CN114805600B - 猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的VLPs及其制备方法与应用 - Google Patents
猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的VLPs及其制备方法与应用 Download PDFInfo
- Publication number
- CN114805600B CN114805600B CN202210320567.1A CN202210320567A CN114805600B CN 114805600 B CN114805600 B CN 114805600B CN 202210320567 A CN202210320567 A CN 202210320567A CN 114805600 B CN114805600 B CN 114805600B
- Authority
- CN
- China
- Prior art keywords
- recombinant
- cap
- vlps
- arg
- porcine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000710198 Foot-and-mouth disease virus Species 0.000 title claims abstract description 58
- 241001670114 Porcine circovirus type 2-D Species 0.000 title claims abstract description 14
- 102000036639 antigens Human genes 0.000 title abstract description 13
- 108091007433 antigens Proteins 0.000 title abstract description 13
- 239000000427 antigen Substances 0.000 title abstract description 11
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 48
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 47
- 208000015181 infectious disease Diseases 0.000 claims abstract description 18
- 229940031626 subunit vaccine Drugs 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 108090000623 proteins and genes Proteins 0.000 claims description 56
- 210000004027 cell Anatomy 0.000 claims description 55
- 241000701447 unidentified baculovirus Species 0.000 claims description 49
- 239000013612 plasmid Substances 0.000 claims description 34
- 102000004169 proteins and genes Human genes 0.000 claims description 29
- 150000001413 amino acids Chemical class 0.000 claims description 27
- 229960005486 vaccine Drugs 0.000 claims description 25
- 238000012546 transfer Methods 0.000 claims description 24
- 239000013598 vector Substances 0.000 claims description 24
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 20
- 241000700605 Viruses Species 0.000 claims description 18
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 12
- 241000282898 Sus scrofa Species 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 11
- 125000003729 nucleotide group Chemical group 0.000 claims description 11
- 230000009977 dual effect Effects 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 241000238631 Hexapoda Species 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 5
- 239000002356 single layer Substances 0.000 claims description 4
- 238000010367 cloning Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 230000001571 immunoadjuvant effect Effects 0.000 claims description 3
- 239000000568 immunological adjuvant Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 230000001804 emulsifying effect Effects 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 238000000856 sucrose gradient centrifugation Methods 0.000 claims 1
- 230000001131 transforming effect Effects 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 abstract description 18
- 230000003053 immunization Effects 0.000 abstract description 16
- 241000282887 Suidae Species 0.000 abstract description 10
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 abstract description 9
- 239000002245 particle Substances 0.000 abstract description 7
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 abstract description 6
- 230000028993 immune response Effects 0.000 abstract description 4
- 208000011580 syndromic disease Diseases 0.000 abstract description 3
- 230000014509 gene expression Effects 0.000 description 24
- 210000002966 serum Anatomy 0.000 description 21
- 235000018102 proteins Nutrition 0.000 description 17
- 238000001514 detection method Methods 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- 238000002649 immunization Methods 0.000 description 13
- 241000269335 Ambystoma laterale x Ambystoma jeffersonianum Species 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 12
- 241001673669 Porcine circovirus 2 Species 0.000 description 12
- 108020004705 Codon Proteins 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 108010028295 histidylhistidine Proteins 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 230000003321 amplification Effects 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 238000003753 real-time PCR Methods 0.000 description 7
- 101710132601 Capsid protein Proteins 0.000 description 6
- 101710197658 Capsid protein VP1 Proteins 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 6
- YKNBJXOJTURHCU-DCAQKATOSA-N Leu-Asp-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YKNBJXOJTURHCU-DCAQKATOSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 101710118046 RNA-directed RNA polymerase Proteins 0.000 description 6
- 101710108545 Viral protein 1 Proteins 0.000 description 6
- 239000013642 negative control Substances 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 108010017949 tyrosyl-glycyl-glycine Proteins 0.000 description 6
- 108010073969 valyllysine Proteins 0.000 description 6
- 238000011725 BALB/c mouse Methods 0.000 description 5
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000005847 immunogenicity Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- 108010002350 Interleukin-2 Proteins 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 238000003306 harvesting Methods 0.000 description 4
- 230000004727 humoral immunity Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 3
- LYILPUNCKACNGF-NAKRPEOUSA-N Ala-Val-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)N LYILPUNCKACNGF-NAKRPEOUSA-N 0.000 description 3
- XPSGESXVBSQZPL-SRVKXCTJSA-N Arg-Arg-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O XPSGESXVBSQZPL-SRVKXCTJSA-N 0.000 description 3
- JGDGLDNAQJJGJI-AVGNSLFASA-N Arg-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)N JGDGLDNAQJJGJI-AVGNSLFASA-N 0.000 description 3
- HJVGMOYJDDXLMI-AVGNSLFASA-N Arg-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CCCNC(N)=N HJVGMOYJDDXLMI-AVGNSLFASA-N 0.000 description 3
- CPSHGRGUPZBMOK-CIUDSAMLSA-N Arg-Asn-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O CPSHGRGUPZBMOK-CIUDSAMLSA-N 0.000 description 3
- NKNILFJYKKHBKE-WPRPVWTQSA-N Arg-Gly-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O NKNILFJYKKHBKE-WPRPVWTQSA-N 0.000 description 3
- YBIAYFFIVAZXPK-AVGNSLFASA-N Arg-His-Arg Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O YBIAYFFIVAZXPK-AVGNSLFASA-N 0.000 description 3
- NVUIWHJLPSZZQC-CYDGBPFRSA-N Arg-Ile-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NVUIWHJLPSZZQC-CYDGBPFRSA-N 0.000 description 3
- GMFAGHNRXPSSJS-SRVKXCTJSA-N Arg-Leu-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O GMFAGHNRXPSSJS-SRVKXCTJSA-N 0.000 description 3
- INXWADWANGLMPJ-JYJNAYRXSA-N Arg-Phe-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CC1=CC=CC=C1 INXWADWANGLMPJ-JYJNAYRXSA-N 0.000 description 3
- APHUDFFMXFYRKP-CIUDSAMLSA-N Asn-Asn-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N APHUDFFMXFYRKP-CIUDSAMLSA-N 0.000 description 3
- JZRLLSOWDYUKOK-SRVKXCTJSA-N Asn-Asp-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)N)N JZRLLSOWDYUKOK-SRVKXCTJSA-N 0.000 description 3
- PBSQFBAJKPLRJY-BYULHYEWSA-N Asn-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N PBSQFBAJKPLRJY-BYULHYEWSA-N 0.000 description 3
- OLISTMZJGQUOGS-GMOBBJLQSA-N Asn-Ile-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N OLISTMZJGQUOGS-GMOBBJLQSA-N 0.000 description 3
- YVXRYLVELQYAEQ-SRVKXCTJSA-N Asn-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N YVXRYLVELQYAEQ-SRVKXCTJSA-N 0.000 description 3
- BYLSYQASFJJBCL-DCAQKATOSA-N Asn-Pro-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O BYLSYQASFJJBCL-DCAQKATOSA-N 0.000 description 3
- YSYTWUMRHSFODC-QWRGUYRKSA-N Asn-Tyr-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O YSYTWUMRHSFODC-QWRGUYRKSA-N 0.000 description 3
- 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 3
- BKOIIURTQAJHAT-GUBZILKMSA-N Asp-Pro-Pro Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 BKOIIURTQAJHAT-GUBZILKMSA-N 0.000 description 3
- 101150044789 Cap gene Proteins 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- OFPWCBGRYAOLMU-AVGNSLFASA-N Gln-Asp-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)N)N)O OFPWCBGRYAOLMU-AVGNSLFASA-N 0.000 description 3
- NSNUZSPSADIMJQ-WDSKDSINSA-N Gln-Gly-Asp Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O NSNUZSPSADIMJQ-WDSKDSINSA-N 0.000 description 3
- 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 3
- MBOAPAXLTUSMQI-JHEQGTHGSA-N Gly-Glu-Thr Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MBOAPAXLTUSMQI-JHEQGTHGSA-N 0.000 description 3
- GDOZQTNZPCUARW-YFKPBYRVSA-N Gly-Gly-Glu Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O GDOZQTNZPCUARW-YFKPBYRVSA-N 0.000 description 3
- LCRDMSSAKLTKBU-ZDLURKLDSA-N Gly-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN LCRDMSSAKLTKBU-ZDLURKLDSA-N 0.000 description 3
- 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 3
- PROLDOGUBQJNPG-RWMBFGLXSA-N His-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC2=CN=CN2)N)C(=O)O PROLDOGUBQJNPG-RWMBFGLXSA-N 0.000 description 3
- STOOMQFEJUVAKR-KKUMJFAQSA-N His-His-His Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)C1=CNC=N1 STOOMQFEJUVAKR-KKUMJFAQSA-N 0.000 description 3
- FHKZHRMERJUXRJ-DCAQKATOSA-N His-Ser-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 FHKZHRMERJUXRJ-DCAQKATOSA-N 0.000 description 3
- XVZJRZQIHJMUBG-TUBUOCAGSA-N His-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC1=CN=CN1)N XVZJRZQIHJMUBG-TUBUOCAGSA-N 0.000 description 3
- JJQQGCMKLOEGAV-OSUNSFLBSA-N Ile-Thr-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)O)N JJQQGCMKLOEGAV-OSUNSFLBSA-N 0.000 description 3
- DLFAACQHIRSQGG-CIUDSAMLSA-N Leu-Asp-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O DLFAACQHIRSQGG-CIUDSAMLSA-N 0.000 description 3
- IDGZVZJLYFTXSL-DCAQKATOSA-N Leu-Ser-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IDGZVZJLYFTXSL-DCAQKATOSA-N 0.000 description 3
- FPFOYSCDUWTZBF-IHPCNDPISA-N Leu-Trp-Leu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H]([NH3+])CC(C)C)C(=O)N[C@@H](CC(C)C)C([O-])=O)=CNC2=C1 FPFOYSCDUWTZBF-IHPCNDPISA-N 0.000 description 3
- MPOHDJKRBLVGCT-CIUDSAMLSA-N Lys-Ala-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)N MPOHDJKRBLVGCT-CIUDSAMLSA-N 0.000 description 3
- ISHNZELVUVPCHY-ZETCQYMHSA-N Lys-Gly-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O ISHNZELVUVPCHY-ZETCQYMHSA-N 0.000 description 3
- PLDJDCJLRCYPJB-VOAKCMCISA-N Lys-Lys-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PLDJDCJLRCYPJB-VOAKCMCISA-N 0.000 description 3
- YSPZCHGIWAQVKQ-AVGNSLFASA-N Lys-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN YSPZCHGIWAQVKQ-AVGNSLFASA-N 0.000 description 3
- NYTDJEZBAAFLLG-IHRRRGAJSA-N Lys-Val-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(O)=O NYTDJEZBAAFLLG-IHRRRGAJSA-N 0.000 description 3
- OXIWIYOJVNOKOV-SRVKXCTJSA-N Met-Met-Arg Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@H](C(O)=O)CCCNC(N)=N OXIWIYOJVNOKOV-SRVKXCTJSA-N 0.000 description 3
- NSMXRFMGZYTFEX-KJEVXHAQSA-N Met-Thr-Tyr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CCSC)N)O NSMXRFMGZYTFEX-KJEVXHAQSA-N 0.000 description 3
- PESQCPHRXOFIPX-UHFFFAOYSA-N N-L-methionyl-L-tyrosine Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 PESQCPHRXOFIPX-UHFFFAOYSA-N 0.000 description 3
- 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 3
- MPGJIHFJCXTVEX-KKUMJFAQSA-N Phe-Arg-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O MPGJIHFJCXTVEX-KKUMJFAQSA-N 0.000 description 3
- KIEPQOIQHFKQLK-PCBIJLKTSA-N Phe-Asn-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KIEPQOIQHFKQLK-PCBIJLKTSA-N 0.000 description 3
- HTKNPQZCMLBOTQ-XVSYOHENSA-N Phe-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CC=CC=C1)N)O HTKNPQZCMLBOTQ-XVSYOHENSA-N 0.000 description 3
- FMMIYCMOVGXZIP-AVGNSLFASA-N Phe-Glu-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O FMMIYCMOVGXZIP-AVGNSLFASA-N 0.000 description 3
- UAMFZRNCIFFMLE-FHWLQOOXSA-N Phe-Glu-Tyr Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N UAMFZRNCIFFMLE-FHWLQOOXSA-N 0.000 description 3
- XNMYNGDKJNOKHH-BZSNNMDCSA-N Phe-Ser-Tyr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XNMYNGDKJNOKHH-BZSNNMDCSA-N 0.000 description 3
- SHUFSZDAIPLZLF-BEAPCOKYSA-N Phe-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N)O SHUFSZDAIPLZLF-BEAPCOKYSA-N 0.000 description 3
- RGMLUHANLDVMPB-ULQDDVLXSA-N Phe-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N RGMLUHANLDVMPB-ULQDDVLXSA-N 0.000 description 3
- MWQXFDIQXIXPMS-UNQGMJICSA-N Phe-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC1=CC=CC=C1)N)O MWQXFDIQXIXPMS-UNQGMJICSA-N 0.000 description 3
- 241000202347 Porcine circovirus Species 0.000 description 3
- LNLNHXIQPGKRJQ-SRVKXCTJSA-N Pro-Arg-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H]1CCCN1 LNLNHXIQPGKRJQ-SRVKXCTJSA-N 0.000 description 3
- LEIKGVHQTKHOLM-IUCAKERBSA-N Pro-Pro-Gly Chemical compound OC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 LEIKGVHQTKHOLM-IUCAKERBSA-N 0.000 description 3
- XSXABUHLKPUVLX-JYJNAYRXSA-N Pro-Ser-Trp Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O XSXABUHLKPUVLX-JYJNAYRXSA-N 0.000 description 3
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 3
- MQQBBLVOUUJKLH-HJPIBITLSA-N Ser-Ile-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MQQBBLVOUUJKLH-HJPIBITLSA-N 0.000 description 3
- KZPRPBLHYMZIMH-MXAVVETBSA-N Ser-Phe-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KZPRPBLHYMZIMH-MXAVVETBSA-N 0.000 description 3
- GZGFSPWOMUKKCV-NAKRPEOUSA-N Ser-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO GZGFSPWOMUKKCV-NAKRPEOUSA-N 0.000 description 3
- KQNDIKOYWZTZIX-FXQIFTODSA-N Ser-Ser-Arg Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCNC(N)=N KQNDIKOYWZTZIX-FXQIFTODSA-N 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- DIPIPFHFLPTCLK-LOKLDPHHSA-N Thr-Gln-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N)O DIPIPFHFLPTCLK-LOKLDPHHSA-N 0.000 description 3
- CRZNCABIJLRFKZ-IUKAMOBKSA-N Thr-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N CRZNCABIJLRFKZ-IUKAMOBKSA-N 0.000 description 3
- URPSJRMWHQTARR-MBLNEYKQSA-N Thr-Ile-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O URPSJRMWHQTARR-MBLNEYKQSA-N 0.000 description 3
- UQCNIMDPYICBTR-KYNKHSRBSA-N Thr-Thr-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UQCNIMDPYICBTR-KYNKHSRBSA-N 0.000 description 3
- XGFYGMKZKFRGAI-RCWTZXSCSA-N Thr-Val-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N XGFYGMKZKFRGAI-RCWTZXSCSA-N 0.000 description 3
- QNXZCKMXHPULME-ZNSHCXBVSA-N Thr-Val-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N)O QNXZCKMXHPULME-ZNSHCXBVSA-N 0.000 description 3
- KXIQQAWIPDDVOE-BPUTZDHNSA-N Trp-Pro-Cys Chemical compound O=C([C@H](CC=1C2=CC=CC=C2NC=1)N)N1CCC[C@H]1C(=O)N[C@@H](CS)C(O)=O KXIQQAWIPDDVOE-BPUTZDHNSA-N 0.000 description 3
- YLHFIMLKNPJRGY-BVSLBCMMSA-N Tyr-Arg-Trp 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](CC1=CNC2=C1C=CC=C2)C(O)=O YLHFIMLKNPJRGY-BVSLBCMMSA-N 0.000 description 3
- NRFTYDWKWGJLAR-MELADBBJSA-N Tyr-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N)C(=O)O NRFTYDWKWGJLAR-MELADBBJSA-N 0.000 description 3
- FDKDGFGTHGJKNV-FHWLQOOXSA-N Tyr-Phe-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N FDKDGFGTHGJKNV-FHWLQOOXSA-N 0.000 description 3
- JHDZONWZTCKTJR-KJEVXHAQSA-N Tyr-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JHDZONWZTCKTJR-KJEVXHAQSA-N 0.000 description 3
- AEOFMCAKYIQQFY-YDHLFZDLSA-N Tyr-Val-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 AEOFMCAKYIQQFY-YDHLFZDLSA-N 0.000 description 3
- KLOZTPOXVVRVAQ-DZKIICNBSA-N Tyr-Val-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 KLOZTPOXVVRVAQ-DZKIICNBSA-N 0.000 description 3
- ZLFHAAGHGQBQQN-GUBZILKMSA-N Val-Ala-Pro Natural products CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O ZLFHAAGHGQBQQN-GUBZILKMSA-N 0.000 description 3
- ZQGPWORGSNRQLN-NHCYSSNCSA-N Val-Asp-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N ZQGPWORGSNRQLN-NHCYSSNCSA-N 0.000 description 3
- FOADDSDHGRFUOC-DZKIICNBSA-N Val-Glu-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N FOADDSDHGRFUOC-DZKIICNBSA-N 0.000 description 3
- URIRWLJVWHYLET-ONGXEEELSA-N Val-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C URIRWLJVWHYLET-ONGXEEELSA-N 0.000 description 3
- DVLWZWNAQUBZBC-ZNSHCXBVSA-N Val-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N)O DVLWZWNAQUBZBC-ZNSHCXBVSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 108010062796 arginyllysine Proteins 0.000 description 3
- 108010068265 aspartyltyrosine Proteins 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000007969 cellular immunity Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 3
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 3
- 108010001064 glycyl-glycyl-glycyl-glycine Proteins 0.000 description 3
- 108010087823 glycyltyrosine Proteins 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 235000014304 histidine Nutrition 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 229940031551 inactivated vaccine Drugs 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 108010057821 leucylproline Proteins 0.000 description 3
- 108010025153 lysyl-alanyl-alanine Proteins 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 108010082795 phenylalanyl-arginyl-arginine Proteins 0.000 description 3
- 108010083476 phenylalanyltryptophan Proteins 0.000 description 3
- 108010089164 prolyl-leucyl-asparagyl-prolyl-lysine Proteins 0.000 description 3
- 108010087846 prolyl-prolyl-glycine Proteins 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 108010005834 tyrosyl-alanyl-glycine Proteins 0.000 description 3
- 108010071635 tyrosyl-prolyl-arginine Proteins 0.000 description 3
- 108010003137 tyrosyltyrosine Proteins 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 2
- SLQQPJBDBVPVQV-JYJNAYRXSA-N Arg-Phe-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O SLQQPJBDBVPVQV-JYJNAYRXSA-N 0.000 description 2
- NGYHSXDNNOFHNE-AVGNSLFASA-N Arg-Pro-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O NGYHSXDNNOFHNE-AVGNSLFASA-N 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- KWUSGAIFNHQCBY-DCAQKATOSA-N Gln-Arg-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O KWUSGAIFNHQCBY-DCAQKATOSA-N 0.000 description 2
- ZJICFHQSPWFBKP-AVGNSLFASA-N Glu-Asn-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZJICFHQSPWFBKP-AVGNSLFASA-N 0.000 description 2
- DJTXYXZNNDDEOU-WHFBIAKZSA-N Gly-Asn-Cys Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN)C(=O)N DJTXYXZNNDDEOU-WHFBIAKZSA-N 0.000 description 2
- FGPLUIQCSKGLTI-WDSKDSINSA-N Gly-Ser-Glu Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O FGPLUIQCSKGLTI-WDSKDSINSA-N 0.000 description 2
- CNHSMSFYVARZLI-YJRXYDGGSA-N His-His-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CNHSMSFYVARZLI-YJRXYDGGSA-N 0.000 description 2
- XHQYFGPIRUHQIB-PBCZWWQYSA-N His-Thr-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC1=CN=CN1 XHQYFGPIRUHQIB-PBCZWWQYSA-N 0.000 description 2
- OUUCIIJSBIBCHB-ZPFDUUQYSA-N Ile-Leu-Asp Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O OUUCIIJSBIBCHB-ZPFDUUQYSA-N 0.000 description 2
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 2
- MJOZZTKJZQFKDK-GUBZILKMSA-N Leu-Ala-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(N)=O MJOZZTKJZQFKDK-GUBZILKMSA-N 0.000 description 2
- QDSKNVXKLPQNOJ-GVXVVHGQSA-N Leu-Gln-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O QDSKNVXKLPQNOJ-GVXVVHGQSA-N 0.000 description 2
- RRVCZCNFXIFGRA-DCAQKATOSA-N Leu-Pro-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O RRVCZCNFXIFGRA-DCAQKATOSA-N 0.000 description 2
- FZIJIFCXUCZHOL-CIUDSAMLSA-N Lys-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN FZIJIFCXUCZHOL-CIUDSAMLSA-N 0.000 description 2
- SUZVLFWOCKHWET-CQDKDKBSSA-N Lys-Tyr-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O SUZVLFWOCKHWET-CQDKDKBSSA-N 0.000 description 2
- RIPJMCFGQHGHNP-RHYQMDGZSA-N Lys-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCCN)N)O RIPJMCFGQHGHNP-RHYQMDGZSA-N 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 101100382437 Porcine circovirus 2 Cap gene Proteins 0.000 description 2
- 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 2
- QZKVWWIUSQGWMY-IHRRRGAJSA-N Val-Ser-Phe Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QZKVWWIUSQGWMY-IHRRRGAJSA-N 0.000 description 2
- 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 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 108010020755 prolyl-glycyl-glycine Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 210000002845 virion Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- HHGYNJRJIINWAK-FXQIFTODSA-N Ala-Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HHGYNJRJIINWAK-FXQIFTODSA-N 0.000 description 1
- AWAXZRDKUHOPBO-GUBZILKMSA-N Ala-Gln-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O AWAXZRDKUHOPBO-GUBZILKMSA-N 0.000 description 1
- YHBDGLZYNIARKJ-GUBZILKMSA-N Ala-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N YHBDGLZYNIARKJ-GUBZILKMSA-N 0.000 description 1
- MMGCRPZQZWTZTA-IHRRRGAJSA-N Arg-His-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N MMGCRPZQZWTZTA-IHRRRGAJSA-N 0.000 description 1
- RKQRHMKFNBYOTN-IHRRRGAJSA-N Arg-His-Lys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N RKQRHMKFNBYOTN-IHRRRGAJSA-N 0.000 description 1
- YQNBILXAUIAUCF-CIUDSAMLSA-N Asn-Cys-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(=O)N)N YQNBILXAUIAUCF-CIUDSAMLSA-N 0.000 description 1
- MRQQMVZUHXUPEV-IHRRRGAJSA-N Asp-Arg-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O MRQQMVZUHXUPEV-IHRRRGAJSA-N 0.000 description 1
- QOJJMJKTMKNFEF-ZKWXMUAHSA-N Asp-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(O)=O QOJJMJKTMKNFEF-ZKWXMUAHSA-N 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LURQDGKYBFWWJA-MNXVOIDGSA-N Gln-Lys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(=O)N)N LURQDGKYBFWWJA-MNXVOIDGSA-N 0.000 description 1
- LPIKVBWNNVFHCQ-GUBZILKMSA-N Gln-Ser-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LPIKVBWNNVFHCQ-GUBZILKMSA-N 0.000 description 1
- VEYGCDYMOXHJLS-GVXVVHGQSA-N Gln-Val-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O VEYGCDYMOXHJLS-GVXVVHGQSA-N 0.000 description 1
- MWTGQXBHVRTCOR-GLLZPBPUSA-N Glu-Thr-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MWTGQXBHVRTCOR-GLLZPBPUSA-N 0.000 description 1
- FZQLXNIMCPJVJE-YUMQZZPRSA-N Gly-Asp-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O FZQLXNIMCPJVJE-YUMQZZPRSA-N 0.000 description 1
- HDNXXTBKOJKWNN-WDSKDSINSA-N Gly-Glu-Asn Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O HDNXXTBKOJKWNN-WDSKDSINSA-N 0.000 description 1
- HQRHFUYMGCHHJS-LURJTMIESA-N Gly-Gly-Arg Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N HQRHFUYMGCHHJS-LURJTMIESA-N 0.000 description 1
- WNGHUXFWEWTKAO-YUMQZZPRSA-N Gly-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN WNGHUXFWEWTKAO-YUMQZZPRSA-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
- GUXQAPACZVVOKX-AVGNSLFASA-N His-Lys-Gln Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N GUXQAPACZVVOKX-AVGNSLFASA-N 0.000 description 1
- YJRSIJZUIUANHO-NAKRPEOUSA-N Ile-Val-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)O)N YJRSIJZUIUANHO-NAKRPEOUSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- QQUJSUFWEDZQQY-AVGNSLFASA-N Lys-Gln-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CCCCN QQUJSUFWEDZQQY-AVGNSLFASA-N 0.000 description 1
- NNCDAORZCMPZPX-GUBZILKMSA-N Lys-Gln-Ser Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N NNCDAORZCMPZPX-GUBZILKMSA-N 0.000 description 1
- WAIHHELKYSFIQN-XUXIUFHCSA-N Lys-Ile-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O WAIHHELKYSFIQN-XUXIUFHCSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- DVOCGBNHAUHKHJ-DKIMLUQUSA-N Phe-Ile-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O DVOCGBNHAUHKHJ-DKIMLUQUSA-N 0.000 description 1
- 101710182846 Polyhedrin Proteins 0.000 description 1
- AHXPYZRZRMQOAU-QXEWZRGKSA-N Pro-Asn-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H]1CCCN1)C(O)=O AHXPYZRZRMQOAU-QXEWZRGKSA-N 0.000 description 1
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 1
- MCWHYUWXVNRXFV-RWMBFGLXSA-N Pro-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 MCWHYUWXVNRXFV-RWMBFGLXSA-N 0.000 description 1
- IIRBTQHFVNGPMQ-AVGNSLFASA-N Pro-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1 IIRBTQHFVNGPMQ-AVGNSLFASA-N 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000519995 Stachys sylvatica Species 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- ZUUDNCOCILSYAM-KKHAAJSZSA-N Thr-Asp-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ZUUDNCOCILSYAM-KKHAAJSZSA-N 0.000 description 1
- JAJOFWABAUKAEJ-QTKMDUPCSA-N Thr-Pro-His Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O JAJOFWABAUKAEJ-QTKMDUPCSA-N 0.000 description 1
- DLZKEQQWXODGGZ-KWQFWETISA-N Tyr-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DLZKEQQWXODGGZ-KWQFWETISA-N 0.000 description 1
- GFHYISDTIWZUSU-QWRGUYRKSA-N Tyr-Asn-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GFHYISDTIWZUSU-QWRGUYRKSA-N 0.000 description 1
- HIINQLBHPIQYHN-JTQLQIEISA-N Tyr-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 HIINQLBHPIQYHN-JTQLQIEISA-N 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- ZLFHAAGHGQBQQN-AEJSXWLSSA-N Val-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N ZLFHAAGHGQBQQN-AEJSXWLSSA-N 0.000 description 1
- XTAUQCGQFJQGEJ-NHCYSSNCSA-N Val-Gln-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N XTAUQCGQFJQGEJ-NHCYSSNCSA-N 0.000 description 1
- WBAJDGWKRIHOAC-GVXVVHGQSA-N Val-Lys-Gln Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O WBAJDGWKRIHOAC-GVXVVHGQSA-N 0.000 description 1
- VPGCVZRRBYOGCD-AVGNSLFASA-N Val-Lys-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O VPGCVZRRBYOGCD-AVGNSLFASA-N 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 208000010399 Wasting Syndrome Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940031416 bivalent vaccine Drugs 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000003631 expected effect Effects 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 239000010419 fine particle Substances 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
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 108010078274 isoleucylvaline Proteins 0.000 description 1
- 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 description 1
- 239000002502 liposome Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000003260 vortexing Methods 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
-
- 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
- A61K39/12—Viral antigens
-
- 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
-
- 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
- A61P31/20—Antivirals for DNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
-
- 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
- A61K2039/70—Multivalent vaccine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14141—Use of virus, viral particle or viral elements as a vector
- C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10022—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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10023—Virus like particles [VLP]
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10051—Methods of production or purification of viral material
- C12N2750/10052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
-
- 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
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32122—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
- 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
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32123—Virus like particles [VLP]
-
- 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
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- 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
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32151—Methods of production or purification of viral material
- C12N2770/32152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
-
- 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
- C12N2800/10—Plasmid DNA
- C12N2800/103—Plasmid DNA for invertebrates
- C12N2800/105—Plasmid DNA for invertebrates for insects
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Communicable Diseases (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明属于分子生物学技术领域，具体涉及猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的病毒样颗粒及其制备方法与应用。所述VLPs由猪圆环2d型框架嵌合猪O型口蹄疫病毒单个表位或不同抗原表位组合后得到的重组蛋白组装而成，所述重组蛋白的氨基酸序列如SEQ ID No.5、SEQ ID No.6或SEQ ID No.7所示。利用上述VLPs制备亚单位疫苗，免疫BALB/c雌鼠后，可诱导机体产生特异性免疫反应，能有效预防PCV2d和FMDV的感染，对控制和净化我国猪O型口蹄疫和断奶仔猪多系统衰竭综合征具有重要意义。
Description
技术领域
本发明属于分子生物技术领域，具体涉及猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的VLPs及其制备方法与应用。
背景技术
口蹄疫(Foot-and-mouth disease，FMD)是由口蹄疫病毒(Foot-and-mouthdisease virus，FMDV)引起的一种高度接触性传染病，对社会经济造成巨大损失。目前，预防和控制该病的重要手段是接种疫苗，但市场上的灭活疫苗具有热稳定性差、免疫持续时间短、安全性低等缺点。因此，研究更安全、低成本、效率高的新型疫苗对FMD的防控具有重要意义。
目前，我国主要流行猪O型口蹄疫，其中研究最多并效果最好的FMDV抗原表位主要位于VP1蛋白。Pan等[Pan Q,Wang H,Ouyang W,et al.Immunogenicity of adenovirus-derived porcine parvovirus-like particles displaying B and T cell epitopes offoot-and-mouth disease[J].Vaccine,2016,34(4):578-585.]利用FMDV VP1第21～40aa、141～160aa和200～213aa插入PPV的VP2外环中，构建重组腺病毒来表达PPV：VLPs(FMDV)，在实验动物中发现无佐剂下可诱导高水平的FMDV特异性体液和细胞免疫。Fang等[Fang M,Li J,Wang H,et al.Correlation between efficacy and structure of recombinantepitope vaccines against bovine type O foot and mouth disease virus[J].Biotechnology Letters,2012,34(5):839-847.]构建了3个片段，选择O型FMDV VP1结构蛋白中B细胞表位第130～140aa和第141～160aa，以及T细胞表位第16～44aa。以不同方式串联6种多肽，结果发现抗原表位的排序对体内产生中和抗体水平是有相关性的。大量研究结果表明，抗原表位对亚单位疫苗的研发具有广阔前景。本课题组前期研究表明，选用猪O型FMDV O/BY/CHA/2010毒株中VP1蛋白的2个B细胞优势抗原表位(129～160aa和200～213aa)和1个T细胞优势抗原表位(16～44aa)串联后表达的重组蛋白能刺激动物机体产生特异性抗体。
猪圆环病毒2型(Porcine circovirus type 2，PCV2)是引起断奶仔猪多系统衰竭综合征(PMWS)的主要病原。大量研究表明，在PCV2 Cap蛋白C端插入外源表位能够同时诱导针对Cap和外源表位的免疫反应。此外，PCV2 Cap蛋白可以在体外自组装形成二十面体对称的病毒衣壳结构。目前我国流行的PCV2流行毒株以PCV2d亚型为主，现有的PCV2a/PCV2b疫苗不能做到有效控制PCV2d毒株的流行。
近年来，杆状病毒表达载体因其具有操作简单、安全性高，可容纳大的目的基因，表达外源蛋白效率高，有翻译后修饰的作用，表达蛋白的免疫原性、生物活性与天然的蛋白相似等优点，能够很好的弥补传统的原核表达系统表达的蛋白以包涵体形式存在，且原核表达系统翻译后加工修饰体系不完善，表达产物的生物活性较低等不足。
病毒样颗粒(Virus-like particles，VLPs)不含病毒核酸，与自然病毒粒子相似，能诱导类似病毒感染体内引起的免疫反应。此外，抗原表位是蛋白抗原中的短氨基酸序列，更容易被MHC分子识别，与整个蛋白抗原相比，能直接诱导有效的免疫。VLPs可直接当作疫苗，是近年来发现的免疫原性强、效果好且具有广阔应用前景的基因工程疫苗。因此，开发一种安全有效并可预防PCV2d、FMDV的新型基因工程二联疫苗是十分必要的。
发明内容
为了克服现有技术的不足和缺点，本发明的目的在于提供一种猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的VLPs及其制备方法与应用。利用制备的VLPs制备亚单位疫苗，免疫BALB/c雌鼠后，可诱导机体产生特异性免疫反应，能有效预防PCV2d和FMDV的感染，对控制和净化我国猪O型口蹄疫和断奶仔猪多系统衰竭综合征具有重要意义。
本发明的第一个目的在于提供一种猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的重组蛋白，所述重组蛋白由猪圆环2d型Cap蛋白嵌合如下猪O型口蹄疫病毒抗原表位组合后得到；
当所述猪O型口蹄疫病毒抗原表位为T细胞表位第16～44位氨基酸，所述重组蛋白的氨基酸序列如SEQ ID No.5所示；
或者，所述猪O型口蹄疫病毒抗原表位为T细胞表位第16～44位氨基酸与B细胞表位第200～213位氨基酸串联，所述重组蛋白的氨基酸序列如SEQ ID No.6所示；
或者，所述猪O型口蹄疫病毒抗原表位为B细胞表位第129～160位氨基酸与T细胞表位第16～44位氨基酸串联，所述重组蛋白的氨基酸序列如SEQ ID No.7所示。
所述T细胞表位第16～44位氨基酸序列如SEQ ID No.8所示；所述猪O型口蹄疫病毒抗原表位为T细胞表位第16～44位氨基酸与B细胞表位第200～213位氨基酸串联，氨基酸序列如SEQ ID No.11所示；所述猪O型口蹄疫病毒抗原表位为B细胞表位第129～160位氨基酸与T细胞表位第16～44位氨基酸串联，氨基酸序列如SEQ ID No.12所示。
本发明的第二个目的在于提供一种上述重组蛋白的编码基因，当所述重组蛋白的氨基酸序列为SEQ ID No.5，其编码基因的核苷酸序列如SEQ ID No.2所示；或者，所述重组蛋白的氨基酸序列为SEQ ID No.6，其编码基因的核苷酸序列如SEQ ID No.3所示；或者，所述重组蛋白的氨基酸序列为SEQ ID No.7，其编码基因的核苷酸序列如SEQ ID No.4所示。
本发明的第三个目的是提供一种含有上述编码基因的转移载体，所述转移载体是将所述编码基因克隆至表达载体所得，所述表达载体为双启动子pFastBacTM Dual载体。
本发明的第四个目的在于提供一种含有上述编码基因的重组病毒，是将含有上述编码基因的转移载体转化感受态细胞，获得重组病毒质粒，将重组病毒质粒转染昆虫细胞，培养后获得重组病毒。该重组病毒能分泌表达串联单个表位或者不同抗原表位组合的重组猪圆环病毒2d型Cap蛋白。
作为本发明优选的技术方案，所述感受态细胞为DH10Bac，所述重组病毒为重组杆状病毒。
优选地，所述昆虫细胞为sf9单层昆虫细胞。
本发明的另一个目的在于提供一种猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的VLPs，由上述的猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的重组蛋白组装而成。
优选地，所述VLPs采用如下方法制备得到：将上述得到的重组病毒，感染到悬浮培养的High-Five昆虫细胞表达重组蛋白，扩大培养后，收集目的蛋白，采用蔗糖梯度离心纯化，即得所述VLPs。
本发明还提供了一种PCV2d和FMDV二联亚单位疫苗，其由上述的VLPs与免疫佐剂乳化或混合制得。
本发明最后提供了上述PCV2d和FMDV二联亚单位疫苗在制备用于治疗和预防PCV2d和FMDV感染的药物中的应用。
本发明采用上述VLPs与免疫佐剂进行乳化后制成亚单位疫苗，免疫BALB/c雌鼠后，可诱导机体产生体液免疫和细胞免疫反应，产生PCV2和FMDV两种特异性抗体。
与现有技术相比，本发明的有益效果体现在：
(1)本发明根据杆状病毒密码子的偏爱性，人工设计合成了三个插入猪O型FMDV单个表位或者不同抗原表位组合的重组猪圆环病毒2d型Cap核苷酸序列，其序列如SEQ IDNO.2、SEQ ID NO.3、SEQ ID NO.4所示，该序列提高了目的基因在杆状病毒中表达水平和免疫原性。
(2)本发明通过生物信息学软件模拟重组蛋白的空间结构，预测其在体外能自组装形成VLPs，避免体外组装VLPs的失败性。通过实施例8验证了VLPs能在体外组装，证明生物信息学软件的预测的有效性。后续研究可以借鉴验证实验设计能否形成VLPs。
(3)本发明将人工合成的重组基因Cap-T、Cap-TB2和Cap-B1T分别置于杆状病毒pH和p10启动子下表达，提高了外源基因的表达量。表达出来的重组蛋白能够与阳性血清发生特异性结合，具有良好的反应原性，且经过表达条件优化的重组蛋白应用于实际生产中可以很大程度上降低生产成本。
(4)本发明的重组VLPs制备亚单位疫苗，免疫BALB/c雌鼠后，可诱导机体产生体液免疫和细胞免疫反应，能预防断奶仔猪多系统衰竭综合征和口蹄疫病毒的感染，对控制和净化我国猪O型口蹄疫和猪圆环2d型具有重要意义。
附图说明
图1是重组蛋白空间结构预测(a、b、c、d分别代表Cap、Cap-T、Cap-TB2、Cap-B1T蛋白的空间结构预测，其中绿色、红色和黄色分别代表插入的T、B1和B2细胞抗原表位)。
图2是重组转移载体构建图。
图3是重组转移质粒的鉴定(a和b分别为Polyhedrin启动子和p10启动子下重组转移质粒鉴定：M：DL2000 DNA marker；1:pFBD-Cap-T；2：pFBD-Cap-TB2；3：pFBD-Cap-B1T)。
图4是重组杆状病毒质粒PCR鉴定方法图示。
图5是重组杆状病毒质粒的鉴定(M：DL5000 DNA marker；1：rBac-Cap-T；2：rBac-Cap-TB2；3：rBac-Cap-B1T)。
图6是重组杆状病毒的包装(a：正常sf9细胞；b、c、d：分别为转染的rBac-Cap-T、rBac-Cap-TB2、rBac-Cap-B1T的sf9细胞)。
图7是标准品质粒pMD-19-T-Ac的双酶切鉴定(M：DL5000 DNA marker；1：质粒pMD-19-T-Ac BamH I/Pst I酶切产物)。
图8是重组蛋白间接免疫荧光检测(a、b、c：白光下Ac-Cap-T、Ac-Cap-TB2、Ac-Cap-B1T组；d：白光下阴性对照组；e、f、g：荧光下Ac-Cap-T、Ac-Cap-TB2、Ac-Cap-B1T组；h：荧光下阴性对照组)。
图9是重组蛋白的Western Blot检测(1：感染Ac-Cap-T的sf9细胞；2：感染Ac-Cap-TB2的sf9细胞；3：感染Ac-Cap-B1T的sf9细胞；4：阴性对照)。
图10是不同表达条件下重组蛋白表达量的趋势(a、b、c分别代表重组蛋白Cap-T、Cap-TB2和Cap-B1T的表达时相分析；1、5、9：感染细胞后48h蛋白表达；2、6、10：感染细胞后72h蛋白表达；3、7、11：感染细胞后96h蛋白表达；4、8、12：感染细胞后120h蛋白表达)。
图11是电镜观察到的VLPs(92000×；标尺：100nm)(a、b、c分别为Cap-T-VLPs；Cap-TB2-VLPs；Cap-B1T-VLPs)。
图12是小鼠血清中PCV2特异性抗体ELISA检测结果(*代表每组间与商品化疫苗组比具有显著差异(p＜0.05)，其中*代表p＜0.05，**代表p＜0.01，***代表p＜0.001，****代表p＜0.0001；ns代表每组间与商品化疫苗组比无显著差异(p＞0.05))。
图13是小鼠血清中FMDV特异性抗体ELISA检测结果(*代表每组间与商品化疫苗组比具有显著差异(p＜0.05)，其中*代表p＜0.05，**代表p＜0.01，***代表p＜0.001，****代表p＜0.0001；ns代表每组间与商品化疫苗组比无显著差异(p＞0.05))。
图14是小鼠脾脏中T淋巴细胞亚群变化(*代表每组间与PBS组比具有显著差异(p＜0.05)，其中*代表p＜0.05，**代表p＜0.01；ns代表每组间与PBS组比无显著差异(p＞0.05))。
图15是小鼠血清中IL-2、IL-4和IFN-γ检测情况(*代表每组间与PBS组比具有显著差异(p＜0.05)，其中*代表p＜0.05，**代表p＜0.01；***代表p＜0.001；****代表p＜0.0001；ns代表每组间与PBS组比无显著差异(p＞0.05))。
具体实施方式
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。本发明实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，均为可从商业途径得到的试剂和材料。
实施例中，表达载体pFastBacTM Dual、大肠杆菌感受态细胞DH10Bac、DH5a均由华南农业大学兽医学院微生物学与免疫学教研室保存；猪圆环病毒2型杆状病毒载体灭活疫苗(Ingelvac CircoFLEX)为勃林格殷格翰公司产品；猪O型口蹄疫灭活疫苗Re-O/MYA98/JSCZ/2013株为金宇保灵生物药品有限公司产品；猪口蹄疫阳性血清和猪PCV2阳性血清由广东永顺生物制药股份有限公司馈赠。
实施例1重组蛋白基因的设计与合成
根据GenBank中登录的猪O型FMDV O/BY/CHA/2010株(GenBank number：JN998085.1)中选取FMDV VP1蛋白中免疫原性好的一个T细胞优势表位(16～44aa)和两个B细胞优势表位(129～160aa、200～213aa)，所述T细胞表位第16～44位氨基酸序列如SEQ IDNo.8所示，所述B细胞表位第129～160位氨基酸序列如SEQ ID No.9所示，所述B细胞表位第200～213位氨基酸序列如SEQ ID No.10所示。
根据GenBank上公布的PCV2d亚型BHD株全基因序列(GenBank number：HM038017)，其核苷酸序列如SEQ ID No.1所示。
于PCV2d Cap蛋白C端引入单个表位或者不同抗原表位组合的FMDV VP1优势抗原表位，以“GGGGS”为连接子，将T细胞表位(16～44aa)连接到猪圆环病毒2d型Cap基因中，在终止密码子前设计编码6个组氨酸(His标签)碱基序列，得到一个重组核苷酸序列Cap-T，一种密码子优化核苷酸序列如SEQ ID No.2所示。以“GGGGS”为连接子，串联T细胞表位(16～44aa)与B细胞表位(200～213aa)，串联基因命名TB2，后再以“GGGGS”为连接子，将串联基因TB2连接到猪圆环病毒2d型Cap基因中，在终止密码子前设计编码6个组氨酸(His标签)碱基序列，得到一个重组核苷酸序列Cap-TB2，一种密码子优化核苷酸序列如SEQ ID No.3所示。以“GGGGS”为连接子，串联B细胞表位(129～160aa)与T细胞表位(16～44aa)，串联基因命名B1T，后再以“GGGGS”为连接子，将串联基因B1T连接到猪圆环病毒2d型Cap基因中，在终止密码子前设计编码6个组氨酸(His标签)碱基序列，得到一个重组核苷酸序列Cap-B1T，一种密码子优化核苷酸序列如SEQ ID No.4所示。
密码子优化的重组蛋白Cap-T，其氨基酸序列如SEQ ID No.5所示。密码子优化的重组蛋白Cap-TB2，其氨基酸序列如SEQ ID No.6所示。密码子优化的重组蛋白Cap-TB2，其氨基酸序列如SEQ ID No.7所示。
实施例2重组蛋白亚单位结构及3D模型分析
为了保证设计的重组蛋白能形成VLPs,本说明运用SWISS-MODEL(http://SWISS-Model.expasy.org/)和Modeller(https://salilab.org/modeller/download.insta-llation.html)生成五种重组蛋白的同源性模型。使用PyMoL软件显示了五种重组蛋白的亚单位和3D结构(http//www.pymol.org/)。结果如图1所示。
实施例3重组杆状病毒转移载体的构建
(1)转移质粒载体pFastBacTM Dual是双启动子表达载体，在P10、PH启动子下可以分别表达两个外源基因，本发明的构建图谱如图2所示。上述设计好的三个重组基因经上海生工生物股份有限公司合成后，会克隆入pUC-19中，获得重组质粒pUC-19-Cap-T；pUC-19-Cap-TB2；pUC-19-B1T。之后使用Primer5设计三对特异性引物(表1)，在PH-Cap-T、PH-Cap-TB2和PH-Cap-B1T中引入EcoR I和Not I酶切位点，在P10-Cap-T、P10-Cap-TB2和P10-Cap-B1T中引入Xho I和Sph I酶切位点。以合成的重组质粒为模板，通过上述引物扩增Cap-T、Cap-TB2、Cap-B1T。PCR体系如表2所示。配好的PCR体系在PCR仪按照以下条件扩增：98℃预变性3min，98℃变性10s，55℃退火20s，72℃延伸30s，共30个循环；72℃延伸5min。反应结束后，加5μL10×loadingbuffer，以110V 30min，进行1％核酸琼脂电泳。
表1引物序列
表2 PCR反应体系
(2)将双酶切后的目的片段和pFastBac TM Dual载体进行连接，并将连接产物转化DH5α感受态细胞，以LB(AMP)固体培养基筛选。挑取阳性克隆进行菌落PCR鉴定，上下游鉴定引物为pFBD1-F/R，并扩增阳性克隆，按Omga质粒提取试剂盒说明步骤提取质粒，并酶切鉴定。最终分别获得重组杆状病毒转移载体pFBD-Cap-T-1、pFBD-Cap-TB2-1、pFBD-Cap-B1T-1。
(3)将步骤(1)回收的带Xho I和Sph I酶切位点的目的片段和重组杆状病毒转移载体pFBD-Cap-T-1、pFBD-Cap-TB2-1、pFBD-Cap-B1T-1分别用Xho I和Sph I限制性核酸内切酶进行双酶切，胶回收后连接、转化、鉴定(鉴定引物为pFBD1-F/R和pFBD2-F/R，具体方法参见步骤(2))，分别获得重组杆状病毒转移载体pFBD-Cap-T-2、pFBD-Cap-TB2-2、pFBD-Cap-B1T-2，于-20℃保存。用于扩增不同基因的引物序列及检测引物信息如表3。
转移载体pFastBac Dual中包含pFBD-2F/R引物结合位点，位于p10启动子下多克隆位点的两侧，重组质粒PCR扩增产物长度应为插入序列总长度+144bp，即重组杆状病毒转移载体pFBD-Cap-T-2PCR扩增产物长度应为822+144＝966bp；重组杆状病毒转移载体pFBD-Cap-TB2-2 PCR扩增产物长度应为870+144＝1014bp；重组杆状病毒转移载体pFBD-Cap-B1T-2PCR扩增产物长度应为924+144＝1068bp。
转移载体pFastBac Dual中包含pFBD-1F/R引物结合位点，位于多角体启动子(PH启动子)下多克隆位点的两侧，可用于重组转移质粒的PCR鉴定，重组质粒PCR扩增产物长度应为插入序列总长度+173bp,即重组杆状病毒转移载体pFBD-Cap-T-1PCR扩增产物长度应为822+173＝995bp；重组杆状病毒转移载体pFBD-Cap-TB2-1 PCR扩增产物长度应为870+173＝1043bp；重组杆状病毒转移载体pFBD-Cap-B1T-1PCR扩增产物长度应为924+173＝1097bp。如图3所示，电泳结果显示扩增产物长度与预期相符。
表3用于扩增不同基因的引物序列及检测引物
(4)对重组转移质粒进行双酶切鉴定，pFBD-Cap-T-2、pFBD-Cap-TB2-2、pFBD-Cap-B1T-2使用限制性内切酶EcoR I/Not I和Xho I/Sph I分别进行双酶切鉴定，酶切产物进行1％的琼脂糖凝胶电泳。
实施例4重组杆状病毒质粒的构建
将鉴定正确的重组杆状病毒转移载体pFBD-Cap-T-2、pFBD-Cap-TB2-2、pFBD-Cap-B1T-2分别转化到DH10Bac感受态细胞中。具体实施方式：①将10μL的重组转移质粒加到DH10Bac感受态细胞，冰上放置30min；②从冰上取出，放置于42℃水浴中热激90s后迅速放在冰上孵育2min；③在超净台中向EP管中加900μL SOC培养基，于37℃细菌培养摇床200r/min振荡培养4h；④在超净台中用SOC培养基对转化菌进行10-1、10-2、10-3梯度稀释后，分别吸取100μL上述稀释梯度的转化菌涂布于含7μg/mL Gen，50μg/mL Kan，10μg/mL Tet，40μg/mLIPTG和100μg/mLX-Gal LB固体培养基上；⑤将平板倒置于37℃细菌培养箱中孵育48h，之后挑选蓝白斑，其中白色菌落即为目标菌落；⑥挑取白色菌落后重新在含7μg/mL Gen，50μg/mL Kan，10μg/mL Tet，40μg/mL IPTG和100μg/mL X-Gal LB平板上进行三区划线纯化，将划好线的平板放置37℃细菌培养箱中培养48h。
该实验原理是借由质粒上的Tn5转座单位和细胞内辅助质粒的功能将目的基因转座到杆状病毒载体Bacmid上(图4)，由于重组后的杆状病毒质粒分子量较大，不能使用常规的酶切鉴定检验外源基因片段是否插入成功，所以使用M13-F和M13-R通用引物(M13F：CCCAGTCACGACGACGTTGTAAAACG，M13R：AGCGGATAACAATTTCACACAGG)对重组杆粒进行PCR鉴定，结果如图5所示。将鉴定正确的重组杆状病毒质粒命名rBac-Cap-T、rBac-Cap-TB2、rBac-Cap-B1T。
实施例5重组杆状病毒的获得
(1)转染细胞病变
利用阳离子脂质体cellfectin II介导转染，将重组杆状病毒质粒rBac-Cap-T、rBac-Cap-TB2、rBac-Cap-B1T分别转染sf9单层细胞(购自武汉大学中国典型物保藏中心)，于27℃培养箱中孵育3～5h；转染完成后，去除细胞孔中的上清，并补充2mL sf900 III培养基，27℃培养箱中静置培养72h以上；
转染后，每隔24h对转染后细胞观察一次，当转染细胞开始出现了典型的细胞病变特征，如细胞停止增长、细胞边缘粗糙、胞内出现大量囊泡等特征(图6)时，表明可能成功拯救出重组杆状病毒。继续培养可观察到细胞开始大量脱壁并裂解破碎(约转染后4～6d)，收获细胞培养物，室温1000rpm离心5min，上清即为重组杆状病毒(P1代)，于收获的病毒液中加入终浓度为2％的FBS，-80℃保存备用。将重组杆粒分别转染后获得的重组杆状病毒分别命名为Ac-Cap-T、Ac-Cap-TB2、Ac-Cap-B1T。由于P1代重组杆状病毒的病毒滴度较低，需要对其进行传代及扩增，以增加病毒滴度，参照Bac-to-Bac Baculoviruse Expression System操作说明书，分别获得第二代重组杆状病毒(P2代)和第三代重组杆状病毒(P3代)。
(2)重组杆状病毒滴度测定
运用Primer 5软件，以杆状病毒DNA为模板，设计一对引物F/R用于实时荧光定量PCR，设计一对引物Ac-F/R用于扩增杆状病毒DNA部分AcMK107序列，引物Ac-F/R扩增区域包含引物F/R扩增片段。引物序列见表4。
表4引物序列
以杆状病毒DNA为模板，Ac-F/Ac-R为引物，扩增目的基因经1％的琼脂糖凝胶电泳，回收纯化目的片段。参照pMD18-T载体的说明书，将胶回收纯化的目的片段和pMD18-T载体进行16℃连接12～16h。反应体系如表5所示：
表5连接反应体系
将连接产物转化DH5α大肠杆菌感受态细胞，将阳性单菌落接种于5mL含Amp的LB液体培养基，放置37℃恒温摇床按200r/min振荡培养16h，按OMEGA质粒提取试剂盒步骤提取质粒，并以BamH I/PstΙ对质粒进行双酶切鉴定(图7)，将鉴定确的质粒送至北京擎科生物科技有限公司进行测序。将测序正确的质粒命名为pMD18T-Ac，于-20℃保存备用。用微量紫外分光光度计测量所提质粒浓度，按照下面公式计算标准质粒DNA拷贝数。
(3)绝对荧光定量PCR
将构建好的标准品质粒进行梯度稀释，稀释为108、107、106、105、104copies/μL，把稀释好的不同梯度的标准品质粒作为模板，每个浓度做三个重复，在绝对荧光定量PCR中以标准品质粒跑出来的曲线作为标准曲线；按照Viral DNA Kit抽提试剂盒提取重组杆状病毒DNA，做好相应标记后，每一个待测的重组杆状病毒做两个梯度稀释，即稀释10倍和100倍，并做三个重复进行检测，本实验以Elution Buffer作为阴性对照，做三个复孔保证实验的一致性。荧光定量PCR按照上海近岸科技有限公司的SYBR qPCR SuperMixPlus产品说明书进行操作，荧光定量PCR反应条件：95℃预变性1min；95℃变性20s，60℃延伸1min，共40个循环反应；60℃采集荧光信号。反应体系如表6所示。
表6荧光定量PCR反应体系
根据荧光定量PCR结果绘制标准曲线，计算重组病毒DNA拷贝数，并按以下公式计算病毒滴度：
重组杆状病毒Ac-Cap-T、Ac-Cap-TB2和Ac-Cap-B1T经连续两代扩增，获得P3代重组杆状病毒各50mL。通过步骤(3)的方法测量重组杆状病毒的滴度，结果显示重组杆状病毒Ac-Cap-T、Ac-Cap-TB2、Ac-Cap-B1T拷贝数分别为1.37×107copies/μL、1.30×107copies/μL和1.03×107copies/μL，计算病毒滴度分别为为2.59×108pfu/mL、2.45×108pfu/mL和1.94×108pfu/mL。
实施例6重组蛋白的鉴定
(1)间接免疫荧光鉴定外源基因的表达
将重组杆状病毒Ac-Cap-T、Ac-Cap-TB2和Ac-Cap-B1T按M.O.I＝1感染单层Sf9细胞，27℃培养72h细胞，72h后进行间接免疫荧光检测多表位重组RBT基因的表达。以His单克隆抗体(购自碧云天公司)为一抗，FITC标记羊抗鼠IgG(购自碧云天公司)为二抗。荧光显微镜下观察，感染Ac-Cap-T、Ac-Cap-TB2、Ac-Cap-B1T的sf9细胞均能检测到特异性荧光，而阴性对照细胞则没有荧光，表明目的蛋白成功在细胞内表达，结果如图8所示。
(2)Western-blot检测重组蛋白表达
将Ac-Cap-T、Ac-Cap-TB2和Ac-Cap-B1T分别以MOI＝1的剂量感染sf9细胞，以野生型杆状病毒作为阴性对照。采用超声破碎裂解细胞，加入相应体积的5×SDS-PAGE上样缓冲液，通过金属浴100℃10min变性蛋白。按照SDS-PAGE凝胶快速配置试剂盒说明书配制SDS-PAGE凝胶，将上述处理后的样品经SDS-PAGE凝胶电泳后，转印PVDF膜后分别以鼠源His标签抗体、猪源FMDV阳性血清和猪源PCV2阳性血清为一抗进行Western blot鉴定。
结果如图9所示，感染Ac-Cap-T重组病毒的细胞样品在32kDa处有特异性条带；感染Ac-Cap-TB2重组病毒的细胞样品在35kDa处有特异性条带；感染Ac-Cap-B1T重组病毒的细胞样品在37kDa处有特异性条带。从图中可见在目的条带附近有多余的杂带，其原因推测为SDS使得多聚体蛋白解聚，而实际测出来的相对分子质量，只是代表寡聚体蛋白的亚基分子质量。综上，以上条带均符合预期大小，而野生杆状病毒并无特异性条带，证明重组病毒构建成功且能正确表达目的蛋白。
实施例7重组蛋白表达条件优化
为了提高表达效率，本实验使用High Five悬浮细胞表达重组蛋白。摸索重组目的蛋白的最优表达条件：从不同收获时间、不同接毒剂量两个方面探究重组目的蛋白的最优表达条件。具体实施方法为：①将悬浮High Five细胞按1×106cells/mL的浓度接种于20mLHigh Five-SFM培养基中，以140r/min在27℃的摇床中悬浮培养至细胞浓度为2×106cells/mL；②将重组杆状病毒Ac-Cap-T、Ac-Cap-TB2、Ac-Cap-B1T分别以MOI为0.1、1.0和10.0接种High Five细胞，以140r/min在27℃的摇床中悬浮培养。分别于接种重组杆状病毒后的第48h、72h、96h和120h取80μL细胞培养悬液做检测用；③样品制备：在每份样品中加入20μl电泳缓冲液，在恒温金属浴中以100℃煮沸10min。样品-20℃保存；④按照SDS-PAGE凝胶快速配置试剂盒说明书配制SDS-PAGE凝胶，将上述处理后的样品经SDS-PAGE凝胶电泳后，转印PVDF膜后分别以鼠源His标签抗体为一抗，对不同收获时间、不同接毒剂量收获的变性处理的蛋白样品通过Western Blot检测重组蛋白的表达水平，通过ImageJ软件分析灰度值，根据灰度值绘制目的蛋白表达量的变化趋势。
结果如图10所示，在细胞裂解液中，第48h后即可检测到三种重组蛋白的表达，三种重组蛋白在三个感染剂量中，都是随着时间的增多先上升后下降，考虑到实际生产过程中的成本问题，本实验未尝试使用更高感染剂量。考虑到实际生产中的成本问题，本发明在实际生产中推荐按MOI＝1.0的感染剂量，于感染后72h收获重组蛋白样品。
实施例8重组病毒样颗粒的电镜观察
(1)收获重组蛋白
按照实施例7推荐的表达条件将P3代Ac-Cap-T、Ac-Cap-TB2和Ac-Cap-B1T按MOI＝1分别接种于浓度为2×106cells/mL的体积为50mL的悬浮High Five细胞中，于感染72h时收集表达重组蛋白；
超声破碎处理细胞：按照重组蛋白收获时间，收集细胞以1000r/min室温离心10min，弃去上清，按照M细胞/VPBS＝1：5比例加入PH＝7.4的PBS，涡旋混匀后，放置冰上进行超声波破碎。超声条件为超声5s，停止9s，超声功率采用40％。
超声结束后，以11000r/min 4℃离心15min，将上清收集到新的离心管中并做好相应标记。
(2)浓缩重组蛋白
使用Millipore超滤管对收集的细胞裂解上清进行离心浓缩，离心条件为4℃，4000r/min离心60min。
(3)蔗糖梯度超速离心
①在12.5mL的超速管中依次加入2mL 80％、50％、30％的蔗糖溶液。
②加入步骤(2)的浓缩样品，体积不足用PBS补足。
③用电子天平将超速管进行严格配平后，在超速离心机使用SW41转子，按照超速离心机的使用说明严格操作，以30000r/min，4℃离心3.5h。
④超速离心结束后，用1mL注射器吸取不同密度梯度的蛋白环进行Western Blot检测重组蛋白，结果发现在各蛋白环都能检测到重组蛋白。
⑤采用Thermo Scientific PierceTMBCA Protein Assay Kit试剂盒测量不同密度梯度的蛋白环的蛋白浓度，发现在30％～50％这一条带中浓度最高。
(4)TEM透射电镜检测
用0.22μm滤器对收集的30％～50％蛋白环溶液过滤后，取10μL用PBS稀释100倍后，用1％醋酸双氧铀负染样品，在TEM透射电镜观察重组病毒粒子组装情况，结果如图11所示，重组VLPs大小在20～30nm左右，大小形态略有不同，颗粒表面呈规则球形状，其形态、大小与文献报道中Cap形成的VLPs相似，本研究表明嵌合猪O型FMDV的B、T细胞表位不影响Cap在High Five细胞中的装配。
应用实验例重组VLPs制备的亚单位疫苗的免疫效力测试
(1)疫苗的制备
将实施例8中收集的重组病毒粒子用PBS将浓度调整到每毫升50μg，并将其分别与ISA 201VG佐剂按照1：1比例进行乳化，相关研究指出核酸佐剂作为免疫刺激分子，可更好的提高疫苗的保护效力。本发明设计VLPs疫苗+核酸佐剂NAA免疫组来验证NAA的加入是否会增强VLPs疫苗的免疫原性，随后进行理化性能的检测。
(2)疫苗质量检测
本次实验制备的疫苗外观为乳白色液体，用注射器吸取少量乳液滴于水中，第一滴会迅速散开，第二滴不会扩散，取疫苗1mL到1.5mL EP管中，以3000r/min，4℃离心15min后，疫苗分层但是不破乳。将制备好的疫苗接种BALB/c小鼠后，在免疫后28d中小鼠全部健活，每组小鼠精神状况良好，免疫部位没有发炎现象。
(3)重组VLPs免疫效力检测
①BALB/c雌鼠鼠分组情况
将45只4周龄的SPF级BALB/c雌鼠随机分成9组，每组5头，采用皮下多点注射相应疫苗，各组免疫时间、剂量和注射试剂见表7。
表7实验动物分组
所有小鼠共免疫2次，每次免疫间隔两周，免疫方式为皮下注射。在首免后第7d、14d、21d和28d眶下采血收集每组小鼠血液，收集的血液量为每只500μL，刚收集的血液做好标记后，先放37℃培养箱中1h，接着在4℃冰箱中过夜斜放收集的小鼠血清，次日将析出的血清分装，做好标记后放-20℃保存，用于ELISA抗体检测。同时观察免疫组小鼠存活情况，接种部位有无异常等以进行亚单位疫苗安全性检查。
②ELISA抗体检测
用FMDV ELISA Antibody Test Kit和PCV2 ELISA Antibody Test Kit(均来自深圳芬德生物技术有限公司)检测待检血清抗体水平。
根据1：10倍稀释待检BALB/c小鼠血清，将HRP标记的羊抗鼠IgG按照1：250的稀释比进行稀释，参照试剂盒说明书检测免疫后各组小鼠血清中PCV2抗体水平。结果见图12所示，在首免7d、14d里，VLPs组较商品化组更早产生抗体，在二免后，除PBS组，其余各实验组小鼠血清中特异性抗体均出现不同程度的提升，其中二免后7d VLPs组特异性抗体水平显著高于商品化疫苗组(p＜0.05)；但在14d中商品化疫苗组产生的特异性抗体水平与VLPs组无显著差异(p＞0.05)。由此推测VLPs疫苗组能在免疫后较短时间诱导较高水平的体液免疫应答。综上，本实验以PCV2d Cap为框架组装的含FMDV抗原表位的VLPs能诱导BALB/c小鼠产生PCV2特异性抗体。
根据1：1000倍稀释待检BALB/c小鼠血清，将HRP标记的羊抗鼠IgG按照1：250的稀释比进行稀释，参照试剂盒说明书检测免疫后各组小鼠血清中FMDV抗体水平，结果如图13所示：首免后28d，FMDV特异性抗体水平达到最高水平，与商品化疫苗组相比，各组间差异显著(p＜0.05)，对照组血清无特异性抗体。
综上，本发明的三个重组VLPs免疫小鼠后，能产生PCV2和FMDV两种特异性抗体，但加入核酸佐剂组没有取得预期效果，查阅相关文献后，其原因可能是是NAA序列是参考对畜禽有免疫增强作用序列而设计的，故对BALB/c小鼠无明显的免疫增强作用，但在猪体上实验是有良好的增强作用的。
③小鼠脾脏T淋巴细胞的流式细胞仪检测
在二免后14d，取免疫组和空白对照组小鼠脾脏淋巴细胞进行CD3+CD4+、CD3+CD8+T细胞水平分析。结果如图14所示，在首次免疫28d后，实验组小鼠的CD3+CD4+和CD3+CD8+T淋巴细胞百分率与PBS组相比有升高趋势，三个重组VLPs中，Cap-B1T-VLPs的CD3+CD4+和CD3+CD8+T淋巴细胞百分率最高。
④细胞因子检测
在二免后14d采集各个实验组小鼠血清，采用按照江苏酶免ELISA试剂盒说明书对小鼠血清中Th1型(IFN-γ和IL-2)和Th2型(IL-4)细胞因子浓度进行检测。结果由图15所示，各免疫组小鼠血清中IFN-γ、IL-2和IL-4分泌水平均高于PBS组。其中，Cap-B1T-VLPs组IFN-γ、IL-2和IL-4分泌水平高于Cap-T-VLPs组和Cap-TB2-VLPs组，表明VLPs作为免疫原能刺激Th1、Th2型细胞因子产生。
综上，本发明重组VLPs均能产生体液免疫和细胞免疫，Cap-B1T-VLPs综合产生的体液免疫和细胞免疫效果优于其他两个VLPs，其原因可能与选取的抗原表位肽段有关。
本发明的上述实施例仅仅是为了清楚地说明本发明技术方案的所作的举例，而并非是对本发明的具体实施方式的限定。凡在本发明权利要求书的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。
序列表
<110> 华南农业大学
<120> 猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的VLPs及其制备方法与应用
<130> ZM221111CS
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 705
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atgacgtatc caaggaggcg tttccgcaga cgaagacacc gcccccgcag ccatcttggc 60
cagatcctcc gccgccgccc ctggctcgtc cacccccgcc accgttaccg ctggagaagg 120
aaaaatggga tcttcaacac ccgcctctcc cgcaccatcg gttatactgt caagaaaacc 180
acagtcagaa cgccctcctg gaatgtggac atgatgagat ttaatattaa tgattttctt 240
cccccaggag ggggctcaaa ccccctcact gtgccctttg aatactacag aataaggaag 300
gttaaggttg aattctggcc ctgctcccca atcacccagg gtgacagggg agtgggctcc 360
actgctgtta ttctagatga taactttgta acaaaggcca atgccctaac ctatgacccc 420
tatgtaaact actcctcccg ccataccata acccagccct tctcctacca ctcccggtac 480
tttaccccga aacctgtcct tgataggaca atcgattact tccaacccaa taacaaaaga 540
aatcaactct ggctgagact acaaactact ggaaatgtag accatgtagg cctcggcact 600
gcgttcgaaa acagtatcta cgaccaggac tacaatatcc gtataaccat gtatgtacaa 660
ttcagggaat ttaatcttaa agacccccca cttaacccta agtga 705
<210> 2
<211> 822
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
atgacctacc cccgccgccg tttccgccgc agacgtcaca ggccccgcag ccacctcggc 60
caaatcctgc gccgccgccc atggctggtg caccctagac accgctaccg ctggcgccgc 120
aagaacggta tcttcaacac ccgcctgtcc cgcaccatcg gttacaccgt caagaagacc 180
accgtgagaa ccccctcctg gaacgtcgac atgatgcgct tcaacatcaa cgacttcctg 240
ccccctggtg gtggctccaa ccctctgacc gtgcccttcg agtactaccg catccgcaag 300
gtgaaggtgg aattttggcc ctgtagtcct atcacccagg gagaccgcgg tgtgggttcc 360
actgctgtga tcctggacga caacttcgtg accaaggcta acgctctgac ctacgaccct 420
tacgtgaact actcctcccg ccacaccatc acccaacctt tctcctacca ctcccgctac 480
ttcaccccca agcctgtgct ggaccgcacc atcgactact tccaacccaa caacaagcgc 540
aaccaactct ggctccgcct ccaaaccacc ggcaacgtcg accacgtcgg tctcggcacc 600
gccttcgaaa actccatcta cgaccaagac tacaacatcc gcatcaccat gtacgtgcaa 660
ttccgcgagt tcaacctcaa ggacccccct ctgaacccta agggtggtgg tggttccgag 720
aactacggcg gcgagaccca ggtgcagcgc cgtcaccaca ccgacgtctc cttcatcctc 780
gacaggttcg tgaaggtgac ccctcaccac caccaccacc ac 822
<210> 3
<211> 870
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
atgacctacc cccgccgccg tttccgccgc agacgtcaca ggccccgcag ccacctcggc 60
caaatcctgc gccgccgccc atggctggtg caccctagac accgctaccg ctggcgccgc 120
aagaacggta tcttcaacac ccgcctgtcc cgcaccatcg gttacaccgt caagaagacc 180
accgtgagaa ccccctcctg gaacgtcgac atgatgcgct tcaacatcaa cgacttcctg 240
ccccctggtg gtggctccaa ccctctgacc gtgcccttcg agtactaccg catccgcaag 300
gtgaaggtgg aattttggcc ctgtagtcct atcacccagg gagaccgcgg tgtgggttcc 360
actgctgtga tcctggacga caacttcgtg accaaggcta acgctctgac ctacgaccct 420
tacgtgaact actcctcccg ccacaccatc acccaacctt tctcctacca ctcccgctac 480
ttcaccccca agcctgtgct ggaccgcacc atcgactact tccaacccaa caacaagcgc 540
aaccaactct ggctccgcct ccaaaccacc ggcaacgtcg accacgtcgg tctcggcacc 600
gccttcgaaa actccatcta cgaccaagac tacaacatcc gcatcaccat gtacgtgcaa 660
ttccgcgagt tcaacctcaa ggacccccct ctgaacccta agggtggtgg tggttccgag 720
aactacggcg gcgagaccca ggtgcagcgc cgtcaccaca ccgacgtctc cttcatcctc 780
gacaggttcg tgaaggtgac ccctggtggt cgccacaagc agaagatcgt ggcccctgtg 840
aagcagtctt tgcaccacca ccaccaccac 870
<210> 4
<211> 924
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
atgacctacc cccgccgccg tttccgccgc agacgtcaca ggccccgcag ccacctcggc 60
caaatcctgc gccgccgccc atggctggtg caccctagac accgctaccg ctggcgccgc 120
aagaacggta tcttcaacac ccgcctgtcc cgcaccatcg gttacaccgt caagaagacc 180
accgtgagaa ccccctcctg gaacgtcgac atgatgcgct tcaacatcaa cgacttcctg 240
ccccctggtg gtggctccaa ccctctgacc gtgcccttcg agtactaccg catccgcaag 300
gtgaaggtgg aattttggcc ctgtagtcct atcacccagg gagaccgcgg tgtgggttcc 360
actgctgtga tcctggacga caacttcgtg accaaggcta acgctctgac ctacgaccct 420
tacgtgaact actcctcccg ccacaccatc acccaacctt tctcctacca ctcccgctac 480
ttcaccccca agcctgtgct ggaccgcacc atcgactact tccaacccaa caacaagcgc 540
aaccaactct ggctccgcct ccaaaccacc ggcaacgtcg accacgtcgg tctcggcacc 600
gccttcgaaa actccatcta cgaccaagac tacaacatcc gcatcaccat gtacgtgcaa 660
ttccgcgagt tcaacctcaa ggacccccct ctgaacccta agggtggtgg tggttccgtg 720
tacaacggta actgcaagta cgctggaggc agcctgccta acgtgagggg cgatctccag 780
gtgctggctc aaaaggctgc tcgccctctg cccggtggag agaactacgg tggtgagacc 840
caagtgcaac gtcgccacca cactgacgtg tccttcatcc tggaccgttt cgtgaaggtg 900
acccctcacc accaccacca ccac 924
<210> 5
<211> 274
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 5
Met Thr Tyr Pro Arg Arg Arg Phe Arg Arg Arg Arg His Arg Pro Arg
1 5 10 15
Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro
20 25 30
Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg
35 40 45
Leu Ser Arg Thr Ile Gly Tyr Thr Val Lys Lys Thr Thr Val Arg Thr
50 55 60
Pro Ser Trp Asn Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu
65 70 75 80
Pro Pro Gly Gly Gly Ser Asn Pro Leu Thr Val Pro Phe Glu Tyr Tyr
85 90 95
Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr
100 105 110
Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn
115 120 125
Phe Val Thr Lys Ala Asn Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr
130 135 140
Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr
145 150 155 160
Phe Thr Pro Lys Pro Val Leu Asp Arg Thr Ile Asp Tyr Phe Gln Pro
165 170 175
Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Thr Gly Asn
180 185 190
Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp
195 200 205
Gln Asp Tyr Asn Ile Arg Ile Thr Met Tyr Val Gln Phe Arg Glu Phe
210 215 220
Asn Leu Lys Asp Pro Pro Leu Asn Pro Lys Gly Gly Gly Gly Ser Glu
225 230 235 240
Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp Val
245 250 255
Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro His His His His
260 265 270
His His
<210> 6
<211> 290
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
Met Thr Tyr Pro Arg Arg Arg Phe Arg Arg Arg Arg His Arg Pro Arg
1 5 10 15
Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro
20 25 30
Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg
35 40 45
Leu Ser Arg Thr Ile Gly Tyr Thr Val Lys Lys Thr Thr Val Arg Thr
50 55 60
Pro Ser Trp Asn Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu
65 70 75 80
Pro Pro Gly Gly Gly Ser Asn Pro Leu Thr Val Pro Phe Glu Tyr Tyr
85 90 95
Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr
100 105 110
Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn
115 120 125
Phe Val Thr Lys Ala Asn Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr
130 135 140
Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr
145 150 155 160
Phe Thr Pro Lys Pro Val Leu Asp Arg Thr Ile Asp Tyr Phe Gln Pro
165 170 175
Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Thr Gly Asn
180 185 190
Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp
195 200 205
Gln Asp Tyr Asn Ile Arg Ile Thr Met Tyr Val Gln Phe Arg Glu Phe
210 215 220
Asn Leu Lys Asp Pro Pro Leu Asn Pro Lys Gly Gly Gly Gly Ser Glu
225 230 235 240
Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp Val
245 250 255
Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro Gly Gly Arg His
260 265 270
Lys Gln Lys Ile Val Ala Pro Val Lys Gln Ser Leu His His His His
275 280 285
His His
290
<210> 7
<211> 308
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 7
Met Thr Tyr Pro Arg Arg Arg Phe Arg Arg Arg Arg His Arg Pro Arg
1 5 10 15
Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro
20 25 30
Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg
35 40 45
Leu Ser Arg Thr Ile Gly Tyr Thr Val Lys Lys Thr Thr Val Arg Thr
50 55 60
Pro Ser Trp Asn Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu
65 70 75 80
Pro Pro Gly Gly Gly Ser Asn Pro Leu Thr Val Pro Phe Glu Tyr Tyr
85 90 95
Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr
100 105 110
Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn
115 120 125
Phe Val Thr Lys Ala Asn Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr
130 135 140
Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr
145 150 155 160
Phe Thr Pro Lys Pro Val Leu Asp Arg Thr Ile Asp Tyr Phe Gln Pro
165 170 175
Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Thr Gly Asn
180 185 190
Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp
195 200 205
Gln Asp Tyr Asn Ile Arg Ile Thr Met Tyr Val Gln Phe Arg Glu Phe
210 215 220
Asn Leu Lys Asp Pro Pro Leu Asn Pro Lys Gly Gly Gly Gly Ser Val
225 230 235 240
Tyr Asn Gly Asn Cys Lys Tyr Ala Gly Gly Ser Leu Pro Asn Val Arg
245 250 255
Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu Pro Gly
260 265 270
Gly Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr
275 280 285
Asp Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro His His
290 295 300
His His His His
305
<210> 8
<211> 29
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 8
Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp
1 5 10 15
Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro
20 25
<210> 9
<211> 32
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 9
Val Tyr Asn Gly Asn Cys Lys Tyr Ala Gly Gly Ser Leu Pro Asn Val
1 5 10 15
Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu Pro
20 25 30
<210> 10
<211> 14
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 10
Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Ser Leu
1 5 10
<210> 11
<211> 45
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 11
Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp
1 5 10 15
Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro Gly Gly Arg
20 25 30
His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Ser Leu
35 40 45
<210> 12
<211> 63
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 12
Val Tyr Asn Gly Asn Cys Lys Tyr Ala Gly Gly Ser Leu Pro Asn Val
1 5 10 15
Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu Pro
20 25 30
Gly Gly Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His
35 40 45
Thr Asp Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro
50 55 60
Claims (3)
1.一种猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的VLPs，其特征在于，由猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的重组蛋白组装而成，所述重组蛋白由猪圆环2d型Cap蛋白嵌合如下猪O型口蹄疫病毒抗原表位组合后得到；
所述猪O型口蹄疫病毒抗原表位为B细胞表位第129～160位氨基酸与T细胞表位第16～44位氨基酸串联，所述重组蛋白的氨基酸序列如SEQ ID No.7所示，其编码基因的核苷酸序列如SEQ ID No.4所示；
所述VLPs采用如下方法制备得到：将所述编码基因克隆至双启动子pFastBacTM Dual表达载体中得到转移载体，将转移载体转化至感受态细胞DH10Bac，获得重组病毒质粒，再转染sf9单层昆虫细胞，培养后获得重组杆状病毒，将重组杆状病毒感染到悬浮培养的High-Five昆虫细胞表达重组蛋白，扩大培养后，收集目的蛋白，采用蔗糖梯度离心纯化，即得所述VLPs。
2.一种PCV2d和FMDV二联亚单位疫苗，其特征在于，其由权利要求1所述的VLPs与免疫佐剂乳化或混合制得。
3.权利要求2所述疫苗在制备用于治疗和预防PCV2d和FMDV感染的药物中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210320567.1A CN114805600B (zh) | 2022-03-29 | 2022-03-29 | 猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的VLPs及其制备方法与应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210320567.1A CN114805600B (zh) | 2022-03-29 | 2022-03-29 | 猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的VLPs及其制备方法与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114805600A CN114805600A (zh) | 2022-07-29 |
| CN114805600B true CN114805600B (zh) | 2024-03-29 |
Family
ID=82530305
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210320567.1A Active CN114805600B (zh) | 2022-03-29 | 2022-03-29 | 猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的VLPs及其制备方法与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114805600B (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501186A (zh) * | 2020-11-26 | 2021-03-16 | 浙江鼎持生物制品有限公司 | 猪圆环病毒2d型CAP蛋白及其在亚单位疫苗制备中的应用 |
-
2022
- 2022-03-29 CN CN202210320567.1A patent/CN114805600B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501186A (zh) * | 2020-11-26 | 2021-03-16 | 浙江鼎持生物制品有限公司 | 猪圆环病毒2d型CAP蛋白及其在亚单位疫苗制备中的应用 |
Non-Patent Citations (2)
| Title |
|---|
| 以猪圆环病毒2型为载体表达口蹄疫病毒VP1抗原表位的研究;张飞雁;CNKI优秀硕士学位论文全文库;摘要 * |
| 口蹄疫表位疫苗的研究进展;李世芳等;中国畜牧兽医;第44卷(第2期);第398-399页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114805600A (zh) | 2022-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112876570B (zh) | 非洲猪瘟病毒疫苗及其制备方法 | |
| CN112076315A (zh) | 新冠病毒s蛋白和铁蛋白亚基融合的纳米抗原颗粒、新冠疫苗及其制备方法和应用 | |
| CN111218459A (zh) | 一种以人复制缺陷腺病毒为载体的重组新型冠状病毒疫苗 | |
| CN108371710B (zh) | 猫传染性鼻结膜炎和猫泛白细胞减少症二联疫苗及制备方法 | |
| US20230285535A1 (en) | Recombinant Pseudorabies Virus and Vaccine Composition thereof | |
| CN113461788B (zh) | 猫冠状病毒重组抗原、其基因工程亚单位疫苗及应用 | |
| WO2023051850A1 (zh) | 一类来源于新型冠状病毒s2蛋白hr区域的重组融合蛋白及其应用 | |
| CN113416236B (zh) | 猪圆环病毒3型病毒样颗粒及其制备方法与应用 | |
| CN107227311B (zh) | 重组猪细小病毒样粒子及其制备方法和应用 | |
| CN110256539B (zh) | O型口蹄疫病毒新型基因工程亚单位疫苗 | |
| CN111825768A (zh) | 基于自组装铁蛋白纳米抗原颗粒及流感疫苗和制备方法 | |
| CN114703204B (zh) | 猫细小病毒vp2蛋白及所得自主组装病毒样颗粒 | |
| CN117143923A (zh) | 一种猪圆环病毒2型和/或3型亚单位疫苗及其建立方法和应用 | |
| CN113862284B (zh) | 一种编码重组禽流感病毒ha蛋白的基因、病毒样颗粒、疫苗及制备与应用 | |
| CN114805599B (zh) | 一种基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的VLPs及应用 | |
| CN112442131B (zh) | 基于自组装铁蛋白纳米抗原颗粒及由其制备的传染性法氏囊病疫苗和应用 | |
| CN112442130B (zh) | 基于自组装铁蛋白纳米抗原颗粒及由其制备的狂犬疫苗和应用 | |
| CN107868131A (zh) | 一种猪细小病毒病亚单位疫苗及其制备方法 | |
| CN116102660B (zh) | 一种猪细小病毒基因工程表位疫苗及其制备方法 | |
| CN111454989A (zh) | 一种嵌合型基因i型乙型脑炎病毒病毒样颗粒疫苗及其制备方法和应用 | |
| CN114805600B (zh) | 猪圆环2d型框架嵌合猪O型口蹄疫病毒抗原表位的VLPs及其制备方法与应用 | |
| CN112439057B (zh) | 基于自组装铁蛋白纳米抗原颗粒及由其制备的猪瘟疫苗和应用 | |
| CN111729078B (zh) | 鸡传染性贫血病毒基因工程疫苗 | |
| CN113827714A (zh) | 一种h7n9亚型禽流感病毒样颗粒疫苗制剂及制备和应用 | |
| CN102643335A (zh) | 重组轮状病毒vp6载体蛋白及其制备 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |