H3N8亚型马流感病毒内蒙古株全基因序列分析及NS1基因的原核表达
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摘要
马流感(Equine influenza,EI)是由马流感病毒引起的一种急性、高度接触性的马属动物呼吸道传染病。近年来,随着国际马术赛事的蓬勃兴起以及马属动物流动范围扩大等原因,致使EI在全球上频繁暴发、流行并由此对赛马业等构成了严重威胁。因此,从分子水平上阐明EIV H3N8亚型的流行规律,不仅在病毒学研究领域中有重要的学术意义,而且对EI防控也具有实用价值。
本研究先将内蒙古株A/equine/Inner Mongolia/8/08(H3N8)在SPF鸡胚中增殖,提取病毒核酸,根据EIV 8个基因片段的3’末端高度保守的核苷酸序列特点,设计一条反转录引物,根据GenBank已发表的H3N8亚型EIV 8个基因的cDNA序列,利用Oligo6.0设计并合成14对特异性引物,通过RT-PCR方法扩增出8个基因的序列片段,将它们连接到pMD18-T载体上。序列测定及分析结果显示,EIV 8个基因的cDNA包含该病毒完整的开放阅读框,与美洲株Kentucky/02的同源率最高,HA,NA,NP,NS1,NS2,M1,M2,PA,PBl,PB2基因核苷酸序列的同源性分别为98.7%,99.0%,99.1%,99.5%,99.4%,97.5%,97.4%,98.7%,98.8%,99.2%,两者各基因有着相似的遗传演化过程。
本研究将全长700bp的NS1基因亚克隆到原核表达载体pET-32a中,经双酶切鉴定为阳性的重组质粒再转化到感受态细胞BL21(DE3)中,挑选含重组质粒pET-NS1的菌落,经SDS-PAGE分析表明,用IPTG(终浓度为0.8mmol/L)诱导培养5h,pET-NS1融合蛋白表达量达到高峰,以包涵体形式存在,目的蛋白与载体上的标签蛋白融合后大小约为46kDa,与理论值相符。Western-blot分析表明,融合蛋白能与H3N8亚型马流感病毒感染马阳性血清发生特异性反应,具有良好的抗原性。
Equine influenza (EI) is an acute contagious respiratory infectious disease which is common for horses.In recent years, with the international horse racing increasing and the scope enlarging,it seriously threats horse racing because of the outbreak and epidemic ofequine influenza.It is very significant not only on virology and veterinary medicine study but also on prevent and control equine infuenza that master the epidemic regularity of EIV subtype H3N8 on molecular level.
In this study. Virus of the local isolate A/equine/Inner Mongolia/8/08(H3N8) was propagated in SPF chicken embryo and nudeic acid was extracted. According to the conservation sequence of 3’terminus of eight gene fragment from EIV, a piece of primer was designed to reverse transcript RNA. Accordmg to the cDNA sequence from H3N8 EIV published in GenBank, fourteen pairs of specific primers were designed to amplify all of the eight gene by software Oligo6.0 and the products of RT-PCR was cloned into pMD18-T vector. The sequencing results show that all the cDNA has a complete open reading frame.By comparing with some strains of the same subtype published in GenBank,there is the highest percent homology of nucleotide with American strain Kentucky/02.The homology of nucleotide sequence of H A , N A , N P , N S 1 , N S 2 , M 1 , M 2 , P A , P B l a n d P B 2 i s 98.7%,99.0%,99.1%,99.5%,99.4%,97.5%,97.4%,98.7%,98.8% and 99.2% respectively,comparing with the sequence of the strain Kentucky/02.It showed that there is the same evolutional process of all the gene and this strain and the strain Kentucky/02 may derive from the same ancestor.
The NS1 gene,700bp, was subcloned into prokaryotic expression vector pET-32a, and the positive recombinant plasmid was transformed into competence cell BL21 (DE3). The colony containing the positive plasmid pET-32a was screened and induced by 0.8mmol/L IPTG and analyzed by SDS-PAGE. The result in gel showed that the targat protein was mostly in inclusions when induced by IPTG,and the product was 46kDa fused with tag protein.The Western-blot result showed that the recombinant protein can specific bind with EIV(H3N8) positive serum.
本研究先将内蒙古株A/equine/Inner Mongolia/8/08(H3N8)在SPF鸡胚中增殖,提取病毒核酸,根据EIV 8个基因片段的3’末端高度保守的核苷酸序列特点,设计一条反转录引物,根据GenBank已发表的H3N8亚型EIV 8个基因的cDNA序列,利用Oligo6.0设计并合成14对特异性引物,通过RT-PCR方法扩增出8个基因的序列片段,将它们连接到pMD18-T载体上。序列测定及分析结果显示,EIV 8个基因的cDNA包含该病毒完整的开放阅读框,与美洲株Kentucky/02的同源率最高,HA,NA,NP,NS1,NS2,M1,M2,PA,PBl,PB2基因核苷酸序列的同源性分别为98.7%,99.0%,99.1%,99.5%,99.4%,97.5%,97.4%,98.7%,98.8%,99.2%,两者各基因有着相似的遗传演化过程。
本研究将全长700bp的NS1基因亚克隆到原核表达载体pET-32a中,经双酶切鉴定为阳性的重组质粒再转化到感受态细胞BL21(DE3)中,挑选含重组质粒pET-NS1的菌落,经SDS-PAGE分析表明,用IPTG(终浓度为0.8mmol/L)诱导培养5h,pET-NS1融合蛋白表达量达到高峰,以包涵体形式存在,目的蛋白与载体上的标签蛋白融合后大小约为46kDa,与理论值相符。Western-blot分析表明,融合蛋白能与H3N8亚型马流感病毒感染马阳性血清发生特异性反应,具有良好的抗原性。
Equine influenza (EI) is an acute contagious respiratory infectious disease which is common for horses.In recent years, with the international horse racing increasing and the scope enlarging,it seriously threats horse racing because of the outbreak and epidemic ofequine influenza.It is very significant not only on virology and veterinary medicine study but also on prevent and control equine infuenza that master the epidemic regularity of EIV subtype H3N8 on molecular level.
In this study. Virus of the local isolate A/equine/Inner Mongolia/8/08(H3N8) was propagated in SPF chicken embryo and nudeic acid was extracted. According to the conservation sequence of 3’terminus of eight gene fragment from EIV, a piece of primer was designed to reverse transcript RNA. Accordmg to the cDNA sequence from H3N8 EIV published in GenBank, fourteen pairs of specific primers were designed to amplify all of the eight gene by software Oligo6.0 and the products of RT-PCR was cloned into pMD18-T vector. The sequencing results show that all the cDNA has a complete open reading frame.By comparing with some strains of the same subtype published in GenBank,there is the highest percent homology of nucleotide with American strain Kentucky/02.The homology of nucleotide sequence of H A , N A , N P , N S 1 , N S 2 , M 1 , M 2 , P A , P B l a n d P B 2 i s 98.7%,99.0%,99.1%,99.5%,99.4%,97.5%,97.4%,98.7%,98.8% and 99.2% respectively,comparing with the sequence of the strain Kentucky/02.It showed that there is the same evolutional process of all the gene and this strain and the strain Kentucky/02 may derive from the same ancestor.
The NS1 gene,700bp, was subcloned into prokaryotic expression vector pET-32a, and the positive recombinant plasmid was transformed into competence cell BL21 (DE3). The colony containing the positive plasmid pET-32a was screened and induced by 0.8mmol/L IPTG and analyzed by SDS-PAGE. The result in gel showed that the targat protein was mostly in inclusions when induced by IPTG,and the product was 46kDa fused with tag protein.The Western-blot result showed that the recombinant protein can specific bind with EIV(H3N8) positive serum.
引文
1郭元吉,程小雯.流行性感冒病毒及其实验技术,中国三峡出版社,1997:3-120.
2殷震,刘景华.动物病毒学(第二版)[M].北京:科学出版社,1997:704 -728.
3 WEBSTER, R. G.. Are equine 1 influenza viruses still present in horses? Equine Veterinary Journal ,1993,25:537-538.
4 Fields B N, Knipe D M, Howley P M, et al. Fields virology[M].Lippincott-Raven publishers, phildelphia , 1996:1397-1445.
5金奇.医学分子病毒学[M].北京:科学出版社,2001:633-643.
6 Franklin, R. M. and E. Wecker: Inactivation of some animal viruses by hydroxylamine and the structure of ribonucleic acid. 1959, Nature 84:343-345.
7 Laver W. G: The structure of influenza viruses. II. Disruption of the virus particle and separation of neuraminidase activity. Virology 1963, 20:251-262.
8 Klenk HD, Rott R. The molecular biology of influenza virus pathogenicity.[J] Adv. Virus Res,1988, 34:247-281.
9 Steinhauer D A.Role of hemagglutinin cleavage for the pathogenicity of influenza virus . [J]. Virology, 1999. 258(1):1-20.
10 Biswas S K,Nayak D P.Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein[J].J Virol.1994.68:1819-1826.
11 Nakagawa Y,Kimura N,Toyoda T,et al.the RNA polymerase PB2 subunit is not for replication of the influenza virus genome but is involved in capped mRNA synthesis[J] Virology. 1995. 65:245-253.
12 Davey, J., N. J. Dimmock, and A. Colman. Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes. [J]. Cell,1985. 40:667-675.
13 Neumann G, Castrucci M R, Kawaoka Y. Nuclear import and export of influenza virus nucleoprotein [J]. J Virol. 1997,71(12): 9690-9700.
14 Wang P., P. Palese, R. O’Neill. The NPI-1/NPI-3 (karyopherin a) binding site on the influenza A virus nucleoprotein NP is a nonconventional nuclear localization signal. [J].J Virol.1997.71:1850-1856.
15 Shimizu K,. Handa H, Nakada S,et al. Regulation of influenza virus RNA polymerase activity by cellular and viral factors.[J].Nucleic acids Res, 1994,22(23):5047-5053.
16 Biswas S K., Boutz P, Nayak D P. Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins. [J]. J Viro1,1998, 72(7): 5493-5501.
17 Yewdell J W, Bennink J R, et al. Influenza A virus nucleoprotein is a major targetantigen for cross-reactive anti-influenza A virus cytotoxic T lymhocytes. [J].Proc NatlAcad Sci USA, 1985, 820:1785-1789.
18 Christensen J P, Doherty P C, Branum K C, Riberdy J M. Profound protection against respiratory challenge with a lethal H7N7 influenza A virus by increasing the magnitude of CD8+T' cell memory. [J]. J Virol, 2000, 74:11690-11696.
19 Sakaguchi T,Tu Q,Pinto L H,et al.The active aligomeric state of theminimalistic Influenza virus M2 channel is a tetramer[J].Proc Natl Acad Sci USA. 1997.94:5000-5004.
20 Holsingwe L J, Nichani D, Pinto L H,et a1.Influenza A virus M2 channel protein: a structue-function analysis [J].J Virol, 1994, 68:1551-1563.
21 Helenius A. Unpacking the incoming influenza virus[J].Ce11.1992.69:577-578.
22 Watanbe K,Handa H,Mizumoto K,et a1.Mechanism for inhibition of Influenza A virus RNA polymerase activity by matrix protein[J].J Viro1.1996.70:241-247.
23 Sha B, Luo M. Structure of a bifunctional membrane RNA binding protein:influenza virus matrix protein M1[J].Nat Structboi1.1997.4:239-244.
24 Elster C,Larsen K,Gagno J,et a1.Inlluenza virus M1 protein binds to RNA through its nuclear localization signal[J].J Gen Viro1.1997.78:1589-1596.
25 Hankins R,Nagata K,Kato A,et al. Mechanism of Influenza virus transcription inhibition ivity by matrix(M1) protein[J].Res Viro1.1990.141:305-314.
26 Sakaguchi T,Lesser G P,Lamb R A.The ion channel activity of Influenza virus M2 protein affects transport through the Golgi apparatus [J].J CELL Bio1.1996.133:733-747.
27 Birch-Machin I, Rowan A, Pick J,et al.Expression of the nonstructural protein NS1 of equine influenza A virus: detection of anti-NS1 antibody in post infection equine sera [J]. J Virol Methods,1997,65(2):255-263.
28 Ozaki H, Sugiura T, Sugita S,et al. Detection of anti-bodies to the nonstructural protein (NS1) of influenza A virus allows distinction between vaccinated and infected horses [J].Vet Microbiol,2001,82:111-119.
29 Ludwig S.,Pleschka S.,WolffT.A fatal relationshipd—influenza virus interactions with the host cell.Viral Immunol,1999,12(3):175-196.
30 Lommer B.S.,Luo M.Structural plasticity in influenza virus protein NS2(NEP).J Biol Chem,2002,277(9):7108-7117.
31 Cassetti M C, Noah D L, Montelione G t, et al. Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5' untranslated region[J],Virology, 2001, 289 (2):180-185.
32 Li, Y, Yamakita Y, Krug R M, et al. Regulation of a nuclear export signal by an adjacent inhibitory sequence :The effector domain of the influenza virus NS1 protein[J].Proc. Natl. Acad. Sci.USA, 1998, 95:4864-4869.
33 Yuan W, AraminiJ M,Montelione G T, et al.Structural basis for ubiquitin-like 1SG15 protein binding to the NS. protein of influenza B virus:a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS 1 protein of influenza A virus[J].Virology, 2002, 304 (2):291-301.
34 Chien C Y, Xu Y, Xiao R, et al. Biophysical characterization of the complex between double-stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus: evidence for a novel RNA-binding mode [J].Biochemistry, 2004, 43(7):1950-1962.
35李先茜,杜培革,傅桂莲,等.应用定点突变技术进行流感病毒NS1蛋白功能的研究[J]北华大学学报,2004, 5 (4):316-320.
36 Geiss G K, Salvatnre M, Tumpey T M, et a1. Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells:The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza[J].Proc Natl Acad Sci USA,2002,99(16):10736-10741.
37 Seo S H, Webster K G. Tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells[J].J Virol, 2002, 76(3):1071-1076.
38 0'Neill R. E, Talon J, Palese P, et al.The influenza virus ENP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins [J].EMBO J,1998,17:288-296.
39 Oxburgh, L. M. Berg, B. Klingeborn, E Emmoth, and T. Linne: Evolution of H3N8 equine influenza Virus from 1963 to 1991. Virus Res. 1994.34:153-165.
40 Cook RF, Sinclair R, Mumford JA: Detection of influenza nucleoprotein antigen in nasal secretions from horses infected with A/equine influenza (H3N8) viruses. J Virol Meth 1988;20:1-12.
41 Oxburgh,L.,L.Akerblom,T.Fridberger, B. Klingeborn,and T. Linne: Identification of two antigenically and genetically distinct lineages of H3N8 equine influenza virus in Sweden. Epidemiol. Infect. 1998, 120:61-70.
42 J.萨姆布鲁克, D. W.拉塞尔.分子克隆实验指南(第三版)[M],科学出版社,2002:98.
43 Studier FW, Rosenberg A H, Dunn J J, et al. Use of T7 RNA polymerase to direct expression of cloned genes[J]. Methods Enzymol.1990, 85:60-89.
44 Robert Mierendorf, Keith Yeager, et al. The pET system:Your chioce for expression[J]. Advanced Products and Protocols for Molecular Biology Reseach, 1994,1(1):32-36.
45 Marston FA, Hartley D L. Solubilization of protein aggregates[J].Methods Enzymol,1990,182:264-276.
46 Dubendorff, Studier. Creation of a T7 autogene Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter[J]. J Mol Biol.1991,219(1):61-68.
47 Vito M, Gabriella E, Nicola D,et al.An outbreak of equine influenza virus in vaccinated horses in Italy is due to an H3N8 strain closely related to recent North American representatives of the Florida sub-lineage[J].Vet Microbiol. 2007, 121 (31):56-63.
48 Borchers K., Daly J., Stiens G.,et al. Characterisation of three equine influenza A H3N8 viruses from Germany (2000 and 2002): evidence for frozen evolution[J]. Vet. Microbiol.2005,107:13-21.
49 Daly J. M., A. Lai C. K., Binns M. M.,et al.Antigenic and genetic evolution of equine H3N8 influenza A viruses[J]. J Gen Virol,1996,77:661-671.
50 Lai A.C., Chambers T.M., Holland R.E.,et al. Diverged evolution of recent equine-2 influenza (H3N8) viruses in the Western Hemisphere[J]. Arch. Virol. 2001, 146(6): 1063-1074.
51 Manuguerra J.C., Zientara S,. Sailleau C.,et al.Evidence for evolutionary stasis and genetic drift by genetic analysis of two equine infuenza H3 viruses isolated in France[J]. Vet. Microbiology,2000,74:59-70.
52 Oxburgh, L., Berg, M., Klingeborn, B.,et al. Equine influenza virus from the 1991 Swedish epizootic shows major genetic and antigenic divergence from the prototype virus[J]. Virus Res., 1993,28(3), 263-272.
53 Oxburgh L,Hagstro E. A PCR based method for the identification of equine influenza virus from clinical samples[J].Vet Microbiol,1999(67):161-174.
2殷震,刘景华.动物病毒学(第二版)[M].北京:科学出版社,1997:704 -728.
3 WEBSTER, R. G.. Are equine 1 influenza viruses still present in horses? Equine Veterinary Journal ,1993,25:537-538.
4 Fields B N, Knipe D M, Howley P M, et al. Fields virology[M].Lippincott-Raven publishers, phildelphia , 1996:1397-1445.
5金奇.医学分子病毒学[M].北京:科学出版社,2001:633-643.
6 Franklin, R. M. and E. Wecker: Inactivation of some animal viruses by hydroxylamine and the structure of ribonucleic acid. 1959, Nature 84:343-345.
7 Laver W. G: The structure of influenza viruses. II. Disruption of the virus particle and separation of neuraminidase activity. Virology 1963, 20:251-262.
8 Klenk HD, Rott R. The molecular biology of influenza virus pathogenicity.[J] Adv. Virus Res,1988, 34:247-281.
9 Steinhauer D A.Role of hemagglutinin cleavage for the pathogenicity of influenza virus . [J]. Virology, 1999. 258(1):1-20.
10 Biswas S K,Nayak D P.Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein[J].J Virol.1994.68:1819-1826.
11 Nakagawa Y,Kimura N,Toyoda T,et al.the RNA polymerase PB2 subunit is not for replication of the influenza virus genome but is involved in capped mRNA synthesis[J] Virology. 1995. 65:245-253.
12 Davey, J., N. J. Dimmock, and A. Colman. Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes. [J]. Cell,1985. 40:667-675.
13 Neumann G, Castrucci M R, Kawaoka Y. Nuclear import and export of influenza virus nucleoprotein [J]. J Virol. 1997,71(12): 9690-9700.
14 Wang P., P. Palese, R. O’Neill. The NPI-1/NPI-3 (karyopherin a) binding site on the influenza A virus nucleoprotein NP is a nonconventional nuclear localization signal. [J].J Virol.1997.71:1850-1856.
15 Shimizu K,. Handa H, Nakada S,et al. Regulation of influenza virus RNA polymerase activity by cellular and viral factors.[J].Nucleic acids Res, 1994,22(23):5047-5053.
16 Biswas S K., Boutz P, Nayak D P. Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins. [J]. J Viro1,1998, 72(7): 5493-5501.
17 Yewdell J W, Bennink J R, et al. Influenza A virus nucleoprotein is a major targetantigen for cross-reactive anti-influenza A virus cytotoxic T lymhocytes. [J].Proc NatlAcad Sci USA, 1985, 820:1785-1789.
18 Christensen J P, Doherty P C, Branum K C, Riberdy J M. Profound protection against respiratory challenge with a lethal H7N7 influenza A virus by increasing the magnitude of CD8+T' cell memory. [J]. J Virol, 2000, 74:11690-11696.
19 Sakaguchi T,Tu Q,Pinto L H,et al.The active aligomeric state of theminimalistic Influenza virus M2 channel is a tetramer[J].Proc Natl Acad Sci USA. 1997.94:5000-5004.
20 Holsingwe L J, Nichani D, Pinto L H,et a1.Influenza A virus M2 channel protein: a structue-function analysis [J].J Virol, 1994, 68:1551-1563.
21 Helenius A. Unpacking the incoming influenza virus[J].Ce11.1992.69:577-578.
22 Watanbe K,Handa H,Mizumoto K,et a1.Mechanism for inhibition of Influenza A virus RNA polymerase activity by matrix protein[J].J Viro1.1996.70:241-247.
23 Sha B, Luo M. Structure of a bifunctional membrane RNA binding protein:influenza virus matrix protein M1[J].Nat Structboi1.1997.4:239-244.
24 Elster C,Larsen K,Gagno J,et a1.Inlluenza virus M1 protein binds to RNA through its nuclear localization signal[J].J Gen Viro1.1997.78:1589-1596.
25 Hankins R,Nagata K,Kato A,et al. Mechanism of Influenza virus transcription inhibition ivity by matrix(M1) protein[J].Res Viro1.1990.141:305-314.
26 Sakaguchi T,Lesser G P,Lamb R A.The ion channel activity of Influenza virus M2 protein affects transport through the Golgi apparatus [J].J CELL Bio1.1996.133:733-747.
27 Birch-Machin I, Rowan A, Pick J,et al.Expression of the nonstructural protein NS1 of equine influenza A virus: detection of anti-NS1 antibody in post infection equine sera [J]. J Virol Methods,1997,65(2):255-263.
28 Ozaki H, Sugiura T, Sugita S,et al. Detection of anti-bodies to the nonstructural protein (NS1) of influenza A virus allows distinction between vaccinated and infected horses [J].Vet Microbiol,2001,82:111-119.
29 Ludwig S.,Pleschka S.,WolffT.A fatal relationshipd—influenza virus interactions with the host cell.Viral Immunol,1999,12(3):175-196.
30 Lommer B.S.,Luo M.Structural plasticity in influenza virus protein NS2(NEP).J Biol Chem,2002,277(9):7108-7117.
31 Cassetti M C, Noah D L, Montelione G t, et al. Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5' untranslated region[J],Virology, 2001, 289 (2):180-185.
32 Li, Y, Yamakita Y, Krug R M, et al. Regulation of a nuclear export signal by an adjacent inhibitory sequence :The effector domain of the influenza virus NS1 protein[J].Proc. Natl. Acad. Sci.USA, 1998, 95:4864-4869.
33 Yuan W, AraminiJ M,Montelione G T, et al.Structural basis for ubiquitin-like 1SG15 protein binding to the NS. protein of influenza B virus:a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS 1 protein of influenza A virus[J].Virology, 2002, 304 (2):291-301.
34 Chien C Y, Xu Y, Xiao R, et al. Biophysical characterization of the complex between double-stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus: evidence for a novel RNA-binding mode [J].Biochemistry, 2004, 43(7):1950-1962.
35李先茜,杜培革,傅桂莲,等.应用定点突变技术进行流感病毒NS1蛋白功能的研究[J]北华大学学报,2004, 5 (4):316-320.
36 Geiss G K, Salvatnre M, Tumpey T M, et a1. Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells:The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza[J].Proc Natl Acad Sci USA,2002,99(16):10736-10741.
37 Seo S H, Webster K G. Tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells[J].J Virol, 2002, 76(3):1071-1076.
38 0'Neill R. E, Talon J, Palese P, et al.The influenza virus ENP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins [J].EMBO J,1998,17:288-296.
39 Oxburgh, L. M. Berg, B. Klingeborn, E Emmoth, and T. Linne: Evolution of H3N8 equine influenza Virus from 1963 to 1991. Virus Res. 1994.34:153-165.
40 Cook RF, Sinclair R, Mumford JA: Detection of influenza nucleoprotein antigen in nasal secretions from horses infected with A/equine influenza (H3N8) viruses. J Virol Meth 1988;20:1-12.
41 Oxburgh,L.,L.Akerblom,T.Fridberger, B. Klingeborn,and T. Linne: Identification of two antigenically and genetically distinct lineages of H3N8 equine influenza virus in Sweden. Epidemiol. Infect. 1998, 120:61-70.
42 J.萨姆布鲁克, D. W.拉塞尔.分子克隆实验指南(第三版)[M],科学出版社,2002:98.
43 Studier FW, Rosenberg A H, Dunn J J, et al. Use of T7 RNA polymerase to direct expression of cloned genes[J]. Methods Enzymol.1990, 85:60-89.
44 Robert Mierendorf, Keith Yeager, et al. The pET system:Your chioce for expression[J]. Advanced Products and Protocols for Molecular Biology Reseach, 1994,1(1):32-36.
45 Marston FA, Hartley D L. Solubilization of protein aggregates[J].Methods Enzymol,1990,182:264-276.
46 Dubendorff, Studier. Creation of a T7 autogene Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter[J]. J Mol Biol.1991,219(1):61-68.
47 Vito M, Gabriella E, Nicola D,et al.An outbreak of equine influenza virus in vaccinated horses in Italy is due to an H3N8 strain closely related to recent North American representatives of the Florida sub-lineage[J].Vet Microbiol. 2007, 121 (31):56-63.
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