CN109608552A - 一种小反刍兽疫重组融合蛋白及其制备方法和应用 - Google Patents
一种小反刍兽疫重组融合蛋白及其制备方法和应用 Download PDFInfo
- Publication number
- CN109608552A CN109608552A CN201811605308.3A CN201811605308A CN109608552A CN 109608552 A CN109608552 A CN 109608552A CN 201811605308 A CN201811605308 A CN 201811605308A CN 109608552 A CN109608552 A CN 109608552A
- Authority
- CN
- China
- Prior art keywords
- des petits
- peste des
- petits ruminants
- antigen
- fusion protein
- 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.)
- Withdrawn
Links
- 208000007634 Peste-des-Petits-Ruminants Diseases 0.000 title claims abstract description 84
- 238000005215 recombination Methods 0.000 title claims abstract description 34
- 230000006798 recombination Effects 0.000 title claims abstract description 34
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 29
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 108091007433 antigens Proteins 0.000 claims abstract description 48
- 102000036639 antigens Human genes 0.000 claims abstract description 38
- 239000000427 antigen Substances 0.000 claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 230000000890 antigenic effect Effects 0.000 claims abstract description 8
- 108020004705 Codon Proteins 0.000 claims abstract description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 4
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 4
- 238000004153 renaturation Methods 0.000 claims abstract description 4
- 108700005078 Synthetic Genes Proteins 0.000 claims abstract description 3
- 238000005267 amalgamation Methods 0.000 claims abstract description 3
- 238000012546 transfer Methods 0.000 claims abstract description 3
- 230000001939 inductive effect Effects 0.000 claims description 13
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 10
- 238000002604 ultrasonography Methods 0.000 claims description 10
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 9
- 230000006698 induction Effects 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 7
- 238000001042 affinity chromatography Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 238000005227 gel permeation chromatography Methods 0.000 claims description 2
- 238000004255 ion exchange chromatography Methods 0.000 claims description 2
- 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 1
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 210000004027 cell Anatomy 0.000 abstract description 6
- 238000005457 optimization Methods 0.000 abstract description 6
- 238000011161 development Methods 0.000 abstract description 2
- 210000001236 prokaryotic cell Anatomy 0.000 abstract description 2
- 238000013461 design Methods 0.000 description 35
- 108020004414 DNA Proteins 0.000 description 34
- 108010061238 threonyl-glycine Proteins 0.000 description 12
- PYTZFYUXZZHOAD-WHFBIAKZSA-N Gly-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)CN PYTZFYUXZZHOAD-WHFBIAKZSA-N 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- ZJBWJHQDOIMVLM-WHFBIAKZSA-N Cys-Cys-Gly Chemical compound SC[C@H](N)C(=O)N[C@@H](CS)C(=O)NCC(O)=O ZJBWJHQDOIMVLM-WHFBIAKZSA-N 0.000 description 8
- 108010047495 alanylglycine Proteins 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000002994 raw material Substances 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 230000004087 circulation Effects 0.000 description 6
- 108010016616 cysteinylglycine Proteins 0.000 description 6
- 108010084264 glycyl-glycyl-cysteine Proteins 0.000 description 6
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 6
- OXOQBEVULIBOSH-ZDLURKLDSA-N Cys-Gly-Thr Chemical compound [H]N[C@@H](CS)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O OXOQBEVULIBOSH-ZDLURKLDSA-N 0.000 description 5
- IDOGEHIWMJMAHT-BYPYZUCNSA-N Gly-Gly-Cys Chemical compound NCC(=O)NCC(=O)N[C@@H](CS)C(O)=O IDOGEHIWMJMAHT-BYPYZUCNSA-N 0.000 description 5
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 4
- VNBNZUAPOYGRDB-ZDLURKLDSA-N Gly-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)CN)O VNBNZUAPOYGRDB-ZDLURKLDSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- SMYXEYRYCLIPIL-ZLUOBGJFSA-N Cys-Cys-Cys Chemical compound SC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O SMYXEYRYCLIPIL-ZLUOBGJFSA-N 0.000 description 3
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-N 0.000 description 3
- CEXINUGNTZFNRY-BYPYZUCNSA-N Gly-Cys-Gly Chemical compound [NH3+]CC(=O)N[C@@H](CS)C(=O)NCC([O-])=O CEXINUGNTZFNRY-BYPYZUCNSA-N 0.000 description 3
- SLUWOCTZVGMURC-BFHQHQDPSA-N Thr-Gly-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O SLUWOCTZVGMURC-BFHQHQDPSA-N 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 108010004073 cysteinylcysteine Proteins 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 239000012149 elution buffer Substances 0.000 description 3
- 210000003000 inclusion body Anatomy 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 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 2
- AEJSNWMRPXAKCW-WHFBIAKZSA-N Cys-Ala-Gly Chemical compound SC[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O AEJSNWMRPXAKCW-WHFBIAKZSA-N 0.000 description 2
- URDUGPGPLNXXES-WHFBIAKZSA-N Cys-Gly-Cys Chemical compound SC[C@H](N)C(=O)NCC(=O)N[C@@H](CS)C(O)=O URDUGPGPLNXXES-WHFBIAKZSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- JQFILXICXLDTRR-FBCQKBJTSA-N Gly-Thr-Gly Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)NCC(O)=O JQFILXICXLDTRR-FBCQKBJTSA-N 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- 108010079364 N-glycylalanine Proteins 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- IGROJMCBGRFRGI-YTLHQDLWSA-N Thr-Ala-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O IGROJMCBGRFRGI-YTLHQDLWSA-N 0.000 description 2
- DGOJNGCGEYOBKN-BWBBJGPYSA-N Thr-Cys-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)O)N)O DGOJNGCGEYOBKN-BWBBJGPYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 2
- 150000002460 imidazoles Chemical class 0.000 description 2
- 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 2
- 229960002064 kanamycin sulfate Drugs 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- OJHZNMVJJKMFGX-RNWHKREASA-N (4r,4ar,7ar,12bs)-9-methoxy-3-methyl-1,2,4,4a,5,6,7a,13-octahydro-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one;2,3-dihydroxybutanedioic acid Chemical compound OC(=O)C(O)C(O)C(O)=O.O=C([C@@H]1O2)CC[C@H]3[C@]4([H])N(C)CC[C@]13C1=C2C(OC)=CC=C1C4 OJHZNMVJJKMFGX-RNWHKREASA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- -1 4 μ l Substances 0.000 description 1
- UWQJHXKARZWDIJ-ZLUOBGJFSA-N Ala-Ala-Cys Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(O)=O UWQJHXKARZWDIJ-ZLUOBGJFSA-N 0.000 description 1
- DECCMEWNXSNSDO-ZLUOBGJFSA-N Ala-Cys-Ala Chemical compound C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O DECCMEWNXSNSDO-ZLUOBGJFSA-N 0.000 description 1
- KRHRBKYBJXMYBB-WHFBIAKZSA-N Ala-Cys-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CS)C(=O)NCC(O)=O KRHRBKYBJXMYBB-WHFBIAKZSA-N 0.000 description 1
- FUSPCLTUKXQREV-ACZMJKKPSA-N Ala-Glu-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O FUSPCLTUKXQREV-ACZMJKKPSA-N 0.000 description 1
- LJFNNUBZSZCZFN-WHFBIAKZSA-N Ala-Gly-Cys Chemical compound N[C@@H](C)C(=O)NCC(=O)N[C@@H](CS)C(=O)O LJFNNUBZSZCZFN-WHFBIAKZSA-N 0.000 description 1
- VGPWRRFOPXVGOH-BYPYZUCNSA-N Ala-Gly-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)NCC(O)=O VGPWRRFOPXVGOH-BYPYZUCNSA-N 0.000 description 1
- OKIKVSXTXVVFDV-MMWGEVLESA-N Ala-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C)N OKIKVSXTXVVFDV-MMWGEVLESA-N 0.000 description 1
- IPZQNYYAYVRKKK-FXQIFTODSA-N Ala-Pro-Ala Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IPZQNYYAYVRKKK-FXQIFTODSA-N 0.000 description 1
- KUFVXLQLDHJVOG-SHGPDSBTSA-N Ala-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C)N)O KUFVXLQLDHJVOG-SHGPDSBTSA-N 0.000 description 1
- UFBURHXMKFQVLM-CIUDSAMLSA-N Arg-Glu-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O UFBURHXMKFQVLM-CIUDSAMLSA-N 0.000 description 1
- AUFHLLPVPSMEOG-YUMQZZPRSA-N Arg-Gly-Glu Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AUFHLLPVPSMEOG-YUMQZZPRSA-N 0.000 description 1
- RCENDENBBJFJHZ-ACZMJKKPSA-N Asn-Asn-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O RCENDENBBJFJHZ-ACZMJKKPSA-N 0.000 description 1
- FTCGGKNCJZOPNB-WHFBIAKZSA-N Asn-Gly-Ser Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FTCGGKNCJZOPNB-WHFBIAKZSA-N 0.000 description 1
- SDHFVYLZFBDSQT-DCAQKATOSA-N Asp-Arg-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N SDHFVYLZFBDSQT-DCAQKATOSA-N 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- HYKFOHGZGLOCAY-ZLUOBGJFSA-N Cys-Cys-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O HYKFOHGZGLOCAY-ZLUOBGJFSA-N 0.000 description 1
- KOHBWQDSVCARMI-BWBBJGPYSA-N Cys-Cys-Thr Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KOHBWQDSVCARMI-BWBBJGPYSA-N 0.000 description 1
- CVLIHKBUPSFRQP-WHFBIAKZSA-N Cys-Gly-Ala Chemical compound [H]N[C@@H](CS)C(=O)NCC(=O)N[C@@H](C)C(O)=O CVLIHKBUPSFRQP-WHFBIAKZSA-N 0.000 description 1
- DZLQXIFVQFTFJY-BYPYZUCNSA-N Cys-Gly-Gly Chemical compound SC[C@H](N)C(=O)NCC(=O)NCC(O)=O DZLQXIFVQFTFJY-BYPYZUCNSA-N 0.000 description 1
- ALNKNYKSZPSLBD-ZDLURKLDSA-N Cys-Thr-Gly Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O ALNKNYKSZPSLBD-ZDLURKLDSA-N 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- JESJDAAGXULQOP-CIUDSAMLSA-N Gln-Arg-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)CN=C(N)N JESJDAAGXULQOP-CIUDSAMLSA-N 0.000 description 1
- SNLOOPZHAQDMJG-CIUDSAMLSA-N Gln-Glu-Glu Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SNLOOPZHAQDMJG-CIUDSAMLSA-N 0.000 description 1
- XFAUJGNLHIGXET-AVGNSLFASA-N Gln-Leu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XFAUJGNLHIGXET-AVGNSLFASA-N 0.000 description 1
- DYVMTEWCGAVKSE-HJGDQZAQSA-N Gln-Thr-Arg Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O DYVMTEWCGAVKSE-HJGDQZAQSA-N 0.000 description 1
- ILGFBUGLBSAQQB-GUBZILKMSA-N Glu-Glu-Arg Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ILGFBUGLBSAQQB-GUBZILKMSA-N 0.000 description 1
- VNCNWQPIQYAMAK-ACZMJKKPSA-N Glu-Ser-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O VNCNWQPIQYAMAK-ACZMJKKPSA-N 0.000 description 1
- QXUPRMQJDWJDFR-NRPADANISA-N Glu-Val-Ser Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O QXUPRMQJDWJDFR-NRPADANISA-N 0.000 description 1
- GQGAFTPXAPKSCF-WHFBIAKZSA-N Gly-Ala-Cys Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(=O)O GQGAFTPXAPKSCF-WHFBIAKZSA-N 0.000 description 1
- VXKCPBPQEKKERH-IUCAKERBSA-N Gly-Arg-Pro Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)N1CCC[C@H]1C(O)=O VXKCPBPQEKKERH-IUCAKERBSA-N 0.000 description 1
- KKBWDNZXYLGJEY-UHFFFAOYSA-N Gly-Arg-Pro Natural products NCC(=O)NC(CCNC(=N)N)C(=O)N1CCCC1C(=O)O KKBWDNZXYLGJEY-UHFFFAOYSA-N 0.000 description 1
- NMROINAYXCACKF-WHFBIAKZSA-N Gly-Cys-Cys Chemical compound NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O NMROINAYXCACKF-WHFBIAKZSA-N 0.000 description 1
- BULIVUZUDBHKKZ-WDSKDSINSA-N Gly-Gln-Asn Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O BULIVUZUDBHKKZ-WDSKDSINSA-N 0.000 description 1
- FIQQRCFQXGLOSZ-WDSKDSINSA-N Gly-Glu-Asp Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O FIQQRCFQXGLOSZ-WDSKDSINSA-N 0.000 description 1
- CCQOOWAONKGYKQ-BYPYZUCNSA-N Gly-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)CN CCQOOWAONKGYKQ-BYPYZUCNSA-N 0.000 description 1
- UQJNXZSSGQIPIQ-FBCQKBJTSA-N Gly-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)CN UQJNXZSSGQIPIQ-FBCQKBJTSA-N 0.000 description 1
- FFJQHWKSGAWSTJ-BFHQHQDPSA-N Gly-Thr-Ala Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O FFJQHWKSGAWSTJ-BFHQHQDPSA-N 0.000 description 1
- ZKJZBRHRWKLVSJ-ZDLURKLDSA-N Gly-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN)O ZKJZBRHRWKLVSJ-ZDLURKLDSA-N 0.000 description 1
- FNXSYBOHALPRHV-ONGXEEELSA-N Gly-Val-Lys Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN FNXSYBOHALPRHV-ONGXEEELSA-N 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- VQPPIMUZCZCOIL-GUBZILKMSA-N Leu-Gln-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O VQPPIMUZCZCOIL-GUBZILKMSA-N 0.000 description 1
- HFBCHNRFRYLZNV-GUBZILKMSA-N Leu-Glu-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HFBCHNRFRYLZNV-GUBZILKMSA-N 0.000 description 1
- HPBCTWSUJOGJSH-MNXVOIDGSA-N Leu-Glu-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HPBCTWSUJOGJSH-MNXVOIDGSA-N 0.000 description 1
- GCXGCIYIHXSKAY-ULQDDVLXSA-N Leu-Phe-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GCXGCIYIHXSKAY-ULQDDVLXSA-N 0.000 description 1
- IWWMPCPLFXFBAF-SRVKXCTJSA-N Lys-Asp-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O IWWMPCPLFXFBAF-SRVKXCTJSA-N 0.000 description 1
- 101710175243 Major antigen Proteins 0.000 description 1
- QAHFGYLFLVGBNW-DCAQKATOSA-N Met-Ala-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN QAHFGYLFLVGBNW-DCAQKATOSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- IHCXPSYCHXFXKT-DCAQKATOSA-N Pro-Arg-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O IHCXPSYCHXFXKT-DCAQKATOSA-N 0.000 description 1
- MGDFPGCFVJFITQ-CIUDSAMLSA-N Pro-Glu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MGDFPGCFVJFITQ-CIUDSAMLSA-N 0.000 description 1
- 208000006257 Rinderpest Diseases 0.000 description 1
- CAJFZCICSVBOJK-SHGPDSBTSA-N Thr-Ala-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAJFZCICSVBOJK-SHGPDSBTSA-N 0.000 description 1
- UKBSDLHIKIXJKH-HJGDQZAQSA-N Thr-Arg-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O UKBSDLHIKIXJKH-HJGDQZAQSA-N 0.000 description 1
- QWMPARMKIDVBLV-VZFHVOOUSA-N Thr-Cys-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O QWMPARMKIDVBLV-VZFHVOOUSA-N 0.000 description 1
- LYGKYFKSZTUXGZ-ZDLURKLDSA-N Thr-Cys-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)NCC(O)=O LYGKYFKSZTUXGZ-ZDLURKLDSA-N 0.000 description 1
- UZJDBCHMIQXLOQ-HEIBUPTGSA-N Thr-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O UZJDBCHMIQXLOQ-HEIBUPTGSA-N 0.000 description 1
- WYKJENSCCRJLRC-ZDLURKLDSA-N Thr-Gly-Cys Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)O WYKJENSCCRJLRC-ZDLURKLDSA-N 0.000 description 1
- KBBRNEDOYWMIJP-KYNKHSRBSA-N Thr-Gly-Thr Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O KBBRNEDOYWMIJP-KYNKHSRBSA-N 0.000 description 1
- MXDOAJQRJBMGMO-FJXKBIBVSA-N Thr-Pro-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O MXDOAJQRJBMGMO-FJXKBIBVSA-N 0.000 description 1
- TZQWJCGVCIJDMU-HEIBUPTGSA-N Thr-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)O)N)O TZQWJCGVCIJDMU-HEIBUPTGSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- MIAZWUMFUURQNP-YDHLFZDLSA-N Val-Tyr-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N MIAZWUMFUURQNP-YDHLFZDLSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000011162 core material Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000012502 diagnostic product Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 208000003265 stomatitis Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18422—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Urology & Nephrology (AREA)
- Pulmonology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种小反刍兽疫重组融合蛋白，涉及生物医药领域，包括小反刍兽疫抗原和小反刍兽疫单链抗体；一种小反刍兽疫重组融合蛋白的制备方法，包括以下步骤:(1)查找小反刍兽疫中含有主要抗原决定簇的N(425‑525aa)抗原；(2)优化小反刍兽疫N(425‑525aa)抗原基因的密码子，合成核酸序列并进行表达；(3)构建针对小反刍兽疫N(13‑20aa)抗原的单链抗体，调取单链抗体scFv的基因；(4)重组优化的小反刍兽疫N(425‑525aa)抗原基因和针对小反刍兽疫N(13‑20aa)抗原的单链抗体scFv的基因，并进行融合表达；(5)纯化及复性小反刍兽疫重组融合蛋白。本发明在开发单克隆抗体的基础上又进行重组抗体技术开发单链抗体，易于在细胞中表达，可以在真核或者原核细胞中表达，可大量生产，成本较低。
Description
技术领域
本发明涉及生物医药技术领域，特别涉及一种小反刍兽疫重组融合蛋白及其制备方法和应用。
背景技术
小反刍兽疫(peste des petits ruminants,PPR)俗称羊瘟,又名小反刍兽假性牛瘟、肺肠炎、口炎肺肠炎复合症,是由小反刍兽疫病毒引起的一种急性病毒性传染病。1978年印度发现PPR,2007年，中国西藏地区爆发PPR。由于其广泛的传播性、对经济的影响性及其在全球消除牛瘟的活动中所扮演的重要作用,PPR越来越受到重视。
体外诊断核心原料的开发是开发相应诊断试剂的前提，只有开发出具有高活性、高性能的抗原抗体，体外诊断试剂产品才能成为现实。因此，针对小反刍兽疫抗体和抗原开发出能够用以生产诊断试剂的原料迫在眉睫，从某种程度上说，原料的开发也必将成为降低小反刍兽疫病蔓延的首要解决问题。
发明内容
本发明的目的在于，提供一种小反刍兽疫重组融合蛋白及其制备方法和应用。
本发明的技术方案:一种小反刍兽疫重组融合蛋白，包括小反刍兽疫抗原和小反刍兽疫单链抗体；
所述小反刍兽疫抗原为含有主要抗原决定簇的N(425-525aa)抗原；
所述小反刍兽疫单链抗体为scFv。
一种小反刍兽疫重组融合蛋白的制备方法，包括以下步骤:
(1)查找小反刍兽疫中含有主要抗原决定簇的N(425-525aa)抗原；
(2)优化小反刍兽疫N(425-525aa)抗原基因的密码子，合成核酸序列并进行表达；
(3)构建针对小反刍兽疫N(13-20aa)抗原的单链抗体，调取单链抗体scFv的基因；
(4)重组优化的小反刍兽疫N(425-525aa)抗原基因和针对小反刍兽疫N(13-20aa)抗原的单链抗体scFv的基因，并进行融合表达；
(5)采用超声的方式破碎诱导表达之后的细菌，将破碎条件定为200w，每次超声6s，间隔3s超声一次，共180次，纯化及复性小反刍兽疫重组融合蛋白。
前述的一种小反刍兽疫重组融合蛋白的制备方法中，所述步骤(2)和步骤(4)在大肠杆菌***中表达。
在大肠杆菌中表达蛋白时，不同密码子使用的频率有较大区别，为了使重组蛋白的表达量达到最大，需将小反刍兽疫N(425-525aa)抗原基因序列进行密码子优化，然后合成优化后的核酸序列并进行表达。
前述的一种小反刍兽疫重组融合蛋白的制备方法，所述步骤(2)和步骤(4)表达时的诱导温度为20℃、25℃、28℃或37℃，诱导转速为250rpm，诱导的IPTG浓度为0.1mM。
前述的一种小反刍兽疫重组融合蛋白的制备方法，所述诱导温度为25℃或37℃。
前述的一种小反刍兽疫重组融合蛋白的制备方法和应用中，所述诱导温度为25℃。
前述的一种小反刍兽疫重组融合蛋白的制备方中，所述步骤(5)的纯化方式选自离子交换层析、凝胶过滤层析或亲和层析。
前述的一种小反刍兽疫重组融合蛋白的制备方法中，所述步骤(5)的纯化方式为亲和层析。
为了使得重组蛋白更好地保持活性位点，本发明选择比较温和的培养和诱导条件，其表达时的诱导温度为20℃、25℃、28℃、37℃，进一步优选的诱导温度为25℃、37℃，更进一步优选的诱导温度为25℃。诱导转速为250rpm，诱导的IPTG(异丙基-β-D-硫代半乳糖苷)浓度为0.1mM。这样使得重组蛋白有了更加缓慢的表达，有充分的时间进行空间构象的形成。
本发明采用超声的方式破碎细菌。在破碎菌体的时候，为了避免剧烈条件，将破碎条件定为200w，每次超声6s，间隔3s超声一次，共180次。
一种小反刍兽疫重组融合蛋白的应用，所述重组蛋白用于制备小反刍兽疫检测试剂盒。
与现有技术相比，本发明具有以下优点及有益效果:
(1)首次开发针对小反刍兽疫主要抗原N的单链抗体scFv。
本发明在开发单克隆抗体的基础上又进行重组抗体技术开发单链抗体，单链抗体具有以下优点:1)具有完整的抗原结合位点，不含抗体恒定区；2)分子量小，减少了抗体的非特异性结合，降低了诊断产品的假阳性风险；3)结构简单，易于基因工程改造；4)易于在细胞中表达，可以在真核或者原核细胞中表达，可大量生产，成本较低。
(2)国内外首次开发小反刍兽疫主要抗原表位及针对N抗原的scFv重组蛋白。
由于感染小反刍兽疫初期不产生抗体，致使在此段时间我们检测不到相应的小反刍兽疫抗体，从而导致漏检，为了解决此问题必须在检测小反刍兽疫抗体的同时增加对抗原的检测，开发针对小反刍兽疫N抗原的抗体成为关键。
(3)制备了同时结合小反刍兽疫抗体和抗原的高活性、高灵敏度和高特异性的重组蛋白。
在主要抗原表位和针对小反刍兽疫N抗原的抗体开发出后，利用基因工程技术将筛选出的高效抗原表位(小反刍兽疫N抗原的425-525aa)和针对小反刍兽疫N抗原的单链抗体scFv(13-20aa)重组，制备成具有同时结合小反刍兽疫抗体和抗原的高活性、高灵敏度和高特异性的重组蛋白，该重组蛋白具有以下优点:1)在快诊试剂条中只需要使用一种原料即可，这样可以避免多种原料间的交叉反应；2)在原料的生产上，只需要稳定一种原料的生产工艺即可，这样可以减少生产成本的投入，缩短原料生产周期，降低生产批间差；3)节省了开发小反刍兽疫诊断试剂的成本。
具体实施方式
下面结合实施例对本发明作进一步的说明，但并不作为对本发明限制的依据。
实施例:一种小反刍兽疫重组融合蛋白，包括小反刍兽疫抗原和小反刍兽疫单链抗体；
所述小反刍兽疫抗原为含有主要抗原决定簇的N(425-525aa)抗原；
所述小反刍兽疫单链抗体为scFv。
其制备方法，包括如下步骤:
1.小反刍兽疫N抗原基因的密码子优化及表达载体构建；
从NCBI查找N(425-525aa)抗原基因GenBank:FJ905304.1，蛋白质序列如下:
GGGESSAPATREGVKAAIPNGSEERDRKQTRPGRPRGETPGQLLLEIMPEDEVSRESGQNPREAQRSAEALFRLQAMAKILEDQEEGEDNNQVYNDKDLLG
经过密码子优化后序列如下:
GGCGGCGGCGAATCCTCCGCGCCGGCGACCCGTGAAGGCGTGAAAGCGGCGATT CCGAATGGCTCCGAAGAACGTGATCGTAAACAGACCCGTCCGGGCCGTCCGCGTGGCGAAACCCCGGGCCAGCTGCTGCTGGAAATTATGCCGGAAGATGAAGTGTCCCGTGAATCCGGCCAGAATCCGCGTGAAGCGCA
CGTTCCGCGGAAGCGCTGTTTCGTCTGCAGGCGATGGCGAAAATTCTGGAAGATCAGGAAGAAGGCGAAGATAATAATCAGGTGTATAATGATAAAGATCTGCTGGGC
设计引物如下:
P1u:GCCCATATGGGCGGCGGCGAATCCTCCGCGCCGGCGACCCGTGAAGGCGT GAAAGCGGC
P1d:ATCACGTTCTTCGGAGCCATTCGGAATCGCCGCTTTCACGCCT
P2u:TCCGAAGAACGTGATCGTAAACAGACCCGTCCGGGCCGTCCGCGTGGCGA AACC
P2d:TTCATCTTCCGGCATAATTTCCAGCAGCAGCTGGCCCGGGGTTTCGCCAC GCGGACGG
P3u:ATGCCGGAAGATGAAGTGTCCCGTGAATCCGGCCAGAATCCGCGTGAAGC GCA
P3d:CGCCATCGCCTGCAGACGAAACAGCGCTTCCGCGGAACGCTGCGCTTCAC GCGGA
P4u:CGCCATCGCCTGCAGACGAAACAGCGCTTCCGCGGAACGCTGCGCTTCAC GCGGA
P4d:GCCCTCGAGGCCCAGCAGATCTTTATCATTATACACCTGATTATTATCTT CGCCTTCTTC
实验方法如下:
将引物P1u,P1d,P2u,P2d混合，进行PCR反应，所得产物命名为W1；将引物P3u,P3d,P4u,P4d混合，进行PCR反应，所得产物命名为W2；然后将上述两个产物W1,W2混合做为模板，用引物P1u,P4d进行PCR反应即得到优化后小反刍兽疫的N(425-525aa)基因。上述四个PCR(TOYOBO公司产品，货号:02510D1)反应条件如下:94℃，2分钟X1个循环；(98℃，15秒，55℃，30秒，68℃，15秒)X30个循环；72℃，7分钟X1个循环。测序证明序列正确后，将此PCR产物用NdeI和XhoI限制性内切酶(购自NEB公司)进行双酶切之后，***到用相同两个酶切处理的pET30a(Novagen产品,货号69909-3)载体中。
2.构建针对小反刍兽疫核心蛋白小反刍兽疫N抗原的单链抗体：
(1)单克隆抗体(只针对结合N抗原蛋白13-20aa的抗体)细胞株的获得；
a.将构建pET30a-N(1-525aa)载体转化至BL21表达菌种表达纯化后获得N抗原蛋白；
b.用获得的N(1-525aa)抗原蛋白免疫小鼠；
c.用单克隆抗体技术筛选到只针对结合N(1-525aa)抗原蛋白13-20aa的抗体细胞株；
(2)细胞RNA的提取
用TRIzol LS(invitrogen产品,货号10296-010)试剂，按照说明书操作步骤提取细胞总RNA。
cDNA第一链的合成：
取1μg RNA做逆转录模板，Oligo(d)T 1μl做逆转录引物，
加DEPC水至总体积12μl，混匀；
↓
70℃变性5min，迅速冰浴冷却；
↓
依次加入5×缓冲液4μl，RNase抑制剂1μl，dNTP(10mM each)
2μl，混匀；
37℃孵育5min；
↓
加入AMV逆转录酶1μl，
37℃反转录60min；
↓
70℃10min终止反应；
↓
冰上冷却。
(3)以逆转录cDNA为模板，按照以下引物，用RT-PCR扩增重链轻链基因。
mHVu1:GATGTGAAGCTTCAGGAGTC
mHVu2:CAGGTGCAGCTGAAGGAGTC
mHVu3:CAGGTGCAGCTGAAGCAGTC
mHVu4:CAGGTTACTCTGAAAGAGTC
mHVu5:AAGGTCCAGCTGCAACAATC
mHVu6:GAGGTCCAGCTGCAGCAGTC
mHVu7:CAGGTCCAACTGCAGCAGCC
mHVu8:GAGGTGAAGCTGGTGGAGTC
mHVu9:GAGGTGAAGCTGGTGGAATC
mHVu10:GATGTGAACTTGGAAGTGTC
mHVd1:TGCAGAGACAGTGACCAGAGT
mHVd2:TGAGGAGACTGTGAGAGTGGT
mHVd3:TGAGGAGACGGTGACTGAGGT
mHVd4:TGAGGAGACGGTGACCGTGGT
mKVu1:GATGTTTTGATGACCCAAACT
mKVu2:GATATTGTGATGACGCAGGCT
mKVu3:GATATTGTGATAACCCAG
mKVu4:GACATTGTGCTGACCCAATCT
mKVu5:GACATTGTGATGACCCAGTCT
mKVu6:GATATTGTGCTAACTCAGTCT
mKVu7:GATATCCAGATGACACAGACT
mKVu8:GACATCCAGCTGACTCAGTCT
mKVu9:CAAATTGTTCTCACCCAGTCT
mKVd1:CCGTTTCAGCTCCAGCTTG
mKVd2:CCGTTTTATTTCCAGCTTGGT
mKVd3:CCGTTTTATTTCCAACTTTG
用引物mHVu1—mHVu10分别与mHVd1—mHVd4组合；mKVu1—mKVu9分别与mKVd1—mKVd4组合做PCR反应。
PCR反应体系25μl，其中cDNA 0.5μl、上游引物0.25μl、下游引物0.25μl和Taq酶0.2μl dNTP 2μl 10×buffer 2.5μl。
PCR(TOYOBO公司产品，货号:02510D1)，PCR条件为:94℃，2分钟X1个循环；(98℃，5秒，55℃，30秒，68℃，15秒)X30个循环；72℃，7分钟X1个循环。
经测序验证后确定重链和轻链基因，然后通过递归PCR将重链和轻链基因连接起来，此重组基因为rscFv。
3.含有小反刍兽疫N抗原基因(425-525aa)和针对小反刍兽疫N抗原的单链抗体基因(13-20aa)重组，并进行表达:
(1)通过递归PCR将有小反刍兽疫N抗原基因(425-525aa)和针对小反刍兽疫N抗原的单链抗体基因(13-20aa)连接在一起，用NdeI和XhoI限制性内切酶(购自NEB公司)进行双酶切之后，***到用相同两个酶切处理的pET30a(Novagen产品，货号69909-3)载体中。
(2)将上述重组蛋白基因的表达载体转化至大肠杆菌BL21中，涂布于含100ug/ml硫酸卡那霉素(上海生工生物工程服务有限公司，货号:KB0286)的LB平板上，37℃过夜培养，挑取单克隆菌落，用含有相同浓度的硫酸卡那霉素的300mlLB培养基37℃培养至OD600达0.6左右，用浓度为0.1mM的IPTG(生工，货号:IB0168)进行诱导表达，诱导条件为:37℃，转速250rpm，5h。诱导之后，将培养液4℃5000rpm离心20min收集菌体。
4.含有小反刍兽疫重组蛋白的纯化及复性:
将菌体用50ml包涵体抽提液(20mM Tris-HCl，0.5MUrea pH 7.5，0.5M NaCl,2％Triton X-100)重悬，然后超声破碎，条件为每次超声3s，间隔6s，共180次，12000rpm，4℃离心收集包涵体沉淀。用上样缓冲液Binding Buffer(50mM Tris，8M Urea，0.5M Nacl，20mM咪唑pH8.0)50ml破碎包涵体，上柱纯化，用洗脱缓冲液Elution Buffer(50mM Tris，8MUrea，0.5M Nacl，300mM咪唑pH8.0)洗脱目的蛋白。将纯化后的重组蛋白用透析缓冲液(1mM氧化型谷胱甘肽GSSH，2mM还原型谷胱甘肽GSH，2mMEDTA，20mM Tris,pH8.5)透析,每隔48h换一次透析液。取出透析后的蛋白液，经据乙醇-20000进行浓缩，于-20℃保存备用。
序列表
<110> 杭州亿米诺生物科技有限公司
<120> 一种小反刍兽疫重组融合蛋白及其制备方法和应用
<130> 2
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 101
<212> PRT
<213> N(425-525aa抗原基因GenBank：FJ905304.1的蛋白质序列)
<400> 1
Gly Gly Gly Glu Ser Ser Ala Pro Ala Thr Arg Glu Gly Val Lys Ala
1 5 10 15
Ala Ile Pro Asn Gly Ser Glu Glu Arg Asp Arg Lys Gln Thr Arg Pro
20 25 30
Gly Arg Pro Arg Gly Glu Thr Pro Gly Gln Leu Leu Leu Glu Ile Met
35 40 45
Pro Glu Asp Glu Val Ser Arg Glu Ser Gly Gln Asn Pro Arg Glu Ala
50 55 60
Gln Arg Ser Ala Glu Ala Leu Phe Arg Leu Gln Ala Met Ala Lys Ile
65 70 75 80
Leu Glu Asp Gln Glu Glu Gly Glu Asp Asn Asn Gln Val Tyr Asn Asp
85 90 95
Lys Asp Leu Leu Gly
100
<210> 2
<211> 303
<212> PRT
<213> N(425-525aa抗原基因GenBank：FJ905304.1经过密码子优化后的蛋白质序列)
<400> 2
Gly Gly Cys Gly Gly Cys Gly Gly Cys Gly Ala Ala Thr Cys Cys Thr
1 5 10 15
Cys Cys Gly Cys Gly Cys Cys Gly Gly Cys Gly Ala Cys Cys Cys Gly
20 25 30
Thr Gly Ala Ala Gly Gly Cys Gly Thr Gly Ala Ala Ala Gly Cys Gly
35 40 45
Gly Cys Gly Ala Thr Thr Cys Cys Gly Ala Ala Thr Gly Gly Cys Thr
50 55 60
Cys Cys Gly Ala Ala Gly Ala Ala Cys Gly Thr Gly Ala Thr Cys Gly
65 70 75 80
Thr Ala Ala Ala Cys Ala Gly Ala Cys Cys Cys Gly Thr Cys Cys Gly
85 90 95
Gly Gly Cys Cys Gly Thr Cys Cys Gly Cys Gly Thr Gly Gly Cys Gly
100 105 110
Ala Ala Ala Cys Cys Cys Cys Gly Gly Gly Cys Cys Ala Gly Cys Thr
115 120 125
Gly Cys Thr Gly Cys Thr Gly Gly Ala Ala Ala Thr Thr Ala Thr Gly
130 135 140
Cys Cys Gly Gly Ala Ala Gly Ala Thr Gly Ala Ala Gly Thr Gly Thr
145 150 155 160
Cys Cys Cys Gly Thr Gly Ala Ala Thr Cys Cys Gly Gly Cys Cys Ala
165 170 175
Gly Ala Ala Thr Cys Cys Gly Cys Gly Thr Gly Ala Ala Gly Cys Gly
180 185 190
Cys Ala Gly Cys Gly Thr Thr Cys Cys Gly Cys Gly Gly Ala Ala Gly
195 200 205
Cys Gly Cys Thr Gly Thr Thr Thr Cys Gly Thr Cys Thr Gly Cys Ala
210 215 220
Gly Gly Cys Gly Ala Thr Gly Gly Cys Gly Ala Ala Ala Ala Thr Thr
225 230 235 240
Cys Thr Gly Gly Ala Ala Gly Ala Thr Cys Ala Gly Gly Ala Ala Gly
245 250 255
Ala Ala Gly Gly Cys Gly Ala Ala Gly Ala Thr Ala Ala Thr Ala Ala
260 265 270
Thr Cys Ala Gly Gly Thr Gly Thr Ala Thr Ala Ala Thr Gly Ala Thr
275 280 285
Ala Ala Ala Gly Ala Thr Cys Thr Gly Cys Thr Gly Gly Gly Cys
290 295 300
<210> 3
<211> 59
<212> DNA
<213> 设计引物(P1u)
<400> 3
gcccatatgg gcggcggcga atcctccgcg ccggcgaccc gtgaaggcgt gaaagcggc 59
<210> 4
<211> 43
<212> DNA
<213> 设计引物(P1d)
<400> 4
atcacgttct tcggagccat tcggaatcgc cgctttcacg cct 43
<210> 5
<211> 54
<212> DNA
<213> 设计引物(P2u)
<400> 5
tccgaagaac gtgatcgtaa acagacccgt ccgggccgtc cgcgtggcga aacc 54
<210> 6
<211> 58
<212> DNA
<213> 设计引物(P2d)
<400> 6
ttcatcttcc ggcataattt ccagcagcag ctggcccggg gtttcgccac gcggacgg 58
<210> 7
<211> 53
<212> DNA
<213> 设计引物(P3u)
<400> 7
atgccggaag atgaagtgtc ccgtgaatcc ggccagaatc cgcgtgaagc gca 53
<210> 8
<211> 55
<212> DNA
<213> 设计引物(P3d)
<400> 8
cgccatcgcc tgcagacgaa acagcgcttc cgcggaacgc tgcgcttcac gcgga 55
<210> 9
<211> 55
<212> DNA
<213> 设计引物(P4u)
<400> 9
cgccatcgcc tgcagacgaa acagcgcttc cgcggaacgc tgcgcttcac gcgga 55
<210> 10
<211> 60
<212> DNA
<213> 设计引物(P4d)
<400> 10
gccctcgagg cccagcagat ctttatcatt atacacctga ttattatctt cgccttcttc 60
<210> 11
<211> 20
<212> DNA
<213> 设计引物(mHVu1)
<400> 11
gatgtgaagc ttcaggagtc 20
<210> 12
<211> 20
<212> DNA
<213> 设计引物(mHVu2)
<400> 12
caggtgcagc tgaaggagtc 20
<210> 13
<211> 20
<212> DNA
<213> 设计引物(mHVu3)
<400> 13
caggtgcagc tgaagcagtc 20
<210> 14
<211> 20
<212> DNA
<213> 设计引物(mHVu4)
<400> 14
caggttactc tgaaagagtc 20
<210> 15
<211> 20
<212> DNA
<213> 设计引物(mHVu5)
<400> 15
aaggtccagc tgcaacaatc 20
<210> 16
<211> 20
<212> DNA
<213> 设计引物(mHVu6)
<400> 16
gaggtccagc tgcagcagtc 20
<210> 17
<211> 20
<212> DNA
<213> 设计引物(mHVu7)
<400> 17
caggtccaac tgcagcagcc 20
<210> 18
<211> 20
<212> DNA
<213> 设计引物(mHVu8)
<400> 18
gaggtgaagc tggtggagtc 20
<210> 19
<211> 20
<212> DNA
<213> 设计引物(mHVu9)
<400> 19
gaggtgaagc tggtggaatc 20
<210> 20
<211> 20
<212> DNA
<213> 设计引物(mHVu10)
<400> 20
gatgtgaact tggaagtgtc 20
<210> 21
<211> 21
<212> DNA
<213> 设计引物(mHVd1)
<400> 21
tgcagagaca gtgaccagag t 21
<210> 22
<211> 21
<212> DNA
<213> 设计引物(mHVd2)
<400> 22
tgaggagact gtgagagtgg t 21
<210> 23
<211> 21
<212> DNA
<213> 设计引物(mHVd3)
<400> 23
tgaggagacg gtgactgagg t 21
<210> 24
<211> 21
<212> DNA
<213> 设计引物(mHVd4)
<400> 24
tgaggagacg gtgaccgtgg t 21
<210> 25
<211> 21
<212> DNA
<213> 设计引物(mKVu1)
<400> 25
gatgttttga tgacccaaac t 21
<210> 26
<211> 21
<212> DNA
<213> 设计引物(mKVu2)
<400> 26
gatattgtga tgacgcaggc t 21
<210> 27
<211> 18
<212> DNA
<213> 设计引物(mKVu3)
<400> 27
gatattgtga taacccag 18
<210> 28
<211> 21
<212> DNA
<213> 设计引物(mKVu4)
<400> 28
gacattgtgc tgacccaatc t 21
<210> 29
<211> 21
<212> DNA
<213> 设计引物(mKVu5)
<400> 29
gacattgtga tgacccagtc t 21
<210> 30
<211> 21
<212> DNA
<213> 设计引物(mKVu6)
<400> 30
gatattgtgc taactcagtc t 21
<210> 31
<211> 21
<212> DNA
<213> 设计引物(mKVu7)
<400> 31
gatatccaga tgacacagac t 21
<210> 32
<211> 21
<212> DNA
<213> 设计引物(mKVu8)
<400> 32
gacatccagc tgactcagtc t 21
<210> 33
<211> 21
<212> DNA
<213> 设计引物(mKVu9)
<400> 33
caaattgttc tcacccagtc t 21
<210> 34
<211> 19
<212> DNA
<213> 设计引物(mKVd1)
<400> 34
ccgtttcagc tccagcttg 19
<210> 35
<211> 21
<212> DNA
<213> 设计引物(mKVd2)
<400> 35
ccgttttatt tccagcttgg t 21
<210> 36
<211> 20
<212> DNA
<213> 设计引物(mKVd3)
<400> 36
ccgttttatt tccaactttg 20
Claims (9)
1.一种小反刍兽疫重组融合蛋白，其特征在于:包括小反刍兽疫抗原和小反刍兽疫单链抗体:
所述小反刍兽疫抗原为含有主要抗原决定簇的N(425-525aa)抗原；
所述小反刍兽疫单链抗体为scFv。
2.一种基于权利要求1所述的一种小反刍兽疫重组融合蛋白的制备方法，其特征在于:包括以下步骤:
(1)查找小反刍兽疫中含有主要抗原决定簇的N(425-525aa)抗原；
(2)优化小反刍兽疫N(425-525aa)抗原基因的密码子，合成核酸序列并进行表达；
(3)构建针对小反刍兽疫N(13-20aa)抗原的单链抗体，调取单链抗体scFv的基因；
(4)重组优化的小反刍兽疫N(425-525aa)抗原基因和针对小反刍兽疫N(13-20aa)抗原的单链抗体scFv的基因，并进行融合表达；
(5)采用超声的方式破碎诱导表达之后的细菌，将破碎条件定为200w，每次超声6s，间隔3s超声一次，共180次，纯化及复性小反刍兽疫重组融合蛋白。
3.根据权利要求2所述的一种小反刍兽疫重组融合蛋白的制备方法，其特征在于:所述步骤(2)和步骤(4)在大肠杆菌***中表达。
4.根据权利要求2所述的一种小反刍兽疫重组融合蛋白的制备方法，其特征在于:所述步骤(2)和步骤(4)表达时的诱导温度为20℃、25℃、28℃或37℃，诱导转速为250rpm，诱导的IPTG浓度为0.1mM。
5.根据权利要求2所述的一种小反刍兽疫重组融合蛋白的制备方法，其特征在于:所述诱导温度为25℃或37℃。
6.根据权利要求2所述的一种小反刍兽疫重组融合蛋白的制备方法，其特征在于:所述诱导温度为25℃。
7.根据权利要求2所述的一种小反刍兽疫重组融合蛋白的制备方法，其特征在于:所述步骤(5)的纯化方式选自离子交换层析、凝胶过滤层析或亲和层析。
8.根据权利要求2所述的一种小反刍兽疫重组融合蛋白及其制备方法，其特征在于:所述步骤(5)的纯化方式为亲和层析。
9.一种基于权利要求1所述的一种小反刍兽疫重组融合蛋白的应用，其特征在于:所述重组蛋白用于制备小反刍兽疫检测试剂盒。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811605308.3A CN109608552A (zh) | 2018-12-26 | 2018-12-26 | 一种小反刍兽疫重组融合蛋白及其制备方法和应用 |
| CN201911286035.5A CN110885378A (zh) | 2018-12-26 | 2019-12-13 | 一种小反刍兽疫重组融合蛋白、其制备方法及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811605308.3A CN109608552A (zh) | 2018-12-26 | 2018-12-26 | 一种小反刍兽疫重组融合蛋白及其制备方法和应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109608552A true CN109608552A (zh) | 2019-04-12 |
Family
ID=66012525
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811605308.3A Withdrawn CN109608552A (zh) | 2018-12-26 | 2018-12-26 | 一种小反刍兽疫重组融合蛋白及其制备方法和应用 |
| CN201911286035.5A Pending CN110885378A (zh) | 2018-12-26 | 2019-12-13 | 一种小反刍兽疫重组融合蛋白、其制备方法及应用 |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911286035.5A Pending CN110885378A (zh) | 2018-12-26 | 2019-12-13 | 一种小反刍兽疫重组融合蛋白、其制备方法及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN109608552A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112213493A (zh) * | 2019-07-11 | 2021-01-12 | 中国兽医药品监察所 | 可用于区分疫苗免疫和自然感染的小反刍兽疫检测试剂盒 |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2316130A1 (en) * | 1999-08-19 | 2001-02-19 | Masanori Fukui | Method for detection or determination of hcv core antigens and reagent for detection or determination for use therein |
| KR100593213B1 (ko) * | 2004-11-05 | 2006-06-28 | 대한민국 | 곤충세포발현 재조합 ｎ 단백질 및 ｎ 단백질에 대한 단클론항체를 이용한 가성우역의 신속진단방법 |
| US20110038935A1 (en) * | 2007-12-06 | 2011-02-17 | Marasco Wayne A | Antibodies against influenza virus and methods of use thereof |
| WO2011041691A1 (en) * | 2009-10-02 | 2011-04-07 | University Of Iowa Research Foundation | Methods of improving vaccine immunogenicity |
| CN104558108B (zh) * | 2013-10-17 | 2018-03-06 | 上海市农业科学院 | 小反刍兽疫病毒n蛋白的线性抗原表位最小基序肽 |
| CN105968175B (zh) * | 2016-04-14 | 2019-11-08 | 中国农业科学院哈尔滨兽医研究所 | 一种重组小反刍兽疫病毒n蛋白及其表达方法和应用 |
| CN107586783A (zh) * | 2016-07-06 | 2018-01-16 | 华中农业大学 | 抗小反刍兽疫病毒n蛋白单克隆抗体及其应用 |
| CN108059684A (zh) * | 2017-12-25 | 2018-05-22 | 杭州亿米诺生物科技有限公司 | 一种牛病毒性腹泻重组蛋白、其制备方法及应用 |
-
2018
- 2018-12-26 CN CN201811605308.3A patent/CN109608552A/zh not_active Withdrawn
-
2019
- 2019-12-13 CN CN201911286035.5A patent/CN110885378A/zh active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112213493A (zh) * | 2019-07-11 | 2021-01-12 | 中国兽医药品监察所 | 可用于区分疫苗免疫和自然感染的小反刍兽疫检测试剂盒 |
| CN112213493B (zh) * | 2019-07-11 | 2023-07-07 | 中国兽医药品监察所 | 可用于区分疫苗免疫和自然感染的小反刍兽疫检测试剂盒 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110885378A (zh) | 2020-03-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111499765B (zh) | 一种冠状病毒融合蛋白及其制备方法与应用 | |
| CN113336844B (zh) | 一种靶向新冠病毒n蛋白的鲨鱼单域抗体及其制备方法和应用 | |
| CN103396481B (zh) | 一种二型登革热病毒ns1蛋白的重链单域抗体及其制备方法和应用 | |
| CN112111006B (zh) | 抗牛结节性皮肤病病毒的抗体、检测试纸及试剂盒 | |
| CN113087792B (zh) | 一种犬瘟热病毒纳米抗体及其应用 | |
| CN107286250A (zh) | 一种布鲁氏菌融合蛋白、其制备方法及应用 | |
| CN108059684A (zh) | 一种牛病毒性腹泻重组蛋白、其制备方法及应用 | |
| CN113740536A (zh) | 一种非洲猪瘟病毒p30阻断ELISA抗体检测试剂盒及其应用 | |
| CN102321179B (zh) | 一种丙型肝炎病毒重组蛋白及基因序列 | |
| CN111944044A (zh) | 一种抗ASFV-p30蛋白的纳米抗体及其制备方法和应用 | |
| CN109608552A (zh) | 一种小反刍兽疫重组融合蛋白及其制备方法和应用 | |
| CN104710529A (zh) | 一种抗鱼类病毒性出血性败血症病毒的单链抗体 | |
| CN109593121A (zh) | 一种i型牛疱疹病毒重组蛋白及其制备方法和应用 | |
| CN114163521B (zh) | 一种鉴别猪瘟病毒2.1亚型强毒株及其抗体的单克隆抗体 | |
| Groves et al. | Affinity maturation of phage display antibody populations using ribosome display | |
| CN113687073B (zh) | 一种非洲猪瘟病毒p54阻断ELISA抗体检测试剂盒及其应用 | |
| CN118240070A (zh) | 高亲和力的抗鸡传染性法氏囊病毒的scFv抗体的制备及其应用 | |
| CN109734783A (zh) | 一种猫艾滋病毒重组蛋白及其制备方法和应用 | |
| CN114195889A (zh) | 一种SARS-Cov-2-N纳米抗体及其衍生蛋白和应用 | |
| CN103159853A (zh) | 全人源抗丙型肝炎病毒核心抗原的基因工程Fab抗体及其制备方法和应用 | |
| CN109485725A (zh) | 一种包虫重组蛋白及其制备方法和应用 | |
| CN112342198A (zh) | PAT/pat单抗杂交瘤细胞株、产生的抗体及其制备方法 | |
| CN112679607A (zh) | 一种肌钙蛋白i e13单链抗体的制备方法 | |
| CN104628851A (zh) | 一种抗狂犬病毒的基因工程抗体、其制备方法和应用 | |
| CN105859879B (zh) | 一种抗鱼类淋巴囊肿病毒的单链抗体 |
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 | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20190412 |
|
| WW01 | Invention patent application withdrawn after publication |