CN109136267A - BVDV ErnsThe method of the ox of albumen and antibody and screening BVDV infection - Google Patents
BVDV ErnsThe method of the ox of albumen and antibody and screening BVDV infection Download PDFInfo
- Publication number
- CN109136267A CN109136267A CN201811095791.5A CN201811095791A CN109136267A CN 109136267 A CN109136267 A CN 109136267A CN 201811095791 A CN201811095791 A CN 201811095791A CN 109136267 A CN109136267 A CN 109136267A
- Authority
- CN
- China
- Prior art keywords
- bvdv
- rns
- albumen
- antibody
- kit
- 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
- 241000710780 Bovine viral diarrhea virus 1 Species 0.000 title claims abstract description 188
- PPBOKXIGFIBOGK-BDTUAEFFSA-N bvdv Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)C(C)C)[C@@H](C)CC)C1=CN=CN1 PPBOKXIGFIBOGK-BDTUAEFFSA-N 0.000 title claims abstract description 185
- 238000000034 method Methods 0.000 title claims abstract description 40
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 24
- 238000012216 screening Methods 0.000 title claims abstract description 16
- 210000002966 serum Anatomy 0.000 claims abstract description 34
- 230000014509 gene expression Effects 0.000 claims abstract description 28
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 24
- 241000238631 Hexapoda Species 0.000 claims abstract description 18
- 241000701447 unidentified baculovirus Species 0.000 claims abstract description 18
- 229940031626 subunit vaccine Drugs 0.000 claims abstract description 15
- 108010008529 bovine viral diarrhea virus gp53 Proteins 0.000 claims abstract description 14
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 13
- 102000004190 Enzymes Human genes 0.000 claims description 40
- 108090000790 Enzymes Proteins 0.000 claims description 40
- 229940088598 enzyme Drugs 0.000 claims description 40
- 238000002965 ELISA Methods 0.000 claims description 32
- 241000700605 Viruses Species 0.000 claims description 26
- 230000000903 blocking effect Effects 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 26
- 238000001514 detection method Methods 0.000 claims description 18
- 239000013642 negative control Substances 0.000 claims description 18
- 239000013604 expression vector Substances 0.000 claims description 17
- 238000008157 ELISA kit Methods 0.000 claims description 13
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 13
- 239000000126 substance Substances 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 12
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 10
- 239000000427 antigen Substances 0.000 claims description 10
- 102000036639 antigens Human genes 0.000 claims description 10
- 108091007433 antigens Proteins 0.000 claims description 10
- 230000008901 benefit Effects 0.000 claims description 8
- 239000003550 marker Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- 238000002372 labelling Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 229960002685 biotin Drugs 0.000 claims description 5
- 235000020958 biotin Nutrition 0.000 claims description 5
- 239000011616 biotin Substances 0.000 claims description 5
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 claims description 4
- 102000013563 Acid Phosphatase Human genes 0.000 claims description 4
- 108010051457 Acid Phosphatase Proteins 0.000 claims description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 4
- 108010015776 Glucose oxidase Proteins 0.000 claims description 4
- 239000004366 Glucose oxidase Substances 0.000 claims description 4
- 102000009617 Inorganic Pyrophosphatase Human genes 0.000 claims description 4
- 108010009595 Inorganic Pyrophosphatase Proteins 0.000 claims description 4
- 108060001084 Luciferase Proteins 0.000 claims description 4
- 239000005089 Luciferase Substances 0.000 claims description 4
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 claims description 4
- 229960004373 acetylcholine Drugs 0.000 claims description 4
- 238000003149 assay kit Methods 0.000 claims description 4
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 4
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 4
- 229940116332 glucose oxidase Drugs 0.000 claims description 4
- 235000019420 glucose oxidase Nutrition 0.000 claims description 4
- 239000013641 positive control Substances 0.000 claims description 4
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims description 2
- 102100022624 Glucoamylase Human genes 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 239000000523 sample Substances 0.000 claims description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims 2
- 241000710914 Totivirus Species 0.000 abstract description 12
- 229940031551 inactivated vaccine Drugs 0.000 abstract description 7
- 230000009385 viral infection Effects 0.000 abstract description 5
- 239000000243 solution Substances 0.000 description 32
- 210000004027 cell Anatomy 0.000 description 25
- 241000283690 Bos taurus Species 0.000 description 17
- 239000013598 vector Substances 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 16
- 238000002474 experimental method Methods 0.000 description 14
- 230000009182 swimming Effects 0.000 description 10
- 206010051511 Viral diarrhoea Diseases 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 8
- 239000000872 buffer Substances 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 230000003472 neutralizing effect Effects 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 231100000614 poison Toxicity 0.000 description 6
- 239000002574 poison Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 4
- 241000710777 Classical swine fever virus Species 0.000 description 4
- 241000282898 Sus scrofa Species 0.000 description 4
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 4
- -1 azomethine Chemical compound 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000010166 immunofluorescence Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 102000016938 Catalase Human genes 0.000 description 3
- 108010053835 Catalase Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 125000000853 cresyl group Chemical class C1(=CC=C(C=C1)C)* 0.000 description 3
- LXWYCLOUQZZDBD-LIYNQYRNSA-N csfv Chemical compound C([C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LXWYCLOUQZZDBD-LIYNQYRNSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- VGIRNWJSIRVFRT-UHFFFAOYSA-N 2',7'-difluorofluorescein Chemical compound OC(=O)C1=CC=CC=C1C1=C2C=C(F)C(=O)C=C2OC2=CC(O)=C(F)C=C21 VGIRNWJSIRVFRT-UHFFFAOYSA-N 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241001118702 Border disease virus Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 206010011732 Cyst Diseases 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000710781 Flaviviridae Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 241000710778 Pestivirus Species 0.000 description 2
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 2
- 108010053210 Phycocyanin Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 238000013320 baculovirus expression vector system Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical group OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 208000031513 cyst Diseases 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 2
- 229960002963 ganciclovir Drugs 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound 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 SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- XNGIFLGASWRNHJ-UHFFFAOYSA-N phthalic acid Chemical compound OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 210000002845 virion Anatomy 0.000 description 2
- CZWUESRDTYLNDE-UHFFFAOYSA-N (2z)-2-[(2e,4e,6e)-7-[1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindol-1-ium-2-yl]hepta-2,4,6-trienylidene]-1-ethyl-3,3-dimethylindole-5-sulfonate Chemical compound CC1(C)C2=CC(S([O-])(=O)=O)=CC=C2N(CC)\C1=C/C=C/C=C/C=C/C1=[N+](CCCCCC(O)=O)C2=CC=C(S(O)(=O)=O)C=C2C1(C)C CZWUESRDTYLNDE-UHFFFAOYSA-N 0.000 description 1
- QGKMIGUHVLGJBR-UHFFFAOYSA-M (4z)-1-(3-methylbutyl)-4-[[1-(3-methylbutyl)quinolin-1-ium-4-yl]methylidene]quinoline;iodide Chemical compound [I-].C12=CC=CC=C2N(CCC(C)C)C=CC1=CC1=CC=[N+](CCC(C)C)C2=CC=CC=C12 QGKMIGUHVLGJBR-UHFFFAOYSA-M 0.000 description 1
- IDLISIVVYLGCKO-UHFFFAOYSA-N 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein Chemical compound O1C(=O)C2=CC=C(C(O)=O)C=C2C21C1=CC(OC)=C(O)C(Cl)=C1OC1=C2C=C(OC)C(O)=C1Cl IDLISIVVYLGCKO-UHFFFAOYSA-N 0.000 description 1
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 1
- XJGFWWJLMVZSIG-UHFFFAOYSA-N 9-aminoacridine Chemical compound C1=CC=C2C(N)=C(C=CC=C3)C3=NC2=C1 XJGFWWJLMVZSIG-UHFFFAOYSA-N 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 241000238421 Arthropoda Species 0.000 description 1
- 241000701412 Baculoviridae Species 0.000 description 1
- 241000537222 Betabaculovirus Species 0.000 description 1
- 241000283725 Bos Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000282994 Cervidae Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- MQIUGAXCHLFZKX-UHFFFAOYSA-N Di-n-octyl phthalate Natural products CCCCCCCCOC(=O)C1=CC=CC=C1C(=O)OCCCCCCCC MQIUGAXCHLFZKX-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 244000283207 Indigofera tinctoria Species 0.000 description 1
- LSDPWZHWYPCBBB-UHFFFAOYSA-N Methanethiol Chemical compound SC LSDPWZHWYPCBBB-UHFFFAOYSA-N 0.000 description 1
- 206010029174 Nerve compression Diseases 0.000 description 1
- 206010029803 Nosocomial infection Diseases 0.000 description 1
- AWZJFZMWSUBJAJ-UHFFFAOYSA-N OG-514 dye Chemical compound OC(=O)CSC1=C(F)C(F)=C(C(O)=O)C(C2=C3C=C(F)C(=O)C=C3OC3=CC(O)=C(F)C=C32)=C1F AWZJFZMWSUBJAJ-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- GYDJEQRTZSCIOI-UHFFFAOYSA-N Tranexamic acid Chemical compound NCC1CCC(C(O)=O)CC1 GYDJEQRTZSCIOI-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 108010004469 allophycocyanin Proteins 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229960001441 aminoacridine Drugs 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- BJQHLKABXJIVAM-UHFFFAOYSA-N bis(2-ethylhexyl) phthalate Chemical compound CCCCC(CC)COC(=O)C1=CC=CC=C1C(=O)OCC(CC)CCCC BJQHLKABXJIVAM-UHFFFAOYSA-N 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 125000001295 dansyl group Chemical group [H]C1=C([H])C(N(C([H])([H])[H])C([H])([H])[H])=C2C([H])=C([H])C([H])=C(C2=C1[H])S(*)(=O)=O 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- IINNWAYUJNWZRM-UHFFFAOYSA-L erythrosin B Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C([O-])=C(I)C=C21 IINNWAYUJNWZRM-UHFFFAOYSA-L 0.000 description 1
- 229940011411 erythrosine Drugs 0.000 description 1
- 235000012732 erythrosine Nutrition 0.000 description 1
- 239000004174 erythrosine Substances 0.000 description 1
- VYXSBFYARXAAKO-UHFFFAOYSA-N ethyl 2-[3-(ethylamino)-6-ethylimino-2,7-dimethylxanthen-9-yl]benzoate;hydron;chloride Chemical compound [Cl-].C1=2C=C(C)C(NCC)=CC=2OC2=CC(=[NH+]CC)C(C)=CC2=C1C1=CC=CC=C1C(=O)OCC VYXSBFYARXAAKO-UHFFFAOYSA-N 0.000 description 1
- GWQVMPWSEVRGPY-UHFFFAOYSA-N europium cryptate Chemical compound [Eu+3].N=1C2=CC=CC=1CN(CC=1N=C(C=CC=1)C=1N=C(C3)C=CC=1)CC(N=1)=CC(C(=O)NCCN)=CC=1C(N=1)=CC(C(=O)NCCN)=CC=1CN3CC1=CC=CC2=N1 GWQVMPWSEVRGPY-UHFFFAOYSA-N 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 208000027686 extensive ulcer Diseases 0.000 description 1
- FPVGTPBMTFTMRT-NSKUCRDLSA-L fast yellow Chemical compound [Na+].[Na+].C1=C(S([O-])(=O)=O)C(N)=CC=C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 FPVGTPBMTFTMRT-NSKUCRDLSA-L 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- VYNDHICBIRRPFP-UHFFFAOYSA-N pacific blue Chemical compound FC1=C(O)C(F)=C2OC(=O)C(C(=O)O)=CC2=C1 VYNDHICBIRRPFP-UHFFFAOYSA-N 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- IEQIEDJGQAUEQZ-UHFFFAOYSA-N phthalocyanine Chemical compound N1C(N=C2C3=CC=CC=C3C(N=C3C4=CC=CC=C4C(=N4)N3)=N2)=C(C=CC=C2)C2=C1N=C1C2=CC=CC=C2C4=N1 IEQIEDJGQAUEQZ-UHFFFAOYSA-N 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 150000002910 rare earth metals Chemical class 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 102220029906 rs907610 Human genes 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000036413 temperature sense Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- C07K16/08—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
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
-
- 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
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- General Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of BVDV ErnsThe method of the ox of albumen and antibody and screening BVDV infection, is related to field of biotechnology, the BVDV ErnsAlbumen is expressed using baculovirus insect cell system carrier system, the albumen of expression and with the protein bound monoclonal antibody, and include the ErnsAlbumen and/or the kit of monoclonal antibody can be widely used in the correlative study of BVDV.When ox is after BVDV E2 subunit vaccine is immune, can may be not present in the presence of the antibody in conjunction with BVDV E2 albumen in the serum of ox can be with BVDV ErnsThe antibody that albumen combines.When ox is by the totivirus infection of BVDV or after the inactivated vaccine of totivirus is immune, can exist in the serum of ox can be with BVDV ErnsThe antibody that albumen combines.Therefore the present invention using both between difference, come judge the ox after BVDV E2 subunit vaccine is immune whether by BVDV virus infection, it is convenient and efficient.
Description
Technical field
The present invention relates to field of biotechnology, more particularly, to a kind of BVDV ErnsAlbumen and antibody and screening BVDV infection
Ox method.
Background technique
Bovine viral diarrhea virus is a kind of Niu Chuanran based on diarrhea, cough and fever in New York first discovery
Property disease, referred to as bovine viral diarrhoea (BVD).Find that a kind of disease incidence is low and the death rate is high again afterwards, with the extensive ulcer of gastrointestinal tract
For the disease of the ox of obvious lesion, referred to as mucosal disease (Mucosaldisease, MD).American Veterinary association in 1971 is by bovine viral
Property diarrhea (BVD) and mucosal disease (MD) Uniform Name be bovine viral diarrhoea/mucosal disease, abbreviation BVD/MD.
Bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) is flaviviridae
(Flaviviridae) representative species of pestivirus (Pestivirus), the BVDV and swine fever virus (Classical in category
Swine fever virus, CSFV) and sheep border disease virus (Border disease virus, BDV) there are antigen correlation
Property, there is cross reactions in serology, and can break through host specificity and cross-infection occurs, therefore BVDV is not only
One of important pathogen of ox, and sheep, goat, pig, deer and other ruminants can be infected.
BVDV virion is subsphaeroidal, and diameter is about 40-60nm, and nucleocapsid diameter is 25-30nm, there is cyst membrane.In CsCl
Middle buoyant density is 1.14g/cm3, sedimentation coefficient S20=140.Virus is sensitive to ether, chloroform and pH3.0.Virus is thermo-labile,
56 DEG C can make its inactivation, and MgCl2 does not shield to virus.Virus is stable at low temperature, and the virus of vacuum freeze-drying can be -60
The long period is saved at~-70 DEG C.
BVDV genome is single-stranded positive RNA, and Genome Size is about 12.5kb, is divided into 5 ' end non-translational regions, opening is read
Read framework region and 3 ' end non-translational regions.Wherein E2 albumen be determine the antigenic main portions of BVDV, and with anti-BVDV antibody
In conjunction with the main portions of, mediated immunity neutralization reaction and host cell identification, absorption.The gene of coding E2 albumen is located at ORF's
It 2077-3198, is made of 374 amino acid residues.Non-glycosylated protein molecular weight is about 42kDa.
BVDV E2 subunit vaccine will list, but still infect wild poison generation for identifying immune vaccine generation antibody
Effective detection method of antibody is not yet established.And the relevant albumen research of BVDV is less in current research, lacks research
The GAP-associated protein GAP and antibody of BVDV still infects wild poison generation antibody so that lacking foundation and identifying immune vaccine generation antibody
The technical foundation of effective detection method.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is the provision of a kind of expression BVDV ErnsThe expression vector of albumen alleviates existing skill
BVDV E can be expressed by lacking one kind present in artrnsThe problem of carrier and system of albumen.
The second object of the present invention is the provision of a kind of BVDV E of above-mentioned expression vector expressionrnsAlbumen, which can
To be widely used in the correlative study of BVDV, material base has been established for the relevant molecular biology experiment of BVDV.
The third object of the present invention is the provision of a kind of and above-mentioned BVDV ErnsProtein bound monoclonal antibody, the list
Clonal antibody can be widely used in the correlative study of BVDV, establish substance base for the relevant molecular biology experiment of BVDV
Plinth.
The fourth object of the present invention is the provision of a kind of kit, which includes BVDV ErnsAlbumen and/or with
BVDV ErnsProtein bound monoclonal antibody can be applied in the molecular biology experiment of a variety of research BVDV.
The fifth object of the present invention is the provision of a kind of above-mentioned BVDV ErnsAlbumen and above-mentioned BVDV ErnsProtein binding
Monoclonal antibody or kit detection BVDV in purposes, this is widely used, suitable for detection BVDV and BVDV include
Albumen or anti-BVDV antibody.
The sixth object of the present invention is the provision of a kind of method of ox for screening the wild virus strain infection of BVDV, alleviates existing
Have present in technology, lack it is a kind of from through BVDV E2 subunit vaccine be immunized ox in, filter out and infected the wild poison of BVDV
The problem of method of the ox of strain.
In order to solve the above technical problems, spy of the present invention adopts the following technical scheme that
A kind of expression BVDV ErnsThe expression vector of albumen, the expression vector express the sequence as shown in SEQ ID NO.1
The E of codingrnsAlbumen；The expression vector is baculoviral；
Further, the BVDV ErnsAlbumen is expressed by baculovirus insect cell system carrier system.
The present invention also provides a kind of BVDV E of above-mentioned expression vector expressionrnsAlbumen；
Further, the BVDV ErnsThe labeled substance markers of albumen；
Further, the label includes fluorescent marker, enzyme label or biotin labeling.
The present invention also provides a kind of and above-mentioned BVDV ErnsProtein bound monoclonal antibody；
Further, the labeled substance markers of the monoclonal antibody；
Further, the label includes fluorescent marker, enzyme label or biotin labeling.
The present invention also provides a kind of kit, the kit includes above-mentioned BVDV ErnsAlbumen and/or above-mentioned Dan Ke
Grand antibody.
Further, the kit is ELISA kit；The ELISA kit includes labeled substance markers
BVDV ErnsAlbumen；And/or labeled substance markers with BVDV ErnsProtein bound monoclonal antibody；
Further, the BVDV ErnsAlbumen is the albumen of enzyme label；And/or the BVDV ErnsThe Dan Ke of albumen
Grand antibody is the antibody of enzyme label；
Further, enzyme used in the enzyme label includes horseradish peroxidase, acid phosphatase, alkaline phosphatase
Enzyme, beta-D-galactosidase, glucose oxidase, glucoamylase, enzyme acetylcholine, inorganic pyrophosphatase, luciferase or
Catalase；
Further, the ELISA kit further includes antigen coated microplate, positive control, negative control, sample dilution
Liquid, cleaning solution, developing solution and terminate liquid.
Further, the ELISA kit is to block ELISA antibody assay kit；The blocking ELISA antibody inspection
BVDV E in test agent boxrnsAlbumen is as coating plate antigen, the BVDV ErnsThe monoclonal antibody of albumen is horseradish peroxidating
The antibody of object enzyme label；
Further, negative control is BVDV E in the kitrnsNegative serum.
The present invention also provides a kind of above-mentioned BVDV ErnsAlbumen, said monoclonal antibody or mentioned reagent box are detecting
Purposes in BVDV.
The present invention also provides a kind of methods of ox for screening the wild virus strain infection of BVDV, and the method includes detection oxen
It whether there is and BVDV E as claimed in claim 2 in serumrnsThe antibody that albumen combines；
If existing and the BVDV E in the serum of oxrnsThe antibody that albumen combines, then the ox is by the wild poison of BVDV
The ox of strain infection；
The ox is immune through BVDV E2 subunit vaccine；
Further, described to be detected as detecting using mentioned reagent box.
Further, being detected in serum by ELISA method whether there is and BVDV E as claimed in claim 2rnsAlbumen
The antibody combined；
Wherein, the antibody that enzyme marks in ELISA is as claimed in claim 3 and BVDV ErnsProtein bound monoclonal is anti-
Body；
Further, the antibody of the enzyme label is the antibody of horseradish peroxidase-labeled.
Further, the ELISA method is blocking-up type ELISA detection method；
Further, the blocking-up type ELISA detection method includes the following steps: for sample to be tested to be incubated in coating
BVDV ErnsIn the ELISA reaction plate of albumen, the antibody of the enzyme label is then added, detects OD after being incubated for colour developing450Value,
Then according to OD450Value calculate sample blocking rate；Wherein,
Blocking rate is negative sample in the sample of 0%-30%；
Blocking rate is positive sample in the sample of 35%-100%；
Blocking rate is suspicious sample in the sample of 30%-35%；
Further, the blocking rate calculates as follows:
Blocking rate=(OD average value-sample OD average value of negative control)/negative control OD average value ×
100%.
Compared with prior art, the invention has the following beneficial effects:
Expression BVDV E provided by the inventionrnsThe expression vector of albumen uses baculovirus insect cell system carrier system
System expression, with high security, the vector construction period is short and the high advantage of protein expression level.
The E of above-mentioned expression vector expression provided by the inventionrnsAlbumen has neutralizing epitope, the neutralizing antibody tool of generation thereon
There is the ability for neutralizing BVDV and CSFV.ErnsWith dsRNA and RNase activity is combined, the activity is to the letter for inhibiting dsRNA to mediate
It number is required.Therefore the albumen is a kind of highly important albumen in the experiment of research BVDV.The albumen can also pass through
Further modification is applied in different kinds of molecules biological experiment, has established substance base for the relevant molecular biology experiment of BVDV
Plinth.
Provided by the invention and above-mentioned BVDV ErnsProtein bound monoclonal antibody, the monoclonal antibody can be extensive
Applied to the correlative study of BVDV, material base has been established for the relevant molecular biology experiment of BVDV.
Kit provided by the invention, the kit include BVDV ErnsAlbumen and/or with BVDV ErnsIt is protein bound
Monoclonal antibody can be applied in the molecular biology experiment of a variety of research BVDV for example can be applied to immunoblotting, exempt from
In epidemic disease group, enzyme linked immunoassay or immunofluorescence technique.
Above-mentioned BVDV E provided by the inventionrnsAlbumen and above-mentioned BVDV ErnsProtein bound monoclonal antibody or reagent
Purposes of the box in detection BVDV, this is widely used, suitable for detecting the albumen or anti-BVDV that BVDV and BVDV include
Antibody.
The method of the ox of the screening wild virus strain infection of BVDV provided by the invention, can be from through BVDV E2 subunit vaccine
In immune ox, the ox for having infected the wild strain of BVDV is filtered out.When ox is after BVDV E2 subunit vaccine is immune, the blood of ox
Can may be not present in the presence of the antibody in conjunction with BVDV E2 albumen in clear can be with BVDV ErnsThe antibody that albumen combines.When ox quilt
The totivirus of BVDV infects or after the inactivated vaccine of totivirus is immune, and can exist in the serum of ox can be with BVDV Erns
The antibody that albumen combines.Therefore for the present invention using the difference between both, i.e., whether there is in the serum of sample to be tested can
With with BVDV ErnsThe antibody that albumen combines, to judge the ox after BVDV E2 subunit vaccine is immune whether again by BVDV
Virus infection.This method utilizes molecular biology method, and sample need to only acquire the serum of ox, utilize the spy between antigen and antibody
Whether anisotropic reaction can be detected out the sample for providing the serum by BVDV virus infection, therefore this method is with easy to operate
And the big advantage of flux.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the flow chart for the expression vector establishment that the embodiment of the present invention 1 provides；
Fig. 2 is the target gene for carrier construction for the amplification that the embodiment of the present invention 1 provides；
Fig. 3 is the segment for the target gene that the infection 4 days Sf-9 insect cells that the embodiment of the present invention 1 provides amplify；
Fig. 4 is E in the embodiment of the present invention 1rnsThe Western-blot testing result of protein expression.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with embodiment and attached drawing, it is clear that retouched
The embodiment stated is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field
Those of ordinary skill's every other embodiment obtained without making creative work, belongs to protection of the present invention
Range.
The present invention provides a kind of expression BVDV ErnsThe expression vector of albumen, the expression vector expression such as SEQ ID
The E of sequential coding shown in NO.1rnsAlbumen；The expression vector is baculoviral.
The present invention expresses BVDV E using baculovirus insect cell system carrier systemrnsAlbumen, baculoviral belong to bar
Shape Viraceae (Baculoviridae), is divided into nucleopolyhedrosis virus category and Granulovirus, and virion is in the shape of a rod, is one
Class single infection arthropod and the virus with cyst membrane.The exogenous DNA that baculoviral can accommodate larger segment is inserted into
For the ideal carrier for expressing large fragment DNA.
In some preferred embodiments, the BVDV ErnsAlbumen is by baculovirus insect cell system carrier system
Expression.Baculovirus insect cell system carrier system (Insect cell baculovirus expression vector
System, IC-BEVS) there is traditional advantage possessed by eukaryotic expression system, while having both prokaryotic expression system recombinant protein
The high feature of expression quantity.Baculovirus insect cell system carrier system has the advantage that 1. baculoviral has good
Safety.2. system vector construction period compared with mammalian cell expression system is short.3. baculoviral can accommodate big point
The Insert Fragment of son.4. baculovirus expression system obtains the high-caliber expression of recombinant protein.5. baculovirus expression carries
System system has in the same intracellular ability for expressing multiple genes simultaneously.
ErnsAlbumen is important one of the structural proteins of BVDV, and body can be induced to generate neutralizing antibody, therefore expression system
In it is particularly significant to the process of the modification after protein translation, if expression albumen lack posttranslational modification processing, it will make table
There are antigenic specificities with native protein for the albumen reached, and easily form inclusion body.Baculovirus insect cell system carrier system
Unite the E expressedrnsAlbumen it is available with natural structure similar in albumen, thus possess epitope similar with native protein.
The albumen expressed using baculovirus insect cell system carrier system, the antibody that induction body generates are connect with natural antibody
Closely, be conducive to experiment and application in molecular biology in this way, it is possible to reduce experimental error.
The present invention also provides a kind of BVDV E of above-mentioned expression vector expressionrnsAlbumen.ErnsIt is in BVDV coding albumen
The conservative albumen of height, there is neutralizing epitope thereon, and the neutralizing antibody of generation has the ability for neutralizing BVDV and CSFV.ErnsWith knot
DsRNA and RNase activity is closed, which is required to the signal for inhibiting dsRNA to mediate.Therefore the albumen is research BVDV's
It is a kind of highly important albumen in experiment.The albumen can also be real applied to different kinds of molecules biology by further modifying
In testing, such as it can be used for anti-BVDV E in sizing or quantitative detection sample to be testedrnsThe antibody of albumen, such as can be used as point
Standard items in sub- biological experiment, such as be also used as preparing anti-BVDV ErnsThe antigen etc. of the antibody of albumen, the albumen
Material base has been established for the relevant molecular biology experiment of BVDV.
The present invention also provides a kind of and BVDV ErnsProtein bound monoclonal antibody, the monoclonal antibody can be with
It is widely used in the correlative study of BVDV, has established material base for the relevant molecular biology experiment of BVDV, such as can be with
Applied in immunoblotting, immunohistochemistry, enzyme linked immunoassay or immunofluorescence technique.The BVDV E of the present inventionrnsEgg
The monoclonal antibody of white combination is the antibody obtained using conventional method, such as uses BVDV ErnsAfter protein immunization mouse, so
Above-mentioned antibody is obtained by preparing hybridoma afterwards.The present invention to the preparation method of the monoclonal antibody with no restrictions, as long as
It can be with the BVDV ErnsProtein binding.
In some optional embodiments of the present invention, the BVDV ErnsAlbumen and/or the monoclonal antibody are through marking
Remember substance markers.In some alternative embodiments, it is described label for example can be but be not limited to fluorescent marker, enzyme label or
Person's biotin labeling.
In some alternative embodiments, the fluorescent marker is typical but non-limiting uses following fluorescent dye mark
Note: Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminacrine,
BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5- carboxylic
Base -4 ', 5 '-two chloro- 2 ', 7 '-dimethoxyfluoresceins, 5- carboxyl -2 ', 4 ', 5 ', 7 '-tetrachlorofluoresceins, 5- carboxyl fluorescence
Element, 5- carboxyrhodamine, 6- carboxyrhodamine, 6- carboxyl tetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7,
6-FAM, dansyl Cl, fluorescein, HEX, 6-JOE, NBD (7- nitro benzo -2- oxa- -1,3- diazole), Oregon Green
488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid (TPA), isophthalic
Dioctyl phthalate, cresols consolidate purple, cresols royal purple, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, yellow fast
Purine, succinylfluoresceins, rare earth metal cryptate, three pairs of pyridyl group diamines europiums, europium cryptate or chelate, two
Amine, dicyanin, La Jolla indigo plant dyestuff, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, sulphur
Amine, algae red green white, phycoerythrin R, REG, rhodamine be green, rhodamine isothiocyanates, rhodamine are red, ROX, TAMRA, TET,
One of TRIT (the different mercaptan of tetramethylrhodamine), tetramethylrhodamine and texas Red are a variety of.
In some alternative embodiments, the enzyme marks the typical but non-limiting enzyme mark using following type
Note: horseradish peroxidase, acid phosphatase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucose form sediment
One of powder, enzyme acetylcholine, inorganic pyrophosphatase, luciferase or catalase are a variety of.
The present invention also provides a kind of kit, the kit includes the BVDV ErnsAlbumen and/or it is described with
BVDV ErnsProtein bound monoclonal antibody.
Albumen and antibody are common reagents in molecular biology experiment, therefore contain the BVDV ErnsAlbumen and/
Or described and BVDV ErnsThe kit of protein bound monoclonal antibody serves many purposes, such as can be applied to Diagnosis of Sghistosomiasis
In mark, immunohistochemistry, enzyme linked immunoassay or immunofluorescence technique.
In some alternative embodiments, the kit is ELISA kit；The ELISA kit includes warp
Mark the BVDV E of substance markersrnsAlbumen；And/or the BVDV E of labeled substance markersrnsThe monoclonal antibody of albumen.
Such as in a kind of optional embodiment, the ELISA kit includes for being coated on reaction plate
BVDV ErnsAlbumen, and the BVDV E marked using enzymernsCompetitive ELISA can be used in the monoclonal antibody of albumen, the kit
Detect the BVDV E in sample to be testedrnsAntibody；In another example in another optional embodiment, the ELISA kit
BVDV E including including enzyme labelrnsThe monoclonal antibody of albumen, the method that double crush syndrome can be used are detected to test sample
BVDV E in thisrnsAlbumen.
In some optionally embodiments, the BVDV ErnsAlbumen is the albumen of enzyme label；And/or the BVDV
ErnsThe monoclonal antibody of albumen is the antibody of enzyme label.Optionally, enzyme used in enzyme label for example can be but unlimited
It forms sediment in for horseradish peroxidase, acid phosphatase, alkaline phosphatase, beta-D-galactosidase, glucose oxidase, glucose
Powder, enzyme acetylcholine, inorganic pyrophosphatase, luciferase or catalase.It can be understood that the present invention does not limit ELISA
The type of enzyme in middle enzyme label labeling antibody, can be, it is preferable to use horseradish peroxidase-labeled as long as meeting ELISA experimental principle
Antibody.
In some alternative embodiments, the kit further include antigen coated microplate, positive control, negative control,
Sample diluting liquid, cleaning solution, developing solution and terminate liquid.
In some alternative embodiments, the ELISA kit is to block ELISA antibody assay kit；It is described
Block BVDV E in ELISA antibody assay kitrnsAlbumen is as coating plate antigen, the BVDV ErnsThe monoclonal of albumen
Antibody is the antibody of horseradish peroxidase-labeled；
The kit also includes negative control, and the negative control is BVDV ErnsNegative antibody serum.
The present invention also provides a kind of above-mentioned BVDV ErnsAlbumen, above-mentioned BVDV ErnsMonoclonal antibody or mentioned reagent
Purposes of the box in detection BVDV.In some alternative embodiments, the purposes include detection sample to be tested whether by
BVDV infection；Whether sample to be tested generates the antibody of anti-BVDV after BVDV totivirus puts out a fire vaccine immunity；It is in sample to be tested
It is no that there are associated antibodies of BVDV etc..
The present invention also provides a kind of methods of ox for screening the wild virus strain infection of BVDV, and this method is for detecting through BVDV
Whether the immune ox of E2 subunit vaccine infects BVDV again；
The method includes detecting in the serum of ox to whether there is and the BVDV ErnsThe antibody that albumen combines；If ox
Serum in exist and the BVDV ErnsThe antibody that albumen combines, then the ox is by the ox of the wild virus strain infection of BVDV.
When ox is after BVDV E2 subunit vaccine is immune, can exist in the serum of ox combined with BVDV E2 albumen it is anti-
Body may be not present can be with BVDV ErnsThe antibody that albumen combines.When ox is by the totivirus infection of BVDV or through totivirus
Inactivated vaccine it is immune after, can exist in the serum of ox can be with BVDV ErnsThe antibody that albumen combines.Therefore present invention benefit
With the difference between both, i.e., whether there is in the serum of sample to be tested can be with BVDV ErnsThe antibody that albumen combines,
To judge the ox after BVDV E2 subunit vaccine is immune whether again by BVDV virus infection.This method utilizes molecular biology
Method, sample need to only acquire the serum of ox, reacting to can be detected out and provide the blood using the specificity between antigen and antibody
Whether clear sample is by BVDV virus infection, therefore this method has the advantages that easy to operate and flux is big.
In some alternative embodiments, being detected in serum by ELISA method whether there is and above-mentioned BVDV Erns
The antibody that albumen combines；Wherein, enzyme target antibody is described with BVDV E in ELISArnsThe monoclonal that albumen combines is anti-
Body；The antibody of the enzyme label is preferably the antibody of horseradish peroxidase-labeled.
In one preferred embodiment, the ELISA method is blocking-up type ELISA detection method.Some optional
Embodiment in, this method comprises the following steps: sample to be tested and negative control being incubated in and have been coated with BVDV ErnsAlbumen
ELISA reaction plate in, be then added the enzyme target antibody, microplate reader detects OD after being incubated for colour developing450Value, then basis
OD450Value calculate sample blocking rate；Wherein, blocking rate is negative sample in the sample of 0%-30%；Blocking rate is in 35%-
100% sample is positive sample；Blocking rate is suspicious sample in the sample of 30%-35%.
In some preferred embodiments, the blocking rate calculates as follows:
Blocking rate=(OD average value-sample OD average value of negative control)/negative control OD average value ×
100%.
In an optional embodiment, the blocking-up type ELISA detection method is as described below: coating BVDV ErnsEgg
Test serum and each hole 50 μ l/ of negative control is added after washing closing in white (0.1 μ g/mL), is incubated at room temperature 1h, PBST washing 6
It is secondary；The BVDV E of horseradish peroxidase-labeled is addedrnsThe monoclonal antibody of albumen is incubated at room temperature 1h, and PBST is washed 6 times, added
100 μ l of substrate developing solution is reacted at room temperature 5 minutes；In each reacting hole plus 100 μ l of 1mol/L hydrochloric acid terminates reaction.
Beneficial effects of the present invention are further illustrated below with reference to preferred embodiment.
1 vector construction of embodiment
Experimental material:
Reagent: the BVDV E of synthesisrns, primer, Pfu archaeal dna polymerase, dNTPs, PCR buffer,Vector, NotI restriction endonuclease, competent cell, QIAguick PCR purification kit, LB-kana
Culture solution, BaculoDirectTMBaculovirus Expression System kit, SF900II serum-free medium,
The secondary antibody of the rabbit-anti pig of reagent needed for Western-blot, bovine viral diarrhoea Erns positive serum, FITC label.
Cell: Sf-9 insect cell is purchased from U.S. Invitrogen Life Technologies, Inc.
Bovine viral diarrhoea Erns recombinant baculovirus construction method process is as shown in Figure 1.
A)BVDV ErnsThe amplification of gene:
BVDV ErnsGene is using the sequence as shown in SEQ ID NO.1 synthesized as template, with high-fidelity PfuDNA polymerase
Expand its BVDV ErnsGene, amplimer include BacERNS-F1 and BacERNS-R1, and wherein BacERNS-F1 has such as SEQ
Sequence shown in ID NO.2, BacERNS-R1 have the sequence as shown in SEQ ID NO.3,
Primer sequence is as follows:
BacERNS-F1:5'-CACCATGGTTGGATCCATGAAAATAGTGCCC-3'；
BacERNS-R1:5’-TATAAGCTTAGCCGCATATGCTCC-3’。
In addition, primer introduces at 5 ' ends of PCR product in present embodimentCloningsite (CACC) and
Kozak transcriptional initiation sequence (ACCATGG) comprising ATG.
Wherein PCR detailed step is as described below: taking the BVDV E synthesizedrnsFor template, primer (10 μM) each 1 μ l is added,
5 μ l dNTPs (2.5mM), 5 μ 10 × PCR of l buffer, 1 μ l Pfu polymerase, 36 μ l distilled waters.First with 95 DEG C of initial denaturations 3
Minute, it is denaturalized to anneal within 30 seconds, 57 DEG C 30 seconds, 72 DEG C to extend then at 94 DEG C and carries out 35 circulations under 1 minute program, last 72 DEG C
It re-extends 10 minutes.Electrophoretic analysis is carried out in 100 volts of voltages with 1.0% Ago-Gel after reaction, electrophoresis result is such as
Shown in Fig. 2, wherein road 1 is gene template forever, swimming lane 2 is negative control, and swimming lane 3 is DNA Marker 2000.Target gene is big
Small about 946bp can be seen that Successful amplification goes out the segment of target gene size according to fig. 2.Target fragment uses QIAguickTM
The recycling of PCR purification kit, detailed step refer to purification kit operation manual.After purifying through 1.0% agargel electrophoresis into
After row confirmation, its OD value, conversion recycling DNA concentration are measured in wavelength 260nm using spectrophotometer, -20 DEG C or less refrigerators are protected
It deposits spare.
B) the building of baculovirus transfer vector
a)Cloning reaction: test method refers to Invitrogen operation manual.Reaction system is as follows: 0.5-4 μ l
Purified blunt-end PCR product (wherein including 5-10ng DNA), 1 μ l salting liquid (1.2mol/LNaCl, 0.06mol/
LMgCl2), 1 μ lCarrier, adding distilled water makes 6 μ l of total volume.Postposition room temperature 15 is mixed to divide
Zhong Hou sets reaction tube on ice, spare.
B) plasmid converts: taking in 2 μ l step a)Cloning reaction solution adds to One
TOP10Chemically Competent E.coli (Invitrogen) is acted in pipe, and patting tube wall is uniformly mixed it, ice
It is upper to place 30 minutes, thermal shock 30 seconds in 42 DEG C of water-baths, reaction tube is put back into cooled on ice 2 minutes rapidly later, then to reaction
The 250 μ l SOC culture solutions risen again are added in pipe, are put into 37 DEG C with revolving speed 200r/min shaken cultivation 60 minutes.Take 50 μ l
Above-mentioned bacterium solution is uniformly applied in LB-kana screening flat board (containing 50 μ g/ml kanamycin), and face up placement 30 minutes,
It is cultured after base absorbs completely after bacterium solution and is inverted culture dish, overnight incubation in 37 DEG C of incubators.
c)BVDV ErnsThe screening of recombinant transfer vector: by PCR method, BVDV E is carried outrnsAt the beginning of recombinant transfer vector
Step screening.With sterile 10 μ l white pipette tips, 24 monoclonals of picking, are respectively seeded to 1ml LB- in LB-kana screening flat board
In kana culture solution, in 37 DEG C of constant-temperature shaking incubators, stayed overnight with revolving speed 200r/min shaken cultivation.
PCR reaction solution is prepared first and is distributed into 24 pipes, contains BacERNS-F1 and BacERNS-R1 in every pipe PCR reaction solution
Primer (10 μM) μ 10 × PCR of l of each 0.5 μ l, 2.5 μ l dNTPs (2.5mM), 2.5 buffer, 0.5 μ l Pfu DNA polymerization
Enzyme, 16.5 μ l distilled waters.2 μ l bacterium solutions are as PCR reaction template.
PCR reaction process is as previously described.Electrophoretic analysis is carried out in 100 volts of voltages with 1.0% agar gel after reaction.If
There is expected product, then chooses 4-5 corresponding bacterium solutions, 1 ‰ ratios are seeded in 3ml LB-Kana culture solution, in 37 DEG C
In constant-temperature shaking incubator, stayed overnight with revolving speed 200r/min shaken cultivation.
Use QIAprepTMThe extraction of Spin Plasmid Kit progress recombinant vector: by 3ml bacterium solution with revolving speed 10000r/
After min is centrifuged 4 minutes, supernatant is removed, 250 μ l buffer P1 (100mg/mL of A containing RNase) are added, thallus is made to suspend
Afterwards, mixed liquor is moved in 1.5ml microcentrifugal tube, adds 250 μ l buffer P2, and spun upside down and become for several times to liquid
It is transparent sticky, 350 μ l buffer N3, slow shaken several times, with revolving speed 12000r/min centrifugation 10 minutes are added.Draw supernatant
It is added in QIAprep spin column, with revolving speed 12000r/min centrifugation 1 minute, abandons filtrate, 750 μ l buffer are added
PE washes away extra salt into column, with revolving speed 12000r/min centrifugation 1 minute, abandons filtrate, then be centrifuged 2 minutes to keep away
Exempt from liquid residual, finally set column in new microcentrifugal tube, 20 μ l distilled waters are added, standing 1 minute fills carrier DNA
Divide dissolution, with revolving speed 12000r/min centrifugation 1 minute, collects filtrate, be stored in -20 DEG C or less.Recombinant vector is through limiting inscribe
After enzyme NotI cutting, electrophoretic analysis is carried out in 100 volts of voltages with 1.0% Ago-Gel.If being consistent with expected primer size,
Wherein 2 extracted recombinant vectors are then chosen, withPrimer (the M13F/ of vector itself
M13R), sequence verification is carried out.
D) preservation of recombinant transfer vector: the E.coli strain inoculated containing recombinant transfer vector is trained in 5ml LB-kana
In nutrient solution (containing 50 μ g/ml kanamycin), 37 DEG C of constant-temperature shaking incubators are set, it is small with revolving speed 200r/min shaken cultivation 16
When, 20% glycerol is added, dispenses, sets -70 DEG C or less preservations.
C) LR recombining reaction
Recombinant transfer vector is after sequence verification is correct, by the recombinant vector of extraction, with spectrophotometer at wavelength 260nm
Survey the nucleic acid concentration of recombinant vector.Again with " BaculoDirectTMBaculovir μ s Expression System " carry out LR
Recombining reaction.Concrete operations are as follows: sterile working is by 1 μ l recombinant transfer vector (100-300ng), 4 μ l
Reaction buffer and 1 μ l distilled water are separately added into 10 μ l BaculoDirectTMIn Linear DNA (300ng).From-
LR is taken out in 70 DEG C of refrigeratorsEnzyme mix is set rise again on ice 2 minutes after, vibrate 2 times, 2 seconds every time, take 4 μ l LREnzyme mix is added in above-mentioned mixed liquor, and patting tube wall for several times is uniformly mixed it, can not acutely vibrate in order to avoid
Baculovirus DNA fracture.After 25 DEG C of senses are made 18 hours, 2 μ l Proteinase K solution are added, set 37 DEG C of effects 10
Minute, interrupt LR recombining reaction.
D it) transfects
Take 6 porocyte culture plates, every hole inoculation 1.5 × 106The Sf-9 of a activity good (cell survival rate is greater than 95%)
Cell repeats 2 holes.Cell is set 26-28 DEG C to cultivate 1 hour, cell is made to adhere completely to culture plate bottom.It needs first to match before transfection
Make following mixed liquor, transfection cocktail A:20 μ l LR recombining reaction liquid (containing the baculovirus DNA recombinated) and 200 μ l
SF900II serum-free insect cell culture solution；Transfection cocktail B:12 μ lReagent and 200 μ l
SF900II serum-free insect cell culture solution.Transfection cocktail A is mixed with B, patting tube wall makes its mixing, sets room temperature
Sense is made 45 minutes.Attached Sf-9 cell is first cleaned carefully with SF900II serum-free insect cell culture solution, then by A, B
Mixed liquor is divided into two pipes, and every pipe is added 800 μ l SF900II serum-free insect cell culture solutions, spins upside down for several times.Finally
Culture solution in 6 orifice plates is drained as far as possible, then is slowly added to A, B mixed liquor in 2 holes along tube wall.After tissue culture plate is sealed,
26-28 DEG C is cultivated 5 hours.Mixed liquor is taken out, the Sf- that 3ml contains antibiotic and 100 μM of ganciclovir is added in every hole
900 II SFM, are finally sealed with adhesive tape, are set in 26-28 DEG C of constant incubator and are cultivated 5.Successful baculoviral is not recombinated
Genosome, the thymidine kinase gene with herpes simplex virus, in the culture solution of addition ganciclovir, nothing
Method carries out the duplication of DNA, therefore reduces the generation for the baculoviral not recombinated.
E)BVDV ErnsThe harvest and identification of recombinant baculovirus
A) virus harvest: virus liquid is collected into 50ml centrifuge tube, thin with revolving speed 3000r/min centrifugation removal in 5 minutes
10% fetal calf serum is added in born of the same parents' fragment in virus liquid, later will be sick to reduce destruction of the protease that may be present to virus
4 DEG C or less spare, or -70 DEG C or less long-term preservations are kept in dark place in venom.For transfect after separation primary when, with 0.45 μm of filter
Film filtering, then be sub-packed in 1.5ml centrifuge tube.
B) viruses indentification:
The identification of gene: extracting the DNA infected in Sf-9 insect cell supernatant on the 4th, using primer BacERNS-F1 and
BacERNS-R1 expands the DNA in supernatant, as a result as shown in Figure 3, wherein swimming lane 1 is the sf9 cell of infection, swimming lane M
For DNA Marker200, swimming lane 2 is negative control, and swimming lane 3 is cell controls, and swimming lane 4 is positive control.It can from Fig. 3
Out, the sf9 cellular nucleic acid of infection is extracted, after reverse transcription and PCR amplification, electrophoresis result, which can be seen that, amplifies target gene size
There is target gene in segment, the sf9 of infection, tentatively illustrate the sf9 of infection for the positive into the cell.
ErnsProtein expression analysis: BVDV E is verified by Western-blotrnsThe expression of albumen and reactionogenicity.First
SDS-PAGE is carried out, then the protein band on SDS-PAGE glue is transferred on pvdf membrane, sets the skimmed milk power containing 5%
In PBST, room temperature is closed 30 minutes, is cleaned 5 times, and primary antibody BVDV E is addedrnsPositive serum, room temperature act on 1 hour, clean 5 times,
The secondary antibody of the rabbit-anti pig containing horseradish peroxidase-labeled is added, room temperature acts on 1 hour, cleans 5 times, in carrying out DAB in darkroom
Colour developing.As a result as shown in Figure 4, wherein swimming lane 1 is cell controls, and swimming lane 2 is the albumen of expression, and swimming lane M is albumen Marker.
As seen from Figure 4, protein expression carries out western-blot detection, occurs the band of expected about 35kDa, explanation as the result is shown
The albumen of expression has reactionogenicity.
Immunofluorescence: the BVDV E that will be builtrnsRecombinant baculovirus is inoculated in Sf-9 cell, sets 26-28 DEG C of constant temperature training
It supports every hole after cultivating 7,7 in case to be washed 6 times with PBST, 100 μ l formaldehyde is then added, fix 10 minutes, primary antibody BVDV is added
ErnsPositive serum (1 ︰ 1000), sense are made 1 hour, the IgG secondary antibody of the rabbit-anti pig of FITC label are added, in inverted fluorescence microscope
Whether there is or not fluorescence signals for lower observation.
Embodiment 3 is the method for the ox that blocking-up type ELISA screens the wild virus strain infection of BVDV
This method comprises the following steps: by BVDV ErnsAlbumen (0.1 μ g/mL) is coated in ELISA reaction plate, washing closing
Each hole 50 μ l/ of test serum sample is added afterwards, is incubated at room temperature 1h, PBST is washed 3-6 times, and horseradish peroxidase-labeled is added
BVDV ErnsThe monoclonal antibody of albumen is incubated at room temperature 1h, and PBST is washed 6 times, adds 100 μ l of substrate developing solution, reacts at room temperature 5 points
Clock；In each reacting hole plus 100 μ l of 1mol/L hydrochloric acid terminates reaction.
Wherein each sample to be tested includes following sample: the cow's serum of immune BVDV inactivated vaccine (totivirus)；Infect BVDV
The cow's serum of totivirus；The cow's serum of immune E2 subunit vaccine；Swine fever virus ErnsPositive Sera.
Reaction plate is placed in microplate reader to the OD for detecting each reacting hole450Value, calculate blocking rate；
Blocking rate is calculated according to following formula: blocking rate=(OD average value-sample OD average value of negative control)/yin
Property control OD average value × 100%.
Blocking rate is negative sample in the sample of 0%-30%；
Blocking rate is positive sample in the sample of 35%-100%；
Blocking rate is suspicious sample in the sample of 30%-35%；
Its result is as follows: E in the cow's serum of immune BVDV inactivated vaccine (totivirus)rnsThe positive rate of antibody positive is
93.7%；
Infect E in the cow's serum of BVDV totivirusrnsThe positive rate of antibody positive is 100%；
The cow's serum E of immune E2 subunit vaccinernsThe positive rate of antibody positive is 0%；
Swine fever virus ErnsPositive Sera ErnsThe positive rate of antibody positive is 0%.
Embodiment 4 screens the application of the method for the ox of the wild virus strain infection of BVDV
200 parts of cow's serums of screening acquisition before immune are coated with Erns albumen (0.1 μ g/mL) after purification, after washing closing
50 hole μ l/ of test serum is added, is incubated at room temperature 1h, PBST is washed 6 times；Enzyme is added and marks monoclonal antibody, is incubated at room temperature 1h, PBST washing
6 times, add 100 μ l of substrate developing solution, reacts at room temperature 5 minutes；In each reacting hole plus 100 μ l of 1mol/L hydrochloric acid terminates reaction.ErnsIt is anti-
Body positive rate is 15.5%,
By the ox isolated rearing of Erns negative antibody, wherein 80 immune bovine viral diarrhoea/mucosal virus inactivated vaccines
(totivirus), 80 immune bovine viral diarrhoeas/mucosal virus E2 subunit vaccine, another 9 are not immunized, and take a blood sample and survey after 35 days
ErnsAntibody is detected according to the above method, and bovine viral diarrhoea/mucosal virus inactivated vaccine (totivirus) Erns antibody is immunized
Positive rate 93.7% attacks recall rate 100% after poison；Immune bovine viral diarrhoea/mucosal virus E2 subunit vaccine, non-immune group
Ox Erns antibody positive rate 0% attacks recall rate 100% after poison, it was demonstrated that the detection method has good sensibility and special
Property, it can be used for detecting bovine viral diarrhoea/mucosal virus Erns antibody, can be used for screening the ox of the wild virus strain infection of BVDV.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
SEQUENCE LISTING
<110>Tian Kang Biological Co., Ltd.
<120>method of the ox of BVDV Erns albumen and antibody and screening BVDV infection
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 921
<212> DNA
<213>artificial sequence
<400> 1
atgaaaatag tgcccaaaga gtctgagaaa gacagcaaga ctaaaccgcc agatgctacg 60
atagtggtag atggagttaa ataccaggta aagaagaagg gaaaagtcaa gagtaaaaat 120
acgcaggacg gtttatatca taacaaaaat aagccgccag aatcacgcaa gaaactagag 180
aaagcattat tggcatgggc aatattggct gcagtcttga ttcaagttac aatgggtgaa 240
aatataacac agtggaacct acaggataat gggacagaag ggatacaacg ggcaatgttc 300
caaagggggg tgaacagaag tctacacggg atctggccag gaaaaatctg tacaggtgtc 360
ccttcccatc tagccaccga tatggaacta aaaacgatcc atggtatgat ggatgcaagt 420
gaaaagacca actatacgtg ttgcagactt caacgccatg agtggaacaa gcatggttgg 480
tgcaactggt acaatattga accttggatt ttactcatga atagaaccca agccaatctt 540
actgagggtc aaccaccaag agaatgcgcg gtcacttgta ggtatgatag agatagtgat 600
ctgaatgtgg taacacaagc tagagatagc cccacaccat tgacaggctg taagaaaggg 660
caaaattttt cttttgcagg catattgatg cggggtccct gcaacttcga aatagctgcg 720
agcgatgtgt tgttcaaaga agatgaatgc accagcatgt ttcaggatac tgcgcattac 780
ctcgttgacg ggatgaccaa ttccctggaa agtgccagac aagggaccgc taaactgacg 840
acctggttag gcaagcagct tgggatacta ggaaaaaaat tggaaaacaa gagcaagaca 900
tggtttggag catatgcggc t 921
<210> 2
<211> 31
<212> DNA
<213>artificial sequence
<400> 2
caccatggtt ggatccatga aaatagtgcc c 31
<210> 3
<211> 24
<212> DNA
<213>artificial sequence
<400> 3
tataagctta gccgcatatg ctcc 24
Claims (10)
1. a kind of expression BVDV ErnsThe expression vector of albumen, which is characterized in that the expression vector expression such as SEQ ID NO.1
The E of shown sequential codingrnsAlbumen；The expression vector is baculoviral；
Preferably, the BVDV ErnsAlbumen is expressed by baculovirus insect cell system carrier system.
2. a kind of BVDV E of expression vector expression described in claim 1rnsAlbumen；
Preferably, the BVDV ErnsThe labeled substance markers of albumen；
Preferably, the label includes fluorescent marker, enzyme label or biotin labeling.
3. a kind of and BVDV E as claimed in claim 2rnsProtein bound monoclonal antibody；
Preferably, the labeled substance markers of the monoclonal antibody；
Preferably, the label includes fluorescent marker, enzyme label or biotin labeling.
4. a kind of kit, which is characterized in that the kit includes BVDV E as claimed in claim 2rnsAlbumen and/or power
Benefit require 3 described in monoclonal antibody.
5. kit according to claim 4, which is characterized in that the kit is ELISA kit；The ELISA
Kit includes the BVDV E of labeled substance markersrnsAlbumen；And/or labeled substance markers with BVDV ErnsIt is protein bound
Monoclonal antibody；
Preferably, the BVDV ErnsAlbumen is the albumen of enzyme label；And/or the BVDV ErnsThe monoclonal antibody of albumen
For the antibody of enzyme label；
Preferably, enzyme used in the enzyme label includes horseradish peroxidase, acid phosphatase, alkaline phosphatase, β-D-
Galactosidase, glucose oxidase, glucoamylase, enzyme acetylcholine, inorganic pyrophosphatase, luciferase or hydrogen peroxide
Enzyme；
Preferably, the ELISA kit further includes antigen coated microplate, positive control, negative control, sample diluting liquid, washing
Liquid, developing solution and terminate liquid.
6. kit according to claim 5, which is characterized in that the ELISA kit is to block ELISA antibody test
Kit；BVDV E in the blocking ELISA antibody assay kitrnsAlbumen is as coating plate antigen, the BVDV ErnsEgg
White monoclonal antibody is the antibody of horseradish peroxidase-labeled；
Preferably, negative control is BVDV E in the kitrnsNegative serum.
7. a kind of BVDV E as claimed in claim 2rnsAlbumen, monoclonal antibody as claimed in claim 3 or claim 4-6
Any one of described in kit detection BVDV in purposes.
8. a kind of method for the ox for screening the wild virus strain infection of BVDV, which is characterized in that the method includes detecting the serum of ox
In whether there is and BVDV E as claimed in claim 2rnsThe antibody that albumen combines；
If existing and the BVDV E in the serum of oxrnsThe antibody that albumen combines, then the ox is by the wild strain sense of BVDV
The ox of dye；
The ox is immune through BVDV E2 subunit vaccine；
Preferably, described to be detected as detecting using kit described in any one of claim 4-6.
9. according to the method described in claim 8, it is characterized in that, being detected in serum by ELISA method whether there is and power
Benefit require 2 described in BVDV ErnsThe antibody that albumen combines；
Wherein, the antibody that enzyme marks in ELISA is as claimed in claim 3 and BVDV ErnsProtein bound monoclonal antibody；
Preferably, the antibody of the enzyme label is the antibody of horseradish peroxidase-labeled.
10. according to the method described in claim 9, it is characterized in that, the ELISA method is blocking-up type ELISA detection method；
Preferably, the blocking-up type ELISA detection method, which includes the following steps: for sample to be tested to be incubated in, has been coated with BVDV Erns
In the ELISA reaction plate of albumen, the antibody of the enzyme label is then added, detects OD after being incubated for colour developing450Value, then basis
OD450Value calculate sample blocking rate；Wherein,
Blocking rate is negative sample in the sample of 0%-30%；
Blocking rate is positive sample in the sample of 35%-100%；
Blocking rate is suspicious sample in the sample of 30%-35%；
Preferably, the blocking rate calculates as follows:
Blocking rate=(OD average value-sample OD average value of negative control)/negative control OD average value × 100%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811095791.5A CN109136267B (en) | 2018-09-19 | 2018-09-19 | BVDV Erns proteins and antibodies and methods for screening BVDV infected cattle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811095791.5A CN109136267B (en) | 2018-09-19 | 2018-09-19 | BVDV Erns proteins and antibodies and methods for screening BVDV infected cattle |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109136267A true CN109136267A (en) | 2019-01-04 |
| CN109136267B CN109136267B (en) | 2023-03-31 |
Family
ID=64814943
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811095791.5A Active CN109136267B (en) | 2018-09-19 | 2018-09-19 | BVDV Erns proteins and antibodies and methods for screening BVDV infected cattle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109136267B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057132A (en) * | 2019-10-16 | 2020-04-24 | 四川农业大学 | Bovine viral diarrhea virus E0 protein amino acid and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965348A (en) * | 2009-04-28 | 2013-03-13 | 辉瑞大药厂 | Bovine viral diarrhea-mucosal virus with modified erns protein |
| US9291624B2 (en) * | 2011-08-24 | 2016-03-22 | Zoetis Services Llc | Vaccine diagnostics |
| CN108456663A (en) * | 2018-03-26 | 2018-08-28 | 中国农业科学院兰州兽医研究所 | 1 type bovine viral diarrhea virus sample particle of one kind and its preparation and application |
-
2018
- 2018-09-19 CN CN201811095791.5A patent/CN109136267B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965348A (en) * | 2009-04-28 | 2013-03-13 | 辉瑞大药厂 | Bovine viral diarrhea-mucosal virus with modified erns protein |
| US9291624B2 (en) * | 2011-08-24 | 2016-03-22 | Zoetis Services Llc | Vaccine diagnostics |
| CN108456663A (en) * | 2018-03-26 | 2018-08-28 | 中国农业科学院兰州兽医研究所 | 1 type bovine viral diarrhea virus sample particle of one kind and its preparation and application |
Non-Patent Citations (2)
| Title |
|---|
| GREGO, E. ET AL.: "Development and application of an enzyme-linked immunosorbent assay for detection of bovine viral diarrhea antibody based on Erns glycoprotein expressed in a baculovirus system", 《JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION》 * |
| ILSE U. SILVA-KROTT ET AL.: "Cloning, sequencing, and in vitro expression of glycoprotein gp48 of a noncytopathogenic strain of bovine viral diarrhea virus", 《VETERINARY MICROBIOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057132A (en) * | 2019-10-16 | 2020-04-24 | 四川农业大学 | Bovine viral diarrhea virus E0 protein amino acid and preparation method thereof |
| CN111057132B (en) * | 2019-10-16 | 2022-01-07 | 四川农业大学 | Bovine viral diarrhea virus E0 protein amino acid and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109136267B (en) | 2023-03-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107022548B (en) | Anti- AQP4 autoantibody detection material of a kind of human body and preparation method thereof | |
| CN105973854B (en) | A kind of indirect immunofluorescence kit of the 4 type aviadenovirus antibody of detection based on F2 albumen | |
| CN112724208A (en) | SADS-CoV recombinant S protein extracellular segment and preparation method and application thereof | |
| CN104725490A (en) | Porcine circovirus 2 type ELISA antibody detection kit | |
| CN109001450A (en) | A kind of kit and preparation method detecting chicken Mycoplasma synoviae antibody | |
| CN105273065B (en) | 1 type duck hepatitis A virus VP2 recombinant protein of one kind, ELISA kit and preparation method thereof | |
| CN109402069A (en) | A kind of pseudovirion and its preparation method and application | |
| CN109913423A (en) | A kind of the recombination Vero cell line and application of stable expression pig Delta coronavirus N protein | |
| CN106755110B (en) | Preparation method of sensitive cell subcloned Vero/Slam/V for enhancing PPRV replication | |
| CN108776225A (en) | Pig parvoviral VLPs antibody assay kits and preparation method thereof, application | |
| CN105200014B (en) | Duck tembusu virus infection clones attenuated vaccine strain and its preparation method and application | |
| CN114230660A (en) | Monoclonal antibody for resisting micropterus salmoides iridovirus LMBV and application thereof | |
| CN109136267A (en) | BVDV ErnsThe method of the ox of albumen and antibody and screening BVDV infection | |
| CN102533663B (en) | Foot-and-mouth disease hybridoma cell line, monoclonal antibody, detection reagent and test kit | |
| CN109959789B (en) | Rabies virus antibody detection paper and preparation method and detection method thereof | |
| CN110156878B (en) | Porcine pseudorabies virus gE-gI protein, expression plasmid thereof, preparation method and application | |
| CN106018780B (en) | A kind of indirect immunofluorescence kit of the type aviadenovirus antibody of detection 4 based on F1 albumen | |
| CN112362880A (en) | IFA (IFA neutralizing antibody) detection method for PRRSV (porcine reproductive and respiratory syndrome Virus) | |
| CN112379105A (en) | Method for detecting IPMA (ionic polymer antigen) neutralizing antibody of PRRSV (porcine reproductive and respiratory syndrome Virus) | |
| CN103983782A (en) | ELISA kit for detecting hog cholera virus Erns IgM antibody | |
| CN109187970B (en) | It is a kind of quickly to detect aquatic products disease gold mark nucleic acid test strip and preparation method thereof | |
| CN116023506B (en) | ASFV nonstructural protein dominant antigen epitope fusion protein, kit and application thereof | |
| CN104031145A (en) | Monoclonal antibody against duck hepatitis A virus type A and application thereof | |
| CN106929487A (en) | A kind of foot and mouth disease virus Hybrid virus like particles and preparation method thereof | |
| CN108866008A (en) | The monoclonal antibody and its cell strain of anti-Koi herpesvirus and application |
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 | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230406 Address after: Workstation 6-013, 6th Floor, No. 88 Modern Avenue, Industrial Park, Suzhou City, Jiangsu Province, 215000 (Cluster Registration) Patentee after: Tiankang Pharmaceutical (Suzhou) Co.,Ltd. Address before: 830000 No. 528 Changchun South Road, Urumqi high tech Industrial Development Zone, Xinjiang Uygur Autonomous Region Patentee before: TECON BIOLOGICAL Co.,Ltd. |
|
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: Workstation 6-013, 6th Floor, No. 88 Modern Avenue, Industrial Park, Suzhou City, Jiangsu Province, 215000 (Cluster Registration) Patentee after: Tiankang Pharmaceutical Co.,Ltd. Address before: Workstation 6-013, 6th Floor, No. 88 Modern Avenue, Industrial Park, Suzhou City, Jiangsu Province, 215000 (Cluster Registration) Patentee before: Tiankang Pharmaceutical (Suzhou) Co.,Ltd. |