg-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O HPSVTWMFWCHKFN-GARJFASQSA-N 0.000 description 1
- GMFAGHNRXPSSJS-SRVKXCTJSA-N Arg-Leu-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O GMFAGHNRXPSSJS-SRVKXCTJSA-N 0.000 description 1
- XSPKAHFVDKRGRL-DCAQKATOSA-N Arg-Pro-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O XSPKAHFVDKRGRL-DCAQKATOSA-N 0.000 description 1
- YJRORCOAFUZVKA-FXQIFTODSA-N Asn-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N)CN=C(N)N YJRORCOAFUZVKA-FXQIFTODSA-N 0.000 description 1
- COUZKSSMBFADSB-AVGNSLFASA-N Asn-Glu-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)N)N COUZKSSMBFADSB-AVGNSLFASA-N 0.000 description 1
- DMLSCRJBWUEALP-LAEOZQHASA-N Asn-Glu-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O DMLSCRJBWUEALP-LAEOZQHASA-N 0.000 description 1
- NLRJGXZWTKXRHP-DCAQKATOSA-N Asn-Leu-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NLRJGXZWTKXRHP-DCAQKATOSA-N 0.000 description 1
- XJQRWGXKUSDEFI-ACZMJKKPSA-N Asp-Glu-Asn Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O XJQRWGXKUSDEFI-ACZMJKKPSA-N 0.000 description 1
- 235000011331 Brassica Nutrition 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- BUIYOWKUSCTBRE-CIUDSAMLSA-N Cys-Arg-Gln Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O BUIYOWKUSCTBRE-CIUDSAMLSA-N 0.000 description 1
- WTNLLMQAFPOCTJ-GARJFASQSA-N Cys-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CS)N)C(=O)O WTNLLMQAFPOCTJ-GARJFASQSA-N 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- XUMFMAVDHQDATI-DCAQKATOSA-N Gln-Pro-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O XUMFMAVDHQDATI-DCAQKATOSA-N 0.000 description 1
- PAQUJCSYVIBPLC-AVGNSLFASA-N Glu-Asp-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PAQUJCSYVIBPLC-AVGNSLFASA-N 0.000 description 1
- ZYRXTRTUCAVNBQ-GVXVVHGQSA-N Glu-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZYRXTRTUCAVNBQ-GVXVVHGQSA-N 0.000 description 1
- SOYWRINXUSUWEQ-DLOVCJGASA-N Glu-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O SOYWRINXUSUWEQ-DLOVCJGASA-N 0.000 description 1
- YZACQYVWLCQWBT-BQBZGAKWSA-N Gly-Cys-Arg Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O YZACQYVWLCQWBT-BQBZGAKWSA-N 0.000 description 1
- PAWIVEIWWYGBAM-YUMQZZPRSA-N Gly-Leu-Ala Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O PAWIVEIWWYGBAM-YUMQZZPRSA-N 0.000 description 1
- HAOUOFNNJJLVNS-BQBZGAKWSA-N Gly-Pro-Ser Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O HAOUOFNNJJLVNS-BQBZGAKWSA-N 0.000 description 1
- PYFHPYDQHCEVIT-KBPBESRZSA-N Gly-Trp-Gln Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(N)=O)C(O)=O PYFHPYDQHCEVIT-KBPBESRZSA-N 0.000 description 1
- DCRODRAURLJOFY-XPUUQOCRSA-N His-Ala-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)NCC(O)=O DCRODRAURLJOFY-XPUUQOCRSA-N 0.000 description 1
- CNHSMSFYVARZLI-YJRXYDGGSA-N His-His-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CNHSMSFYVARZLI-YJRXYDGGSA-N 0.000 description 1
- SKYULSWNBYAQMG-IHRRRGAJSA-N His-Leu-Arg Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SKYULSWNBYAQMG-IHRRRGAJSA-N 0.000 description 1
- CWSZWFILCNSNEX-CIUDSAMLSA-N His-Ser-Asn Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N CWSZWFILCNSNEX-CIUDSAMLSA-N 0.000 description 1
- GQKSJYINYYWPMR-NGZCFLSTSA-N Ile-Gly-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N1CCC[C@@H]1C(=O)O)N GQKSJYINYYWPMR-NGZCFLSTSA-N 0.000 description 1
- GVKKVHNRTUFCCE-BJDJZHNGSA-N Ile-Leu-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)O)N GVKKVHNRTUFCCE-BJDJZHNGSA-N 0.000 description 1
- REXAUQBGSGDEJY-IGISWZIWSA-N Ile-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N REXAUQBGSGDEJY-IGISWZIWSA-N 0.000 description 1
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- HYIFFZAQXPUEAU-QWRGUYRKSA-N Leu-Gly-Leu Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(C)C HYIFFZAQXPUEAU-QWRGUYRKSA-N 0.000 description 1
- FBNPMTNBFFAMMH-UHFFFAOYSA-N Leu-Val-Arg Natural products CC(C)CC(N)C(=O)NC(C(C)C)C(=O)NC(C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-UHFFFAOYSA-N 0.000 description 1
- VKVDRTGWLVZJOM-DCAQKATOSA-N Leu-Val-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O VKVDRTGWLVZJOM-DCAQKATOSA-N 0.000 description 1
- PBLLTSKBTAHDNA-KBPBESRZSA-N Lys-Gly-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PBLLTSKBTAHDNA-KBPBESRZSA-N 0.000 description 1
- DRRXXZBXDMLGFC-IHRRRGAJSA-N Lys-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN DRRXXZBXDMLGFC-IHRRRGAJSA-N 0.000 description 1
- MFDDVIJCQYOOES-GUBZILKMSA-N Met-Val-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCSC)N MFDDVIJCQYOOES-GUBZILKMSA-N 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- METZZBCMDXHFMK-BZSNNMDCSA-N Phe-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N METZZBCMDXHFMK-BZSNNMDCSA-N 0.000 description 1
- 241000219998 Philenoptera violacea Species 0.000 description 1
- NHDVNAKDACFHPX-GUBZILKMSA-N Pro-Arg-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O NHDVNAKDACFHPX-GUBZILKMSA-N 0.000 description 1
- BNBBNGZZKQUWCD-IUCAKERBSA-N Pro-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 BNBBNGZZKQUWCD-IUCAKERBSA-N 0.000 description 1
- GRIRJQGZZJVANI-CYDGBPFRSA-N Pro-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H]1CCCN1 GRIRJQGZZJVANI-CYDGBPFRSA-N 0.000 description 1
- ORPZXBQTEHINPB-SRVKXCTJSA-N Pro-Arg-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H]1CCCN1)C(O)=O ORPZXBQTEHINPB-SRVKXCTJSA-N 0.000 description 1
- HJSCRFZVGXAGNG-SRVKXCTJSA-N Pro-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1 HJSCRFZVGXAGNG-SRVKXCTJSA-N 0.000 description 1
- SBVPYBFMIGDIDX-SRVKXCTJSA-N Pro-Pro-Pro Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1N(C(=O)[C@H]2NCCC2)CCC1 SBVPYBFMIGDIDX-SRVKXCTJSA-N 0.000 description 1
- 101710199886 Protein 0.5 Proteins 0.000 description 1
- 101150023114 RNA1 gene Proteins 0.000 description 1
- 101150084101 RNA2 gene Proteins 0.000 description 1
- 101100353432 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PRP2 gene Proteins 0.000 description 1
- FWTFAZKJORVTIR-VZFHVOOUSA-N Thr-Ser-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O FWTFAZKJORVTIR-VZFHVOOUSA-N 0.000 description 1
- QJIODPFLAASXJC-JHYOHUSXSA-N Thr-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N)O QJIODPFLAASXJC-JHYOHUSXSA-N 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- CXPJPTFWKXNDKV-NUTKFTJISA-N Trp-Leu-Ala Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 CXPJPTFWKXNDKV-NUTKFTJISA-N 0.000 description 1
- STKZKWFOKOCSLW-UMPQAUOISA-N Trp-Thr-Val Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)[C@@H](C)O)=CNC2=C1 STKZKWFOKOCSLW-UMPQAUOISA-N 0.000 description 1
- RGJZPXFZIUUQDN-BPNCWPANSA-N Tyr-Val-Ala Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O RGJZPXFZIUUQDN-BPNCWPANSA-N 0.000 description 1
- DDRBQONWVBDQOY-GUBZILKMSA-N Val-Ala-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O DDRBQONWVBDQOY-GUBZILKMSA-N 0.000 description 1
- NMANTMWGQZASQN-QXEWZRGKSA-N Val-Arg-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N NMANTMWGQZASQN-QXEWZRGKSA-N 0.000 description 1
- QGFPYRPIUXBYGR-YDHLFZDLSA-N Val-Asn-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N QGFPYRPIUXBYGR-YDHLFZDLSA-N 0.000 description 1
- FXVDGDZRYLFQKY-WPRPVWTQSA-N Val-Gly-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C FXVDGDZRYLFQKY-WPRPVWTQSA-N 0.000 description 1
- JZWZACGUZVCQPS-RNJOBUHISA-N Val-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N JZWZACGUZVCQPS-RNJOBUHISA-N 0.000 description 1
- RTJPAGFXOWEBAI-SRVKXCTJSA-N Val-Val-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N RTJPAGFXOWEBAI-SRVKXCTJSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 235000021016 apples Nutrition 0.000 description 1
- 108010091092 arginyl-glycyl-proline Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 1
- 108010084264 glycyl-glycyl-cysteine Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000003760 hair shine Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 1
- 229960002064 kanamycin sulfate Drugs 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 208000035824 paresthesia Diseases 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 108010024607 phenylalanylalanine Proteins 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/14011—Bromoviridae
- C12N2770/14021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
Abstract
The invention discloses a kind of antiserum of anti-apple necrosis mosaic virus and preparation method thereof.The sero-fast method provided by the present invention for preparing anti-apple necrosis mosaic virus comprises the following steps：Immunogen immune animal is used as using the coat protein of apple necrosis mosaic virus.The present invention passes through the coat protein gene in E. coli apple necrosis mosaic virus using molecular biology method, it is purified into destination protein, new zealand rabbit is immunized with it, the polyclonal antiserum of obligatory type is prepared, the present invention provides fundamental basis for the prevention and control of the disease in the viral Serologic detection and apple production.
Description
Technical field
The invention belongs to phytovirology and field of immunology, be related to a kind of anti-apple necrosis mosaic virus antiserum and
Its preparation method.
Background technology
Apple is one of important industrial crops in China, and FAO data are shown, 2014, and China's Apple Culture gross area is about
, about 41,000,000 tons of total output, 50% or so of world's apple is accounted for respectively, occupy the whole world first by 2270000 hectares.China's Apple Culture
It is distributed mainly on the province such as Shaanxi, Shandong, Hebei, Henan, Gansu, Shanxi, Liaoning, Xinjiang, it has also become drive the weight of regional development
Approach is wanted, and is increasingly becoming the important component of industry restructuring, economic development.But Apple virus disease is in China's apple
The incidence in fruit producing region is higher, and up to 40-100%, once infecting, fruit tree will be throughout one's life with poison.Wherein, apple mosaic is apple
One of most common Disease in production, seriously governs the sound development of China's Apple Industry.
A nearest research shows, a kind of new virus is detected in apple floral leaf sample --- apple necrosis floral leaf
Viral (Apple necrotic mosaic virus, ApNMV), ApNMV belongs to Bromoviridae (Bromoviridae)
The subgroup (Subgroup 3) of Ilarvirus category (Ilarvirus) the 3rd, its genome structure are triad, including
RNA1, RNA2 and RNA3, wherein RNA3 5 ' end one CP of coding, are made up of, coding section length is 660nt 219aa.In addition,
Recall rates of the new virus ApNMV in China's apple floral leaf sample is up to 82.6%, higher with the generation presence of flower leaf paresthesia
Correlation, it may be possible to cause the important pathogen thing of China's apple mosaic, be a serious threat to China's Apple Culture.
Pathogen test is premise and the basis of virosis prevention and control.At present, the detection method on fruit tree virus mainly has life
Thing, serology, electron microscope observation and molecular biology method.Wherein, biological method detection cycle is long, specific
Difference, workload are big, and require higher to the field practical experience of testing staff；Although electron microscopic observation can be according to virus and including
Body characteristicses and host cell lesion situation, more intuitively carry out the detection of plant virus, but, the inspection the same with molecular biology
Survey method is limited by experiment condition and detection device, and the detection to a large amount of samples is relatively cumbersome.It is different, serum
Method especially ELISA adsorption analysis method (ELISA method) can be quick, sensitive, accurate to the progress of a large amount of samples because of it
Detection, and the advantages that experimental cost is low, method is simple, turn into the important tool of plant virus identification, classification and detection.
But the relevant report of the preparation of ApNMV serum and effective detection is there is no both at home and abroad.
The content of the invention
It is an object of the invention to provide a kind of antiserum of anti-apple necrosis mosaic virus and preparation method thereof.
The sero-fast method provided by the invention for preparing anti-apple necrosis mosaic virus, comprises the following steps：With apple
The coat protein of downright bad mosaic virus is as immunogen immune animal.
Wherein, the coat protein of the apple necrosis mosaic virus is following any：
(A1) amino acid sequence is the protein of the sequence 1 in sequence table；
(A2) by the amino acid sequence shown in sequence in sequence table 1 by one or several amino acid residues substitution and/
Or missing and/or addition and the protein with identical function；
(A3) amino acid sequence limited with (A1) or (A2) has more than 99%, more than 95%, more than 90%, 85%
Above or more than 80% homology and with identical function protein；
(A4) N-terminal of any limited protein and/or C-terminal connect the fusion obtained after label in (A1)-(A3)
Albumen.
In the process, the animal concretely new zealand rabbit.
In the process, the new zealand rabbit being immunized can proceed as follows：By the apple necrosis mosaic virus
Coat protein is emulsified with isometric Freund's complete adjuvant (first) or incomplete Freund's adjuvant (follow-up), uses SPF levels
New zealand rabbit carries out intracutaneous multi-point injection and is immunized, and is rested one week after immune for the first time, 5-6 times (such as 6 times) are immunized altogether, immune every time
The dosage of the coat protein of the apple necrosis mosaic virus is 0.1mg.
In the process, the coat protein as the apple necrosis mosaic virus of immunogene specifically can be according to as follows
Described " preparation method of the coat protein of apple necrosis mosaic virus " prepares.
Present invention also offers the preparation method of the coat protein of apple necrosis mosaic virus, specifically may include to walk as follows
Suddenly：
(1) encoding gene of the coat protein of the apple necrosis mosaic virus is cloned into more grams of prokaryotic expression carrier
Long Weidianchu, obtain recombinant prokaryotic expression vector；
(2) recombinant prokaryotic expression vector is imported into the prokaryotic as acceptor, obtains recombinating prokaryotic；
(3) the restructuring prokaryotic is cultivated, therefrom obtains the coat protein of the apple necrosis mosaic virus.
In the present invention, the prokaryotic expression carrier is specially pET28a (+) carrier.Accordingly, the multiple cloning sites
Specially BamH I and Hind III.The prokaryotic as acceptor is specially e. coli bl21 (DE3).
In step (3), in addition to IPTG is added to final concentration of into the cultivating system of the restructuring prokaryotic
The step of 0.5mM, 37 DEG C of Fiber differentiation 2h.
In the process, the encoding gene of the coat protein of the apple necrosis mosaic virus is concretely following any
DNA molecular：
(B1) DNA molecular in sequence table shown in sequence 2；
(B2) DNA molecular limited under strict conditions with (B1) hybridizes and encodes any shown eggs of as above (A1)-(A4)
The DNA molecular of white matter；
(B3) DNA sequence dna limited with (B1) or (B2) has more than 99%, more than 95%, more than 90%, more than 85%
Or more than 80% homology, and encode as above (A1)-(A4) it is any shown in protein DNA molecule.
The present invention also protects two kinds of products shown in following (A) and (B) respectively.
(A) the anti-apple being prepared using " the sero-fast method for preparing anti-apple necrosis mosaic virus " as described above
The antiserum of downright bad mosaic virus.
(B) apple being prepared using " preparation method of the coat protein of apple necrosis mosaic virus " as described above is bad
The coat protein of dead mosaic virus.
The present invention also protects four kinds of applications shown in following (C)-(F) respectively.
(C) apple being prepared using " preparation method of the coat protein of apple necrosis mosaic virus " as described above is bad
The coat protein of dead mosaic virus and record " the sero-fast method for preparing anti-apple necrosis mosaic virus " as described above
Readable carrier prepare be used to producing in the antiserum of anti-apple necrosis mosaic virus or the kit of polyclonal antibody should
With.
(D) apple being prepared using " preparation method of the coat protein of apple necrosis mosaic virus " as described above is bad
Application of the coat protein of dead mosaic virus in the antiserum that anti-apple necrosis mosaic virus is prepared as immunogene.
(E) the anti-apple being prepared using " the sero-fast method for preparing anti-apple necrosis mosaic virus " as described above
Application of the antiserum of downright bad mosaic virus in apple necrosis mosaic virus is detected.
(F) the anti-apple being prepared using " the sero-fast method for preparing anti-apple necrosis mosaic virus " as described above
Application of the antiserum of downright bad mosaic virus in apple necrosis mosaic virus is prevented and treated.
The present invention is passed through in the outer of E. coli apple necrosis mosaic virus using molecular biology method
Coat protein gene, destination protein is purified into, two SPF (Specific pathogen Free) level new zealand rabbit is immunized with it
Son, the polyclonal antiserum of obligatory type is prepared, the prevention and control for the disease in the viral Serologic detection and apple production carry
For theoretical foundation.
Brief description of the drawings
Fig. 1 is that RT-PCR expands cp genes.M：D2000Marker；1-2：It is AM75 Apple Leaves samples.Two swimming lanes
In have specific band that is bright, single and meeting expected purpose gene size (675bp).
Fig. 2 is BamH I and the double digestion product electrophoresis detection figures of Hind III.M：DNA Marker DL10000；1：BamH I
With Hind III double digestion cp gene glue reclaim products；2：BamH I and Hind III double digestions pET-28a (+) plasmid；3：Not
PET-28a (+) plasmid of digestion.
Fig. 3 is that recombinant protein expresses SDS-PAGE and Western blot analyses in a small amount.A is SDS-PAGE analysis results；
Wherein, 1：37 DEG C are incubated overnight lysate；2：37 DEG C are incubated overnight sediment fraction；3：37 DEG C are incubated overnight supernatant fraction；4：Not
Induced through IPTG；5：Whole lysates are induced through IPTG；6：Through IPTG induced precipitations part；7：Supernatant fraction is induced through IPTG；
8：BSA 0.5μg；9：BSA 1.0μg；10：BSA 2.0μg.B is Western blot analysis results；Wherein, 1：37 DEG C overnight
Cultivate lysate；2：37 DEG C are incubated overnight sediment fraction；3：37 DEG C are incubated overnight supernatant fraction；4：Induced without IPTG；5：Through
IPTG induces whole lysates；6：Through IPTG induced precipitations part；7：Supernatant fraction is induced through IPTG；8：Negative control；9：Sun
Property control；Anti- His antibody tests.
Fig. 4 analyzes for recombinant protein great expression SDS-PAGE and Western blot.A is SDS-PAGE analysis results；
Wherein, 1：The μ g of purifying protein 1；2：BSA 0.5μg；3：BSA 1.0μg；4：BSA 2.0μg.B is Western blot analysis knots
Fruit；The μ g of purifying protein 0.5, anti-His antibody tests.
Fig. 5 reacts for polyclonal antiserum bioactivity.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Main agents：Polysaccharide polyphenol plant total RNA extraction reagent box (Polysaccharides＆Polyphenolics-
Rich RNAprep Pure) Tiangeng biotechnology (Beijing) company, restriction enzyme are purchased from essentially from precious biotech firm
(TaKaRa), RT-PCR amplification needed for reagent essentially from Pu Luomaige (Beijing) Bioisystech Co., Ltd (PROMEGA),
AxyPrep DNA gels QIAquick Gel Extraction Kit and the small extraction reagent kit of AxyPrep DNAs are purchased from Axygen Reagent Companies, protokaryon table
Given up to carrier pET-28a (+) by Plant Protection institute, Chinese Academy of Agricultral Sciences Zhao Tingchang researcher, chemiluminescence nitrite ion
With pvdf membrane Beijing Bo Ermai biotechnologys are purchased from purchased from Millipore companies (U.S.), goat-anti rabbit ELIAS secondary antibody (article No. 458)
Co., Ltd.
Key instrument：Bio-Rad PCR instruments, Bio-Rad transferring films instrument, KCBIO-2800 gel imaging systems, DYY-6B types
It is voltage stabilizing electrophoresis apparatus, ATTO AE-6200 Vertial electrophorestic tanks (Japan), new TS-1 decolorization swinging tables, thermostat water bath, incubated
Case, micro ultraviolet specrophotometerND-1000UV-Vis Spectrophotometer (NanoDrop,
America), AIRTECH SW-CJ-1FD superclean benches, the multi-function microplate readers of Synergy 4 (America).
Primer synthesis is sequenced by raw work bioengineering (Shanghai) stock by Beijing Liuhe Huada Genomics Technology Co., Ltd
Part Co., Ltd completes.
Embodiment 1, the prokaryotic expression of the coat protein of apple necrosis mosaic virus and identification
First, recombinant prokaryotic expression vector pET-ApNMVCP structure
1st, plant Total RNAs extraction
Plant Total RNAs extraction uses polysaccharide polyphenol plant total RNA extraction reagent box (Tiangeng), and concrete operation method is illustratively
Book is carried out.Originally it is the Apple Leaves for having infected apple necrosis mosaic virus AM75 for sample.
2nd, the design of ApNMV cp gene orders specific amplification primer
The apple necrosis mosaic virus AM75 amplified based on inventor's early stage place team RNA3 sequences, design cp bases
Because of sequence amplification primer, the end of forward primer 5 ' introduces BamH I, Hind III digestions site and corresponding protection base (table 1).
The ApNMV CP sequence amplification specific primers of table 1
Note：* underline part and represent restriction endonuclease sites.
3rd, RT-PCR expands cp genes and double digestion structure prokaryotic expression recombinant plasmid
Reverse transcription system：
Mixed liquor is mixed, 37 DEG C of incubation 1h, then enters performing PCR reaction (reaction system is as follows) or -20 DEG C saves backup.
PCR reaction cycle parameters：98 DEG C of pre-degeneration 30sec, 98 DEG C of denaturation 10sec, 65 DEG C of annealing 30sec, 72 DEG C extend
30sec, circulate 32 times；72 DEG C re-extend 10min, 4 DEG C of preservations.PCR primer is analyzed with 1.5% agarose gel electrophoresis,
Electrophoresis 30min or so under DYY-6B type voltage stabilizing electrophoresis apparatuses, 120V, running gel shines in KCBIO-2800 gel image analysers
Phase, and purpose band (Fig. 1) is cut, the purifying that amplified production is carried out using AxyPrep DNA gels QIAquick Gel Extraction Kit is reclaimed.
It is formulated as follows double digestion reaction system：
After mixing, 37 DEG C of incubation 15h, 5 μ 10 × loading of l buffer are added to terminate endonuclease reaction, digestion products are with 1%
Agarose gel electrophoresis is analyzed, electrophoresis 30min or so under DYY-6B type voltage stabilizing electrophoresis apparatuses, 120V, running gel in
Taken a picture (Fig. 2) in KCBIO-2800 gel image analysers, and cut purpose band, reclaimed and tried using AxyPrep DNA gels
Agent box carries out the purifying recovery of amplified production.
It is formulated as follows coupled reaction system：
After flicking mixing, centrifugation, 4 DEG C of connections overnight.Connection liquid is transformed into 50 μ l DH5 α competent cells, coated plate, 37
DEG C culture after, picking monoclonal in 1ml Kana (kanamycin sulfate, 100mg/L) resistance LB fluid nutrient mediums, 37 DEG C shake
Swing after shaking bacterium, bacterium solution PCR method identifies positive recombinant, and send raw work bioengineering (Shanghai) share limited positive colony
Company is sequenced, and verifies the correctness of catenation sequence and frame.It will show through sequencing by the restriction enzyme site of pET-28a (+) carrier
Small fragment between BamH I and Hind III replaces with the restructuring in sequence table after DNA fragmentation shown in the 1-657 positions of sequence 2
Plasmid is named as pET-ApNMVCP.Wherein, DNA fragmentation shown in sequence 2 is the shell of present invention gained apple necrosis mosaic virus
Encoding gene (i.e. cp genes) sequence of albumen, the shell egg of the apple necrosis mosaic virus in polynucleotide shown in sequence 1
(i.e. CP albumen) in vain.
Then, select the correct bacterium solution of frame and expand culture, use the small extraction reagent kit (AxyPrepTM of DNA
Plasmid Miniprep Kit 250-prep AxyPrep) extraction recombinant plasmid pET-ApNMVCP, -20 DEG C save backup.
Certainly, can be with DNA pieces shown in direct labor's composition sequence 2 when Prepare restructuring plasmid pET-ApNMVCP
Section, and by common molecular cloning approach insert it into pET-28a (+) carrier restriction enzyme site BamH I and Hind III it
Between.
2nd, CP Primary structures and protein purification
Frame correct recombinant plasmid pET-ApNMVCP and pET-28a (+) prepared by step 1 is converted into large intestine bar respectively
Bacterium expression bacterial strain BL21 (DE3), 37 DEG C are incubated overnight, and picking single bacterium colony, are incubated overnight, and preserve glycerol stock and press overnight bacterium solution
1:20 (volume ratio) ratios are inoculated into fresh Kana resistances LB culture mediums, a small amount of expression.Great expression after expressing successfully in a small amount, connects
100 μ l glycerol stocks of kind are incubated overnight into 50ml Kana resistance LB culture mediums, then by 1:20 (volume ratio) ratios turn to be inoculated into
In 1000ml culture mediums, 37 DEG C, 160rpm, shaken cultivation.When OD600 reaches 0.6 or so, added into the bacterium solution of culture
0.5mM IPTG, inducible protein expression, 37 DEG C, 160rpm, shaken cultivation, 2h.4 DEG C, 8000rpm, 20min centrifugation recovery bacterium
Body.For purifying protein with Ni Sepharose 6Fast Flow, the albumen finally reclaimed concentrates on 250mM imidazoles/8M Urea
In PBS, for follow-up more anti-immunities.
3rd, SDS-PAGE the and Western blot detection and analysis of expression product
When CP protein mini-expressions are expressed, thalline is collected by centrifugation, adds 1/10 volume sample buffer solution, concussion is suspended, and 100 DEG C are boiled
5min is boiled, SDS-PAGE analyses, concentrate glue voltage 8V/cm, separation gel 15V/cm are carried out with 12% glue.Coomassie brilliant blue
After R-250 dyeing 1h, decolourize 3h on room temperature shaker.The Western blot analyses of expression product refer to Towbin et al.
(Towbin H,Staehelin T,Gordon J.Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets:procedure and some applications
[J].Proceedings of the National Academy of Sciences of the United States of
America,1979,76(9):Method 4350-4354.), using the His-tag antibody of preservation as primary antibody, goat-anti rabbit enzyme labelled antibody
For secondary antibody, finally developed the color with TMB.Great expression detection is then using being loading sample by albumen after purification, and remaining is the same as a small amount of
Express SDS-PAGE and Western blot analyses.
The result for carrying out SDS-PAGE and Western blot detection and analysis to a small amount of expression samples is shown (Fig. 3), is passed through
After IPTG induced expressions, about have in molecular weight at 25kDa and be expected purpose band appearance of the same size with recombinant protein, without
Then as a result illustrate cp genes by success induced expression without the band for significantly meeting expected size in the swimming lane of IPTG inductions.
After a large amount of induced expressions, the sediment fraction after being cracked to Escherichia coli is entered with Ni Sepharose 6Fast Flow
Row protein purification, the albumen by recovery are concentrated in 250mM imidazoles/8M Urea PBS, concentration 1.2mg/ml, volume 2ml,
Copurification obtains recombinant protein 2.4mg.In addition, obtained purifying protein is subjected to SDS-PAGE and Western blot detections point
Analyse (Fig. 4), about have the appearance of main purpose protein band near 25kDa in molecular weight, illustrate that experiment is purified into satisfactory mesh
Albumen.
The sero-fast preparation and application of embodiment 2, anti-apple necrosis mosaic virus
First, sero-fast preparation and potency evaluation
First blood 2mL is taken to prepare a small amount of normal serum in SPF new zealand rabbit ear veins, as negative control.Afterwards, to implementation
Isometric Freund's complete adjuvant (first) or incomplete Freund's adjuvant are added in purified protein product prepared by example 1
(follow-up) is emulsified, and intracutaneous multi-point injection is carried out under barrier environment using 2 SPF levels new zealand rabbits is immunized.Enter on every Fridays
Row is immune, is rested one week after immune for the first time, is immunized altogether 6 times, and the coat protein of the apple necrosis mosaic virus is immunized every time
Dosage be 0.1mg.Complete immune latter week blood sampling 2ml and carry out ELISA bioactivities, as potency not enough needs supplementary immunization.Such as
Potency is up to standard, then is taken a blood sample entirely and ELISA is detected.The sodium azide of the filtered addition 0.09% of antiserum of preparation, 4 DEG C of guarantors
Deposit, be standby.In addition, gained antiserum is subjected to 5 times of spacing gradient dilutions, using the purpose fusion protein of expression as antigen coat,
Utilize the potency of indirect ELISA detection method measure serum.
2 SPF level new zealand rabbits are immunized in the destination protein of purifying, finally obtain ApNMV specific antisera.With
The purpose fusion protein of expression is antigen coat, and sero-fast potency is determined using indirect ELISA detection method.As a result show
(Fig. 5), remains to show obvious positive reaction after 62500 times of the blood sampling serum-dilution of 2 rabbits, at the same with preimmune serum,
Blank control does not have obvious serological reaction.The result illustrates that the antiserum quality that the present invention prepares is good.
2nd, indirect ELISA detection Apple Leaves sample
(" graceful tinkling of pieces of jade Brassica 2 et 4s monoclonal antibody preparation and disease are applied with reference to Shi Manling, Zheng's generation tinkling of pieces of jade academic dissertation method
Virus gene group variation research [Ph.D. Dissertation] Hangzhou:Zhejiang University, 2005. " and " Zheng's generation tinkling of pieces of jade Wheat in China mosaic disease
Functional analysis [Ph.D. Dissertation] the Hangzhou of malicious infectious clone structure and motor protein:Zhejiang University, 2012. "), slightly
Change, the ApNMV polyclonal antiserums prepared using step 1 is primary antibodies, indirect ELISA (ID-ELISA) method detection field apple
Leaf sample.Concrete operations are as follows：
1st, weigh the fresh blade of 0.2g or so apples (removing master pulse), after liquid nitrogen grinding, then add 2mL coating dilutions
(10mL/g) is fully ground, 5000rpm room temperatures centrifugation 3min；
2nd, the μ L of supernatant 100 are drawn and add ELISA Plate, 4 DEG C of overnight or 37 DEG C of insulating boxs placement 2h；
3rd, PBST washings three times, stand 3min, are patted on paper handkerchief, dry residual every time；
4th, the skimmed milk powers of 150 μ L 3%, 37 DEG C of closing 1h are added；
5th, confining liquid is use up, pats, dry residual liquid, 100 μ L is added and dilutes the ApNMV Anti-TNF-α blood after 400 times
Clearly, 37 DEG C of placement 1h；
6th, washing is the same as step 3；
7th, the skimmed milk powers of 100 μ L 3% are added and dilute the goat-anti rabbit ELIAS secondary antibody after 8000 times, room temperature places 1h；
8th, washing is the same as step 3；
9th, the nitrite ion after 100 μ L mixings, color development at room temperature 20-30min are added；
10th, 100 μ L 2M sulfuric acid terminating reactions, ELIASA survey OD450nm, and record.
School zero is carried out as blank control to be not added with antigen, according to critical value=testing sample OD values/negative control (healthy apple
Fruit leaf sample) OD values are judged, if critical value>2, then it is assumed that sample band poison, otherwise without poison.
In order to further verify the accuracy of ELISA testing results, control is used as using RT-PCR methods simultaneously in experiment.RT-
The primer that PCR is used is as follows：
ApNMV-F：5’-ATGGTGTGCAATCGCTGTCA-3’；
ApNMV-R：5’-CATCGACCATAAGGATATCA-3’.
With reference to Noda H, Yamagishi N, Yaegashi H, Xing F, Xie J P, Li S F, Zhou T, Ito T,
Yoshikawa N.Apple necrotic mosaic virus,a novel ilarvirus from mosaic-
diseased apple trees in Japan and China[J].Journal of General Plant
Pathology,2017,83:83-90.。
The testing result of sample shows (table 2) that sample 1-6 P/N values are all higher than 2, is judged as the ApNMV positives；7-9 samples
P/N values are respectively less than 2, are judged as ApNMV feminine genders.The ELISA testing results are consistent with RT-PCR testing results early stage, show this hair
The ApNMV antiserums of bright preparation can be applied to Apple Leaves ApNMV detection, and detection sensitivity is high, specificity is good.
The sample indirect ELISA of table 2 determines ApNMV
Note:OD is compareed in table450nmData are the average of three health this life cigarette samples, sample OD450nmMeasure does two
Repeat；P/N values are testing sample and control sample OD450nmRatio, if P/N values>2, then it is assumed that sample band poison, otherwise without poison.
<110>Plant Protection institute, Chinese Academy of Agricultral Sciences
<120>A kind of antiserum of anti-apple necrosis mosaic virus and preparation method thereof
<130> GNCLN171369
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 219
<212> PRT
<213>Apple necrosis mosaic virus
<400> 1
Met Val Cys Asn Arg Cys His His Thr His Ala Gly Gly Cys Arg Ser
1 5 10 15
Cys Arg Gln Cys His Pro Arg Asp Ala Ala Pro Pro Pro Pro Arg Ala
20 25 30
Arg Ala Arg Ala Gln Asn Val Val Ala Arg Gly Leu Ala Arg Pro Glu
35 40 45
Thr Ser Ala Arg Glu Pro Arg Arg Leu Gln Trp Thr Val Ile Gly Pro
50 55 60
Asn Glu Val Pro Arg Val Pro Arg Gly Tyr Val Ala His Ser Asn Arg
65 70 75 80
Glu Val Val Ala Thr Ser Ala Gly Lys Phe Leu His Val Asn Phe Ser
85 90 95
Thr Thr Phe Pro Gln Leu Leu Gly Leu Asn Leu Arg Ile Leu Ser Val
100 105 110
Val Val Arg Ala Ser Cys Leu Val Ser Ala Gly Trp Val Gly Met Leu
115 120 125
Glu Asp Phe Asp Glu Asn His Leu Arg Gly Pro Ser Ala Leu Ser Arg
130 135 140
Lys Gly Phe Arg Gln Asp Gln Pro Arg Gly Trp Gln Trp Leu Ala Pro
145 150 155 160
Ser Asp Leu Glu Tyr Asp Thr Phe Ala Asn Ser His Arg Leu Val Phe
165 170 175
Glu Val Lys Asn Glu Phe Ala Ala Gly Ala Lys Val Leu Val Arg Asp
180 185 190
Ile Tyr Ile Val Val Asn Asp Leu Pro Arg Ile Val Ile Pro Asn Asp
195 200 205
Ile Leu Met Val Asp Glu Asp Leu Leu Asp Val
210 215
<210> 2
<211> 660
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
atggtgtgca atcgctgtca tcacactcac gctggtgggt gtcggtcctg ccgacagtgc 60
cacccgagag atgctgctcc accaccaccg agagctcgag ctagagctca aaacgtggta 120
gcgcgaggtt tagcgcgacc agagacttca gctagggagc cgaggaggct tcaatggacc 180
gtgataggtc cgaatgaggt accacgagta ccgaggggat acgtggcaca tagcaacaga 240
gaagttgttg cgactagcgc tgggaagttt ctacatgtga acttcagcac gactttcccg 300
caactattag gattgaatct taggattctc tccgtggtag ttcgagccag ctgcctggta 360
tccgctgggt gggtgggaat gttggaggac tttgatgaga atcatctcag aggtccgagt 420
gccttgtcca ggaagggttt tcgccaagac caaccgagag gttggcaatg gttggctcct 480
tccgatttag aatacgatac gtttgcgaac tcgcaccgtt tagtattcga agtcaagaac 540
gaattcgcgg caggcgcgaa agttcttgtg agggacatct atatagtggt aaatgattta 600
ccacgaattg tgatcccgaa tgatatcctt atggtcgatg aagacctttt ggatgtctag 660
Claims (10)
1. a kind of sero-fast method for preparing anti-apple necrosis mosaic virus, comprises the following steps：With apple necrosis mosaic disease
The coat protein of poison is as immunogen immune animal.
2. according to the method for claim 1, it is characterised in that：The coat protein of the apple necrosis mosaic virus is as follows
It is any：
(A1) amino acid sequence is the protein of the sequence 1 in sequence table；
(A2) amino acid sequence shown in sequence in sequence table 1 by the substitution of one or several amino acid residues and/or is lacked
Lose and/or add and there is the protein of identical function；
(A3) amino acid sequence limited with (A1) or (A2) has more than 99%, more than 95%, more than 90%, more than 85%
Or more than 80% homology and with identical function protein；
(A4) N-terminal of any limited protein and/or C-terminal connect the fusion protein obtained after label in (A1)-(A3).
3. method according to claim 1 or 2, it is characterised in that：The animal is new zealand rabbit.
4. according to any described method in claim 1-3, it is characterised in that：The apple necrosis floral leaf as immunogene
The coat protein of virus prepares according to any methods described in claim 5-7.
5. the preparation method of the coat protein of apple necrosis mosaic virus, comprises the following steps：
(1) encoding gene of the coat protein of apple necrosis mosaic virus is cloned into the multiple cloning sites of prokaryotic expression carrier
Place, obtains recombinant prokaryotic expression vector；
(2) recombinant prokaryotic expression vector is imported into the prokaryotic as acceptor, obtains recombinating prokaryotic；
(3) the restructuring prokaryotic is cultivated, therefrom obtains the coat protein of the apple necrosis mosaic virus.
6. according to the method for claim 5, it is characterised in that：The prokaryotic expression carrier is pET28a (+) carrier；As
The prokaryotic of acceptor is e. coli bl21 (DE3).
7. the method according to claim 5 or 6, it is characterised in that：In step (3), in addition to it is thin to the restructuring protokaryon
In the cultivating system of born of the same parents the step of addition IPTG to final concentration of 0.5mM, 37 DEG C of Fiber differentiation 2h.
8. according to any described method in claim 5-7, it is characterised in that：The shell egg of the apple necrosis mosaic virus
White encoding gene is following any DNA molecular：
(B1) DNA molecular in sequence table shown in sequence 2；
(B2) (A1)-(A4) is any shown in the DNA molecular hybridization limited under strict conditions with (B1) and coding claim 2
Protein DNA molecule；
(B3) with (B1) or (B2) limit DNA sequence dna have more than 99%, more than 95%, more than 90%, more than 85% or
More than 80% homology, and encode any shown protein DNA molecules of (A1)-(A4) in claim 2.
9. product, for following (A) or (B)：
(A) antiserum of the anti-apple necrosis mosaic virus that any described method is prepared in claim 1-4 is utilized；
(B) coat protein of the apple necrosis mosaic virus that any described method is prepared in claim 5-8 is utilized.
10. application, it is following any：
(C) using the coat protein for the apple necrosis mosaic virus that any described method is prepared in claim 5-8 and
The readable carrier for recording any described method in claim 1-4 is being prepared for producing anti-apple necrosis mosaic virus
Antiserum or polyclonal antibody kit in application；
(D) existed using the coat protein of the apple necrosis mosaic virus that any described method is prepared in claim 5-8
The application in the antiserum of anti-apple necrosis mosaic virus is prepared as immunogene；
(E) existed using the antiserum of the anti-apple necrosis mosaic virus that any described method is prepared in claim 1-4
Detect the application in apple necrosis mosaic virus；
(F) existed using the antiserum of the anti-apple necrosis mosaic virus that any described method is prepared in claim 1-4
Prevent and treat the application in apple necrosis mosaic virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710790136.0A CN107446041B (en) | 2017-09-05 | 2017-09-05 | Antiserum for resisting apple necrosis mosaic virus and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710790136.0A CN107446041B (en) | 2017-09-05 | 2017-09-05 | Antiserum for resisting apple necrosis mosaic virus and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107446041A true CN107446041A (en) | 2017-12-08 |
| CN107446041B CN107446041B (en) | 2021-04-06 |
Family
ID=60495235
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710790136.0A Active CN107446041B (en) | 2017-09-05 | 2017-09-05 | Antiserum for resisting apple necrosis mosaic virus and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107446041B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113234694A (en) * | 2021-04-29 | 2021-08-10 | 山东农业大学 | Application of apple MdBT2 in prevention and treatment of apple mosaic disease |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH046466A (en) * | 1990-04-24 | 1992-01-10 | Sumitomo Chem Co Ltd | Antiserum used in certification of garlic mosaic disease virus |
-
2017
- 2017-09-05 CN CN201710790136.0A patent/CN107446041B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH046466A (en) * | 1990-04-24 | 1992-01-10 | Sumitomo Chem Co Ltd | Antiserum used in certification of garlic mosaic disease virus |
Non-Patent Citations (3)
| Title |
|---|
| HIROKI NODA: "《Apple necrotic mosaic virus, a novel ilarvirus from mosaic-diseased apple trees in Japan and China》", 《J GEN PLANT PATHOL》 * |
| NCBI GENBANK DATABASE: "《Apple necrotic mosaic virus gene for coat protein, partial cds, strain: KO, Accession No. LC276940》", 《GENBANK DATABASE》 * |
| 怀晓: "《苹果茎沟病毒外壳蛋白基因的克隆、原核表达及抗血清制备》", 《植物保护学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113234694A (en) * | 2021-04-29 | 2021-08-10 | 山东农业大学 | Application of apple MdBT2 in prevention and treatment of apple mosaic disease |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107446041B (en) | 2021-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111499765B (en) | Coronavirus fusion protein and preparation method and application thereof | |
| CN104994740B (en) | For treating and controlling the method and composition of plant disease | |
| CN110105436B (en) | ELISA detection kit for porcine circovirus type 3 antibody and preparation method and application thereof | |
| CN112094340A (en) | Application of plant as host in expression of novel coronavirus pneumonia neutralizing antibody B38 antibody and/or H4 antibody | |
| CN110194790A (en) | The plant immune activator protein FoPII1 of Fusarium oxysporum secretion and its application | |
| CN107056898A (en) | 3 type of carp herpesviral, 1301 plants of ORF136 DNA recombinant expressions albumen, antibody and its application | |
| CN111808176B (en) | Bovine herpes virus antigen compositions and uses thereof | |
| CN104211784B (en) | It is used to prepare the protein and method of Hepatitis E virus sample particle | |
| CN110938118B (en) | Plant immune activation protein PC2 secreted by phytophthora infestans and application thereof | |
| CN108776229A (en) | A kind of sugarcane streak mosaic virus double antibody sandwich enzyme immunity detection reagent and preparation and detection method | |
| CN111487417A (en) | MCR-1 drug-resistant protein double-antibody sandwich E L ISA detection kit and detection method | |
| CN105541977B (en) | Riemerella anatipestifer OmpH intercept recombinant protein and preparation method and application | |
| CN113604438B (en) | Monoclonal antibody for resisting tilapia lake virus, cell strain and application thereof | |
| Tian et al. | An immunochromatographic test strip for on-site detection of Ralstonia solanacearum in tobacco leaves and soil | |
| CN101875980B (en) | Kit and method for detecting Macrobrachium rosenbergii Nodavirus | |
| CN107446041A (en) | A kind of antiserum of anti-apple necrosis mosaic virus and preparation method thereof | |
| CN101367870A (en) | Bovine rotavirus recombinant VP6 protein antigen and method of preparing the same | |
| CN103059117A (en) | High throughput screening of important schistosoma japonicum antigens and application of antigens in schistosomiasis diagnosis | |
| CN111487418A (en) | NDM-1 drug-resistant protein double-antibody sandwich E L ISA detection kit and detection method | |
| CN105018510B (en) | A method of it improves immune soluble with fmd protein | |
| CN115109131B (en) | Application of polyclonal antibody in preparation of reagent for identifying microsporidian | |
| CN111254155A (en) | Method for expressing virus vaccine by using plant as host | |
| CN106243199B (en) | There is the Vip3Aa11 protein mutant of high activity to beet armyworm | |
| CN113512098B (en) | Indirect ELISA (enzyme-Linked immuno sorbent assay) method for identifying swine fever virus and bovine viral diarrhea virus serum antibodies and application thereof | |
| CN104862331B (en) | A kind of method of solubility expression Rhodococcus equi Disease-causing gene VapA albumen |
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 |