鸡传染性支气管炎病毒S1和NP基因的表达及NP-ELISA方法的建立
|
摘要
IBV血清型众多,不同血清型间交叉保护差,给诊断与防制带来很大的困难;为建立一种更为简便、快速、群特异性、能用于大面积检测抗体的方法,来补充中和试验和HI试验,以评价疫苗的免疫效果,本研究将对NP-ELISA和S1-ELISA进行探讨。
本研究利用RT-PCR技术成功地扩增了传染性支气管炎病毒M41株的NP基因,克隆并重组于原核表达载体pET-28a,经PCR、酶切鉴定及序列测定成功地构建了重组表达载体pET-NP。重组菌pET-NP经IPTG诱导,获得了高效表达,分子量约为49kDa,且以可溶性蛋白形式存在。重组菌pET-NP经大量诱导表达后,收集菌体,超声波裂解,取上清透析后,采用Ni亲和层析柱纯化重组NP蛋白,经12%的SDS-PAGE分析,表明获得了纯度较高的重组NP蛋白。用纯化的NP蛋白作包被抗原,建立了检测IBV抗体的NP-ELISA。抗原最佳包被浓度为1.45μg/孔,待检血清最佳稀释度为1:80,酶标二抗最佳工作浓度为1:800。确定阳性判定标准为0D450≥0.270。NP-ELISA与AIV H9、H5、H7、IBD、ND、EDS_(76)的标准阳性血清不发生交叉反应,具有良好的特异性,且灵敏度高、重复性好,但试验表明与HI试验、攻毒保护试验结果没有相关性,故不能用于IB免疫效果的评价方法。
本研究利用RT-PCR技术成功地获得了完整的S1基因(S1-1),片段大小为1614bp。在分析全长S1基因的基础上,又扩增了去掉信号肽及包含主要抗原位点的S1-2和S1-3片段,大小分别为1533bp和801bp。将三个片段分别克隆并重组于原核表达载体pGEX-6P-1,构建了pGEX-S1-1、pGEX-S1-2、pGEX-S1-3重组原核表达载体,经PCR、酶切鉴定及序列测定证明三个基因片段均正确地插入到表达载体中,且阅读框正确。三种重组质粒分别转化于宿主菌BL21(DE3),经IPTG进行诱导表达,表达产物经12%的SDS-PAGE电泳分析,结果表明,重组质粒pGEX-S1-1和pGEX-S1-3没有得到有效表达,而重组质粒pGEX-S1-2获得了表达,表达产物以包涵体形式存在,表达产物的分子量约为80kDa,与理论上推测的分子量大小一致。表达产物经Western-blotting分析表明,该表达蛋白能与M41株标准阳性血清反应,说明此表达蛋白具有良好的生物学活性。S1蛋白是最重要的保护性抗原,可诱导机体产生中和、血凝抑制及保护性抗体,通过实现S1蛋白的有效表达,并建立S1-ELISA,探讨S1-ELISA效价与攻毒保护间的关系,有望替代HI试验和中和试验,用于免疫后抗体的检测和疫苗质量的评价,S1蛋白的表达也可为S1蛋白结构与功能的深入研究和基因工程疫苗的研制奠定良好基础。
IBV has many different serotypes and clinical situations. Weak cross protection appear between different serotypes. Resently, IBV seriously hinder the development of poulty industry with the occurrence of new variant strains and serotypes. In order to affords strong specificity, high sensitivity ,excellent coincidence and can apply to detect the field sera samples in bulk and procvide an effective method for diagnosis and immunal monitoring of IB ,and also can incomplement VN test and HI test to evaluate the effect of the vaccine. we will do the research on NP-ELISA and S1-ELISA.
The NP gene was obtained by RT-PCR , which nucleotide sequence is 1614bp in length .Then NP gene was cloned and recombined to the pET28a to contrast the expressed vector pET-NP. pET-NP was proved to be right and was induced by IPTG in bulk and was clearaged by ultrasonic wave to obtained the solubily N protein, The expessed N protein was purified by HisTrap HP Kit. The result demonsrated that the purified NP was suitable to be ELISA antigen. The purified N protein was used to establish an indirect ELISA assay for the detection of antibody againt IBV. The optimal concentration of coating antigen was1.45μg/mL, and the optimal dilution of serum and HRP labeled rabbit anti-chicken was 1:80 and 1:800. The border of negative and positive serum in OD_(450) value was 0.270,when the OD_(450) value was equal or over 0.27,it was positive, or else negative. NP-ELISA had no reaction to the positive serums of AIV H9、H5、H7、ND、IBD and EDS_(76) ,which directed that the expressed N protein was only reacted to the serum of IBV. The result have no relativity bewent NP-ELISA and HI test. The results above indicated the NP-ELISA not only affords strong specificity, high sensitivity .excellent coincidence and can apply to detect the field sera samples in bulk.
Complete S1 gene of Avian infectious bronchitis Virus M41 strain was amplified by RT-PCR, which nucleotide sequence is 1614bp in length. According to analysis of S1 pprotein, two pairs of primers were designed to obtain the 1533bp and 801bp of S1 gene, which one was deletaded the signal peptide and another included important antibody sites. The four fragments were cloned into the pGEM-T easy vector to construct the recombinant clone plasmids. The plasmids were proved to be true by PCR, restriction endonuclease analysis and the analysis of nucleotide sequences. Then the four fragments were recombinated into prokaryotic expression vectors pGEX-6P-1 to construct the expression vectors pGEX-S1-2、pGEX-S1-3, which were identified to be true by PCR, restriction endonuclease analysis and the analysis of nucleotide sequences, and then transformated into E.coli BL21 (DE3). The recombinant expression plasmid was induced by 1mmol/L IPTG at 37℃for 5 hours and analyzed by 12% SDS-PAGE. The results described that the recombined vector (pGEX-S1-2) was lowerly expressed in inclusion body which molecular weight was about 80 KDAa as expected by the analysis of SDS-PAGE. The result of western-blot demensrated the fusion proteins of S1 have partical antigen, which indicats the expression products have clinical value as the diagnosis antigen and gene engineering vaccine.
本研究利用RT-PCR技术成功地扩增了传染性支气管炎病毒M41株的NP基因,克隆并重组于原核表达载体pET-28a,经PCR、酶切鉴定及序列测定成功地构建了重组表达载体pET-NP。重组菌pET-NP经IPTG诱导,获得了高效表达,分子量约为49kDa,且以可溶性蛋白形式存在。重组菌pET-NP经大量诱导表达后,收集菌体,超声波裂解,取上清透析后,采用Ni亲和层析柱纯化重组NP蛋白,经12%的SDS-PAGE分析,表明获得了纯度较高的重组NP蛋白。用纯化的NP蛋白作包被抗原,建立了检测IBV抗体的NP-ELISA。抗原最佳包被浓度为1.45μg/孔,待检血清最佳稀释度为1:80,酶标二抗最佳工作浓度为1:800。确定阳性判定标准为0D450≥0.270。NP-ELISA与AIV H9、H5、H7、IBD、ND、EDS_(76)的标准阳性血清不发生交叉反应,具有良好的特异性,且灵敏度高、重复性好,但试验表明与HI试验、攻毒保护试验结果没有相关性,故不能用于IB免疫效果的评价方法。
本研究利用RT-PCR技术成功地获得了完整的S1基因(S1-1),片段大小为1614bp。在分析全长S1基因的基础上,又扩增了去掉信号肽及包含主要抗原位点的S1-2和S1-3片段,大小分别为1533bp和801bp。将三个片段分别克隆并重组于原核表达载体pGEX-6P-1,构建了pGEX-S1-1、pGEX-S1-2、pGEX-S1-3重组原核表达载体,经PCR、酶切鉴定及序列测定证明三个基因片段均正确地插入到表达载体中,且阅读框正确。三种重组质粒分别转化于宿主菌BL21(DE3),经IPTG进行诱导表达,表达产物经12%的SDS-PAGE电泳分析,结果表明,重组质粒pGEX-S1-1和pGEX-S1-3没有得到有效表达,而重组质粒pGEX-S1-2获得了表达,表达产物以包涵体形式存在,表达产物的分子量约为80kDa,与理论上推测的分子量大小一致。表达产物经Western-blotting分析表明,该表达蛋白能与M41株标准阳性血清反应,说明此表达蛋白具有良好的生物学活性。S1蛋白是最重要的保护性抗原,可诱导机体产生中和、血凝抑制及保护性抗体,通过实现S1蛋白的有效表达,并建立S1-ELISA,探讨S1-ELISA效价与攻毒保护间的关系,有望替代HI试验和中和试验,用于免疫后抗体的检测和疫苗质量的评价,S1蛋白的表达也可为S1蛋白结构与功能的深入研究和基因工程疫苗的研制奠定良好基础。
IBV has many different serotypes and clinical situations. Weak cross protection appear between different serotypes. Resently, IBV seriously hinder the development of poulty industry with the occurrence of new variant strains and serotypes. In order to affords strong specificity, high sensitivity ,excellent coincidence and can apply to detect the field sera samples in bulk and procvide an effective method for diagnosis and immunal monitoring of IB ,and also can incomplement VN test and HI test to evaluate the effect of the vaccine. we will do the research on NP-ELISA and S1-ELISA.
The NP gene was obtained by RT-PCR , which nucleotide sequence is 1614bp in length .Then NP gene was cloned and recombined to the pET28a to contrast the expressed vector pET-NP. pET-NP was proved to be right and was induced by IPTG in bulk and was clearaged by ultrasonic wave to obtained the solubily N protein, The expessed N protein was purified by HisTrap HP Kit. The result demonsrated that the purified NP was suitable to be ELISA antigen. The purified N protein was used to establish an indirect ELISA assay for the detection of antibody againt IBV. The optimal concentration of coating antigen was1.45μg/mL, and the optimal dilution of serum and HRP labeled rabbit anti-chicken was 1:80 and 1:800. The border of negative and positive serum in OD_(450) value was 0.270,when the OD_(450) value was equal or over 0.27,it was positive, or else negative. NP-ELISA had no reaction to the positive serums of AIV H9、H5、H7、ND、IBD and EDS_(76) ,which directed that the expressed N protein was only reacted to the serum of IBV. The result have no relativity bewent NP-ELISA and HI test. The results above indicated the NP-ELISA not only affords strong specificity, high sensitivity .excellent coincidence and can apply to detect the field sera samples in bulk.
Complete S1 gene of Avian infectious bronchitis Virus M41 strain was amplified by RT-PCR, which nucleotide sequence is 1614bp in length. According to analysis of S1 pprotein, two pairs of primers were designed to obtain the 1533bp and 801bp of S1 gene, which one was deletaded the signal peptide and another included important antibody sites. The four fragments were cloned into the pGEM-T easy vector to construct the recombinant clone plasmids. The plasmids were proved to be true by PCR, restriction endonuclease analysis and the analysis of nucleotide sequences. Then the four fragments were recombinated into prokaryotic expression vectors pGEX-6P-1 to construct the expression vectors pGEX-S1-2、pGEX-S1-3, which were identified to be true by PCR, restriction endonuclease analysis and the analysis of nucleotide sequences, and then transformated into E.coli BL21 (DE3). The recombinant expression plasmid was induced by 1mmol/L IPTG at 37℃for 5 hours and analyzed by 12% SDS-PAGE. The results described that the recombined vector (pGEX-S1-2) was lowerly expressed in inclusion body which molecular weight was about 80 KDAa as expected by the analysis of SDS-PAGE. The result of western-blot demensrated the fusion proteins of S1 have partical antigen, which indicats the expression products have clinical value as the diagnosis antigen and gene engineering vaccine.
引文
[1]卡尔尼克Bw.禽病学(第10版)[M].高福,苏敬良主译.北京:中国农业出版社,19%653-665.
[2]殷震,刘景华.动物病毒学(第2版)[M].北京:科学出版社,1997,675-680
[3]何永强,周继勇,于涟,等.应用RT2PCR和RFLP分析肾型鸡传染性支气管炎病毒基因型[J]浙江农业学报,2000,12(3):117-120
[4]Kusters JG,Niesters GM,Bleum,et al.Molecular epidemiologyof infectious bronchitis virus in the Netherlands[J].J Gen Yirol,1987,68:343-352.
[5]常维山.肾型鸡传染性支气管炎病毒的鉴定及其S_1基因核酸分析[D].南京农业大学硕士学位论文,1997,12
[6]陈红英,李新生,崔沛,等.鸡传染性支气管炎肠型病毒的分离与鉴定[J].畜牧兽医进展,1999,18(3):7-10
[7]王红宁,魏雨,周生,等.禽传染性支气管炎病毒中国四川株S1基因的克隆与序列分析.中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集,成都,2002,269-271
[8]Isman MM,Cho KO,Hasoksuz Met al.Antigenic and genomic relatedness of turkey origin toronaviruses,bovine coronaviruses,and infectious bronchitis virus of chickens[J].Yirus Researse,2001,45:978-984
[9]Bingham RW,MadgeMH,Tyrrell DA,et al.Haemagglutination by avian infectious bronchitis virus coronavirus[J].J Gen Virol,1975,28(3):381-90
[10]陈福勇.鸡传染性支气管炎的病原学研究[J].中国兽医杂志,1998,24(11):8-10
[11]吴延功.中国畜牧兽医学会家传染病分会教学专业委员会学术会论文集,1994,66-70
[12]步志高,陈万芳,卢景良.传染性支气管炎病毒结构及免疫原性.国外兽医学一畜禽传染病,1998,75(1):7-10
[13]宁晓檬,金卫红,关智慧,等.鸡传染性支气管炎病毒(IBV)干扰新城疫病毒(NDV)增殖现象的研究[J].中国预防兽医学报,1999,21(2):136-138
[14]赖平安,赵玉喜.鸡肾型传染性支气管炎野毒可干扰新城疫活苗的免疫效果[J].中国预防兽医学报,1995,(3):24-16
[15]江国托,刘思国,康丽娟等.我国鸡传染性支气管炎病毒地方流行毒S1基因的RT-PCR 扩增及其克隆与鉴定,中国兽医杂志,1999,25(4):325.
[16]金梅林,陈焕春,何启盖,等.同胚增殖鸡新城疫病毒和传染性支气管炎病毒的效果观察及免疫应答反应[J].畜牧兽医学报,1999,30(1):1-5
[17]JOHNSON R B,MARQUARDT WW.The neutralizing characteristics of strains of infectious bronchitis virus as measured by the constant virus variable serum method in chicken tracheal culture[J].Avian Dis,1975,19:82,90.
[18]IGNIATOVIC J.Immune responses to structural protein infectious bronchitis virus[J].Avian Pathology,1995,24:313-332
[19]武志强,杨奇伟,付朝阳,等.单抗ELISA在鸡传染性支气管炎病毒分型中的应用[J].中国畜禽传染病,1997,19(3):48-50
[20]江国托,李祥瑞,卢景良.畜禽重大疫病防制研究进展[M].北京:中国农业科技出版社,1996,111-118.
[21]吴全忠,王红宁,廖德惠,等.鸡传染性支气管炎病毒(IBV)的血清学分型研究[J].畜牧兽医学报,1997,28(4):366-371
[22]刘兴友,甘孟侯,李文刚.用间接荧光抗体试验检测鸡肾型传染性支气管炎抗原的研究[J].中国兽医科技,1997,27(6):3-5
[23]吕化广,张洪勇,于中泽,等.禽传染性支气管炎病毒分离毒株的血清型鉴定与免疫防制研究[J].中国兽医杂志,1997,(6):3-4
[24]陈福勇.鸡传染性支气管炎病原学研究[J].中国兽医杂志,1998,24(1):8-10
[25]康佐文,杨增歧,赵余放,等.鸡传染性支气管炎地方分离株血清型的研究[J].中国兽医科技,2000,(5):8-10
[26]王红宁,甘孟侯,王林川,等.禽传染性支气管炎(新变型)综合防治研[J].中国家禽,2003,25(23):49-50
[27]杜元钊,范银成,朱万广,等.产蛋鸡传染性支气管炎病毒变异株的分离鉴定[J].中国畜禽传染病,1995,(6):1-4
[28]荣骏弓,康丽娟,谷守林,等.腺胃病变型鸡传染性支气管炎病毒的分离鉴定[J].中国预防兽医学报,1999,21(2):124-127
[29]马凤龙,傅兴伦,陈国柱,等.腺胃病变型鸡传染性支气管炎病毒的分离鉴定及免疫试验[J].中国兽医学报,2004,24(3):225-227
[30]倪斌,张彦明,王晶钰.鸡肾型传染性支气管炎病毒陕西地方株的分离与鉴定[J].中国动物检疫,2005,22(6):27-28
[31]江国托,刘思国,康丽娟,等.中国鸡传染性支气管炎病毒的变异[J].中国畜禽传染病,1998,20(6):321-323.
[32]王红宁,黄勇,吴全忠,等.鸡传染性支气管炎病毒多毒株交叉保护率及S1基因的比较研究[C].中国畜牧兽医学会禽病学分会第五届会员代表大会暨第九次学术研讨会论文集.北京:中国畜牧兽医学会禽病学分会,1998:51-54.
[33]尹燕博,邹勇,邰友华,等.RT-PCR和RFLP对我国部分传染性支气管炎病毒分离株分型[J].中国兽医学报,1999,(6):535-538
[34]磨美兰,李康然,韦平.RT-PCR及RFLP分析技术对鸡传染性支气管炎病毒的诊断和Sl 基因分型的研究[J].中国预防兽医学报,2001,(4):277-281
[35]Brown TD,Boursnell ME,Binns MM,et al.Cloning and sequencing of 5' terminal sequences from avian infectious bronchitis virus genomic RNA[J].J Gen Virol,1986,67(Pt 2):221-228.
[36]Boursnell ME,Binns MM,et al.Sequencing of coronavirus IBV genomic RNA:three open reading frames in the 5' unique region of mRNA[J].J Gen Virol,1985,66(Pt 10):2253-2258
[37]Lai MM,Liao CL,Lin YJ,et al.Coronavirus:how a large RNA viral genome is replicated and transcribed[J].Infect Agents Dis,1994,3(2-3):98-105
[38]Sutou S,SatoS,Okabe T,et al.Cloning and sequencing of genesencoding structural proteins of avian infectious bronchitis virus[J].Virology,1988,165(2):589-595
[39]张德勇.基于IBV重组表达蛋白的抗体检测技术及基因免疫的研究[D].[博士论文].浙江大学,2005
[40]Cavanagh D,Davis PJ.Coronavirus IBV:removal of spike glycopolypeptidue S1by urea abolishes infectivity and haemagglutination but not attachment to cells[3].3Gen Virol,1986a,67(Pt 7):1443-1448
[41]Cavanagh D,Davis PJ,Darbyshire JH,et al.Coronavirus IBV:virus retaining spike glycopolypeptidu S2 but not S1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody,or induce chicken tracheal protection[J].J Gen Virol,1986b,67(Pt7):1435-1442
[42]谢红梅.鸡传染性支气管炎S1基因在大肠杆菌中的表达及生物活性的初步鉴定[D]山东农业大学硕士学位论文,2006,6
[43]Cavanagh D,Davis P 3,Cook J K A,et al.Location of the amino acid differences in the S1 spike glyco-protein subunit of closely related serotypes of infectious bronchitis virus[J].Avlan Pathol,1992b,21:33-43
[44]亓丽红,李晓红,王春,等.鸡传染性支气管炎病毒的分子生物学研究进展[J].家禽科学,2005,7:45-48
[45]吴清民,汪明.动物冠状病毒的研究进展[J].中国农业科技导报,2003,(4):17-23
[46]Cavanagh D.Coronavirus IBV:Structural characterization of the spike protein[J].y Gen Virol,1983,64:2577-2583
[47]徐晓静,乌日罕,王立声.鸡传染性支气管炎分子生物学研究进展[J].畜牧与饲料科学,2005,:228-30
[48]刘胜旺.鸡传染性支气管炎病毒LX4株mRNA5和mRNA6 cDNA的分子特征[J].中国病毒学,2003,18(3):265-270
[49]Shen S,Law Y C,Liu D X.A single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into Virions at the nonpermissive temperature[J].Virology,2004,326:288-298
[50]Kusters JG,Jager EJ,Niesters HG,et al.Sequences evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virusv[J].Yaccine,1990,8(11):605-608
[51]Collisson E W,Parr R L,Li W,et al.An overview of the molec μ Lar characteristics of avian infectious bronchitis virus[J].Poμ Ltry Science Rev.1992,(4):41-55
[52]Kant A,Koch G,Van R D,et al.Location of antigenic sites defined by neutralizing monoclonal antibodies on the S1 avian infectious bronchitis virus glycopolypeptide[J].Gen Virol,1992,73(3):591-596
[53]刘美丽,张兴晓,杨灵芝,等.禽传染性支气管炎病毒S基因的研究进展[J].动物医学进展,2004,25(5):15-17
[54]Koch G.Antigenic domains on the poplomer protein of avian infectious bronchitis virus:correlation with biological functions[J],J Gen Vrol.1990,71:1923-1935
[55]Kuster JG,Niesters HG,Lenstra JA,et al.Phylogeny of antigenic variants of avian coronavirus IBV[J].Viro10gy,1989,169(1):217-221
[56]KaracaK,Naqi S.A monoclonal antibody blocking ELISA to detect serotype-specific infectious bronchitis virus antibodies[J].Vet Microbiol.1993 Mar;34(3):249-57.
[57]Kant A,Koch G,van Roozelaar DJ,et al.Location of antigenic sites defined by neutralizing monoclonal antibodies on the S1 avian infectious bronchitis virus glycopolypeptide[J].J Gen Virol.1992 Mar;73(Pt 3):591-6.
[58]吕广化,张红勇,于中泽,等.中国兽医杂志[J].1997,19(16):3-4
[59]Cavanagh D,Mawditt K,Adzhar A,et al.Does IBV change slowly despite the capacity of the spike protein to vary greatly?[J]Adv Exp Med Biol.1998;440:729-734.
[60]Keeler CL Jr,Reed KL,Nix WA,et al.Serotype identification of avian infectious bronchitis virus by RT-PCR of the peplomer(S-1)gene[J].Avian Dis.1998 Apr-Jun;42(2):275-84.
[61]Kwon HM,Jackwood MW.Molecular cloning and sequence comparison of the S1 glycoprotein of the Gray and JMK strains of avian infectious bronchitis virus[J].Virus Genes.1995Feb;9(3):219-29.
[62]Huang YP,Lee HC,Cheng MC,et al.S1 and N gene analysis of avian infectious bronchitis viruses in Taiwan[J].Avian Dis.2004 Sep;48(3):581-9.
[63]柏佳,马同锁,赵宝华,等.鸡传染性支气管炎X毒株S1基因的克隆与真核表达[J].中国兽医学报,2006,26,(2):122-125
[64]焦红梅,焦新安,殷月兰,等.鸡传染性支气管炎病毒S1基因的真核表达及其产物的 免疫原性[J].扬州大学学报(农业与生命科学版),2005,27(2):44-47
[65]Wang CH,Hong CC,SeakJC,etal.AnELISAforantibodies against infectious bronchitis virus usingan S1 spikepolypeptide[J].VetMicrobiol.2002Apr2;85(4):333-42.
[66]Seah JN,Yu L,Kwang J.Localization of linear B-cell epitopes on infectious bronchitis virus nucleocapsid protein[J].Vet Microbiol,2000,75(1):11-16
[67]刘娟,吉文汇,鸡传染支气管炎研究进展[J].畜禽业,2007,214(2:8-9
[68]Zhou M,Williams Ak,Chung SI,et al.The infectious bronchitis virus nucleocapsid proteins binds RNA sequence in the 3' terminus of genome[J].Virol,1996,217(1):191-199.
[69]Ignjatovic J,Ashton DF,Reece R,et al.Pathogenicity of Australian strains of avian infectious bronchitis virus[J].J Comp Paehol,2002,126(2-3):115-123
[70]Boots A M,Van Lierop MJ.MHC class Ⅱ-restricted T-cell hybridomas recognizing the nucleocapsid protein of avian coronavirus IBV[J].Immunol,1991,72(1):10-14
[71]Seo SH,Collisson EW.Specific cytotoxic T lymphocytes are involved in in vivo clearance of infectious bronchitis virus[J].J Virol,1997,71(7):5173-5177
[72]Collisson EW,Pei J,Dzielawa J,et al.Cytotoxic T lymphocytes are critical in the control of infectious bronchitis virus in poultry[J].Dev Comp Immunol,2000,24(2-3):187-200
[73]王宏俊,陈福勇.利用IBV重组N蛋白建立EL ISA抗体检测试剂盒的研究[J]中国兽医杂志,2003,39(9):3-6
[74]刘思国,康丽娟,江国托,等.鸡传染性支气管炎病毒的快速检测[J].中国预防兽医学报,2002,24(5):386-387
[75]朱建国,张斌,陆苹.检测感染鸡体内传染性支气管炎病毒反转录套式PCR方法的建立上海交通大学学报(农业科学版)[J].2004,22(2):126-129
[76]Strauss E G,Strauss J H.RNAviruses:genome structure and evolution[J].Curr Opin Genet Dev,1991,1(4):485-493.
[77]Domingo E.Rapid evolutionof viral RNA genomes[J].J Nutr,1997,127(5):958-961.
[78]张国庆,刘晶,李广兴,等.鸡传染性支气管炎病毒变异机制的研究进展[J].中国病毒学,2004,19(2):192-196
[79]Jia W,Naqi S A.Sequence analysis of gene 3,gene 4 and gene 5 of avian infectious bronchitis virus strain CU-T2[J].Gene,1997,189(2):189-193.
[80]Smati R,Silim A,Guertin C,et al.Molecular characterization of three new avian infectious bronchitis virus(IBV)t rains isolated in Quebec[J].Virus Genes,2002,25(1):85-93
[81]Lee C W,Jackwood MW.Origin and evolution of Georgia 98(GA98),a new serotype of avian infectious bronchitis virus[J].Virus Res,2001,80(1-2):33-39
[82]Moore K M,Bennett J D,Seal B S,et al.Sequence comparison of avian infectious bronchitis virus S1 glycoproteins of the Florida serotype and five variant isolates from Georgia and California[J].Virus Genes,1998,17(1):63-83
[83]Kusters J G,Niesters H G,Bleumink-Pluym NM,et al.Molecular epidemiology of infectious bronchitis virus in The Netherlands[J].J Gen Virol,1987,68(2):343-352.
[84]Sapats S1,Ashton F,Wright PJ,et al.Novel variation in the N protein of avian infectious bronchitis virus[J].Virology,1996,226(2):412-417
[85]Shieh HK,Shien JH,thou HY,et al.Complete nucleotide sequences of S1 and N genes of infectious bronchitis virus isolated in Japan and Taiwan[J].Vet Med Sci,2004,66(5):555-558
[86]Park JY,Pak SI,Sung H W,et al.Variations in t he nucleocapsid protein gene of infectious bronchitis viruses isolated in Korea[J].Yirus Genes,2005,31(2):153-162
[87]Kusters JG,Jager EJ,Niesters HG,et al.Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus[J].Vaccine,1990 8(6):605-8.
[88]Jia W,Karaca K,Parrish C R.et al.Anovel variant of avian infectious bronchitis virus res μ Lting f tom recombination among three different strains[J].Arch Yirol,1995,40:259-271
[89]Wang L,Junker D,Collisson EW.Evidence of natural recombination within the S1 gene of infectious bronchitis virus[J].Virology,1993,192(2):710-716
[90]Yu L,JiangY,LowS,et al.Characterizationof three infectious bronchitis virus isolates from China associated with proventriculus in vaccinated chickens[J].Avian Dis,2001,45(2):416-424
[91]Farsang A,Ros C,Renst rom LH,et al.Molecular epizootiology of infectious bronchitis virus in Sweden indicating t he involvement of a vaccine strain[J].Avian Pat hol,2002,31(3):229-236
[92]陈德胜,潘杰彦,曹瑞兵,等.传染性支气管炎病毒(IBV)国内分离毒株的分子流行病学分析[J].病毒学报,2003,19(2):149-153
[93]马德星,刘胜旺,李广兴.鸡传染性支气管炎病毒HN株5a 5b及N基因的遗传变异分析[J].中国兽医科技,2005,35(5):357-362.
[94]张庆霞,韩宗玺,邵昱昊,等.中国2000-2004年鸡传染性支气管炎病毒地方分离株核蛋白基因的遗传变异分析[J].中国病毒学,2006,21(6):575-580
[95]潘盛,王红宁,周生,等.鸡传染性支气管炎病毒遗传变异研究进展[J].动物医学进展,2006,27(8):8-12
[96]许金俊,朱国强,许益民.鸡传染性腺胃病(暂定名)的初步调查[J].中国兽医杂志,1997,23(11):11-12
[97]Lohr JE.Diagnosis of infectious bronchitis(IB)by examination of tracheal mucus for IB-precipitatingantigens[J].Avian Dis,1981,25(4):1058-1064
[98]GoughRE,Randall CJ,DaglessM,et al.Anew strainof infectious bronchitis virus infecting domestic fowl in Great Britain[J].Vet Rec,1992,130(22):493-494
[99]封文海,陈德威.琼脂凝胶扩散试验检测鸡传染性支气管炎抗体[J].中国兽医杂志,1993,19(12):6-7
[100]王红宁.中国畜牧兽医学会禽病学分会学术会论文集.1996,150
[101]Bingham RW,Madge MH,Tyrrell DA.Haemagglutination by avian infectious bronchitis virus-a coronavirus[J].J Gen Virol,1975,28(3):381-390
[102]Alexander DJ.A standard technique for hemagglutination inhibition test for antibodies to avian Infectious Bronchitis Virus[J].Vet Rec,1983(3):64
[103]曹洪敬,颜世敢,王文志,等.微量血凝抑制试验检测鸡IBV抗体水平的研究[J]山东农业科学,1999,1:44-45
[104]张建武.鸡传染性支气管炎血凝抑制(HI)试验的研究和应用[D]河南农业大学硕士学位论文,2002
[105]封文海,陈德威.应用间接荧光抗体染色法监测鸡传染性支气管炎抗体的研究[J].畜牧兽医学报,1994,25(2):168-73
[106]朱建国,焦新安,张如宽,等.用单克隆抗体介导的荧光抗体技术检测鸡尿囊细胞内传染性支气管炎病毒的研究[J].中国畜禽传染病,1996,1:6-8
[107]刘兴友,甘孟侯.胰酶分散抗原间接荧光技术抗体法检测鸡肾型传染性支气管炎病毒及其抗体的研究[J].中国兽医杂志,1997a,23(5):3-5
[108]刘兴友,甘孟侯.用间接荧光抗体试验检测鸡肾型传染性支气管炎抗原的研究[J].中国兽医科技,1997b,27(6):3-5
[109]刘思国,尹训南,李广兴,等.间接荧光抗体法对鸡传染性支气管炎病毒进行抗原定位[J]预防兽医学报,2000,22(suppl):82-84
[110]李锋.鸡传染性支气管炎间接ELISA方法的研究[J].中国畜禽传染病,1991,(1):29-32
[111]许兰菊.应用间接酶联免疫吸附试验检测鸡传染性支气管炎抗体的研究[J],中国兽医科技,1997,27(2):3-5
[112]王君玮,尹燕博,刘清河,等.鸡传染性支气管炎ELISA抗体检测试剂盒的研制[J].中国动物检疫,2004,21(3):27-29.
[113]Ndifuna A,Waters AK,Zhou M,et al.Recombinant nucleocapsid protein is potentially an inexpensive,effective serodiagnostic reagent for infectious bronchitis virus[J].J Virol Methods,1998,70(1):37-44
[1114]Wang CH,Hong CC,Seak JC.An ELISA for antibodies against infectious bronchitis virus using an S1 spike polypeptide[J].Vet Microbiol,2002,86(4):333-42
[115]柳滨东.单抗夹心ELISA法检测鸡传染性支气管炎病毒的研究[J]中国畜禽传染病,1997,1:36-39
[116]云长晔,郭丙全.Dot-ELISA间接法检测鸡传染性支气管炎病毒的研究[J].山东畜牧兽医,2000,6:7-8
[117]阎芳,李宏全,邱剑阳.ABC-Dot-ELISA检测鸡传染性支气管炎病毒[J].中国兽医科技,2000,30(4):21-23
[118]Darbyshire J H,Rowell J G,Cook J K A,et al.Using neutralization tests in tracheal organ cultures[J].Archives of Virology,1979,61:227-238
[119]吴全忠.鸡传染性支气管炎病毒(IBV)的血清学分型研究[J].畜牧兽医学报,1997,28(4):366-371
[120]卢伟东,王正党,李晓成,等.斑点免疫金银染色法检测鸡传染性支气管炎病毒抗原的研究[J].中国兽医科技,1999,(4):3-4
[121]王泽霖.用单抗介导的交叉ELISA对IBV毒株的分型研究[J].河南畜牧兽医,2001,119:52-55
[122]王泽霖,王新卫,刘守川,等.银加强金标免疫技术在鸡传染性支气管炎病毒检测中的应用[J].河南农业大学学报,2005,39(2):201-205
[123]Keeler CL,Reed KL,Nix WA,et al.Serotype identification of infectious bronchitis virus by RT-PCR of the S1 gene[J].Avian Dis,1998,42(2):275-84
[124]王林川,刘福安.禽传染性支气管炎病毒免疫原基因组的RT-PCR研究[J].中国兽医杂志,1994,20(8):3-4
[125]刘滨东,刘思国,袁秀芳,等.PCR结合分子杂交法检测鸡传染性支气管炎病毒[J].中国兽医学报,1997,17(5):431-433
[126]刘思国,江国托,康丽娟,等.PCR方法快速检测鸡传染性支气管炎病毒[J].动物医学进展,1999,20(3):30-33
[127]赵建梅,魏荣,王志亮,等.一步法多重RT-PCR检测新城疫、禽流感、传染性支气管炎病毒试验的研究[J].中国动物检疫,2003,(1):22-24
[128]祝玉卿,曾祥伟,张芳,等.呼吸型鸡传染性支气管炎病毒的抗原定位与动态分布[J].现代畜牧兽医,2005,(4):6-8
[129]Kwon HM,Jackwood WM,Brown TP,et al.Polymerase chain reaction and a biotin-labeled DNA probe for detection of infectious bronchitis virus in chickens[J].Avian Dis,1993,37(1):149-56.
[130]王泽霖,王丽,闰若潜,等.用地高辛标记的核酸探针从尿囊液和组织中检测传染性支气管炎病毒[J].中国预防兽医学报,2000,22(1):64-66
[131]莫胜兰,刘惠莉,陆承平.核酸探针检测鸡传染性支气管炎病毒方法的建立及应用[J].中国兽医学报,2007,27(2):168-171
[132]磨美兰,李康然,韦平,等.RT-PCR及RFLP分析技术对鸡传染性支气管炎病毒的诊断和S1基因分型的研究[J].中国预防兽医学报,2001,23(4):277-281
[133]磨美兰,韦平,李康然,等.应用RT-PCR及RFLP技术对鸡传染性支气管炎病毒进行诊断与分型的研究进展[J].广西农业生物科学,2000,19(3):210-215
[134]Ndiuna N,Arna K W,Zhou M,et al.Recombinant nucleocapsid protein is potentially an inexpensive serodiagnostic reagent for IBV[J].J Virol Meth,1998,70:37-44.
[135]杜恩岐,刘胜旺1,孔宪刚,等.鸡传染性支气管炎病毒核蛋白基因在大肠杆菌中的表达及抗原性初步研究[J]病毒学报,2003,19(4):342-347
[136]杨叶民,林刚,孙涛,等.鸡传染性支气管炎病毒核衣壳蛋白基因的克隆、表达及应用[J]上海交通大学学报(农业科学版),2006,24(3):281-285
[137]沈行燕.传染性支气管炎病毒S基因的克隆及在原核细胞中的表达[D].浙江大学硕士学位论文,2001.6
[138]李中华传染性支气管炎病毒纤突蛋白、核蛋白基因的克隆、表达及其在抗体检测中的初步应用[D].华中农业大学硕士学位论文,2006,6
[139]贾鸿莲,孙效彪,杨子森,等.传染性支气管炎病毒TY1株结构蛋白基因部分片段的表达[J].中国兽医科学,2006,36(2):103-106
[140]谢红梅,张兴晓,朱来华,等.鸡传染性支气管炎病毒S1基因在大肠杆菌中的表达及其抗原性的初步鉴定[J].农业生物技术学报,2007,15(1):107-111
[141]时成波,吕安国.改造稀有密码子提高SEA蛋白表达量[J].生物工程学报,2002,18(4):477-480
[2]殷震,刘景华.动物病毒学(第2版)[M].北京:科学出版社,1997,675-680
[3]何永强,周继勇,于涟,等.应用RT2PCR和RFLP分析肾型鸡传染性支气管炎病毒基因型[J]浙江农业学报,2000,12(3):117-120
[4]Kusters JG,Niesters GM,Bleum,et al.Molecular epidemiologyof infectious bronchitis virus in the Netherlands[J].J Gen Yirol,1987,68:343-352.
[5]常维山.肾型鸡传染性支气管炎病毒的鉴定及其S_1基因核酸分析[D].南京农业大学硕士学位论文,1997,12
[6]陈红英,李新生,崔沛,等.鸡传染性支气管炎肠型病毒的分离与鉴定[J].畜牧兽医进展,1999,18(3):7-10
[7]王红宁,魏雨,周生,等.禽传染性支气管炎病毒中国四川株S1基因的克隆与序列分析.中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集,成都,2002,269-271
[8]Isman MM,Cho KO,Hasoksuz Met al.Antigenic and genomic relatedness of turkey origin toronaviruses,bovine coronaviruses,and infectious bronchitis virus of chickens[J].Yirus Researse,2001,45:978-984
[9]Bingham RW,MadgeMH,Tyrrell DA,et al.Haemagglutination by avian infectious bronchitis virus coronavirus[J].J Gen Virol,1975,28(3):381-90
[10]陈福勇.鸡传染性支气管炎的病原学研究[J].中国兽医杂志,1998,24(11):8-10
[11]吴延功.中国畜牧兽医学会家传染病分会教学专业委员会学术会论文集,1994,66-70
[12]步志高,陈万芳,卢景良.传染性支气管炎病毒结构及免疫原性.国外兽医学一畜禽传染病,1998,75(1):7-10
[13]宁晓檬,金卫红,关智慧,等.鸡传染性支气管炎病毒(IBV)干扰新城疫病毒(NDV)增殖现象的研究[J].中国预防兽医学报,1999,21(2):136-138
[14]赖平安,赵玉喜.鸡肾型传染性支气管炎野毒可干扰新城疫活苗的免疫效果[J].中国预防兽医学报,1995,(3):24-16
[15]江国托,刘思国,康丽娟等.我国鸡传染性支气管炎病毒地方流行毒S1基因的RT-PCR 扩增及其克隆与鉴定,中国兽医杂志,1999,25(4):325.
[16]金梅林,陈焕春,何启盖,等.同胚增殖鸡新城疫病毒和传染性支气管炎病毒的效果观察及免疫应答反应[J].畜牧兽医学报,1999,30(1):1-5
[17]JOHNSON R B,MARQUARDT WW.The neutralizing characteristics of strains of infectious bronchitis virus as measured by the constant virus variable serum method in chicken tracheal culture[J].Avian Dis,1975,19:82,90.
[18]IGNIATOVIC J.Immune responses to structural protein infectious bronchitis virus[J].Avian Pathology,1995,24:313-332
[19]武志强,杨奇伟,付朝阳,等.单抗ELISA在鸡传染性支气管炎病毒分型中的应用[J].中国畜禽传染病,1997,19(3):48-50
[20]江国托,李祥瑞,卢景良.畜禽重大疫病防制研究进展[M].北京:中国农业科技出版社,1996,111-118.
[21]吴全忠,王红宁,廖德惠,等.鸡传染性支气管炎病毒(IBV)的血清学分型研究[J].畜牧兽医学报,1997,28(4):366-371
[22]刘兴友,甘孟侯,李文刚.用间接荧光抗体试验检测鸡肾型传染性支气管炎抗原的研究[J].中国兽医科技,1997,27(6):3-5
[23]吕化广,张洪勇,于中泽,等.禽传染性支气管炎病毒分离毒株的血清型鉴定与免疫防制研究[J].中国兽医杂志,1997,(6):3-4
[24]陈福勇.鸡传染性支气管炎病原学研究[J].中国兽医杂志,1998,24(1):8-10
[25]康佐文,杨增歧,赵余放,等.鸡传染性支气管炎地方分离株血清型的研究[J].中国兽医科技,2000,(5):8-10
[26]王红宁,甘孟侯,王林川,等.禽传染性支气管炎(新变型)综合防治研[J].中国家禽,2003,25(23):49-50
[27]杜元钊,范银成,朱万广,等.产蛋鸡传染性支气管炎病毒变异株的分离鉴定[J].中国畜禽传染病,1995,(6):1-4
[28]荣骏弓,康丽娟,谷守林,等.腺胃病变型鸡传染性支气管炎病毒的分离鉴定[J].中国预防兽医学报,1999,21(2):124-127
[29]马凤龙,傅兴伦,陈国柱,等.腺胃病变型鸡传染性支气管炎病毒的分离鉴定及免疫试验[J].中国兽医学报,2004,24(3):225-227
[30]倪斌,张彦明,王晶钰.鸡肾型传染性支气管炎病毒陕西地方株的分离与鉴定[J].中国动物检疫,2005,22(6):27-28
[31]江国托,刘思国,康丽娟,等.中国鸡传染性支气管炎病毒的变异[J].中国畜禽传染病,1998,20(6):321-323.
[32]王红宁,黄勇,吴全忠,等.鸡传染性支气管炎病毒多毒株交叉保护率及S1基因的比较研究[C].中国畜牧兽医学会禽病学分会第五届会员代表大会暨第九次学术研讨会论文集.北京:中国畜牧兽医学会禽病学分会,1998:51-54.
[33]尹燕博,邹勇,邰友华,等.RT-PCR和RFLP对我国部分传染性支气管炎病毒分离株分型[J].中国兽医学报,1999,(6):535-538
[34]磨美兰,李康然,韦平.RT-PCR及RFLP分析技术对鸡传染性支气管炎病毒的诊断和Sl 基因分型的研究[J].中国预防兽医学报,2001,(4):277-281
[35]Brown TD,Boursnell ME,Binns MM,et al.Cloning and sequencing of 5' terminal sequences from avian infectious bronchitis virus genomic RNA[J].J Gen Virol,1986,67(Pt 2):221-228.
[36]Boursnell ME,Binns MM,et al.Sequencing of coronavirus IBV genomic RNA:three open reading frames in the 5' unique region of mRNA[J].J Gen Virol,1985,66(Pt 10):2253-2258
[37]Lai MM,Liao CL,Lin YJ,et al.Coronavirus:how a large RNA viral genome is replicated and transcribed[J].Infect Agents Dis,1994,3(2-3):98-105
[38]Sutou S,SatoS,Okabe T,et al.Cloning and sequencing of genesencoding structural proteins of avian infectious bronchitis virus[J].Virology,1988,165(2):589-595
[39]张德勇.基于IBV重组表达蛋白的抗体检测技术及基因免疫的研究[D].[博士论文].浙江大学,2005
[40]Cavanagh D,Davis PJ.Coronavirus IBV:removal of spike glycopolypeptidue S1by urea abolishes infectivity and haemagglutination but not attachment to cells[3].3Gen Virol,1986a,67(Pt 7):1443-1448
[41]Cavanagh D,Davis PJ,Darbyshire JH,et al.Coronavirus IBV:virus retaining spike glycopolypeptidu S2 but not S1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody,or induce chicken tracheal protection[J].J Gen Virol,1986b,67(Pt7):1435-1442
[42]谢红梅.鸡传染性支气管炎S1基因在大肠杆菌中的表达及生物活性的初步鉴定[D]山东农业大学硕士学位论文,2006,6
[43]Cavanagh D,Davis P 3,Cook J K A,et al.Location of the amino acid differences in the S1 spike glyco-protein subunit of closely related serotypes of infectious bronchitis virus[J].Avlan Pathol,1992b,21:33-43
[44]亓丽红,李晓红,王春,等.鸡传染性支气管炎病毒的分子生物学研究进展[J].家禽科学,2005,7:45-48
[45]吴清民,汪明.动物冠状病毒的研究进展[J].中国农业科技导报,2003,(4):17-23
[46]Cavanagh D.Coronavirus IBV:Structural characterization of the spike protein[J].y Gen Virol,1983,64:2577-2583
[47]徐晓静,乌日罕,王立声.鸡传染性支气管炎分子生物学研究进展[J].畜牧与饲料科学,2005,:228-30
[48]刘胜旺.鸡传染性支气管炎病毒LX4株mRNA5和mRNA6 cDNA的分子特征[J].中国病毒学,2003,18(3):265-270
[49]Shen S,Law Y C,Liu D X.A single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into Virions at the nonpermissive temperature[J].Virology,2004,326:288-298
[50]Kusters JG,Jager EJ,Niesters HG,et al.Sequences evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virusv[J].Yaccine,1990,8(11):605-608
[51]Collisson E W,Parr R L,Li W,et al.An overview of the molec μ Lar characteristics of avian infectious bronchitis virus[J].Poμ Ltry Science Rev.1992,(4):41-55
[52]Kant A,Koch G,Van R D,et al.Location of antigenic sites defined by neutralizing monoclonal antibodies on the S1 avian infectious bronchitis virus glycopolypeptide[J].Gen Virol,1992,73(3):591-596
[53]刘美丽,张兴晓,杨灵芝,等.禽传染性支气管炎病毒S基因的研究进展[J].动物医学进展,2004,25(5):15-17
[54]Koch G.Antigenic domains on the poplomer protein of avian infectious bronchitis virus:correlation with biological functions[J],J Gen Vrol.1990,71:1923-1935
[55]Kuster JG,Niesters HG,Lenstra JA,et al.Phylogeny of antigenic variants of avian coronavirus IBV[J].Viro10gy,1989,169(1):217-221
[56]KaracaK,Naqi S.A monoclonal antibody blocking ELISA to detect serotype-specific infectious bronchitis virus antibodies[J].Vet Microbiol.1993 Mar;34(3):249-57.
[57]Kant A,Koch G,van Roozelaar DJ,et al.Location of antigenic sites defined by neutralizing monoclonal antibodies on the S1 avian infectious bronchitis virus glycopolypeptide[J].J Gen Virol.1992 Mar;73(Pt 3):591-6.
[58]吕广化,张红勇,于中泽,等.中国兽医杂志[J].1997,19(16):3-4
[59]Cavanagh D,Mawditt K,Adzhar A,et al.Does IBV change slowly despite the capacity of the spike protein to vary greatly?[J]Adv Exp Med Biol.1998;440:729-734.
[60]Keeler CL Jr,Reed KL,Nix WA,et al.Serotype identification of avian infectious bronchitis virus by RT-PCR of the peplomer(S-1)gene[J].Avian Dis.1998 Apr-Jun;42(2):275-84.
[61]Kwon HM,Jackwood MW.Molecular cloning and sequence comparison of the S1 glycoprotein of the Gray and JMK strains of avian infectious bronchitis virus[J].Virus Genes.1995Feb;9(3):219-29.
[62]Huang YP,Lee HC,Cheng MC,et al.S1 and N gene analysis of avian infectious bronchitis viruses in Taiwan[J].Avian Dis.2004 Sep;48(3):581-9.
[63]柏佳,马同锁,赵宝华,等.鸡传染性支气管炎X毒株S1基因的克隆与真核表达[J].中国兽医学报,2006,26,(2):122-125
[64]焦红梅,焦新安,殷月兰,等.鸡传染性支气管炎病毒S1基因的真核表达及其产物的 免疫原性[J].扬州大学学报(农业与生命科学版),2005,27(2):44-47
[65]Wang CH,Hong CC,SeakJC,etal.AnELISAforantibodies against infectious bronchitis virus usingan S1 spikepolypeptide[J].VetMicrobiol.2002Apr2;85(4):333-42.
[66]Seah JN,Yu L,Kwang J.Localization of linear B-cell epitopes on infectious bronchitis virus nucleocapsid protein[J].Vet Microbiol,2000,75(1):11-16
[67]刘娟,吉文汇,鸡传染支气管炎研究进展[J].畜禽业,2007,214(2:8-9
[68]Zhou M,Williams Ak,Chung SI,et al.The infectious bronchitis virus nucleocapsid proteins binds RNA sequence in the 3' terminus of genome[J].Virol,1996,217(1):191-199.
[69]Ignjatovic J,Ashton DF,Reece R,et al.Pathogenicity of Australian strains of avian infectious bronchitis virus[J].J Comp Paehol,2002,126(2-3):115-123
[70]Boots A M,Van Lierop MJ.MHC class Ⅱ-restricted T-cell hybridomas recognizing the nucleocapsid protein of avian coronavirus IBV[J].Immunol,1991,72(1):10-14
[71]Seo SH,Collisson EW.Specific cytotoxic T lymphocytes are involved in in vivo clearance of infectious bronchitis virus[J].J Virol,1997,71(7):5173-5177
[72]Collisson EW,Pei J,Dzielawa J,et al.Cytotoxic T lymphocytes are critical in the control of infectious bronchitis virus in poultry[J].Dev Comp Immunol,2000,24(2-3):187-200
[73]王宏俊,陈福勇.利用IBV重组N蛋白建立EL ISA抗体检测试剂盒的研究[J]中国兽医杂志,2003,39(9):3-6
[74]刘思国,康丽娟,江国托,等.鸡传染性支气管炎病毒的快速检测[J].中国预防兽医学报,2002,24(5):386-387
[75]朱建国,张斌,陆苹.检测感染鸡体内传染性支气管炎病毒反转录套式PCR方法的建立上海交通大学学报(农业科学版)[J].2004,22(2):126-129
[76]Strauss E G,Strauss J H.RNAviruses:genome structure and evolution[J].Curr Opin Genet Dev,1991,1(4):485-493.
[77]Domingo E.Rapid evolutionof viral RNA genomes[J].J Nutr,1997,127(5):958-961.
[78]张国庆,刘晶,李广兴,等.鸡传染性支气管炎病毒变异机制的研究进展[J].中国病毒学,2004,19(2):192-196
[79]Jia W,Naqi S A.Sequence analysis of gene 3,gene 4 and gene 5 of avian infectious bronchitis virus strain CU-T2[J].Gene,1997,189(2):189-193.
[80]Smati R,Silim A,Guertin C,et al.Molecular characterization of three new avian infectious bronchitis virus(IBV)t rains isolated in Quebec[J].Virus Genes,2002,25(1):85-93
[81]Lee C W,Jackwood MW.Origin and evolution of Georgia 98(GA98),a new serotype of avian infectious bronchitis virus[J].Virus Res,2001,80(1-2):33-39
[82]Moore K M,Bennett J D,Seal B S,et al.Sequence comparison of avian infectious bronchitis virus S1 glycoproteins of the Florida serotype and five variant isolates from Georgia and California[J].Virus Genes,1998,17(1):63-83
[83]Kusters J G,Niesters H G,Bleumink-Pluym NM,et al.Molecular epidemiology of infectious bronchitis virus in The Netherlands[J].J Gen Virol,1987,68(2):343-352.
[84]Sapats S1,Ashton F,Wright PJ,et al.Novel variation in the N protein of avian infectious bronchitis virus[J].Virology,1996,226(2):412-417
[85]Shieh HK,Shien JH,thou HY,et al.Complete nucleotide sequences of S1 and N genes of infectious bronchitis virus isolated in Japan and Taiwan[J].Vet Med Sci,2004,66(5):555-558
[86]Park JY,Pak SI,Sung H W,et al.Variations in t he nucleocapsid protein gene of infectious bronchitis viruses isolated in Korea[J].Yirus Genes,2005,31(2):153-162
[87]Kusters JG,Jager EJ,Niesters HG,et al.Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus[J].Vaccine,1990 8(6):605-8.
[88]Jia W,Karaca K,Parrish C R.et al.Anovel variant of avian infectious bronchitis virus res μ Lting f tom recombination among three different strains[J].Arch Yirol,1995,40:259-271
[89]Wang L,Junker D,Collisson EW.Evidence of natural recombination within the S1 gene of infectious bronchitis virus[J].Virology,1993,192(2):710-716
[90]Yu L,JiangY,LowS,et al.Characterizationof three infectious bronchitis virus isolates from China associated with proventriculus in vaccinated chickens[J].Avian Dis,2001,45(2):416-424
[91]Farsang A,Ros C,Renst rom LH,et al.Molecular epizootiology of infectious bronchitis virus in Sweden indicating t he involvement of a vaccine strain[J].Avian Pat hol,2002,31(3):229-236
[92]陈德胜,潘杰彦,曹瑞兵,等.传染性支气管炎病毒(IBV)国内分离毒株的分子流行病学分析[J].病毒学报,2003,19(2):149-153
[93]马德星,刘胜旺,李广兴.鸡传染性支气管炎病毒HN株5a 5b及N基因的遗传变异分析[J].中国兽医科技,2005,35(5):357-362.
[94]张庆霞,韩宗玺,邵昱昊,等.中国2000-2004年鸡传染性支气管炎病毒地方分离株核蛋白基因的遗传变异分析[J].中国病毒学,2006,21(6):575-580
[95]潘盛,王红宁,周生,等.鸡传染性支气管炎病毒遗传变异研究进展[J].动物医学进展,2006,27(8):8-12
[96]许金俊,朱国强,许益民.鸡传染性腺胃病(暂定名)的初步调查[J].中国兽医杂志,1997,23(11):11-12
[97]Lohr JE.Diagnosis of infectious bronchitis(IB)by examination of tracheal mucus for IB-precipitatingantigens[J].Avian Dis,1981,25(4):1058-1064
[98]GoughRE,Randall CJ,DaglessM,et al.Anew strainof infectious bronchitis virus infecting domestic fowl in Great Britain[J].Vet Rec,1992,130(22):493-494
[99]封文海,陈德威.琼脂凝胶扩散试验检测鸡传染性支气管炎抗体[J].中国兽医杂志,1993,19(12):6-7
[100]王红宁.中国畜牧兽医学会禽病学分会学术会论文集.1996,150
[101]Bingham RW,Madge MH,Tyrrell DA.Haemagglutination by avian infectious bronchitis virus-a coronavirus[J].J Gen Virol,1975,28(3):381-390
[102]Alexander DJ.A standard technique for hemagglutination inhibition test for antibodies to avian Infectious Bronchitis Virus[J].Vet Rec,1983(3):64
[103]曹洪敬,颜世敢,王文志,等.微量血凝抑制试验检测鸡IBV抗体水平的研究[J]山东农业科学,1999,1:44-45
[104]张建武.鸡传染性支气管炎血凝抑制(HI)试验的研究和应用[D]河南农业大学硕士学位论文,2002
[105]封文海,陈德威.应用间接荧光抗体染色法监测鸡传染性支气管炎抗体的研究[J].畜牧兽医学报,1994,25(2):168-73
[106]朱建国,焦新安,张如宽,等.用单克隆抗体介导的荧光抗体技术检测鸡尿囊细胞内传染性支气管炎病毒的研究[J].中国畜禽传染病,1996,1:6-8
[107]刘兴友,甘孟侯.胰酶分散抗原间接荧光技术抗体法检测鸡肾型传染性支气管炎病毒及其抗体的研究[J].中国兽医杂志,1997a,23(5):3-5
[108]刘兴友,甘孟侯.用间接荧光抗体试验检测鸡肾型传染性支气管炎抗原的研究[J].中国兽医科技,1997b,27(6):3-5
[109]刘思国,尹训南,李广兴,等.间接荧光抗体法对鸡传染性支气管炎病毒进行抗原定位[J]预防兽医学报,2000,22(suppl):82-84
[110]李锋.鸡传染性支气管炎间接ELISA方法的研究[J].中国畜禽传染病,1991,(1):29-32
[111]许兰菊.应用间接酶联免疫吸附试验检测鸡传染性支气管炎抗体的研究[J],中国兽医科技,1997,27(2):3-5
[112]王君玮,尹燕博,刘清河,等.鸡传染性支气管炎ELISA抗体检测试剂盒的研制[J].中国动物检疫,2004,21(3):27-29.
[113]Ndifuna A,Waters AK,Zhou M,et al.Recombinant nucleocapsid protein is potentially an inexpensive,effective serodiagnostic reagent for infectious bronchitis virus[J].J Virol Methods,1998,70(1):37-44
[1114]Wang CH,Hong CC,Seak JC.An ELISA for antibodies against infectious bronchitis virus using an S1 spike polypeptide[J].Vet Microbiol,2002,86(4):333-42
[115]柳滨东.单抗夹心ELISA法检测鸡传染性支气管炎病毒的研究[J]中国畜禽传染病,1997,1:36-39
[116]云长晔,郭丙全.Dot-ELISA间接法检测鸡传染性支气管炎病毒的研究[J].山东畜牧兽医,2000,6:7-8
[117]阎芳,李宏全,邱剑阳.ABC-Dot-ELISA检测鸡传染性支气管炎病毒[J].中国兽医科技,2000,30(4):21-23
[118]Darbyshire J H,Rowell J G,Cook J K A,et al.Using neutralization tests in tracheal organ cultures[J].Archives of Virology,1979,61:227-238
[119]吴全忠.鸡传染性支气管炎病毒(IBV)的血清学分型研究[J].畜牧兽医学报,1997,28(4):366-371
[120]卢伟东,王正党,李晓成,等.斑点免疫金银染色法检测鸡传染性支气管炎病毒抗原的研究[J].中国兽医科技,1999,(4):3-4
[121]王泽霖.用单抗介导的交叉ELISA对IBV毒株的分型研究[J].河南畜牧兽医,2001,119:52-55
[122]王泽霖,王新卫,刘守川,等.银加强金标免疫技术在鸡传染性支气管炎病毒检测中的应用[J].河南农业大学学报,2005,39(2):201-205
[123]Keeler CL,Reed KL,Nix WA,et al.Serotype identification of infectious bronchitis virus by RT-PCR of the S1 gene[J].Avian Dis,1998,42(2):275-84
[124]王林川,刘福安.禽传染性支气管炎病毒免疫原基因组的RT-PCR研究[J].中国兽医杂志,1994,20(8):3-4
[125]刘滨东,刘思国,袁秀芳,等.PCR结合分子杂交法检测鸡传染性支气管炎病毒[J].中国兽医学报,1997,17(5):431-433
[126]刘思国,江国托,康丽娟,等.PCR方法快速检测鸡传染性支气管炎病毒[J].动物医学进展,1999,20(3):30-33
[127]赵建梅,魏荣,王志亮,等.一步法多重RT-PCR检测新城疫、禽流感、传染性支气管炎病毒试验的研究[J].中国动物检疫,2003,(1):22-24
[128]祝玉卿,曾祥伟,张芳,等.呼吸型鸡传染性支气管炎病毒的抗原定位与动态分布[J].现代畜牧兽医,2005,(4):6-8
[129]Kwon HM,Jackwood WM,Brown TP,et al.Polymerase chain reaction and a biotin-labeled DNA probe for detection of infectious bronchitis virus in chickens[J].Avian Dis,1993,37(1):149-56.
[130]王泽霖,王丽,闰若潜,等.用地高辛标记的核酸探针从尿囊液和组织中检测传染性支气管炎病毒[J].中国预防兽医学报,2000,22(1):64-66
[131]莫胜兰,刘惠莉,陆承平.核酸探针检测鸡传染性支气管炎病毒方法的建立及应用[J].中国兽医学报,2007,27(2):168-171
[132]磨美兰,李康然,韦平,等.RT-PCR及RFLP分析技术对鸡传染性支气管炎病毒的诊断和S1基因分型的研究[J].中国预防兽医学报,2001,23(4):277-281
[133]磨美兰,韦平,李康然,等.应用RT-PCR及RFLP技术对鸡传染性支气管炎病毒进行诊断与分型的研究进展[J].广西农业生物科学,2000,19(3):210-215
[134]Ndiuna N,Arna K W,Zhou M,et al.Recombinant nucleocapsid protein is potentially an inexpensive serodiagnostic reagent for IBV[J].J Virol Meth,1998,70:37-44.
[135]杜恩岐,刘胜旺1,孔宪刚,等.鸡传染性支气管炎病毒核蛋白基因在大肠杆菌中的表达及抗原性初步研究[J]病毒学报,2003,19(4):342-347
[136]杨叶民,林刚,孙涛,等.鸡传染性支气管炎病毒核衣壳蛋白基因的克隆、表达及应用[J]上海交通大学学报(农业科学版),2006,24(3):281-285
[137]沈行燕.传染性支气管炎病毒S基因的克隆及在原核细胞中的表达[D].浙江大学硕士学位论文,2001.6
[138]李中华传染性支气管炎病毒纤突蛋白、核蛋白基因的克隆、表达及其在抗体检测中的初步应用[D].华中农业大学硕士学位论文,2006,6
[139]贾鸿莲,孙效彪,杨子森,等.传染性支气管炎病毒TY1株结构蛋白基因部分片段的表达[J].中国兽医科学,2006,36(2):103-106
[140]谢红梅,张兴晓,朱来华,等.鸡传染性支气管炎病毒S1基因在大肠杆菌中的表达及其抗原性的初步鉴定[J].农业生物技术学报,2007,15(1):107-111
[141]时成波,吕安国.改造稀有密码子提高SEA蛋白表达量[J].生物工程学报,2002,18(4):477-480
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |