表达牛流行热病毒G蛋白的重组狂犬病病毒的构建及鉴定
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摘要
目的以狂犬病病毒(RABV)弱毒株SAD株为载体,构建表达牛流行热病毒(BEFV)G蛋白的重组病毒SAD-BG。方法利用反向遗传技术将BEFV的G蛋白基因ORF插入到SAD株基因组的伪基因区,获得感染性克隆,与辅助质粒共转染BHK-21细胞,获得重组病毒SAD-BG株。结果与结论通过RT-PCR、免疫荧光染色鉴定,表明BEFV G蛋白基因存在于重组病毒基因组,且能表达自身RABV G蛋白与外源BEFV G蛋白。通过BHK-21细胞和NA细胞鉴定重组病毒的生长特性,在NA细胞中重组病毒的滴度高于亲本SAD株,可达1.3×108FFU/ml。成功构建了表达BEFV G蛋白的重组病毒SAD-BG,为进一步研制预防狂犬病和牛流行热(BEF)的二联疫苗奠定了基础。
Objective To construct a recombinant rabies virus strain Street Alabama Dufferin(SAD)-BG expressing the G protein of bovine ephemeral fever virus(BEFV)using SAD as the vector. Methods The main immune antigen G gene ORF of BEFV was inserted into the pseudogene region between G and L of SAD strain by reverse genetics technique.Results and Conclusion The recombinant virus was identified by RT-PCR and IFA. The results showed that BEFV G gene existed in the genome of SAD-BG and was expressed successfully. The SAD-BG was cultured and identified on BHK-21 and NA cell and the titer of the recombinant virus was up to 1.3×108 FFU/ml in NA cells,which was not significantly different from SAD. Recombinant SAD-BG strain is successfully constructed,which can contribute to the future development of a bivalent vaccine against rabies and bovine ephemeral fever(BEF).
Objective To construct a recombinant rabies virus strain Street Alabama Dufferin(SAD)-BG expressing the G protein of bovine ephemeral fever virus(BEFV)using SAD as the vector. Methods The main immune antigen G gene ORF of BEFV was inserted into the pseudogene region between G and L of SAD strain by reverse genetics technique.Results and Conclusion The recombinant virus was identified by RT-PCR and IFA. The results showed that BEFV G gene existed in the genome of SAD-BG and was expressed successfully. The SAD-BG was cultured and identified on BHK-21 and NA cell and the titer of the recombinant virus was up to 1.3×108 FFU/ml in NA cells,which was not significantly different from SAD. Recombinant SAD-BG strain is successfully constructed,which can contribute to the future development of a bivalent vaccine against rabies and bovine ephemeral fever(BEF).
引文
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[33]傅倩芸,燕丽娜,王化磊,等.表达SFTSV Gn蛋白的重组狂犬病病毒的构建、鉴定与免疫反应[J].军事医学,2018,42(5):348-352.
[2]Walker PJ,Klement E.Epidemiology and control of bovine ephemeral fever[J].Vet Res,2015,46:124.
[3]Bakhshesh M,Abdollahi D.Bovine ephemeral fever in Iran:diagnosis,isolation and molecular characterization[J].J Arthropod Borne Dis,2015,9(2):195-203.
[4]Aziz-Boaron O,Klausner Z,Hasoksuz M,et al.Circulation of bovine ephemeral fever in the Middle East--strong evidence for transmission by winds and animal transport[J].Vet Microbiol,2012,158(3-4):300-307.
[5]Kongsuwan K,Cybinski D H,Cooper J,et al.Location of neutralizing epitopes on the G protein of bovine ephemeral fever rhabdovirus[J].J Gen Virol,1998,79(Pt 11):2573-2581.
[6]Hou P,Zhao G,He C,et al.Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library[J].BMC Vet Res,2018,14(1):3.
[7]Osinubi MO,Wu X,Franka R,et al.Enhancing comparative rabies DNA vaccine effectiveness through glycoprotein gene modifications[J].Vaccine,2009,27(51):7214-7218.
[8]Shwiff S,Hampson K,Anderson A.Potential economic benefits of eliminating canine rabies[J].Antiviral Res,2013,98(2):352-356.
[9]Liu Y,Zhang S,Zhao J,et al.Fox-and raccoon-dog-associated rabies outbreaks in northern China[J].Virol Sin,2014,29(5):308-310.
[10]Feng Y,Wang W,Guo J,et al.Disease outbreaks caused by steppe-type rabies viruses in China[J].Epidemiol Infect,2015,143(6):1287-1291.
[11]Schnell MJ,Foley HD,Siler CA,et al.Recombinant rabies virus as potential live-viral vaccines for HIV-1[J].Proc Natl Acad Sci USA,2000,97(7):3544-3549.
[12]Wang FX,Zhang SQ,Zhu HW,et al.Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge[J].Vet Microbiol,2014,174(3-4):362-371.
[13]Wirblich C,Coleman CM,Kurup D,et al.One-health:a safe,efficient,dual-use vaccine for humans and animals against Middle East respiratory syndrome coronavirus and rabies virus[J].J Virol,2017,91(2).pii:e02040-16.doi:10.1128/JVI.02040-16.
[14]Willet M,Kurup D,Papaneri A,et al.Preclinical development of inactivated rabies virus-based polyvalent vaccine against rabies and filoviruses[J].J Infect Dis,2015,212(Suppl 2):S414-S424.
[15]Muller TF,Schroder R,Wysocki P,et al.Spatio-temporal use of oral rabies vaccines in fox rabies elimination programmes in Europe[J].PLoS Negl Trop Dis,2015,9(8):e0003953.
[16]王洪梅,侯佩莉,杨宏军,等.牛流行热病毒山东地方株的分离与鉴定[J].山东农业科学,2013,45(2):31-33.
[17]Li Z,Zheng F,Gao S,et al.Large-scale serological survey of bovine ephemeral fever in China[J].Vet Microbiol,2015,176(1-2):155-160.
[18]Yang D,Yang MS,Rhim H,et al.Analysis of five arboviruses and culicoides distribution on cattle farms in Jeollabuk-do,Korea[J].Korean J Parasitol,2018,56(5):477-485.
[19]Zheng F,Qiu C.Phylogenetic relationships of the glycoprotein gene of bovine ephemeral fever virus isolated from mainland China,Taiwan,Japan,Turkey,Israel and Australia[J].Virol J,2012,9:268.
[20]Pasandideh R,Seyfi Abad Shapouri MR,Beigi Nassiri MT.Immunogenicity of a plasmid DNA vaccine encoding G1 epitope of bovine ephemeral fever virus G glycoprotein in mice[J].Onderstepoort J Vet Res,2018,85(1):e1-e6.
[21]Johal J,Gresty K,Kongsuwan K,et al.Antigenic characterization of bovine ephemeral fever rhabdovirus G and GNS glycoproteins expressed from recombinant baculoviruses[J].Arch Virol,2008,153(9):1657-1665.
[22]Wallace DB,Viljoen GJ.Immune responses to recombinants of the South African vaccine strain of lumpy skin disease virus generated by using thymidine kinase gene insertion[J].Vaccine,2005,23(23):3061-3067.
[23]Hertig C,Pye AD,Hyatt AD,et al.Vaccinia virus-expressed bovine ephemeral fever virus G but not G(NS)glycoprotein induces neutralizing antibodies and protects against experimental infection[J].J Gen Virol,1996,77(Pt 4):631-640.
[24]Ma X,Monroe BP,Cleaton JM,et al.Rabies surveillance in the United States during 2017[J].J Am Vet Med Assoc,2018,253(12):1555-1568.
[25]Brookes VJ,Gill GS,Singh BB,et al.Challenges to human rabies elimination highlighted following a rabies outbreak in bovines and a human in Punjab,India[J].2019,66(3):325-336.
[26]Hudson L C,Weinstock D,Jordan T,et al.Clinical features of experimentally induced rabies in cattle and sheep[J].Zentralbl Veterinarmed B,1996,43(2):85-95.
[27]Zhou M,Wang L,Zhou S,et al.Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs[J].Oncotarget,2015,6(36):38504-38516.
[28]Blaney JE,Marzi A,Willet M,et al.Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine[J].PLoSPathog,2013,9(5):e1003389.
[29]Blaney JE,Wirblich C,Papaneri AB,et al.Inactivated or liveattenuated bivalent vaccines that confer protection against rabies and Ebola viruses[J].J Virol,2011,85(20):10605-10616.
[30]Papaneri AB,Wirblich C,Cann JA,et al.A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence[J].Virology,2012,434(1):18-26.
[31]Papaneri AB,Wirblich C,Cooper K,et al.Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein[J].Vaccine,2012,30(43):6136-6141.
[32]Pfaller CK,Cattaneo R,Schnell MJ.Reverse genetics of Mononegavirales:how they work,new vaccines,and new cancer therapeutics[J].Virology,2015,479-480:331-344.
[33]傅倩芸,燕丽娜,王化磊,等.表达SFTSV Gn蛋白的重组狂犬病病毒的构建、鉴定与免疫反应[J].军事医学,2018,42(5):348-352.