狂犬病病毒P蛋白的原核表达及间接ELISA检测方法的建立
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摘要
本试验旨在建立一种检测狂犬病抗体的方法。通过传代培养BHK21细胞扩增狂犬病毒LEP-Flury株病毒,并根据GenBank发表的狂犬病病毒(Rabies Virus, RV) LEP-Flury株的基因序列设计一对引物,通过RT-PCR扩增得到P基因的全长序列,克隆于pGM-T载体中,获得重组质粒pGM-T-P,将重组质粒用限制性内切酶NotI和EcoRI进行双酶切,酶切产物定向克隆于原核表达载体pET-32a (+)中,构建原核重组表达质粒pET-32a-P,阳性重组表达质粒转化原核表达宿主菌BL21(DE3),用IPTG诱导表达P蛋白。SDS-PAGE和Western-blot分析确定蛋白表达量和特异性。使用镍琼脂糖凝胶FF对P蛋白进行纯化,纯化的蛋白作为诊断抗原,通过对反应条件的优化,初步建立间接ELISA方法用于检测狂犬病抗体。结果扩增得到了RV P基因,构建了克隆质粒pGM-T-P和原核表达质粒pET-32a-P,高效表达了主要以可溶性形式存在的P蛋白,并能与RV阳性血清发生特异性反应,用表达的狂犬病病毒重组P蛋白建立了用于检测RV抗体的间接ELISA方法。
In order to established a kind of method to detect the antibody of rabies virus.Amplied rabies virus of LEP-Flury strain by BHK21.The complete length of P gene from rabies virus was amplified by RT-PCR using a pair of specific primers designed according to the relevant sequences from GenBank.The PCR product was cloned into cloning expression vestor pGM-T to obtain the cloning expressed plasmid pGM-T-P. After double-digested by NotI and EcoRI, the product was transferred into prokaryotic expression vetor pET-32a (+) to abstain the prokaryotically expressed plasmid pET-32a-P. The target gene was then expressed in the E.coli BL21(DE3) cell by inducted with IPTG. The highest expression of target protein was analysed by SDS-PAGE, and the good immunoreactivity to rabies virus antibodies was proved by Western-blot analysis. By using purified protein, indirect ELISA for the detection of rabies virus antibodies in canine serum was applied after management of the optional woking condition.
In order to established a kind of method to detect the antibody of rabies virus.Amplied rabies virus of LEP-Flury strain by BHK21.The complete length of P gene from rabies virus was amplified by RT-PCR using a pair of specific primers designed according to the relevant sequences from GenBank.The PCR product was cloned into cloning expression vestor pGM-T to obtain the cloning expressed plasmid pGM-T-P. After double-digested by NotI and EcoRI, the product was transferred into prokaryotic expression vetor pET-32a (+) to abstain the prokaryotically expressed plasmid pET-32a-P. The target gene was then expressed in the E.coli BL21(DE3) cell by inducted with IPTG. The highest expression of target protein was analysed by SDS-PAGE, and the good immunoreactivity to rabies virus antibodies was proved by Western-blot analysis. By using purified protein, indirect ELISA for the detection of rabies virus antibodies in canine serum was applied after management of the optional woking condition.
引文
[1]Mirjam A. R. Preuss, Marie-Luise Faber, Gene S. Tan, et al. Intravenous Inoculation of a Bat-Associated Rabies Virus Causes Lethal Encephalopathy in Mice through Invasion of the Brain via Neurosecretory Hypothalamic Fibers [J].PLoS Pathog,2009,5(6):e1000485.
[2]李伟,周海梦.狂犬病病毒的分子生物学及其保护性抗原的结构和功能[J].传染病信息,1999,112(4)：167-169.
[3]World Health Organization. Strategies for the control and elimination of rabies in Aisa [M]. GenevaSwitzerland,2001:21.
[4]Pastoret PP, Brochier B. Brochier B Epidemiology and control of fox rabies in Europe [J]. Vaccine,1999,17(13-14):1750.
[5]McColl KA, Tordo N. Aguilar Setien AA Bat lyssavirus infections [J].Rev Sei Tech,2000, 19(1):177.
[6]Rupprecht C. The natural History of Rabies,Antegenic relationship of lyssaviruses [M].New York:CRC Press,1991:69-100.
[7]Johnson N, McElhinney LM, Ali YH, et al. Molecular epidemiology of canid rabies in Sudan:evidence for acommon origin of rabies with Ethiopia [J]. Virus Res,2004,104(2):201-205.
[8]Kuzmin IV, Botvinkin AD, McElhinney LM, et al. Molecular epidemiology of terrestrial rabies in the former Soviet Union [J].JWildl Dis,2004,40 (4):617-631.
[9]Hyun BH, Lee KK, Kim IJ, et al. Molecular epidemiology of rabies virus isolates from South Korea [J].Virus Res,2005,114 (1-2):113-125.
[10]Paezs A, Saad C, Nunez C, et al. Molecular epidemiology of rabies in northern Colombia 1994-2003:Evidence for human and fox rabies associated with dogs [J].Epidemiol Infect, 2005,133(3):529-536.
[11]徐葛林,吴杰等.中国19个狂犬病病毒街毒分离株N基因的序列分析病学分析[J].病毒学报,2002,18(1)：48.
[12]张建明,严延生.狂犬病病毒的基因分型及其分子流行病学研究进展[J].中国人兽共患病学报,2006,22(3)：271-273.
[13]辛志明,王寿昆.狂犬病病毒与狂犬病在人间流行状况[J].福建畜牧兽医增刊,2006：77-82.
[14]Rojjanaporn Pulmanausahakul, Jianwei Li, Matthias J.Schnell, et al. The Glycoprotein and the Matrix Protein of Rabies Virus Affect Pathogenicity by Regulating Viral Replication and Facilitating Cell-to-Cell Spread [J].J Virol,2008,82(5):2330-2338.
[15]邵荣标,郑春早,王海燕,等.人类狂犬病毒隐形感染状况研究[J].中国人兽共患病杂志,2004,20(7)：649-651.
[16]熊成龙,张永振,卢思奇.狂犬病毒生物学特征研究进展[J].中华流行病学杂志,2006,27(10)：913-916.
[17]Bourhy H, Kissi B, Tordo. N Molecular diversity of the Lyssavirus genus [J].Virology,1993, 194(1):70-81.
[18]Skerratt L F, Speare R, Berger L, et al. Lyssaviral infection and lead poisoning in black flying foxes from Queensland [J]. J Wildl Dis,1998,34 (2):355-361.
[19]赵云蛟,钱爱东,李公美,等.狂犬病病毒分子流行病学研究进展[J].经济动物学报,2004,8(3)：178-180.
[20]王晓光,侯勇跃,唐青.狂犬病病毒分子生物学研究进展[J].畜牧与饲料科学,2008,(2)72-75.
[21]袁慧君,王三虎,秦鄂德.狂犬病病毒分子生物学研究进展[J].生物技术通讯,2004,15(6)：596-599.
[22]Luo M, Green TJ, Zhang X, et al. Conserved characteristics of the rhabdovirus nucleoprotein [J]. Virus Res,2007,129(2):46-51.
[23]Perea Arang I, Loaza Rubio E, Rojas Anaya E, et al. Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice [J]. Plant Cell REP,2008,27(4):77-85.
[24]Masatani T, Ito N, Shimizu K, et al. Rabies virus nucleoprotein functions to evade activation of the RIG-I-mediated antiviral response [J].J Virol,2010,84(8):4002-12.
[25]Assenberg R, Delmas O, Ren J, et al. Structure of the nucleoprotein binding domain of Mokola virus phosphpprotein [J]. J Virol,2010,84(2):1089-96.
[26]Houimel M, Dellagi K. Peptide mimotopes of rabies virus glycoprotein with immunogenic activity [J]. Vaccine,2009,27(34):4648-55.
[27]Mendonca RZ, Greco KN, Moraes RH, et al. Study of kinetic parameters for the production of recombinant rabies virus glycoprotein [J].Cytotechnology,2009,60(1-3):143-51.
[28]Ito Y, Ito N, Saito S, et al. Amino acid substitutions at positions 242,255 and 268 in rabies virus glycoprotein affect spread of viral infection [J]. Microbiol Immunol,2010, 54(2):89-97.
[29]Paul S.MasterSt, Amiya K.Banerjeet. Resolution of Multiple Complexes of Phosphoprotein NS with Nucleocapsid Protein N of Vesicular Stomatitis Virus [J]. Journal of Virology, 1988,62(8):2651-2657.
[30]Griffith D.Parks. Mapping of a Region of the Paramyxovirus L Protein Required for the Formation of a Stable Complex with the Viral Phosphoprotein P [J].Journal of Virology, 1994,68(8):4862-4872.
[31]Gene S. Tan, Mirjam A. R. Preuss, John C. Williams,et al. The dynein light chain 8 binding motif of rabies virus phosphoprotein promotes efficient viral transcription [J].PNAS,2007, 104(17):7229-7234.
[32]Finke S, Conzelmann K K. Dissociation of rabies virus matrix protein functions in regulation of viral RNA synthesis and virus assembly [J].J Virol,2003, 77(22):12074-12082.
[33]Anastassia V.Komarova, Eleonore Real, Andrew M. Borman, et al. Rabies virus matrix protein interplay with eIF3,newinsights into rabies virus pathogenesis [J].Nucleic Acids Res, 2007,35(5):1522-1532.
[34]Pulmanausahakul R, Li J, Schnell MJ, et al. The glycoprotein and the matrix protein of rabies virus affect pathogenicity by regulating viral replication and facilitating cell-to-cell spread [J].J Virol,2008,82(5):2330-8.
[35]Wirblich C, Tan GS, Papaneri A, et al. PPEY motif within the rabies virus (RV) matrix protein is essential for efficient virion release and RV pathogenicity [J].J Virol,2008, 82(19):9730-8.
[36]Christoph Wirblich, Gene S.Tan, Amy Papaneri, et al. PPEY Motif within the Rabies Virus (RV) Matrix Protein Is Essential for Efficient Virion Release and RV Pathogenicity [J].J Virol,2008,82(19):9730-9738.
[37]Adriane Marschalek, Stefan Finke, Martin Schwemmle, et al. Attenuation of Rabies Virus Replication and Virulence by Picornavirus Internal Ribosome Entry Site Elements [J]. J Virus,2009,83(4):1911-1919.
[38]Pauline Menager, Pascal Roux, Francoise Megret, et al. Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies [J]. PLoS Pathog,2009, 5(2):e1000315.
[39]Christine Tuffereau, Klaus Schmidt, Christelle Langevin, et al. The Rabies Virus Glycoprotein Receptor p75NTR Is Not Essential for Rabies Virus Infection [J]. J Virol,2007, 81(24):13622-13630.
[40]Lentz TL, Burrage TG, Smithal. Is the acetylcholine receptor a rabies virus [J]. Science, 1982,182-215.
[41]Thoulouzemi, Lafage M, Schacchnier M. The neural cell adhesion molecule is a receptor for rabies [J]. J Virol,1998,71-72.
[42]Supperti F, Tuffereau C, Benejean J, et al. Protein nucleotide low affinity nerve growth factor receptor (P75NTR) can serve as a receptor for rabies virus [J]. EMBOJ,1998, 17(24):7250.
[43]Xavier Lahaye, Aurore Vidy, Carole Pomier, et al. Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells:Evidence that NBs Are Sites of Viral Transcription and Replication [J]. J Virol,2009,83(16):7948-7958.
[44]徐葛林.国内外狂犬病毒实验室检测与诊断技术的研究及进展[J].中国计划免疫,2005,11(2)：147-150.
[45]Danielle B Araujo, Helio Langoni, Marilene F Almeida, Jane Megid. Heminested reverse-transcriptase polymerase chain reaction (hnRT-PCR) as a tool for rabies virus detection in stored and decomposed samples[J]. BMC Res Notes,2008,1-17.
[46]Anthony R.Fooks, Nicholas Johnson, Conrad M.Freuling, et al. Emerging Technologies for the Detection of Rabies Virus:Challenges and Hopes in the 21st Century [J]. PLoS Negl Trop Dis,2009,3(9):e530.
[47]Thomas Muller, Bernhard Dietzschold, Hildegund Ertl, et al. Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans [J]. PLoS Negl Trop Dis, 2009,3(11):e542.
[48]D.Craig Hooper, Timothy W.Phares, Marzena J.Fabis, Anirban Roy. The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System [J]. PLoS Negl Trop Dis,2009,3(10):e535.
[49]Ertl HC. Novel vaccines to human rabies [J]. PLoS Negl Trop Dis,2009,3(9):e515.
[50]Jonathan Cenna, Gene S.Tan, Amy B.Papaneri, et al. Immune Modulating Effect by a Phosphoprotein deleted Rabies Virus Vaccine Vector Expressing Two Copies of the Rabies Virus Glycoprotein Gene[J]. Vaccine,2008,26(50):6405-6414.
[51]Milosz Faber, Jianwei Li, Rhonda B.Kean, et al. Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus [J]. Proc Natl Acad Sci USA,2009,106(27):11300-11305.
[52]Elizabeth J.Faul, Celestine N.Wanjalla, James P.McGettigan, et al. Interferon-β expressed by a rabies virus-based HIV-1 vaccine vector serves as a molecular adjuvant and decreases pathogenicity [J]. Virology,2008,382(2):226-238.
[53]Jesse D.Blanton, Joshua Self, Michael Niezgoda, et al. Oral vaccination of raccoons (Procyon lotor) with genetically modified rabies virus vaccines [J]. Vaccine,2007,25(42): 7296-7300.
[54]Gene S.Tan, Mirjam A.R.Preuss, John C.Williams, et al. The dynein light chain 8 binding motif of rabies virus phosphoprotein promotes efficient viral transcription [J]. Proc Natl Acad Sci USA,2007,104(17):7229-7234.
[55]Aurore Vidy, Jamila El Bougrini, Mounira K.Chelbi-Alix, et al. The Nucleocytoplasmic Rabies Virus P Protein Counteracts Interferon Signaling by Inhibiting both Nuclear Accumulation and DNA Binding of STAT1 [J]. J Virol,2007,81(8):4255-4263.
[56]Kenta shimizu. Sensitivity of rabies virus to type Ⅰ interferon is determined by the phosphoprotein gene [J]. Microbio immunol,2006,50(12):975-978.
[57]Danielle Blondel, Tarick Regad, Nicolas Poisson, et al. Rabies virus P and small P products interact directly with PML and reorganize PML nuclear bodies [J]. Oncogene,2002, 7957-7970.
[58]袁慧君,扈荣良.狂犬病病毒SRV9克隆株核蛋白基因的克隆表达与特性分析[J].中国 预防兽医学报,2003,25(1)：5-8.
[59]Ertl HC, Fischer S, Flam. Phosphorylation of the N and M1 protein of rabies virus [J].J Virol,1985,66:2285-2289.
[60]V M Zaides, L I Krotova, L M Selimova, et al. Reevaluation of the proteins in rabies virus particles[J].J Virol,1979,29:1226-1228.
[61]Chenik M, Schnell M, Conzelmann K K, et al. Mapping the interacting domains between the rabies virus polymerase and phosphoprotein [J].J Virol,1998,72:1925-1930.
[62]萨姆布鲁克J,拉塞尔D W.分组克隆实验指南[M].第3版.黄培堂,王嘉玺,朱厚础,等译.北京：科学出版社,2002：1723-1726.
[2]李伟,周海梦.狂犬病病毒的分子生物学及其保护性抗原的结构和功能[J].传染病信息,1999,112(4)：167-169.
[3]World Health Organization. Strategies for the control and elimination of rabies in Aisa [M]. GenevaSwitzerland,2001:21.
[4]Pastoret PP, Brochier B. Brochier B Epidemiology and control of fox rabies in Europe [J]. Vaccine,1999,17(13-14):1750.
[5]McColl KA, Tordo N. Aguilar Setien AA Bat lyssavirus infections [J].Rev Sei Tech,2000, 19(1):177.
[6]Rupprecht C. The natural History of Rabies,Antegenic relationship of lyssaviruses [M].New York:CRC Press,1991:69-100.
[7]Johnson N, McElhinney LM, Ali YH, et al. Molecular epidemiology of canid rabies in Sudan:evidence for acommon origin of rabies with Ethiopia [J]. Virus Res,2004,104(2):201-205.
[8]Kuzmin IV, Botvinkin AD, McElhinney LM, et al. Molecular epidemiology of terrestrial rabies in the former Soviet Union [J].JWildl Dis,2004,40 (4):617-631.
[9]Hyun BH, Lee KK, Kim IJ, et al. Molecular epidemiology of rabies virus isolates from South Korea [J].Virus Res,2005,114 (1-2):113-125.
[10]Paezs A, Saad C, Nunez C, et al. Molecular epidemiology of rabies in northern Colombia 1994-2003:Evidence for human and fox rabies associated with dogs [J].Epidemiol Infect, 2005,133(3):529-536.
[11]徐葛林,吴杰等.中国19个狂犬病病毒街毒分离株N基因的序列分析病学分析[J].病毒学报,2002,18(1)：48.
[12]张建明,严延生.狂犬病病毒的基因分型及其分子流行病学研究进展[J].中国人兽共患病学报,2006,22(3)：271-273.
[13]辛志明,王寿昆.狂犬病病毒与狂犬病在人间流行状况[J].福建畜牧兽医增刊,2006：77-82.
[14]Rojjanaporn Pulmanausahakul, Jianwei Li, Matthias J.Schnell, et al. The Glycoprotein and the Matrix Protein of Rabies Virus Affect Pathogenicity by Regulating Viral Replication and Facilitating Cell-to-Cell Spread [J].J Virol,2008,82(5):2330-2338.
[15]邵荣标,郑春早,王海燕,等.人类狂犬病毒隐形感染状况研究[J].中国人兽共患病杂志,2004,20(7)：649-651.
[16]熊成龙,张永振,卢思奇.狂犬病毒生物学特征研究进展[J].中华流行病学杂志,2006,27(10)：913-916.
[17]Bourhy H, Kissi B, Tordo. N Molecular diversity of the Lyssavirus genus [J].Virology,1993, 194(1):70-81.
[18]Skerratt L F, Speare R, Berger L, et al. Lyssaviral infection and lead poisoning in black flying foxes from Queensland [J]. J Wildl Dis,1998,34 (2):355-361.
[19]赵云蛟,钱爱东,李公美,等.狂犬病病毒分子流行病学研究进展[J].经济动物学报,2004,8(3)：178-180.
[20]王晓光,侯勇跃,唐青.狂犬病病毒分子生物学研究进展[J].畜牧与饲料科学,2008,(2)72-75.
[21]袁慧君,王三虎,秦鄂德.狂犬病病毒分子生物学研究进展[J].生物技术通讯,2004,15(6)：596-599.
[22]Luo M, Green TJ, Zhang X, et al. Conserved characteristics of the rhabdovirus nucleoprotein [J]. Virus Res,2007,129(2):46-51.
[23]Perea Arang I, Loaza Rubio E, Rojas Anaya E, et al. Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice [J]. Plant Cell REP,2008,27(4):77-85.
[24]Masatani T, Ito N, Shimizu K, et al. Rabies virus nucleoprotein functions to evade activation of the RIG-I-mediated antiviral response [J].J Virol,2010,84(8):4002-12.
[25]Assenberg R, Delmas O, Ren J, et al. Structure of the nucleoprotein binding domain of Mokola virus phosphpprotein [J]. J Virol,2010,84(2):1089-96.
[26]Houimel M, Dellagi K. Peptide mimotopes of rabies virus glycoprotein with immunogenic activity [J]. Vaccine,2009,27(34):4648-55.
[27]Mendonca RZ, Greco KN, Moraes RH, et al. Study of kinetic parameters for the production of recombinant rabies virus glycoprotein [J].Cytotechnology,2009,60(1-3):143-51.
[28]Ito Y, Ito N, Saito S, et al. Amino acid substitutions at positions 242,255 and 268 in rabies virus glycoprotein affect spread of viral infection [J]. Microbiol Immunol,2010, 54(2):89-97.
[29]Paul S.MasterSt, Amiya K.Banerjeet. Resolution of Multiple Complexes of Phosphoprotein NS with Nucleocapsid Protein N of Vesicular Stomatitis Virus [J]. Journal of Virology, 1988,62(8):2651-2657.
[30]Griffith D.Parks. Mapping of a Region of the Paramyxovirus L Protein Required for the Formation of a Stable Complex with the Viral Phosphoprotein P [J].Journal of Virology, 1994,68(8):4862-4872.
[31]Gene S. Tan, Mirjam A. R. Preuss, John C. Williams,et al. The dynein light chain 8 binding motif of rabies virus phosphoprotein promotes efficient viral transcription [J].PNAS,2007, 104(17):7229-7234.
[32]Finke S, Conzelmann K K. Dissociation of rabies virus matrix protein functions in regulation of viral RNA synthesis and virus assembly [J].J Virol,2003, 77(22):12074-12082.
[33]Anastassia V.Komarova, Eleonore Real, Andrew M. Borman, et al. Rabies virus matrix protein interplay with eIF3,newinsights into rabies virus pathogenesis [J].Nucleic Acids Res, 2007,35(5):1522-1532.
[34]Pulmanausahakul R, Li J, Schnell MJ, et al. The glycoprotein and the matrix protein of rabies virus affect pathogenicity by regulating viral replication and facilitating cell-to-cell spread [J].J Virol,2008,82(5):2330-8.
[35]Wirblich C, Tan GS, Papaneri A, et al. PPEY motif within the rabies virus (RV) matrix protein is essential for efficient virion release and RV pathogenicity [J].J Virol,2008, 82(19):9730-8.
[36]Christoph Wirblich, Gene S.Tan, Amy Papaneri, et al. PPEY Motif within the Rabies Virus (RV) Matrix Protein Is Essential for Efficient Virion Release and RV Pathogenicity [J].J Virol,2008,82(19):9730-9738.
[37]Adriane Marschalek, Stefan Finke, Martin Schwemmle, et al. Attenuation of Rabies Virus Replication and Virulence by Picornavirus Internal Ribosome Entry Site Elements [J]. J Virus,2009,83(4):1911-1919.
[38]Pauline Menager, Pascal Roux, Francoise Megret, et al. Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies [J]. PLoS Pathog,2009, 5(2):e1000315.
[39]Christine Tuffereau, Klaus Schmidt, Christelle Langevin, et al. The Rabies Virus Glycoprotein Receptor p75NTR Is Not Essential for Rabies Virus Infection [J]. J Virol,2007, 81(24):13622-13630.
[40]Lentz TL, Burrage TG, Smithal. Is the acetylcholine receptor a rabies virus [J]. Science, 1982,182-215.
[41]Thoulouzemi, Lafage M, Schacchnier M. The neural cell adhesion molecule is a receptor for rabies [J]. J Virol,1998,71-72.
[42]Supperti F, Tuffereau C, Benejean J, et al. Protein nucleotide low affinity nerve growth factor receptor (P75NTR) can serve as a receptor for rabies virus [J]. EMBOJ,1998, 17(24):7250.
[43]Xavier Lahaye, Aurore Vidy, Carole Pomier, et al. Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells:Evidence that NBs Are Sites of Viral Transcription and Replication [J]. J Virol,2009,83(16):7948-7958.
[44]徐葛林.国内外狂犬病毒实验室检测与诊断技术的研究及进展[J].中国计划免疫,2005,11(2)：147-150.
[45]Danielle B Araujo, Helio Langoni, Marilene F Almeida, Jane Megid. Heminested reverse-transcriptase polymerase chain reaction (hnRT-PCR) as a tool for rabies virus detection in stored and decomposed samples[J]. BMC Res Notes,2008,1-17.
[46]Anthony R.Fooks, Nicholas Johnson, Conrad M.Freuling, et al. Emerging Technologies for the Detection of Rabies Virus:Challenges and Hopes in the 21st Century [J]. PLoS Negl Trop Dis,2009,3(9):e530.
[47]Thomas Muller, Bernhard Dietzschold, Hildegund Ertl, et al. Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans [J]. PLoS Negl Trop Dis, 2009,3(11):e542.
[48]D.Craig Hooper, Timothy W.Phares, Marzena J.Fabis, Anirban Roy. The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System [J]. PLoS Negl Trop Dis,2009,3(10):e535.
[49]Ertl HC. Novel vaccines to human rabies [J]. PLoS Negl Trop Dis,2009,3(9):e515.
[50]Jonathan Cenna, Gene S.Tan, Amy B.Papaneri, et al. Immune Modulating Effect by a Phosphoprotein deleted Rabies Virus Vaccine Vector Expressing Two Copies of the Rabies Virus Glycoprotein Gene[J]. Vaccine,2008,26(50):6405-6414.
[51]Milosz Faber, Jianwei Li, Rhonda B.Kean, et al. Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus [J]. Proc Natl Acad Sci USA,2009,106(27):11300-11305.
[52]Elizabeth J.Faul, Celestine N.Wanjalla, James P.McGettigan, et al. Interferon-β expressed by a rabies virus-based HIV-1 vaccine vector serves as a molecular adjuvant and decreases pathogenicity [J]. Virology,2008,382(2):226-238.
[53]Jesse D.Blanton, Joshua Self, Michael Niezgoda, et al. Oral vaccination of raccoons (Procyon lotor) with genetically modified rabies virus vaccines [J]. Vaccine,2007,25(42): 7296-7300.
[54]Gene S.Tan, Mirjam A.R.Preuss, John C.Williams, et al. The dynein light chain 8 binding motif of rabies virus phosphoprotein promotes efficient viral transcription [J]. Proc Natl Acad Sci USA,2007,104(17):7229-7234.
[55]Aurore Vidy, Jamila El Bougrini, Mounira K.Chelbi-Alix, et al. The Nucleocytoplasmic Rabies Virus P Protein Counteracts Interferon Signaling by Inhibiting both Nuclear Accumulation and DNA Binding of STAT1 [J]. J Virol,2007,81(8):4255-4263.
[56]Kenta shimizu. Sensitivity of rabies virus to type Ⅰ interferon is determined by the phosphoprotein gene [J]. Microbio immunol,2006,50(12):975-978.
[57]Danielle Blondel, Tarick Regad, Nicolas Poisson, et al. Rabies virus P and small P products interact directly with PML and reorganize PML nuclear bodies [J]. Oncogene,2002, 7957-7970.
[58]袁慧君,扈荣良.狂犬病病毒SRV9克隆株核蛋白基因的克隆表达与特性分析[J].中国 预防兽医学报,2003,25(1)：5-8.
[59]Ertl HC, Fischer S, Flam. Phosphorylation of the N and M1 protein of rabies virus [J].J Virol,1985,66:2285-2289.
[60]V M Zaides, L I Krotova, L M Selimova, et al. Reevaluation of the proteins in rabies virus particles[J].J Virol,1979,29:1226-1228.
[61]Chenik M, Schnell M, Conzelmann K K, et al. Mapping the interacting domains between the rabies virus polymerase and phosphoprotein [J].J Virol,1998,72:1925-1930.
[62]萨姆布鲁克J,拉塞尔D W.分组克隆实验指南[M].第3版.黄培堂,王嘉玺,朱厚础,等译.北京：科学出版社,2002：1723-1726.