冻干人用狂犬病疫苗(Vero细胞)蛋白结构特性分析
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摘要
目的分析冻干人用狂犬病疫苗(Vero细胞)蛋白的结构特性。方法 SDS-PAGE电泳分离4批冻干人用狂犬病疫苗(Vero细胞)原液,电泳条带经nano LC-MS/MS进行蛋白鉴定,Native-PAGE分析病毒颗粒的完整性,并分析蛋白相对分子质量。同时进行N-末端氨基酸序列分析。结果 SDS-PAGE分析可见5个主条带,分别为狂犬病毒G、M、N、P蛋白及G蛋白二聚体(G-1),经nano LC-MS/MS鉴定,确定相对分子质量分别为74 000、21 885、57 287、40 934及141 067,各结构蛋白均包含在病毒颗粒中,M、N、P、G蛋白的N-末端起始氨基酸序列为分别为MNFLR、MDADR、MSKIF、MVPQA,与预期一致。结论 4批冻干人用狂犬病疫苗(Vero细胞)原液所含结构蛋白源于狂犬病病毒。
Objective To analyze the characteristics of protein structure of freeze-dried rabies vaccine(Vero cells) for human use. Methods Four batches of bulk of freeze-dried rabies vaccine(Vero cells)for human use were separated by SDS-PAGE,and the electrophoretic bands were identified by nano LC-MS/MS,while the integrity of virus particles was analyzed by Native-PAGE,and the relative molecular mass was determined. Meanwhile,the N-terminal amino acid sequence was analyzed. Results Five bands were observed on SDS-PAGE profile,which were identified as the G,M,N,P protein and G protein dimer(G-1)of rabies virus,with relative molecular masses of 74 000,21 885,57 287,40 934 and 141 067,respectively. All the structural proteins were located in the virus particles. The sequences of N-terminal amino acid sequences of M,N,P and G proteins were MNFLR,MDADR,MSKIF and MVPQA respectively,which were consisted with those expected. Conclusion The structural proteins of four batches of bulk of freeze-dried rabies vaccine(Vero cells)for human use were derived from rabies virus.
Objective To analyze the characteristics of protein structure of freeze-dried rabies vaccine(Vero cells) for human use. Methods Four batches of bulk of freeze-dried rabies vaccine(Vero cells)for human use were separated by SDS-PAGE,and the electrophoretic bands were identified by nano LC-MS/MS,while the integrity of virus particles was analyzed by Native-PAGE,and the relative molecular mass was determined. Meanwhile,the N-terminal amino acid sequence was analyzed. Results Five bands were observed on SDS-PAGE profile,which were identified as the G,M,N,P protein and G protein dimer(G-1)of rabies virus,with relative molecular masses of 74 000,21 885,57 287,40 934 and 141 067,respectively. All the structural proteins were located in the virus particles. The sequences of N-terminal amino acid sequences of M,N,P and G proteins were MNFLR,MDADR,MSKIF and MVPQA respectively,which were consisted with those expected. Conclusion The structural proteins of four batches of bulk of freeze-dried rabies vaccine(Vero cells)for human use were derived from rabies virus.
引文
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[12]MASATANI T,ITO N,ITO Y,et al.Importance of rabies virus nucleoprotein in viral evasion of interferon response in the brain[J].Microbiol&Immun,2013,57(7):511-517.
[13]POISSON N,REAL E,GAΜDIN Y,et al.Molecular basis for the interaction between rabies virus phosphoprotein P and the dynein light chain LC8:dissociation of dynein-binding properties and transcriptional functionality of P[J].J General Virol,2001,82(11):2691-2696.
[14]YAMAOKA S,ITO N,OHKA S,et al.Involvement of the rabies virus phosphoprotein gene in neuroinvasiveness[J].J Virol,2013,87(22):12327.
[15]NIΜX,TANG L,TSEGGAI T,et al.Wild-type rabies virus phosphoprotein is associated with viral sensitivity to typeⅠinterferon treatment[J].Arch Virol,2013,158(11):2297-2305.
[16]LUO J Y,MA Z Y.Structure and function of rabies virus G protein[C].Proceedings of the 2009 Annual Conference of the Chinese Society of Animal Husbandry and Veterinary Medicine.Beijing:Chinese Society of Animal Husbandry and Veterinary Medicine,2009.(in Chinese)罗金燕,马志永.狂犬病病毒G蛋白的结构和功能[C].中国畜牧兽医学会2009学术年会论文集(下册).北京:中国畜牧兽医学会,2009.
[17]OSWALD M,GEISSLER S,GOEPFERICH A.Targeting the central nervous system(CNS):a review of rabies virus targeting strategies[J].Mol Pharm,2017,5.PMID:28514853[PubmedPublisher].
[2]MONDIALE DE LA SANTE’O,World Health Organization.Human rabies:2016 updates and call for data[J].Wkly Epidemiol Rec,2017,92(7):77-86.
[3]SPARKES J,MCLEOD S,BALLARD G,et al.Rabies disease dynamics in naive dog populations in Australia[J].Prev Vet Med,2016,131:127-136.
[4]BOURHY H,et al.2016 Revealing the micro-scale signature of endemic zoonotic disease transmission in an African urban setting[J].PLo S Pathog,2016,12(14):e1005525.
[5]BEYER H L,HAMPSON K,LEMBO T,et al.Metapopulation dynamics of rabies and the efficacy of vaccination[J].Proc R Soc B,2011,278(1715):2182-2190.
[6]World Organisation for Animal Health.Rabies is tripartite(WHO-OIE-FAO)priority[J].OIE Bull,2014(3):3-4.
[7]World Organisation for Animal Health.The worldwide rabies situation in domestic and wild animals[J].OIE Bull,2067-2069.
[8]WHO.Expert consultation on rabies.Second report[J].World Health Organ TRS,2013,982:1-139.
[9]WANG W J,HU Y M,HU C Q,et al.Evaluation of immunogenicity and safety of domestic human rabies vaccine(Vero cells)[J].Modern Prevent Med,2014,43(11):2079-2082.(in Chinese)王文娟,胡月梅,胡传强,等.一种国产人用狂犬病疫苗(Vero细胞)的免疫原性与安全性评价[J].现代预防医学,2014,43(11):2079-2082.
[10]FINKE S,CONZELMANN K K.Replication strategies of rabies virus[J].Virus Res,2005,111(2):120.
[11]MASATANI T,ITO N,SHIMIZΜK,et al.Amino acids at positions 273 and 394 in rabies virus nucleoprotein are important for both evasion of host RIG-I-mediated antiviral response and pathogenicity[J].Virus Res,2011,155(1):168-174.
[12]MASATANI T,ITO N,ITO Y,et al.Importance of rabies virus nucleoprotein in viral evasion of interferon response in the brain[J].Microbiol&Immun,2013,57(7):511-517.
[13]POISSON N,REAL E,GAΜDIN Y,et al.Molecular basis for the interaction between rabies virus phosphoprotein P and the dynein light chain LC8:dissociation of dynein-binding properties and transcriptional functionality of P[J].J General Virol,2001,82(11):2691-2696.
[14]YAMAOKA S,ITO N,OHKA S,et al.Involvement of the rabies virus phosphoprotein gene in neuroinvasiveness[J].J Virol,2013,87(22):12327.
[15]NIΜX,TANG L,TSEGGAI T,et al.Wild-type rabies virus phosphoprotein is associated with viral sensitivity to typeⅠinterferon treatment[J].Arch Virol,2013,158(11):2297-2305.
[16]LUO J Y,MA Z Y.Structure and function of rabies virus G protein[C].Proceedings of the 2009 Annual Conference of the Chinese Society of Animal Husbandry and Veterinary Medicine.Beijing:Chinese Society of Animal Husbandry and Veterinary Medicine,2009.(in Chinese)罗金燕,马志永.狂犬病病毒G蛋白的结构和功能[C].中国畜牧兽医学会2009学术年会论文集(下册).北京:中国畜牧兽医学会,2009.
[17]OSWALD M,GEISSLER S,GOEPFERICH A.Targeting the central nervous system(CNS):a review of rabies virus targeting strategies[J].Mol Pharm,2017,5.PMID:28514853[PubmedPublisher].
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