CN110055272A - A kind of targeting antibodies therapeutic agent inhibiting PRRS virus multiplication - Google Patents
A kind of targeting antibodies therapeutic agent inhibiting PRRS virus multiplication Download PDFInfo
- Publication number
- CN110055272A CN110055272A CN201910314591.2A CN201910314591A CN110055272A CN 110055272 A CN110055272 A CN 110055272A CN 201910314591 A CN201910314591 A CN 201910314591A CN 110055272 A CN110055272 A CN 110055272A
- Authority
- CN
- China
- Prior art keywords
- therapeutic agent
- targeting antibodies
- prrs virus
- antibodies therapeutic
- dna
- 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.)
- Pending
Links
- 241000700605 Viruses Species 0.000 title claims abstract description 41
- 230000008685 targeting Effects 0.000 title claims abstract description 40
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 title claims abstract description 39
- 239000003814 drug Substances 0.000 title claims abstract description 39
- 229940124597 therapeutic agent Drugs 0.000 title claims abstract description 36
- 230000002401 inhibitory effect Effects 0.000 title claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 239000013604 expression vector Substances 0.000 claims abstract description 16
- 101800001554 RNA-directed RNA polymerase Proteins 0.000 claims abstract description 11
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 6
- 101800000482 Non-structural protein 9 Proteins 0.000 claims abstract description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 4
- 239000003795 chemical substances by application Substances 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 230000029087 digestion Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 101710120037 Toxin CcdB Proteins 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 230000035755 proliferation Effects 0.000 claims description 3
- 239000006142 Luria-Bertani Agar Substances 0.000 claims description 2
- 238000001042 affinity chromatography Methods 0.000 claims description 2
- 229930189065 blasticidin Natural products 0.000 claims description 2
- 230000006698 induction Effects 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 230000009870 specific binding Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 4
- 230000005764 inhibitory process Effects 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 16
- 238000002560 therapeutic procedure Methods 0.000 description 9
- 244000286779 Hansenula anomala Species 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 229920002401 polyacrylamide Polymers 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108010006654 Bleomycin Proteins 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical class N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 102000009109 Fc receptors Human genes 0.000 description 2
- 108010087819 Fc receptors Proteins 0.000 description 2
- 235000014683 Hansenula anomala Nutrition 0.000 description 2
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 108010009298 lysylglutamic acid Proteins 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229940057059 monascus purpureus Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 101150074370 sp9 gene Proteins 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- DWINFPQUSSHSFS-UVBJJODRSA-N Ala-Arg-Trp Chemical compound N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C12)C(=O)O DWINFPQUSSHSFS-UVBJJODRSA-N 0.000 description 1
- XYTNPQNAZREREP-XQXXSGGOSA-N Ala-Glu-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XYTNPQNAZREREP-XQXXSGGOSA-N 0.000 description 1
- OMMDTNGURYRDAC-NRPADANISA-N Ala-Glu-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OMMDTNGURYRDAC-NRPADANISA-N 0.000 description 1
- MQIGTEQXYCRLGK-BQBZGAKWSA-N Ala-Gly-Pro Chemical compound C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O MQIGTEQXYCRLGK-BQBZGAKWSA-N 0.000 description 1
- IFKQPMZRDQZSHI-GHCJXIJMSA-N Ala-Ile-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O IFKQPMZRDQZSHI-GHCJXIJMSA-N 0.000 description 1
- HHRAXZAYZFFRAM-CIUDSAMLSA-N Ala-Leu-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O HHRAXZAYZFFRAM-CIUDSAMLSA-N 0.000 description 1
- VQBULXOHAZSTQY-GKCIPKSASA-N Ala-Trp-Phe Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O VQBULXOHAZSTQY-GKCIPKSASA-N 0.000 description 1
- FEZJJKXNPSEYEV-CIUDSAMLSA-N Arg-Gln-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O FEZJJKXNPSEYEV-CIUDSAMLSA-N 0.000 description 1
- HPSVTWMFWCHKFN-GARJFASQSA-N Arg-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O HPSVTWMFWCHKFN-GARJFASQSA-N 0.000 description 1
- FVBZXNSRIDVYJS-AVGNSLFASA-N Arg-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCN=C(N)N FVBZXNSRIDVYJS-AVGNSLFASA-N 0.000 description 1
- JOTRDIXZHNQYGP-DCAQKATOSA-N Arg-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N JOTRDIXZHNQYGP-DCAQKATOSA-N 0.000 description 1
- LLQIAIUAKGNOSE-NHCYSSNCSA-N Arg-Val-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N LLQIAIUAKGNOSE-NHCYSSNCSA-N 0.000 description 1
- PDQBXRSOSCTGKY-ACZMJKKPSA-N Asn-Ala-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N PDQBXRSOSCTGKY-ACZMJKKPSA-N 0.000 description 1
- MKJBPDLENBUHQU-CIUDSAMLSA-N Asn-Ser-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O MKJBPDLENBUHQU-CIUDSAMLSA-N 0.000 description 1
- JBDLMLZNDRLDIX-HJGDQZAQSA-N Asn-Thr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O JBDLMLZNDRLDIX-HJGDQZAQSA-N 0.000 description 1
- QIRJQYQOIKBPBZ-IHRRRGAJSA-N Asn-Tyr-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QIRJQYQOIKBPBZ-IHRRRGAJSA-N 0.000 description 1
- JZLFYAAGGYMRIK-BYULHYEWSA-N Asn-Val-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O JZLFYAAGGYMRIK-BYULHYEWSA-N 0.000 description 1
- VZNOVQKGJQJOCS-SRVKXCTJSA-N Asp-Asp-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VZNOVQKGJQJOCS-SRVKXCTJSA-N 0.000 description 1
- WSGVTKZFVJSJOG-RCOVLWMOSA-N Asp-Gly-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O WSGVTKZFVJSJOG-RCOVLWMOSA-N 0.000 description 1
- ILQCHXURSRRIRY-YUMQZZPRSA-N Asp-His-Gly Chemical compound C1=C(NC=N1)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC(=O)O)N ILQCHXURSRRIRY-YUMQZZPRSA-N 0.000 description 1
- MFTVXYMXSAQZNL-DJFWLOJKSA-N Asp-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC(=O)O)N MFTVXYMXSAQZNL-DJFWLOJKSA-N 0.000 description 1
- MNQMTYSEKZHIDF-GCJQMDKQSA-N Asp-Thr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O MNQMTYSEKZHIDF-GCJQMDKQSA-N 0.000 description 1
- JSNWZMFSLIWAHS-HJGDQZAQSA-N Asp-Thr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O JSNWZMFSLIWAHS-HJGDQZAQSA-N 0.000 description 1
- YUELDQUPTAYEGM-XIRDDKMYSA-N Asp-Trp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CC(=O)O)N YUELDQUPTAYEGM-XIRDDKMYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- OHLLDUNVMPPUMD-DCAQKATOSA-N Cys-Leu-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N OHLLDUNVMPPUMD-DCAQKATOSA-N 0.000 description 1
- ALTQTAKGRFLRLR-GUBZILKMSA-N Cys-Val-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N ALTQTAKGRFLRLR-GUBZILKMSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- QYKBTDOAMKORGL-FXQIFTODSA-N Gln-Gln-Asp Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N QYKBTDOAMKORGL-FXQIFTODSA-N 0.000 description 1
- XSBGUANSZDGULP-IUCAKERBSA-N Gln-Gly-Lys Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CCCCN)C(O)=O XSBGUANSZDGULP-IUCAKERBSA-N 0.000 description 1
- PODFFOWWLUPNMN-DCAQKATOSA-N Gln-His-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(O)=O PODFFOWWLUPNMN-DCAQKATOSA-N 0.000 description 1
- UESYBOXFJWJVSB-AVGNSLFASA-N Gln-Phe-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O UESYBOXFJWJVSB-AVGNSLFASA-N 0.000 description 1
- HMIXCETWRYDVMO-GUBZILKMSA-N Gln-Pro-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O HMIXCETWRYDVMO-GUBZILKMSA-N 0.000 description 1
- OSCLNNWLKKIQJM-WDSKDSINSA-N Gln-Ser-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O OSCLNNWLKKIQJM-WDSKDSINSA-N 0.000 description 1
- VLOLPWWCNKWRNB-LOKLDPHHSA-N Gln-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O VLOLPWWCNKWRNB-LOKLDPHHSA-N 0.000 description 1
- ZFBBMCKQSNJZSN-AUTRQRHGSA-N Gln-Val-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZFBBMCKQSNJZSN-AUTRQRHGSA-N 0.000 description 1
- ITYRYNUZHPNCIK-GUBZILKMSA-N Glu-Ala-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O ITYRYNUZHPNCIK-GUBZILKMSA-N 0.000 description 1
- CGYDXNKRIMJMLV-GUBZILKMSA-N Glu-Arg-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O CGYDXNKRIMJMLV-GUBZILKMSA-N 0.000 description 1
- HNVFSTLPVJWIDV-CIUDSAMLSA-N Glu-Glu-Gln Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HNVFSTLPVJWIDV-CIUDSAMLSA-N 0.000 description 1
- FBEJIDRSQCGFJI-GUBZILKMSA-N Glu-Leu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O FBEJIDRSQCGFJI-GUBZILKMSA-N 0.000 description 1
- CQGBSALYGOXQPE-HTUGSXCWSA-N Glu-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O CQGBSALYGOXQPE-HTUGSXCWSA-N 0.000 description 1
- YOTHMZZSJKKEHZ-SZMVWBNQSA-N Glu-Trp-Lys Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@@H](N)CCC(O)=O)=CNC2=C1 YOTHMZZSJKKEHZ-SZMVWBNQSA-N 0.000 description 1
- WGYHAAXZWPEBDQ-IFFSRLJSSA-N Glu-Val-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WGYHAAXZWPEBDQ-IFFSRLJSSA-N 0.000 description 1
- XXGQRGQPGFYECI-WDSKDSINSA-N Gly-Cys-Glu Chemical compound NCC(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CCC(O)=O XXGQRGQPGFYECI-WDSKDSINSA-N 0.000 description 1
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 1
- GNPVTZJUUBPZKW-WDSKDSINSA-N Gly-Gln-Ser Chemical compound [H]NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O GNPVTZJUUBPZKW-WDSKDSINSA-N 0.000 description 1
- GMTXWRIDLGTVFC-IUCAKERBSA-N Gly-Lys-Glu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O GMTXWRIDLGTVFC-IUCAKERBSA-N 0.000 description 1
- ZLCLYFGMKFCDCN-XPUUQOCRSA-N Gly-Ser-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CO)NC(=O)CN)C(O)=O ZLCLYFGMKFCDCN-XPUUQOCRSA-N 0.000 description 1
- XHVONGZZVUUORG-WEDXCCLWSA-N Gly-Thr-Lys Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCCN XHVONGZZVUUORG-WEDXCCLWSA-N 0.000 description 1
- GBYYQVBXFVDJPJ-WLTAIBSBSA-N Gly-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)CN)O GBYYQVBXFVDJPJ-WLTAIBSBSA-N 0.000 description 1
- WMKXFMUJRCEGRP-SRVKXCTJSA-N His-Asn-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N WMKXFMUJRCEGRP-SRVKXCTJSA-N 0.000 description 1
- WEWCEPOYKANMGZ-MMWGEVLESA-N Ile-Cys-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)N1CCC[C@@H]1C(=O)O)N WEWCEPOYKANMGZ-MMWGEVLESA-N 0.000 description 1
- ODPKZZLRDNXTJZ-WHOFXGATSA-N Ile-Gly-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N ODPKZZLRDNXTJZ-WHOFXGATSA-N 0.000 description 1
- QQFSKBMCAKWHLG-UHFFFAOYSA-N Ile-Phe-Pro-Pro Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(NC(=O)C(N)C(C)CC)CC1=CC=CC=C1 QQFSKBMCAKWHLG-UHFFFAOYSA-N 0.000 description 1
- PZWBBXHHUSIGKH-OSUNSFLBSA-N Ile-Thr-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PZWBBXHHUSIGKH-OSUNSFLBSA-N 0.000 description 1
- SAEWJTCJQVZQNZ-IUKAMOBKSA-N Ile-Thr-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N SAEWJTCJQVZQNZ-IUKAMOBKSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- ZTLGVASZOIKNIX-DCAQKATOSA-N Leu-Gln-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZTLGVASZOIKNIX-DCAQKATOSA-N 0.000 description 1
- AXZGZMGRBDQTEY-SRVKXCTJSA-N Leu-Gln-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(O)=O AXZGZMGRBDQTEY-SRVKXCTJSA-N 0.000 description 1
- WMIOEVKKYIMVKI-DCAQKATOSA-N Leu-Pro-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WMIOEVKKYIMVKI-DCAQKATOSA-N 0.000 description 1
- YUTNOGOMBNYPFH-XUXIUFHCSA-N Leu-Pro-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YUTNOGOMBNYPFH-XUXIUFHCSA-N 0.000 description 1
- DPURXCQCHSQPAN-AVGNSLFASA-N Leu-Pro-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DPURXCQCHSQPAN-AVGNSLFASA-N 0.000 description 1
- KIZIOFNVSOSKJI-CIUDSAMLSA-N Leu-Ser-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N KIZIOFNVSOSKJI-CIUDSAMLSA-N 0.000 description 1
- VUBIPAHVHMZHCM-KKUMJFAQSA-N Leu-Tyr-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 VUBIPAHVHMZHCM-KKUMJFAQSA-N 0.000 description 1
- SEOXPEFQEOYURL-PMVMPFDFSA-N Leu-Tyr-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O SEOXPEFQEOYURL-PMVMPFDFSA-N 0.000 description 1
- PNPYKQFJGRFYJE-GUBZILKMSA-N Lys-Ala-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O PNPYKQFJGRFYJE-GUBZILKMSA-N 0.000 description 1
- NFLFJGGKOHYZJF-BJDJZHNGSA-N Lys-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN NFLFJGGKOHYZJF-BJDJZHNGSA-N 0.000 description 1
- KZOHPCYVORJBLG-AVGNSLFASA-N Lys-Glu-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCCN)N KZOHPCYVORJBLG-AVGNSLFASA-N 0.000 description 1
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-N 0.000 description 1
- LUTDBHBIHHREDC-IHRRRGAJSA-N Lys-Pro-Lys Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O LUTDBHBIHHREDC-IHRRRGAJSA-N 0.000 description 1
- JOSAKOKSPXROGQ-BJDJZHNGSA-N Lys-Ser-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JOSAKOKSPXROGQ-BJDJZHNGSA-N 0.000 description 1
- MDDUIRLQCYVRDO-NHCYSSNCSA-N Lys-Val-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN MDDUIRLQCYVRDO-NHCYSSNCSA-N 0.000 description 1
- FWAHLGXNBLWIKB-NAKRPEOUSA-N Met-Ile-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCSC FWAHLGXNBLWIKB-NAKRPEOUSA-N 0.000 description 1
- GHQFLTYXGUETFD-UFYCRDLUSA-N Met-Tyr-Tyr Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N GHQFLTYXGUETFD-UFYCRDLUSA-N 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- AUEJLPRZGVVDNU-UHFFFAOYSA-N N-L-tyrosyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 AUEJLPRZGVVDNU-UHFFFAOYSA-N 0.000 description 1
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- CDNPIRSCAFMMBE-SRVKXCTJSA-N Phe-Asn-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O CDNPIRSCAFMMBE-SRVKXCTJSA-N 0.000 description 1
- CJAHQEZWDZNSJO-KKUMJFAQSA-N Phe-Lys-Cys Chemical compound NCCCC[C@@H](C(=O)N[C@@H](CS)C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 CJAHQEZWDZNSJO-KKUMJFAQSA-N 0.000 description 1
- JDMKQHSHKJHAHR-UHFFFAOYSA-N Phe-Phe-Leu-Tyr Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)CC1=CC=CC=C1 JDMKQHSHKJHAHR-UHFFFAOYSA-N 0.000 description 1
- FGWUALWGCZJQDJ-URLPEUOOSA-N Phe-Thr-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FGWUALWGCZJQDJ-URLPEUOOSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- IWNOFCGBMSFTBC-CIUDSAMLSA-N Pro-Ala-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O IWNOFCGBMSFTBC-CIUDSAMLSA-N 0.000 description 1
- TUYWCHPXKQTISF-LPEHRKFASA-N Pro-Cys-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CS)C(=O)N2CCC[C@@H]2C(=O)O TUYWCHPXKQTISF-LPEHRKFASA-N 0.000 description 1
- NMELOOXSGDRBRU-YUMQZZPRSA-N Pro-Glu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H]1CCCN1 NMELOOXSGDRBRU-YUMQZZPRSA-N 0.000 description 1
- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-N 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 1
- ULVMNZOKDBHKKI-ACZMJKKPSA-N Ser-Gln-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O ULVMNZOKDBHKKI-ACZMJKKPSA-N 0.000 description 1
- QYSFWUIXDFJUDW-DCAQKATOSA-N Ser-Leu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYSFWUIXDFJUDW-DCAQKATOSA-N 0.000 description 1
- LRZLZIUXQBIWTB-KATARQTJSA-N Ser-Lys-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LRZLZIUXQBIWTB-KATARQTJSA-N 0.000 description 1
- VLMIUSLQONKLDV-HEIBUPTGSA-N Ser-Thr-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VLMIUSLQONKLDV-HEIBUPTGSA-N 0.000 description 1
- HNDMFDBQXYZSRM-IHRRRGAJSA-N Ser-Val-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HNDMFDBQXYZSRM-IHRRRGAJSA-N 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- DKDHTRVDOUZZTP-IFFSRLJSSA-N Thr-Gln-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O DKDHTRVDOUZZTP-IFFSRLJSSA-N 0.000 description 1
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 1
- WFAUDCSNCWJJAA-KXNHARMFSA-N Thr-Lys-Pro Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(O)=O WFAUDCSNCWJJAA-KXNHARMFSA-N 0.000 description 1
- VEIKMWOMUYMMMK-FCLVOEFKSA-N Thr-Phe-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 VEIKMWOMUYMMMK-FCLVOEFKSA-N 0.000 description 1
- MROIJTGJGIDEEJ-RCWTZXSCSA-N Thr-Pro-Pro Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 MROIJTGJGIDEEJ-RCWTZXSCSA-N 0.000 description 1
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 1
- KZTLZZQTJMCGIP-ZJDVBMNYSA-N Thr-Val-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KZTLZZQTJMCGIP-ZJDVBMNYSA-N 0.000 description 1
- 241000219793 Trifolium Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- CDEZPHVWBQFJQQ-NKKJXINNSA-N Trp-Trp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CC4=CNC5=CC=CC=C54)N)C(=O)O CDEZPHVWBQFJQQ-NKKJXINNSA-N 0.000 description 1
- SSSDKJMQMZTMJP-BVSLBCMMSA-N Trp-Tyr-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CC=C(O)C=C1 SSSDKJMQMZTMJP-BVSLBCMMSA-N 0.000 description 1
- BURPTJBFWIOHEY-UWJYBYFXSA-N Tyr-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 BURPTJBFWIOHEY-UWJYBYFXSA-N 0.000 description 1
- JBBYKPZAPOLCPK-JYJNAYRXSA-N Tyr-Arg-Met Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O JBBYKPZAPOLCPK-JYJNAYRXSA-N 0.000 description 1
- QYSBJAUCUKHSLU-JYJNAYRXSA-N Tyr-Arg-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O QYSBJAUCUKHSLU-JYJNAYRXSA-N 0.000 description 1
- VXFXIBCCVLJCJT-JYJNAYRXSA-N Tyr-Pro-Pro Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N1CCC[C@H]1C(O)=O VXFXIBCCVLJCJT-JYJNAYRXSA-N 0.000 description 1
- RIVVDNTUSRVTQT-IRIUXVKKSA-N Tyr-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O RIVVDNTUSRVTQT-IRIUXVKKSA-N 0.000 description 1
- QHDXUYOYTPWCSK-RCOVLWMOSA-N Val-Asp-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)O)N QHDXUYOYTPWCSK-RCOVLWMOSA-N 0.000 description 1
- BMGOFDMKDVVGJG-NHCYSSNCSA-N Val-Asp-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N BMGOFDMKDVVGJG-NHCYSSNCSA-N 0.000 description 1
- SCBITHMBEJNRHC-LSJOCFKGSA-N Val-Asp-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N SCBITHMBEJNRHC-LSJOCFKGSA-N 0.000 description 1
- ZIGZPYJXIWLQFC-QTKMDUPCSA-N Val-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](C(C)C)N)O ZIGZPYJXIWLQFC-QTKMDUPCSA-N 0.000 description 1
- OTJMMKPMLUNTQT-AVGNSLFASA-N Val-Leu-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](C(C)C)N OTJMMKPMLUNTQT-AVGNSLFASA-N 0.000 description 1
- SBJCTAZFSZXWSR-AVGNSLFASA-N Val-Met-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N SBJCTAZFSZXWSR-AVGNSLFASA-N 0.000 description 1
- NZYNRRGJJVSSTJ-GUBZILKMSA-N Val-Ser-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NZYNRRGJJVSSTJ-GUBZILKMSA-N 0.000 description 1
- BGTDGENDNWGMDQ-KJEVXHAQSA-N Val-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](C(C)C)N)O BGTDGENDNWGMDQ-KJEVXHAQSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000027645 antigenic variation Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000005101 cell tropism Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 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 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 108010040030 histidinoalanine Proteins 0.000 description 1
- 108010045383 histidyl-glycyl-glutamic acid Proteins 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 108010053725 prolylvaline Proteins 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 108010071097 threonyl-lysyl-proline Proteins 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 108010078580 tyrosylleucine Proteins 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
-
- 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
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- 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
- 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
- C12N2770/10011—Arteriviridae
- C12N2770/10022—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)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Public Health (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Communicable Diseases (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to field of biology, and in particular to a kind of targeting antibodies therapeutic agent of the inhibition PRRS virus multiplication by Yeast system expression, the targeting antibodies therapeutic agent have amino acid sequence shown in SEQ ID NO:1.Compared with prior art, beneficial effects of the present invention: the present invention passes through overlapping pcr, anti- PRRS virus nsp9 nano antibody gene is connected to obtain DNA sequence dna with 1 Fc fragment gene of pig IgG, and the sequence is connected to yeast expression vector, and it is transformed into Pichia pastoris competent cell, targeting antibodies therapeutic agent is prepared in culture, this targeting antibodies therapeutic agent had both had the function into PRRS virus target cell, Nsp9 can be specifically bound again, to realize the effect of targeted inhibition PRRS virus multiplication.
Description
Technical field
The invention belongs to field of biology, and in particular to a kind of target of the inhibition PRRS virus multiplication by Yeast system expression
To Antybody therapy agent.
Background technique
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,
PRRS) be a boar caused by PRRS viral (PRRSV) infection acute infectious disease.Since PRRS virus has antigenic variation
Features, the existing vaccines such as property, antibody-dependent enhancement (ADE) and persistent infection have the protective effect of the disease very much
Limit.PRRSV has very strong cell tropism, and main infection has the mononuclear macrophage of Fc receptor.Therefore, exploitation has targeting
The Antybody therapy agent of mononuclear macrophage with Fc receptor will be helpful to the infection and propagation of control PRRSV.
PRRS virus is single strand plus RNA virus, non-knot of the duplication and transcription of gene dependent on virus itself coding
Structure albumen, the rna polymerase activity that wherein there is non-structural protein Nsp9 RNA to rely on are the most important replicase of virus.It is newest
Research discovery Nsp9 interaction can not only occur with some host cell proteins and virus protein itself, to influence disease to virus
Poison proliferation, and the virulence of Nsp9 and virus also has close relationship.Therefore, Nsp9 is most suitable as antiviral drugs
Target spot.
The drug of existing treatment porcine reproductive and respiratory syndrome has therapeutic effect poor, the disadvantage more than side effect.
Summary of the invention
In view of the deficiency of the prior art, one aspect of the present invention provides a kind of targeting for inhibiting PRRS virus multiplication
Antybody therapy agent and its preparation method and application；On the other hand a kind of DNA sequence dna encoding the targeting antibodies therapeutic agent is provided；This
Invention also provides a kind of yeast expression vector.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of targeting antibodies therapeutic agent inhibiting PRRS virus multiplication, the targeting antibodies therapeutic agent have SEQ ID NO:
Amino acid sequence shown in 1；The targeting antibodies therapeutic agent can be targeted into PRRS virus permissive cell, and by special
Property combination PRRS virus nonstructural protein Nsp9, inhibit proliferation of the PRRS virus in permissive cell.
A kind of DNA sequence dna, the DNA sequence dna have nucleotide sequence shown in SEQ ID NO:2；The DNA sequence dna is compiled
The amino acid sequence of code is included in amino acid sequence shown in SEQ ID NO:1；The DNA sequence dna is by over-lap PCR skill
Anti- PRRS virus nsp9 nano antibody gene is connected to obtain by art with pig IgG 1Fc fragment gene.
A kind of expression vector, the expression vector are by being connected to after the DNA sequence dna digestion in claim 2 complete
It is obtained in red Yeast expression carrier pPICZ α A；
The expression vector can express the targeting antibodies therapeutic agent in claim 1.
A kind of preparation method for the targeting antibodies therapeutic agent inhibiting PRRS virus multiplication, comprising the following steps:
Step 1, by overlapping pcr, by anti-PRRS virus nsp9 nano antibody gene and pig IgG 1Fc fragment gene
Series connection obtains DNA sequence dna as claimed in claim 2；
The DNA sequence dna that step 1 obtains is connected in yeast expression vector pPICZ α A by step 2 after digestion
Obtain the expression vector in claim 3；
Step 3, the expression vector that step 2 is obtained is transformed into Pichia pastoris competent cell X-33, then through making a living
It is coated on the Salt LB agar plate of blasticidin resistance and cultivates after change, picking monoclonal colonies are expressed anti-through methanol induction
Body therapeutic agent obtains targeting antibodies therapeutic agent crude product；
Step 4, the targeting antibodies therapeutic agent crude product that step 3 is obtained are obtained by Protein G affinity chromatography
Purity is greater than 90% targeting antibodies therapeutic agent.
It is a kind of inhibit PRRS virus multiplication targeting antibodies therapeutic agent preparation treatment PRRS drug in application.
Compared with prior art, beneficial effects of the present invention:
The present invention is by overlapping pcr, by anti-PRRS virus nsp9 nano antibody gene and pig IgG 1Fc fragment gene
Series connection obtains DNA sequence dna, and the sequence is connected to yeast expression vector, and be transformed into Pichia pastoris competent cell, trains
It supports and targeting antibodies therapeutic agent is prepared, this targeting antibodies therapeutic agent had not only had the function into PRRS virus target cell, but also can
To specifically bind Nsp9, to realize the effect of targeted inhibition PRRS virus multiplication.
Detailed description of the invention
Fig. 1 (a) is targeting antibodies therapeutic agent nucleic acid electrophoretogram, and swimming lane M is DNA molecular standard；It (b) is targeting antibodies treatment
Agent protein electrophoresis figure, swimming lane M are protein molecule standards.
Fig. 2 is enzyme-linked immunization (ELISA) experimental identification targeting antibodies therapeutic agent and Nsp9 interaction.
Fig. 3 is that indirect immunofluorescence experiment detection targeting antibodies therapeutic agent enters in PAMs cell.
After inoculating cell 48 hours, (a) intracellular PRRS virus N sp9 gene level and (b) are trained Fig. 4 difference PRRSV strain
Support supernatant generation of neutrons virus titer (TCID50)。
Specific embodiment
1 Pichia anomala expression Antybody therapy agent of embodiment:
SEQ ID NO:2 DNA sequence dna is obtained by gene chemical synthesis.Upstream and downstream primer is designed, amplification obtains encoding antibody and controls
Treat the gene of agent.Finished using 1 μ g pPICZ α A (being purchased from Invitrogen company) of EcoR I and Xba I (being purchased from NEB) digestion red
Yeast expression carrier and PCR recovery product connect two segments in 16 DEG C with T4DNA ligase afterwards overnight.Take 10 μ l connection products
Conversion is coated on the less salt LB+ containing 50 μ g/ml bleomycins into 80 μ lDH5a α competent escherichia coli cells after activation
Agar plate, 37 DEG C are incubated overnight, and picking single bacterium is fallen within containing 37 DEG C in 50 μ g/ml bleomycin less salt LB liquid mediums,
220rpm cultivates 12h, and bacterium solution PCR is accredited as positive bacterium solution and carries out sequencing analysis.Extracting sequencing result is positive bacterium solution
After the linearisation of restriction enzyme SacI single endonuclease digestion, electricity is rotated into Pichiapastoris expression strain X-33 competence 10 μ g of plasmid,
The Yeast Cultivation YPDS agar plate containing 100 μ g/ml bleomycins is coated with after activation, picking single colonie PCR is identified after 72h,
Choose positive bacterium colony switching BMGY culture medium transfer again after for 24 hours BMMY culture medium with 0.5% culture medium inducing expression.To yeast
Bacterium induced to the 5th day, and 7500rpm is centrifuged 10min culture supernatant.With 1M Tris-base adjust supernatant pH to 7.5-8.0 it
Between, and with 0.45 μm of membrane filtration, through Protein G resin (being purchased from Nanjing Jin Sirui company) purifying, and will mesh after elution
Albumen dialyse into 0.01M PBS (PH7.2).Antybody therapy agent is concentrated by super filter tube, 0.22 μm of sterilised membrane filter
- 80 DEG C are stored in after filtration sterilization for use.
PCR amplification:
Template: SEQ ID NO:2
Upstream primer SEQ ID NO:3:GGTGAATTCCAGGTCCAGCTGCAGGAG
Downstream primer SEQ ID NO:4:GGGTCTAGATCACTTGCCCTGTGTCTTG
2 enzyme linked immunological of embodiment experiment detection Antybody therapy agent and Nsp9 interaction
Firstly, Nsp9 is coated with elisa plate with PBS (0.01M, pH7.2), every hole 400ng, 4 DEG C overnight.Elisa plate is used
After PBS-T (Tween-20 that PBS contains 0.5% volume) washing 3 times, with confining liquid, (skimmed milk power of 2.5% concentration is dissolved in PBS-
T, 200 holes μ l/) closing 1 hour；37 DEG C of targeting antibodies therapeutic agent incubations 1 hour of various concentration is added in washing 3 times, every hole；It washes
It washs 3 times, 37 DEG C of antibody incubations 1 hour of the anti-pig Fc of HRP label is added in every hole；Washing 3 times,；100 bottoms μ l HRP are added in every hole
Object (TMB) reacts at room temperature 15 minutes, and every hole is added 50 μ l 3M sulfuric acid and terminates reaction, reads every hole at 450 nm with microplate reader
OD value.Testing result is shown in Fig. 1, and the Antybody therapy agent of Pichia anomala expression can be specifically bound with PRRS virus N sp9.
3 targeting antibodies therapeutic agent of embodiment enters cell
It after PAMs cell recovery, is layered in 24 porocyte culture plates with every 1 × 106 density of hole, cultivates 4h, discard old culture
After new 1640 culture medium of serum-free RPMI is added in base, while targeting antibodies therapeutic agent is with final concentration of 10 μM 4 degree of absorption 1h
37 DEG C are transferred to, 5% carbon dioxide incubator detects intracellular targeting antibodies using indirect immunofluorescence experiment and control after culture 1
Treat agent.Specific step is as follows: discarding supernatant, cell is washed 3 times with PBS, adds 4% paraformaldehyde, and it is fixed thin to be placed at room temperature for 15min
Born of the same parents, 0.25%Triton X-100 room temperature rupture of membranes 10min.PBS is washed 3 times, and the anti-pig of goat of FITC label is added after 1%BSA closing
Secondary antibody, 37 DEG C of incubation 1h；DAPI is added to be placed at room temperature for 5min.PBS is washed 3 times, in fluorescence microscopy microscopic observation after mounting.As a result such as
Fig. 3 display targeting Antybody therapy agent is able to enter in PAMs cell.
The experiment of 4 virus infection of embodiment
It after PAMs cell recovery, is layered in 24 porocyte culture plates with every 1 × 106 density of hole, cultivates 4h, discard old culture
Base, is added new culture medium, and inoculation 0.01MOI has the PRRSV virus rSD16/TRS2/Clover of fluorescence labels.After infection
1h, Antybody therapy agent are added in every hole cell culture medium with final concentration of 20 μM.Fluorescence virus number in cell is observed after infecting 48h
Amount.As a result as Fig. 4 shows that targeting antibodies therapeutic agent is able to suppress the PRRSV virus rSD16/TRS2/ with fluorescence labels
Breeding in CloverPAMs cell.
The foregoing is merely the preferred embodiments of invention, are not intended to limit the scope of the invention, all to utilize this
It simply modifies or converts made by description of the invention content, be applied directly or indirectly in other relevant technical fields, same
Reason is included within the scope of the present invention.
Sequence table
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>a kind of targeting antibodies therapeutic agent for inhibiting PRRS virus multiplication
<141> 2019-04-09
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 358
<212> PRT
<213> Pichia pastoris
<400> 1
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Thr Asn Ser
20 25 30
Tyr Arg Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
35 40 45
Val Ala Ala Ile Asn Ser Gly Gly Ser Thr Thr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Gln Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Gly Arg Val Gln Trp Trp Pro Val Leu Arg Ala Leu Asn Glu
100 105 110
Asp Asp Tyr Leu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Gly Thr Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro Gly Cys Glu Val
130 135 140
Ala Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu
145 150 155 160
Met Ile Ser Gln Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
165 170 175
Lys Glu His Ala Glu Val Gln Phe Ser Trp Tyr Val Asp Gly Val Glu
180 185 190
Val His Thr Ala Glu Thr Arg Pro Lys Glu Glu Gln Phe Asn Ser Thr
195 200 205
Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln Asp Trp Leu Lys
210 215 220
Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Val Asp Leu Pro Ala Pro
225 230 235 240
Ile Thr Arg Thr Ile Ser Lys Ala Ile Gly Gln Ser Arg Glu Pro Gln
245 250 255
Val Tyr Thr Leu Pro Pro Pro Ala Glu Glu Leu Ser Arg Ser Lys Val
260 265 270
Thr Val Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile His Val
275 280 285
Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu Gly Asn Tyr Arg Thr
290 295 300
Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Phe Phe Leu Tyr Ser Lys
305 310 315 320
Leu Ala Val Asp Lys Ala Arg Trp Asp His Gly Glu Thr Phe Glu Cys
325 330 335
Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile
340 345 350
Ser Lys Thr Gln Gly Lys
355
<210> 2
<211> 1077
<212> DNA
<213> Artificial Sequence
<400> 2
caggtccagc tgcaggagag cggaggaggc tccgtgcagt ccggaggatc cctgaggctg 60
tcctgtgccg cctccggcta caccatcacc aactcctaca ggatggcctg gttccggcag 120
gctcctggca aggagaggga aggcgtggcc gccatcaatt ccggcggctc caccacctac 180
gccgactccg tgaagggccg gttcaccatc agccaggaca acgcccagaa caccctgtac 240
ctgcagatga actccctgaa agccgaggac accgccatgt actactgtgc tgccggaagg 300
gtgcagtggt ggcctgtgct gagggccctg aacgaggacg actacctgta ctggggccag 360
ggcacccagg tgaccgtgtc ctccggcacc aagaccaagc ccccttgccc tatctgccct 420
ggctgtgagg tggctggccc ctccgtgttc atcttccccc ccaagcccaa ggataccctg 480
atgatcagcc agacacccga ggtgacctgc gtggtggtgg acgtgagcaa ggagcacgcc 540
gaggtgcagt tctcctggta cgtggacggc gtggaggtgc ataccgctga gacaaggcct 600
aaggaggagc agttcaacag cacctaccgg gtggtgtccg tgctgcctat ccagcaccag 660
gactggctga agggcaagga gttcaagtgc aaggtgaaca acgtggacct gcccgcccct 720
attacccgga ccatcagcaa ggccatcggc cagagcaggg agcctcaggt gtacaccctc 780
cctccccctg ccgaagagct gtccaggtcc aaagtgaccg tgacctgtct ggtgatcggc 840
ttctaccccc ccgacattca cgtggagtgg aagagcaacg gacagcccga gcccgagggc 900
aattacagga ccacccctcc ccagcaggat gtggacggca ccttcttcct gtacagcaag 960
ctggccgtgg acaaggctag gtgggatcac ggcgagacct tcgagtgcgc cgtgatgcac 1020
gaggccctcc acaaccacta cacccagaag tccatcagca agacacaggg caagtga 1077
<210> 3
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 3
ggtgaattcc aggtccagct gcaggag 27
<210> 4
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 4
gggtctagat cacttgccct gtgtcttg 28
Claims (5)
1. a kind of targeting antibodies therapeutic agent for inhibiting PRRS virus multiplication, which is characterized in that the targeting antibodies therapeutic agent has
Amino acid sequence shown in SEQ ID NO:1；
The targeting antibodies therapeutic agent can be targeted into PRRS virus permissive cell, and pass through specific binding PRRS virus
Non-structural protein Nsp9 inhibits proliferation of the PRRS virus in permissive cell.
2. a kind of DNA sequence dna, which is characterized in that the DNA sequence dna has nucleotide sequence shown in SEQ ID NO:2；
The amino acid sequence of the DNA sequence encoding is included in amino acid sequence shown in SEQ ID NO:1；
The DNA sequence dna is by overlapping pcr, by anti-PRRS virus nsp9 nano antibody gene and pig IgG 1Fc segment base
Because series connection obtains.
3. a kind of expression vector, which is characterized in that the expression vector be by by after the DNA sequence dna digestion in claim 2,
It is connected in yeast expression vector pPICZ α A and obtains；
The expression vector can express the targeting antibodies therapeutic agent in claim 1.
4. it is a kind of inhibit PRRS virus multiplication targeting antibodies therapeutic agent preparation method, feature the following steps are included:
Step 1 is connected anti-PRRS virus nsp9 nano antibody gene with pig IgG 1Fc fragment gene by overlapping pcr
Obtain DNA sequence dna as claimed in claim 2；
The DNA sequence dna that step 1 obtains is connected in yeast expression vector pPICZ α A after digestion and obtains by step 2
Expression vector in claim 3；
Step 3, the expression vector that step 2 is obtained is transformed into Pichia pastoris competent cell X-33, then after overactivation
It is coated on the Salt LB agar plate of blasticidin resistance and cultivates, picking monoclonal colonies are controlled through methanol induction expression antibody
Agent is treated, targeting antibodies therapeutic agent crude product is obtained；
Step 4, the targeting antibodies therapeutic agent crude product that step 3 is obtained obtain purity by Protein G affinity chromatography
Targeting antibodies therapeutic agent greater than 90%.
5. a kind of application of targeting antibodies therapeutic agent for inhibiting PRRS virus multiplication in the drug of preparation treatment PRRS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910314591.2A CN110055272A (en) | 2019-04-18 | 2019-04-18 | A kind of targeting antibodies therapeutic agent inhibiting PRRS virus multiplication |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910314591.2A CN110055272A (en) | 2019-04-18 | 2019-04-18 | A kind of targeting antibodies therapeutic agent inhibiting PRRS virus multiplication |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110055272A true CN110055272A (en) | 2019-07-26 |
Family
ID=67319623
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910314591.2A Pending CN110055272A (en) | 2019-04-18 | 2019-04-18 | A kind of targeting antibodies therapeutic agent inhibiting PRRS virus multiplication |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110055272A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057145A (en) * | 2019-11-22 | 2020-04-24 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Porcine reproductive and respiratory syndrome virus Nsp2 protein nano antibody and application thereof |
| CN111849763A (en) * | 2020-04-08 | 2020-10-30 | 天津大学 | Light-controlled virus propagation system and construction method and application thereof |
| CN116444682A (en) * | 2023-03-31 | 2023-07-18 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Biological protein degradation targeting chimeric for targeting degradation of PRRSV key replicase and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013107388A1 (en) * | 2012-01-19 | 2013-07-25 | 施怀哲维克生物科技股份有限公司 | Interferon and immune globulin fc section fusion protein |
| CN103265637A (en) * | 2013-06-04 | 2013-08-28 | 江苏众红生物工程创药研究院有限公司 | Recombinant porcine interleukin 4-Fc fusion protein as well as coding gene and expression method thereof |
-
2019
- 2019-04-18 CN CN201910314591.2A patent/CN110055272A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013107388A1 (en) * | 2012-01-19 | 2013-07-25 | 施怀哲维克生物科技股份有限公司 | Interferon and immune globulin fc section fusion protein |
| CN103265637A (en) * | 2013-06-04 | 2013-08-28 | 江苏众红生物工程创药研究院有限公司 | Recombinant porcine interleukin 4-Fc fusion protein as well as coding gene and expression method thereof |
Non-Patent Citations (3)
| Title |
|---|
| HONGLIANG LIU等: "n intracellularly expressed Nsp9-specific nanobody in MARC-145 cells inhibits porcine reproductive and respiratory syndrome virus replication", 《VETERINARY MICROBIOLOGY》 * |
| LIZHEN WANG等: "A Nanobody Targeting Viral Nonstructural Protein 9 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication", 《JOURNAL OF VRIOLOGY》 * |
| 郭万美等: "抗癌胚抗原纳米抗体与人Fc融合蛋白在毕赤酵母和HEK293细胞中的表达、纯化和性质比较", 《科学技术与工程》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057145A (en) * | 2019-11-22 | 2020-04-24 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Porcine reproductive and respiratory syndrome virus Nsp2 protein nano antibody and application thereof |
| CN111057145B (en) * | 2019-11-22 | 2021-10-08 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Porcine reproductive and respiratory syndrome virus Nsp2 protein nano antibody and application thereof |
| CN111849763A (en) * | 2020-04-08 | 2020-10-30 | 天津大学 | Light-controlled virus propagation system and construction method and application thereof |
| CN116444682A (en) * | 2023-03-31 | 2023-07-18 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Biological protein degradation targeting chimeric for targeting degradation of PRRSV key replicase and application thereof |
| CN116444682B (en) * | 2023-03-31 | 2023-12-08 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Biological protein degradation targeting chimeric for targeting degradation of PRRSV key replicase and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111217919B (en) | Novel coronavirus S protein double-region subunit nano vaccine based on pyrococcus ferritin | |
| CN111560074B (en) | Novel coronavirus S protein single-region subunit nano vaccine based on helicobacter pylori ferritin | |
| CN111607002B (en) | Novel coronavirus S protein double-region subunit nano vaccine based on helicobacter pylori ferritin | |
| CN110055272A (en) | A kind of targeting antibodies therapeutic agent inhibiting PRRS virus multiplication | |
| CN113230395A (en) | Beta coronavirus antigen, beta coronavirus bivalent vaccine, and preparation method and application thereof | |
| CN111821433A (en) | mRNA vaccine and synthetic method and kit thereof | |
| CN107586759B (en) | Construction method and application of recombinant Newcastle disease virus | |
| CN102827254B (en) | Cell penetrating peptide hPP10 and use thereof | |
| WO2021184884A1 (en) | Replication type recombinant novel coronavirus based on vesicular stomatitis virus vector, and preparation method therefor and application thereof | |
| CN113861285B (en) | Humanized monoclonal antibody of poxvirus and application thereof | |
| CN116350769B (en) | Combined vaccine of novel coronavirus and influenza virus, preparation method and application thereof | |
| CN112194710A (en) | Recombinant protein coded by tilapia lake virus S8 gene, antibody prepared from recombinant protein and application of recombinant protein | |
| JPH01501203A (en) | Diagnosis and vaccines for Japanese encephalitis virus and similar viruses | |
| CN109111507B (en) | Virus recombinant glycoprotein and eukaryotic cell high-efficiency expression method and application thereof | |
| CN109705212A (en) | A kind of antibody, expression vector and therapeutic agent inhibiting PRRS virus replication | |
| CN105859891B (en) | GFP-CD19 fusion protein and application thereof in cell marking | |
| CN115724956B (en) | RBD-targeted high-neutralization-activity anti-SARS-CoV-2 fully human monoclonal antibody 7B3 and application thereof | |
| CN115724955B (en) | RBD-targeted high-neutralization-activity anti-SARS-CoV-2 fully human monoclonal antibody 14B1 and application thereof | |
| CN104707135B (en) | Recombinant protein vaccine, the recombinant expression carrier containing the gene for encoding the recombinant protein vaccine and its application | |
| CN107216395B (en) | A kind of TM4SF1 specific chimerics antigen receptor and its application | |
| CN106496320A (en) | Hog ISG15 recombinant protein and its encoding gene, recombinant plasmid, recombinant bacterial strain and application | |
| CN105085638A (en) | KSHV virus vIRF4 DNA binding domain and polyclonal antibody thereof, and preparation method of polyclonal antibody | |
| CN114075567A (en) | Optimized nucleotide sequence for expressing novel coronavirus antigen and application thereof | |
| WO2023151446A1 (en) | Betacoronavirus fusion recombinant protein, and preparation method and application thereof | |
| CN111778269B (en) | anti-H5N 1 virus entry antibody PTD-3F and application thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190726 |