胸膜肺炎放线杆菌毒素蛋白的克隆表达及多重荧光PCR诊断方法的研究
|
摘要
猪传染性胸膜肺炎(Porcine contagious pleuropneumonia,PCP)是由胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)引起的一种猪的高度接触性传染病,针对胸膜肺炎放线杆菌血清型众多,现有疫苗缺乏交叉保护力,对规模化养猪业造成巨大经济损失的现状,研究开发具有交叉保护力的新型疫苗迫在眉睫。研究发现Apx毒素是APP的主要保护性抗原,可提高疫苗灭活疫苗的保护作用。天然毒素提取产量低、过程繁琐且成本较高,利用体外克隆表达编码Apx毒素结构蛋白的基因,获得的重组蛋白与天然提取的毒素免疫原性无明显差异,可为下一步新型疫苗多肽成分和ELISA诊断抗原的研究做充分准备。ApxⅣ是近年新发现的一个具有APP种属特异性的抗原,只存在于自然感染的动物体内,不会与其它放线杆菌和革兰氏阴性杆菌发生交叉反应。编码ApxⅣ毒素结构蛋白的apxⅣA基因C端特异性和保守性均很强,是首选的诊断抗原和靶基因。
本研究运用蛋白质软件Anthe 5.0分析了APP的两个关键毒力因子ApxⅠA和ApxⅡA的抗原性和亲水性,截取其中主要抗原位点相对集中的核心区域,分别从胸膜肺炎放线杆菌血清1型国内参考株259和血清7型国内参考株265中扩增了apxⅠA和apxⅡA毒素基因,进行体外克隆,构建了原核表达载体pGEX-4T-1/apxⅠA和pET32a/apxⅡA以及真核表达载体pPICZαA/apxⅠA;并用T4连接酶串连apxⅠA和apxⅡA基因,构建了该融合基因的原核表达载体pGEX-4T-1/apxⅠA-apxⅡA;从胸膜肺炎放线杆菌血清1型国内参考株259中扩增apxⅣA毒素C端特异和保守的一段基因,进行体外克隆,构建了原核表达载体pGEX-4T-1/apxⅣA。
以上四个原核表达载体分别在大肠杆菌DH5α、BL21和Top10'F中以IPTG进行诱导表达,ApxⅠA、ApxⅡA、ApxⅠA-ApxⅡA及ApxⅣA在大肠杆菌中均以包涵体形式表达,表达的重组融合蛋白分别占菌体总蛋白的32%、23%、19%和40%,利用溶菌酶和超声波对重组菌进行分次裂解,除去大部分可溶的菌体蛋白,再用Glutathione Sepharose 4B或Ni-NTA对相应产物进行进一步过柱纯化,最终获得的重组融合蛋白纯度在92%～98%之间,Western-blot的结果表明以上蛋白均具有良好的反应原性,可直接用于ELISA诊断抗原或新型疫苗多肽成分的研究。
ApxⅠA的真核表达载体经甲醇诱导,在毕赤酵母GS115中以低水平分泌表达至培养液上清,经40倍浓缩才能检测到目的蛋白,同时从重组酵母总RNA中检测到apxⅠA基因的mRNA,可以证实目的基因发生了转录。经分析,造成apxⅠA基因在毕赤酵母系统中低水平表达的原因可能是:一、组成目的基因的密码子对于毕赤酵母的偏好指数极低;二、大量的酵母稀有密码子使得目的基因中tRNA丰度较低,导致翻译过程出现瓶颈效应;三、目的基因的高AT含量使酵母表达的转录提前终止。
猪传染性胸膜肺炎和猪圆环病毒Ⅱ型在临床上共感染较为普遍,都以侵害呼吸系统为主,根据胸膜肺炎放线杆菌apxⅣA基因和猪圆环病毒Ⅱ型ORF2基因中最特异最保守的片段分别设计引物和荧光标记TaqMan探针,建立了可同时鉴别诊断猪传染性胸膜肺炎和猪圆环病毒Ⅱ型的实时定量荧光PCR方法,与其它经常发生混合感染的猪繁殖呼吸障碍综合征病毒(PRRSV)、猪瘟病毒(HCV)、伪狂犬病毒(PRV)、副猪嗜血杆菌(HP)、多杀性巴氏杆菌、大肠杆菌、链球菌等病原体均无交叉反应。该方法用于检测APP和PCV2 DNA的敏感度分别达到1×10~1拷贝和1×10~0拷贝,并且在两种病原的模板浓度相差10~5个拷贝时仍可以同时被检出。建立了APP和PCV2两种单重的TaqMan探针荧光实时定量PCR检测方法。所建立的诊断方法快速便捷、特异性好、敏感度高,对早期的快速诊断和筛查以及病原的净化具有很高的实用价值。
Porcine contagious pleuropneumonia is a serious contacted infectious disease of porcine caused by Actinobacillus pleuropneumoniae which owns multitude serotype.Based on the deficiency of cross-protection within the serovars of Actinobacillus pleuropneumoniae,exploit an efficiency novel vaccine to reduce the enormous economic loss from this disease was significant.
Previous studies revealed that Apx-toxins including ApxⅠ,ApxⅡand ApxⅢ, the major protective proteins of APP,could be able to elevate protection of inactivated vaccine.The recombinant proteins of Apx-toxins were found to have the same efficiencies with nature proteins.ApxⅣwas species specific protein of APP which was secreted only by animals infected naturally.It was not cross reacted with other species of Actinobacillus or Gram-Negative bacillus.The structural protein of ApxⅣtoxin encoded by C-terminal fragment gene was considered to be a nice diagnostic antigen by recent researches.Since the yields of nature Apx-toxins was limited and the extraction procedure was also complex that new ways were seek to obtain recombinant proteins by bio-techniques.
Intercepted main antigenic determinant regions fromαρχⅠA andαρχⅡA, amplified them from serovar 1 reference 259 and serovar 7 reference 265 to construct prokaryotic expression vectors named pGEX-4T-1/αρχⅠA and pET32a/αρχⅡA,and eukaryotic expression vector named pPICZαA/αρχⅠA. LigatedαρχⅠA andαρχⅡA gene by using T4 ligase,and constructed the prokaryotic expression vectors named pGEX-4T-1/αρχⅠA-αρχⅡA of fusion gene. The C-fragment ofαρχⅣA from serovar 1 reference 259 was amplified to construct prokaryotic expression vectors named pGEX-4T-1/αρχⅣA.
The above recombinant prokaryotic expression vectors were induced by IPTG in E.coli.SDS-PAGE analysis results show that recombinant ApxⅠA, ApxⅡA,ApxⅠA-ApxⅡA and ApxⅣA were expressed in the form of inclusion body,and the recombinant proteins with 32%,23%,19%,40%,respectively, accumulated total amount of bacterial protein and the purity quotients of the fusion proteins were between 92%～98%.Western-blot results revealed that the recombinant proteins have favorable reactionogenicity and were suitable for the clinical diagnosis and a novel vaccine of Actinobacillus pleuropneumoniae infection.
The above recombinant eukaryotic expression vectors of ApxⅠA was expressed in Pichia pastoris by methanol-induced.SDS-PAGE analysis revealed it only was tested after concentrating the supernatant of culture,and the mRNA coding the ApxⅠA was tested by RT-PCR in yeast induced to confirm the proceeding of transcription.After analysis,the low expression ofαρχⅠA gene in Pichia pastoris has several reasons.First,the codon bias index of the target gene for Pichia pastoris was very lowly.Second,the low abundance of tRNA for target gene was leaded to bottle neck effect in translation procedure.Third,the high AT content of target gene induced to premature termination of the transcription.
Co-infection of Porcine contagious pleuropneumonia and porcine circovirus typeⅡoccurred commonly in clinic,and they both attacked respiratory system mainly.A duplex real-time PCR,based on Taqman hybridization probes technology,were developed and applied to detect anctinobacillus pleuropneumoniae and porcine circovirus type 2 in a reaction system.Two pairs of specific primers and two probes labeled with different report groups were designed for detection a 132bp fragment of APPαρχⅣA gene and a 169bp fragment of PCV2 ORF2 gene.The two fragments were ligated with pMD-18T and transformed to E.coli DH5αfor positive plasmid.Two standard curves were developed,and the sensibility for detection of APP DNA and PCV2 DNA were 1×10~1 copies and 1×10~0 copies.The assay was specific for APP and PCV2,and has no cross-reaction to other organisms.Simultaneously, two single real-time PCR assay for APP and for PCV2 were developed.The diagnostic methods developed were highly specific and sensitive,could be used for rapid diagnosis and screening.
本研究运用蛋白质软件Anthe 5.0分析了APP的两个关键毒力因子ApxⅠA和ApxⅡA的抗原性和亲水性,截取其中主要抗原位点相对集中的核心区域,分别从胸膜肺炎放线杆菌血清1型国内参考株259和血清7型国内参考株265中扩增了apxⅠA和apxⅡA毒素基因,进行体外克隆,构建了原核表达载体pGEX-4T-1/apxⅠA和pET32a/apxⅡA以及真核表达载体pPICZαA/apxⅠA;并用T4连接酶串连apxⅠA和apxⅡA基因,构建了该融合基因的原核表达载体pGEX-4T-1/apxⅠA-apxⅡA;从胸膜肺炎放线杆菌血清1型国内参考株259中扩增apxⅣA毒素C端特异和保守的一段基因,进行体外克隆,构建了原核表达载体pGEX-4T-1/apxⅣA。
以上四个原核表达载体分别在大肠杆菌DH5α、BL21和Top10'F中以IPTG进行诱导表达,ApxⅠA、ApxⅡA、ApxⅠA-ApxⅡA及ApxⅣA在大肠杆菌中均以包涵体形式表达,表达的重组融合蛋白分别占菌体总蛋白的32%、23%、19%和40%,利用溶菌酶和超声波对重组菌进行分次裂解,除去大部分可溶的菌体蛋白,再用Glutathione Sepharose 4B或Ni-NTA对相应产物进行进一步过柱纯化,最终获得的重组融合蛋白纯度在92%～98%之间,Western-blot的结果表明以上蛋白均具有良好的反应原性,可直接用于ELISA诊断抗原或新型疫苗多肽成分的研究。
ApxⅠA的真核表达载体经甲醇诱导,在毕赤酵母GS115中以低水平分泌表达至培养液上清,经40倍浓缩才能检测到目的蛋白,同时从重组酵母总RNA中检测到apxⅠA基因的mRNA,可以证实目的基因发生了转录。经分析,造成apxⅠA基因在毕赤酵母系统中低水平表达的原因可能是:一、组成目的基因的密码子对于毕赤酵母的偏好指数极低;二、大量的酵母稀有密码子使得目的基因中tRNA丰度较低,导致翻译过程出现瓶颈效应;三、目的基因的高AT含量使酵母表达的转录提前终止。
猪传染性胸膜肺炎和猪圆环病毒Ⅱ型在临床上共感染较为普遍,都以侵害呼吸系统为主,根据胸膜肺炎放线杆菌apxⅣA基因和猪圆环病毒Ⅱ型ORF2基因中最特异最保守的片段分别设计引物和荧光标记TaqMan探针,建立了可同时鉴别诊断猪传染性胸膜肺炎和猪圆环病毒Ⅱ型的实时定量荧光PCR方法,与其它经常发生混合感染的猪繁殖呼吸障碍综合征病毒(PRRSV)、猪瘟病毒(HCV)、伪狂犬病毒(PRV)、副猪嗜血杆菌(HP)、多杀性巴氏杆菌、大肠杆菌、链球菌等病原体均无交叉反应。该方法用于检测APP和PCV2 DNA的敏感度分别达到1×10~1拷贝和1×10~0拷贝,并且在两种病原的模板浓度相差10~5个拷贝时仍可以同时被检出。建立了APP和PCV2两种单重的TaqMan探针荧光实时定量PCR检测方法。所建立的诊断方法快速便捷、特异性好、敏感度高,对早期的快速诊断和筛查以及病原的净化具有很高的实用价值。
Porcine contagious pleuropneumonia is a serious contacted infectious disease of porcine caused by Actinobacillus pleuropneumoniae which owns multitude serotype.Based on the deficiency of cross-protection within the serovars of Actinobacillus pleuropneumoniae,exploit an efficiency novel vaccine to reduce the enormous economic loss from this disease was significant.
Previous studies revealed that Apx-toxins including ApxⅠ,ApxⅡand ApxⅢ, the major protective proteins of APP,could be able to elevate protection of inactivated vaccine.The recombinant proteins of Apx-toxins were found to have the same efficiencies with nature proteins.ApxⅣwas species specific protein of APP which was secreted only by animals infected naturally.It was not cross reacted with other species of Actinobacillus or Gram-Negative bacillus.The structural protein of ApxⅣtoxin encoded by C-terminal fragment gene was considered to be a nice diagnostic antigen by recent researches.Since the yields of nature Apx-toxins was limited and the extraction procedure was also complex that new ways were seek to obtain recombinant proteins by bio-techniques.
Intercepted main antigenic determinant regions fromαρχⅠA andαρχⅡA, amplified them from serovar 1 reference 259 and serovar 7 reference 265 to construct prokaryotic expression vectors named pGEX-4T-1/αρχⅠA and pET32a/αρχⅡA,and eukaryotic expression vector named pPICZαA/αρχⅠA. LigatedαρχⅠA andαρχⅡA gene by using T4 ligase,and constructed the prokaryotic expression vectors named pGEX-4T-1/αρχⅠA-αρχⅡA of fusion gene. The C-fragment ofαρχⅣA from serovar 1 reference 259 was amplified to construct prokaryotic expression vectors named pGEX-4T-1/αρχⅣA.
The above recombinant prokaryotic expression vectors were induced by IPTG in E.coli.SDS-PAGE analysis results show that recombinant ApxⅠA, ApxⅡA,ApxⅠA-ApxⅡA and ApxⅣA were expressed in the form of inclusion body,and the recombinant proteins with 32%,23%,19%,40%,respectively, accumulated total amount of bacterial protein and the purity quotients of the fusion proteins were between 92%～98%.Western-blot results revealed that the recombinant proteins have favorable reactionogenicity and were suitable for the clinical diagnosis and a novel vaccine of Actinobacillus pleuropneumoniae infection.
The above recombinant eukaryotic expression vectors of ApxⅠA was expressed in Pichia pastoris by methanol-induced.SDS-PAGE analysis revealed it only was tested after concentrating the supernatant of culture,and the mRNA coding the ApxⅠA was tested by RT-PCR in yeast induced to confirm the proceeding of transcription.After analysis,the low expression ofαρχⅠA gene in Pichia pastoris has several reasons.First,the codon bias index of the target gene for Pichia pastoris was very lowly.Second,the low abundance of tRNA for target gene was leaded to bottle neck effect in translation procedure.Third,the high AT content of target gene induced to premature termination of the transcription.
Co-infection of Porcine contagious pleuropneumonia and porcine circovirus typeⅡoccurred commonly in clinic,and they both attacked respiratory system mainly.A duplex real-time PCR,based on Taqman hybridization probes technology,were developed and applied to detect anctinobacillus pleuropneumoniae and porcine circovirus type 2 in a reaction system.Two pairs of specific primers and two probes labeled with different report groups were designed for detection a 132bp fragment of APPαρχⅣA gene and a 169bp fragment of PCV2 ORF2 gene.The two fragments were ligated with pMD-18T and transformed to E.coli DH5αfor positive plasmid.Two standard curves were developed,and the sensibility for detection of APP DNA and PCV2 DNA were 1×10~1 copies and 1×10~0 copies.The assay was specific for APP and PCV2,and has no cross-reaction to other organisms.Simultaneously, two single real-time PCR assay for APP and for PCV2 were developed.The diagnostic methods developed were highly specific and sensitive,could be used for rapid diagnosis and screening.
引文
[1]斯特劳,阿莱尔,蒙加林。猪病学。第8版,北京,中国农业大学出版社,2000:357-367
[2]刘杰.猪传染性胸膜肺炎油佐剂灭活疫苗。畜牧兽医科技信息,2001。(03):40
[3]逯忠新,赵萍,邵英德,等.猪传染性胸膜肺炎三价灭活疫苗的研究--效力、最小免疫量、免疫持续期和保存期试验.中国兽医科技,2002.(05):8-10
[4]Higgins,R.,Lariviere,S.,Mittal,K.R.,et al.Evaluation of a Killed Vaccine Against Porcine Pleuropneumonia Due to Haemophilus pleuropneumoniae.Can Vet J,1985.26(2):86-89.
[5]Prideaux,C.T.,Pierce,L.,Krywult,J.,et al.Protection of mice against challenge with homologous and heterologous serovars of Actinobacillus pleuropneumoniae after live vaccination.Curr Microbiol,1998.37(5):324-32.
[6]Rosendal,S.,Maclnnes,J.I.Characterization of an attenuated strain of Actinobacillus pleuropneumoniae,serotyoe 1.Am J Vet Res,1990.51(5):711-7.
[7]刘建杰,陈焕春,李冲,等.新型基因工程亚单位菌苗对猪传染性胸膜肺炎的保护效力研究.中国农业科学,2005.(03):596-600。
[8]邵美丽,刘思国,王春来,等.猪传染性胸膜肺炎重组亚单位疫苗诱导小鼠免疫保护效果的观察.畜牧兽医学报,2006.(10):1021-1026
[9]Lee,K.Y.,Kim,D.H.,Kang,T.J.,et al.Induction of protective immune responses against the challenge of Actinobacillus pleuropneumoniae by the oral administration of transgenic tobacco plant expressing ApxⅡA toxin from the bacteria.FEMS Immunol Med Microbiol,2006.48(3):381-9.
[10]Shin,S.J.,Bae,J.L.,Cho,Y.W.,et al.Induction of antigen-specific immune responses by oral vaccination with Saccharomyces cerevisiae expressing Actinobaeillus pleuropneumoniae ApxⅡA.FEMS Immunol Med Microbiol,2005.43(2):155-64.
[11]Boekema,B.K.,Kamp,E.M.,Smits,M.A.,et al.Both ApxⅠ and ApxⅡ of Actinobacillus pleuropneumoniae serotype 1 are necessary for full virulence.Vet Microbiol,2004.100(1-2):17-23.
[12]Tascon,R.I.,Vazquez-Boland,J.A.,Gutierrez-Martin,C.B.,et al.The RTX haemolysins ApxⅠ and ApxⅡ are major virulence factors of the swine pathogen Actinobacillus pleuropneumoniae:evidence from mutational analysis.Mol Microbiol,1994.14(2):207-16.
[13]Kamp,E.M.,Stockhofe-Zurwieden,N.,van Leengoed,L.A.,et al.Endobronchial inoculation with Apx toxins of Actinobacillus pleuropneumoniae leads to pleuropneumonia in pigs.Infect Immun,1997.65(10):4350-4.
[14]Reimer,D.,Frey,J.,Jansen,R.,et al.Molecular investigation of the role of ApxⅠ and ApxⅡ in the virulence of Actinobacillus pleuropneumoniae serotype 5.Microb Pathog,1995.18(3):197-209.
[15]Schaller,A.,Kuhn,R,,Kuhnert,P.,et al.Characterization of apxⅣA,a new RTX determinant of Actinobacillus pleuropneumoniae.Microbiology,1999.145(Pt 8)2105-16.
[16]Schaller,A.,Djordjevic,S.P.,Eamens,G.J.,et al.Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxⅣA.Vet Microbiol,2001.79(1):47-62.
[17]斯劳特,阿莱尔,蒙加林。猪病学.北京,中国农业大学出版社,2000:357-367
[18]杨旭夫,彭发泉。我国猪传染性胸膜肺炎嗜血杆菌感染症的确定和诊断.畜牧兽医学报,1990.(3):243-245.
[19]吴家强,崔锦鹏,张秀美,等.猪接触传染性胸膜肺炎研究进展.畜牧与兽医,2002.(10):36-39.
[20]Vigre,H.,Angen,O.,Barfod,K.,et al.Transmission of Actinobacillus pleuropneumoniae in pigs under field-like conditions:emphasis on tonsillar colonisation and passively acquired colostral antibodies.Vet Microbiol,2002.89(2-3):151-9.
[21]Holmgren,N.,Lundeheim,N.,Wallgren,P.Infections with Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae in fattening pigs.Influence of piglet production systems and influence on production parameters.Zentralbl Veterinarmed B,1999.46(8):535-44.
[22]张耀相,杨会霞,李健强。胸膜肺炎放线杆菌生物学特性概述。动物医学进展,2004.(06):140-141.
[23]Jansen,R.,Bdaire,J.,Kamp,E.M.,et al.The CAMP effect of Actinobacillus pleuropneumoniae is caused by Apx toxins.FEMS Microbiol Lett,1995.126(2):139-43.
[24]Nielsen,R.,Andresen,L.O.,Plambeck,T.,et al.Serological characterization of Actinobacillus pleuropneumoniae biotype 2 strains isolated from pigs in two Danish herds.Vet Microbiol,1997.54(1):35-46.
[25]Jacobsen,M.J.,Nielsen,J.P.,Nielsen,R.Comparison of virulence of different Actinobacillus pleuropneumoniae serotypes and biotypes using an aerosol infection model.Vet Microbiol,1996.49(3-4):159-68.
[26]Dubreuil,J.D.,Jacques,M.,Mittal,K.R.,et al.Actinobacillus pleuropneumoniae surface polysaccharides:their role in diagnosis and immunogenicity.Anim Health Res Rev,2000.1(2):73-93.
[27]Nielsen,R.Haemophilus pleuropneumoniae(Actinobacillus pleuropneumoniae).Serotypes 8,3 and 6.Serological response and cross immunity in pigs.Nord Vet Med,1985.37(4):217-27.
[28]Baltes,N.,Tonpitak,W.,Gerlach,G.F.,et al.Actinobacillus pleuropneumoniae iron transport and urease activity:effects on bacterial virulence and host immune response. Infect Immun,2001.69(1):472-8.
[29]Choi,C.,Kwon,D.,Min,K.,et al.Detection and localization of ApxI,-Ⅱ and -Ⅲ genes of Actinobacillus pleuropneumoniae in natural porcine pleuropneumonia in natural porcine pleuropneumonia by in situ hybridization.Vet Pathol,2001.38(4):390-5.
[30]Frey,J.,Bosse,J.T.,Chang,Y.F.,et al.Actinobacillus pleuropneumoniae RTX-toxins:uniform designation of haemolysins,cytolysins,pleurotoxin and their genes.J Gen Microbiol,1993.139(8):1723-8.
[31]Inzana,T.J.,Todd,J.,Veit,H.P.Safety,stability,and efficacy of noncapsulated mutants of Actinobacillus pleuropneumoniae for use in live vaccines.Infect Immun,1993.61(5):1682-6.
[32]Ward,C.K.,Lawrence,M.L.,Veit,H.P.,et al.Cloning and mutagenesis of a serotype-specific DNA region involved in encapsulation and virulence of Actinobacillus pleuropneumoniae serotype 5a:concomitant expression of serotype 5a and 1 capsular polysacchaddes in recombinant A.pleuropneumoniae serotype 1.Infect Immun,1998.66(7):3326-36.
[33]王春来,刘思国,王牟平,等.猪传染性胸膜肺炎放线杆菌血清型7型25-4株ApxⅡCA基因的克隆和基因缺失;中国预防兽医学报,2004。(02):94-97.
[34]张立昌。猪传染性胸膜肺炎研究进展。养猪,2001.(01):40-42。
[35]Frey,J.Virulence in Actinobacillus pleuropneumoniae and RTX toxins.Trends Microbiol,1995.3(7):257-61.
[36]Gygi,D.,Nicolet,J.,Hughes,C.,et al.Functional analysis of the Ca(2+)-regulated hemolysin Ⅰ operon of Actinobacillus pleuropneumoniae serotype 1.Infect Immun,1992.60(8):3059-64.
[37]Lo,R.Y.Molecular characterization of cytotoxins produced by Haemophilus,Actinobacillus,Pasteurella.Can J Vet Res,1990.54 SupplS33-5.
[38]Schaller,A.,Kuhnert,P.,de la Puente-Redondo,V.A.,et al.Apx toxins in Pasteurellaceae species from animals.Vet Microbiol,2000.74(4):365-76.
[39]Jansen,R.,Briaire,J.,Kamp,E.M.,et al.Structural analysis of the Actinobacillus pleuropneumoniae-RTX-toxin Ⅰ(ApxⅠ)operon.Infect Immun,1993.61(9):3688-95.
[40]Seah,J.N.,Frey,J.,Kwang,J.The N-terminal domain of RTX toxin ApxI of Actinobacillus pleuropneumoniae elicits protective immunity in mice.Infect Immun,2002.70(11):6464-7.
[41]李敏,姜国彦,彭永刚,等.猪传染性猪胸膜肺炎APXⅣA毒素特征的研究进展.畜牧兽医科技信息,2004.(09):11-12
[42]Frey,J.,Kuhnert,P.,Villiger,L.,et al.Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins.Res Microbiol,1996.147(5):351-61.
[43]van den Bosch, H., Frey, J. Interference of outer membrane protein PalA with protective immunity against Actinobacillus pleuropneumoniae infections in vaccinated pigs. Vaccine, 2003. 21 (25-26):3601-7.
[44]Gerlach, G F., Anderson, C, Klashinsky, S., et al. Molecular characterization of a protective outer membrane lipoprotein (OmlA) from Actinobacillus pleuropneumoniae serotype 1. Infect Immun, 1993. 61 (2):565-72.
[45]Van Overbeke, I., Chiers, K., Ducatelle, R., et al. Effect of endobronchial challenge with Actinobacillus pleuropneumoniae serotype 9 of pigs vaccinated with a vaccine containing Apx toxins and transferrin-binding proteins. J Vet Med B Infect Dis Vet Public Health, 2001.48 (1):15-20.
[46]Bosse, J. T., Machines, J. I. Genetic and biochemical analyses of Actinobacillus pleuropneumoniae urease. Infect Immun, 1997. 65 (11):4389-94.
[47]Gottschalk, M., De Lasalle, F., Radacovici, S., et al. Evaluation of long chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 5 for the serodiagnosis of swine pleuropneumonia. Vet Microbiol, 1994. 38 (4):315-27.
[48]Radacovici, S., Gottschalk, M., Dubreuil, J. D. Lipopolysaccharides of Actinobacillus pleuropneumoniae (serotype 1): a readily obtainable antigen for ELISA serodiagnosis of pig pleuropneumonia. Vet Microbiol, 1994.39 (3-4):219-30.
[49]Andresen, L. O., Klausen, J., Barfod, K., et al. Detection of antibodies to Actinobacillus pleuropneumoniae serotype 12 in pig serum using a blocking enzyme-linked immunosorbent assay. Vet Microbiol, 2002. 89 (1):61-7.
[50]Klausen, J., Andresen, L. O., Barfod, K., et al. Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum. Vet Microbiol, 2001. 79 (1):11-8.
[51]Nielsen, R., Plambeck, T., Foged, N. T. Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 8. Vet Microbiol, 1993.34(2): 131-8.
[52]Bosse, J. T., Friendship, R., Rosendal, S., et al. Development and evaluation of a mixed-antigen ELISA for serodiagnosis of Actinobacillus pleuropneumoniae serotypes 1, 5, and 7 infections in commercial swine herds. J Vet Diagn Invest, 1993. 5 (3):359-62.
[53]Grondahl-Hansen, J., Barfod, K., Klausen, J., et al. Development and evaluation of a mixed long-chain lipopolysaccharide based ELISA for serological surveillance of infection with Actinobacillus pleuropneumoniae serotypes 2, 6 and 12 in pig herds. Vet Microbiol, 2003. 96 (1):41-51.
[54]Leiner, G, Franz, B., Strutzberg, K., et al. A novel enzyme-linked immunosorbent assay using the recombinant Actinobacillus pleuropneumoniae ApxII antigen for diagnosis of pleuropneumonia in pig herds. Clin Diagn Lab Immunol, 1999. 6 (4):630-2.
[55]Nielsen, R., van den Bosch, J. F., Plambeck, T., et al. Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae.Vet Microbiol,2000.71(1-2):81-7.
[56]王淑敏,冼琼珍,温广杰。PPA-ELISA检测猪传染性胸膜肺炎抗体方法的建立。黑龙江畜牧兽医,2003。(01):37-38.
[57]逯忠新,鲁炳义,赵萍。用间接血凝试验检测猪传染性胸膜肺炎.中国兽医科技,1999。(10):25-27。
[58]Inzana,T.J.Simplified procedure for preparation of sensitized latex particles to detect capsular polysaecharides:application to typing and diagnosis of Actinobaeillus pleuropneumoniae.J Clin Microbiol,1995.33(9):2297-303.
[59]Mittal,K.R.,Higgins,R.,Lariviere,S.Evaluation of slide agglutination and ring precipitation tests for capsular serotyping of Haemophilus pleuropneumoniae.J Clin Microbiol,1982.15(6):1019-23.
[60]Mittal,K.R.,Higgins,R.,Lariviere,S.An evaluation of agglutination and coagglutination techniques for serotyping of Haemophilus pleuropneumoniae isolates.Am J Vet Res,1987.48(2):219-26.
[61]Chiers,K.,Van Overbeke,I.,Donne,E.,et al.Detection of Actinobacillus pleuropneumoniae in cultures from nasal and tonsillar swabs of pigs by a PCR assay based on the nucleotide sequence of a dsbE-like gene.Vet Mierobiol,2001.83(2):147-59.
[62]Frey,J.,Beck,M.,van den Bosch,J.F,et al.Development of an efficient PCR method for toxin typing of Actinobacillus pleuropnettmoniae swains.Mol Cell Probes,1995.9(4):277-82.
[63]Gram,T.,Ahrens,P Improved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nueleotide sequence of an outer membrane lipoprotein.J Clin Microbiol,1998.36(2):443-8.
[64]Jessing,S.G,Angen,O.,Inzana,T.J.Evaluation of a multiplex PCR test for simultaneous identification and serotyping of Actinobacillus pleuropneumoniae serotypes 2,5,and 6.J Clin Microbiol,2003.41(9):4095-100.
[65]Lo,T.M.,Ward,C.K.,Inzana,T.J.Detection and identification of Actinobacillus pleuropneumoniae serotype 5 by multiplex PCR.J.Clin Microbiol,1998.36(6):1704-10.
[66]Rayamajhi,N.,Shin,S.J.,Kang,S.G.,et al.Development and use of a multiplex polymerase chain reaction assay based on Apx toxin genes for genotyping of Actinobacillus pleuropneumoniae isolates.J Vet Diagn Invest,2005.17(4):359-62.
[67]Savoye,C.,Jobert,J.L.,Berthelot-Herault,F.,et al.A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental conditions.Vet Microbiol,2000.73(4):337-47.
[68]Sthitmatee,N.,Sirinarumitr,T.,Makonkewkeyoon,L.,et al.Identification of the Actinobacillus pleuropneumoniae serotype using PCR based-apx genes.Mol Cell Probes,2003.17(6):301-5.
[69]Sirois,M.,Lemire,E.G.,Levesque,R.C.Construction of a DNA probe and detection of Actinobacillus pleuropneumoniae by using polymerase chain reaction.J Clin Microbiol,1991.29(6):1183-7.
[70]Hennessy,K.J.,Iandolo,J.J.,Fenwick,B.W.Serotype identification of Actinobacillus pleuropneumoniae by arbitrarily primed polymerase chain reaction.J Clin Microbiol,1993.31(5):1155-9.
[71]庞耀珊,谢芝勋,刘加波,等.二温式PCR检测猪传染性胸膜肺炎放线杆菌方法的建立与应用.广西农业科学,2006。(02):203-205.
[72]李树清,易建平,陈志飞,等.复合PCR鉴定胸膜肺炎放线杆菌方法的建立及初步应用.微生物学报,2005.(06):966-969.
[73]李树清,易建平,胡永强,等.猪传染性胸膜肺炎放线杆菌的套式PCR检测.中国预防兽医学报,2005.(03):217-222。
[74]Jaglic,Z.,Svastova,P.,Rychlik,I.,et al.Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxⅣA gene and by ribotyping.Vet Microbiol,2004.103(1-2):63-9.
[75]Tumi,C.,Blackall,P.J.An evaluation of the apxⅣA based PCR-REA method for differentiation of Actinobacillus pleuropneumoniae.Vet Microbiol,2007.121(1-2):163-9.
[76]Angen,O.,Jensen,J.,Lavdtsen,D.T.Evaluation of 5' nuclease assay for detection of Actinobacillus pleuropneumoniae.J Clin Microbiol,2001.39(1):260-5.
[77]李树清,易建平,李健,等.胸膜肺炎放线杆菌的实时荧光PCR检测.畜牧兽医学报,2005.(09):969-973.
[78]Cho,W.S.,Choi,C.,Chae,C.In situ hybridization for the detection of the apxⅣ gene in the lungs of pigs experimentally infected with twelve Actinobacillus pleuropneumoniae serotypes.Vet Res,2002.33(6):653-60.
[79]刘杰。猪传染性胸膜肺炎油剂灭活疫苗.畜牧兽医科技信息,2003。(02):49
[80]Hensel,A.,Huter,V.,Katinger,A.,et al.Intramuscular immunization with genetically inactivated(ghosts)Actinobacillus pleuropneumoniae serotype 9 protects pigs against homologous aerosol challenge and prevents carder state.Vaccine,2000.18(26):2945-55.
[81]Liao,C.W.,Cheng,I.C.,Yeh,K.S.,et al.Release characteristics of microspheres prepared by co-spray drying Actinobacillus pleuropneumoniae antigens and aqueous ethyl-cellulose dispersion.J Microencapsul,2001.18(3):285-97.
[82]Liao,C.W.,Chiou,H.Y.,Yeh,K.S.,et al.Oral immunization using formalin-inactivated Actinobacillus pleuropneumoniae antigens entrapped in microspheres with aqueous dispersion polymers prepared using a co-spray drying process.Prev Vet Med,2003.61(1):1-15.
[83]Prideaux,C.T.,Lenghaus,C.,Krywult,J.,et al.Vaccination and protection of pigs against pleuropneumonia with a vaccine strain of Actinobacillus pleuropneumoniae produced by site-specific mutagenesis of the ApxⅡ operon.Infect Immun,1999.67(4):1962-6.
[84]Tonpitak,W.,Baltes,N.,Hennig-Pauka,I.,et al.Construction of an Actinobacillus pleuropneumoniae serotype 2 prototype live negative-marker vaccine.Infect Immun,2002.70(12):7120-5.
[85]Maas,A.,Meens,J.,Baltes,N.,et al.Development of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae infection.Vaccine,2006.24(49-50):7226-37.
[86]贝为成,陈焕春,何启盖,等。猪传染性胸膜肺炎毒素基因工程弱毒活疫苗的研究,全国规模化猪场疫病控制与净化资料专辑,2002:29-32
[87]贝为成,何启盖,方六荣,等。猪传染性胸膜肺炎放线杆菌毒素apxⅡ基因无药物抗性标记突变株HBC--/GFP-+的构建及其生物学特性研究.生物工程学报。2004。(05):719-724
[88]刘霞,刁有祥,禚宝山.猪传染性胸膜肺炎的研究进展。山东农业大学学报(自然科学版),2006.(01):152-156.
[89]Furesz,S.E.,Mallard,B.A.,Bosse,J.T.,et al.Antibody- and cell-mediated immune responses of Actinobacillus pleuropneumoniae-infected and bacterin-vaecinated pigs.Infect Immun,1997.65(2):358-65.
[90]郭洋,刘思国,王春来等。胸膜肺炎放线杆菌菌研究进展。现代畜牧兽医,2006.06:61-64。
[91]王春来,刘思国,邵美丽等.重组ApxⅡA对胸膜肺炎放线杆菌灭活疫苗免疫效果的影响。中国兽医科学,2006.36(06):434-438.
[92]Goethe,R.,Gonzales,O.F,Lindner,T.,et al.A novel strategy for protective Actinobacillus pleuropneumoniae subunit vaccines:detergent extraction of cultures induced by iron restriction.Vaccine,2000.19(7-8):966-75.
[93]Tumamao,J.Q.,Bowles,R.E.,van den Bosch,H.,et al.An evaluation of the role of antibodies to Actinobacillus pleuropneumoniae serovar 1 and 15 in the protection provided by sub-unit and live streptomycin-dependent pleuropneumonia vaccines.Aust Vet J,2004.82(12):773-80.
[94]Tumamao,J.Q.,Bowles,R.E.,van den Bosch,H.,et al.Comparison of the efficacy of a subunit and a live streptomycin-dependent porcine pleuropneumonia vaccine.Aust Vet J,2004.82(6):370-4.
[95]梁雨,田淑芳,刘文军等。核酸免疫可特异地诱发小鼠对乙型肝炎病毒表面抗原S+PreS1免疫反应.病毒学报,1996.(3):193-199
[96]李建伟,赵晓岩,杨建德等.鸡马立克氏病DNA疫苗免疫的初步研究.中国畜禽传染病,1996。(5):9-12.
[97]陈化兰,于康震.DNA免疫诱导鸡对禽流感病毒的免疫保护反应。中国农业科学,1998。31(5):63-68.
[98]贝为成,严琳,何启盖等.猪传染性胸膜肺炎放线杆菌毒素apx ⅡA基因的真核表达及其核酸疫苗.农业生物技术学报,2005。13(5):616-619。
[99]Frey,J.,Nicolet,J.Immunological properties of Actinobacillus pleuropneumoniae hemolysin I.Vet Microbiol,1991.28(1):61-73.
[100]Byrd,W.,Hooke,A.M.Immunization with temperature-sensitive mutants of Actinobacillus pleuropneumoniae induces protective hemolysin-neutralizing antibodies in mice.Curr Microbiol,1997.34(3):149-54.
[101]Haesebrouck,F.,Chiers,K.,Van Overbeke,I.,et al.Actinobacillus pleuropneumoniae infections in pigs:the role of virulence factors in pathogenesis and protection.Vet Microbiol,1997.58(2-4):239-49.
[102]Fischer,B.,Sumner,I.,Goodenough,P.Isolation and renaturation of bio-active proteins expressed in Escherichia coli as inclusion bodies.Arzneimittelforschung,1992.42(12):1512-5.
[103]Burdyehova,R.,Ryehtera,M.,Horvath,R.,et al.Expression of Aetinobaeillus pleuropneumonia gene coding for Apx Ⅰ protein in Eseherichia coli.FEMS Mierobiol Lett,2004.230(1):9-12.
[104]王冬梅,刘磊,逯忠新等。猪胸膜肺炎放线杆菌apxⅠA基因的克隆及其原核表达.中国兽医科技,2005.35(5):373-376.
[105]王芳,何孔旺,邱索平,等.猪传染性胸膜肺炎放线杆菌毒素Ⅰ基因的克隆、序列分析及表达。中国预防兽医学报,2006.(02):121-123.
[106]Frey,J.,van den Bosch,H.,Segers,R.,et al.Identification of a second hemolysin (HlyⅡ)in Actinobacillus pleuropneumoniae serotype 1 and expression of the gene in Eseherichia coli.Infect Immun,1992.60(4):1671-6.
[107]Shin,S.J.,Bae,J.L.,Cho,Y.W.,et al.Expression of apxlA of Actinobacillus pleuropneumoniae in Saccharomyces cerevisiae.J Vet Sci,2003.4(3):225-8.
[108]Cregg,J.,Cereghino,J.,Shi,J.,et al.Recombinant protein expression in Pichia pastoris.Molecular Biotechnology,2000.16(1):23-52.
[109]赵翔,霍克克。毕赤酵母的密码子用法分析。生物工程学报,2000。16(3):308-311.
[110]Gustafsson,C.,Govindarajan,S.,Minshull,J.Codon bias and heterologous protein expression.Trends Biotechnol,2004.22(7):346-53.
[Ill]Sharp,P.M.,Tuohy,T.M.,Mosurski,K.R.Codon usage in yeast:cluster analysis clearly differentiates highly and lowly expressed genes.Nucleic Acids Res,1986.14(13):5125-43.
[112]钱平,李祥敏,仇华吉等.外源基因在甲醇酵母Pichia pastoris中的表达策略.动物 医学进展,2002.23(6):24-27。
[ll3]Fidler,A.E.,Lin,J.S.,Lun,S.,et al.Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris.J Mol Endocrinol,2003.30(2):213-25.
[114]贝为成,严琳,何启盖,等。猪传染性胸膜肺炎放线杆菌毒素apxⅡA基因的真核表达及其核酸疫苗.农业生物技术学报,2005。(05):616-619
[115]逯忠新,赵卫平,赵萍,等.猪胸膜肺炎放线杆菌apxⅡA基因原核表达载体的构建及其表达.中国兽医科技,2004.(08):36-38。
[116]袁秀芳,王一成,徐丽华,等.猪传染性胸膜肺炎放线杆菌apx Ⅱ基因特异片段的克隆和表达.浙江农业学报,2007.(01):6-10.
[117]黄红亮,周锐,陈美玲,等.胸膜肺炎放线杆菌毒素apxⅣA基因的克隆与表达及间接ELISA方法的建立。生物工程学报,2005.21(2):294-299.
[ll8]Dreyfus,A.,Schaller,A.,Nivollet,S.,et al.Use of recombinant ApxⅣ in serodiagnosis of Actinobacillus pleuropneumoniae infections,development and prevalidation of the ApxⅣ ELISA.Vet Mierobiol,2004.99(3-4):227-38.
[119]徐义刚,崔丽春,葛俊伟,等.猪瘟病毒T细胞表位E290多肽与猪细小病毒Vp2蛋白在干酪乳杆菌中的共表达及免疫小鼠特异性抗体的测定.微生物学报,2007。(04):667-672.
[120]范京惠,左玉柱,王玉玲,等.猪瘟病毒T细胞表位与猪细小病毒Vp2融合基因在重组杆状病毒中的表达。中国兽医科学,2007.(08):661-665.
[121]曾黎平,王萍,乌慧玲,等。新城疫病毒F和hn核心片段在杆状病毒中的表达.江西农业大学学报,2005。(01):114-118.
[122]郭设平,刘思国,王春来,等.牛分枝杆菌mpb64和Ag85B融合基因在大肠埃希氏菌中的原核表达.中国兽医科学,2005.(12):946-949
[123]郭设平,刘思国,张秀华,等.牛分枝杆菌抗原mpb70、Mpb83和esat-6的融合表达及重组蛋白的初步应用.畜牧兽医学报,2006.(07)。676-680
L124]吴波,张书环,邓铨涛,等.四种牛分枝杆菌特异性蛋白融合表达及在牛结核病诊断中的临床应用.中国奶牛,2007.(10):16-19.
[125]Cockle,P.J.,Gordon,S.V.,Hewinson,R.G,et al.Field evaluation of a novel differential diagnostic reagent for detection of Mycobacterium bovis in cattle.Clin Vaccine Immunol,2006.13(10):1119-24.
[126]刁有祥,丁家波,姜世金,等。猪传染性胸膜肺炎病料中pcv-2和pnsv的Pcr检测.微生物学报,2005。(03):397-400。
[127]赵朝霞.一起猪圆环病毒、繁殖与呼吸综合征、传染性胸膜肺炎混合感染的病例报 道.中国动物检疫,2007.(01):36-37.
[128]施开创,郭鑫,杨汉春,等.猪繁殖与呼吸综合征病毒和猪圆环病毒2型基因重组质粒实时荧光定量Pcr标准曲线的构建.中国兽医杂志,2007。(04):10-12。
[129]精编分子生物学实验指南.北京,科学出版社,1998:39
[130]花群义,金宁一,周晓黎,等。应用实时荧光定量TaqManRT-PCR检测口蹄疫病毒。中国兽医学报,2005.(02):116-119
[131]罗玉均,张桂红,陈建红,等.Taqman探针荧光定量PCR快速检测猪圆环病毒2型.中国兽医杂志,2007.(01):77-79
[132]石立立,顾潮江,张倩,等.应用改良的实时TaqMan荧光定量RT-PCR技术检测口蹄疫病毒及其3D基因转录水平.中国病毒学,2006。(05):449-453
[133]宋志军,宋长绪,杨增岐,等。猪生殖与呼吸综合征病毒TaqMan荧光定量RT-PCR 检测方法的建立.中国兽医科学,2006.(02):98-102
[134]孙晓智,石鑫,吴绍强.荧光定量PCR技术及其在动物检疫中的应用.中国动物检疫,2006.(12):46-48
[135]索化夷,汤承,岳华,等。实时荧光定量TaqMan-PCR检测鸭瘟病毒方法的建立。中国预防兽医学报,2006.(03):332-335
[136]王忠发,邵俊斌,沃健儿,等.Taqman实时荧光定量PCR快速检测白斑综合症病毒的方法研究。中国卫生检验杂志,2005.(06):663-665
[137]张贺,李波,周虚,等。实时荧光定量PCR技术研究进展及应用。动物医学进展,2006.(S1):665-668
[138]张险朋,兰婷妹,赖笑娴。实时荧光PCR技术在动物疫病诊断中的应用.动物医学进展,2006.(03):102-105
[139]张福良,宋长绪,杨鸣琦,等.猪圆环病毒2型荧光定量PCR检测方法的建立.中国兽医学报,2006。(03):248-250.
[140]张鹤晓,赖平安,高志强,等.荧光RT-PCR检测活禽和禽产品中新城疫病毒中强毒株的研究。中国兽医杂志,2006.(03):1-5
[141]秦智锋,吕建强,肖性龙,等.禽流感H5、H7、H9亚型多重实时荧光RT-PCR检测方法的建立。病毒学报,2006。(02):131-136
[142]陈博文,罗宝正,陈竞帆,等。多重荧光PCR检测Ⅱ型猪链球菌方法的建立及应用。中国兽医学报,2007.(03):355-358。
[143]张蔚,潘劲草,孟冬梅,等.多重实时PCR检测产毒素性霍乱弧菌和副溶血弧菌.微生物学通报,2007。(05):950-954.
[2]刘杰.猪传染性胸膜肺炎油佐剂灭活疫苗。畜牧兽医科技信息,2001。(03):40
[3]逯忠新,赵萍,邵英德,等.猪传染性胸膜肺炎三价灭活疫苗的研究--效力、最小免疫量、免疫持续期和保存期试验.中国兽医科技,2002.(05):8-10
[4]Higgins,R.,Lariviere,S.,Mittal,K.R.,et al.Evaluation of a Killed Vaccine Against Porcine Pleuropneumonia Due to Haemophilus pleuropneumoniae.Can Vet J,1985.26(2):86-89.
[5]Prideaux,C.T.,Pierce,L.,Krywult,J.,et al.Protection of mice against challenge with homologous and heterologous serovars of Actinobacillus pleuropneumoniae after live vaccination.Curr Microbiol,1998.37(5):324-32.
[6]Rosendal,S.,Maclnnes,J.I.Characterization of an attenuated strain of Actinobacillus pleuropneumoniae,serotyoe 1.Am J Vet Res,1990.51(5):711-7.
[7]刘建杰,陈焕春,李冲,等.新型基因工程亚单位菌苗对猪传染性胸膜肺炎的保护效力研究.中国农业科学,2005.(03):596-600。
[8]邵美丽,刘思国,王春来,等.猪传染性胸膜肺炎重组亚单位疫苗诱导小鼠免疫保护效果的观察.畜牧兽医学报,2006.(10):1021-1026
[9]Lee,K.Y.,Kim,D.H.,Kang,T.J.,et al.Induction of protective immune responses against the challenge of Actinobacillus pleuropneumoniae by the oral administration of transgenic tobacco plant expressing ApxⅡA toxin from the bacteria.FEMS Immunol Med Microbiol,2006.48(3):381-9.
[10]Shin,S.J.,Bae,J.L.,Cho,Y.W.,et al.Induction of antigen-specific immune responses by oral vaccination with Saccharomyces cerevisiae expressing Actinobaeillus pleuropneumoniae ApxⅡA.FEMS Immunol Med Microbiol,2005.43(2):155-64.
[11]Boekema,B.K.,Kamp,E.M.,Smits,M.A.,et al.Both ApxⅠ and ApxⅡ of Actinobacillus pleuropneumoniae serotype 1 are necessary for full virulence.Vet Microbiol,2004.100(1-2):17-23.
[12]Tascon,R.I.,Vazquez-Boland,J.A.,Gutierrez-Martin,C.B.,et al.The RTX haemolysins ApxⅠ and ApxⅡ are major virulence factors of the swine pathogen Actinobacillus pleuropneumoniae:evidence from mutational analysis.Mol Microbiol,1994.14(2):207-16.
[13]Kamp,E.M.,Stockhofe-Zurwieden,N.,van Leengoed,L.A.,et al.Endobronchial inoculation with Apx toxins of Actinobacillus pleuropneumoniae leads to pleuropneumonia in pigs.Infect Immun,1997.65(10):4350-4.
[14]Reimer,D.,Frey,J.,Jansen,R.,et al.Molecular investigation of the role of ApxⅠ and ApxⅡ in the virulence of Actinobacillus pleuropneumoniae serotype 5.Microb Pathog,1995.18(3):197-209.
[15]Schaller,A.,Kuhn,R,,Kuhnert,P.,et al.Characterization of apxⅣA,a new RTX determinant of Actinobacillus pleuropneumoniae.Microbiology,1999.145(Pt 8)2105-16.
[16]Schaller,A.,Djordjevic,S.P.,Eamens,G.J.,et al.Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxⅣA.Vet Microbiol,2001.79(1):47-62.
[17]斯劳特,阿莱尔,蒙加林。猪病学.北京,中国农业大学出版社,2000:357-367
[18]杨旭夫,彭发泉。我国猪传染性胸膜肺炎嗜血杆菌感染症的确定和诊断.畜牧兽医学报,1990.(3):243-245.
[19]吴家强,崔锦鹏,张秀美,等.猪接触传染性胸膜肺炎研究进展.畜牧与兽医,2002.(10):36-39.
[20]Vigre,H.,Angen,O.,Barfod,K.,et al.Transmission of Actinobacillus pleuropneumoniae in pigs under field-like conditions:emphasis on tonsillar colonisation and passively acquired colostral antibodies.Vet Microbiol,2002.89(2-3):151-9.
[21]Holmgren,N.,Lundeheim,N.,Wallgren,P.Infections with Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae in fattening pigs.Influence of piglet production systems and influence on production parameters.Zentralbl Veterinarmed B,1999.46(8):535-44.
[22]张耀相,杨会霞,李健强。胸膜肺炎放线杆菌生物学特性概述。动物医学进展,2004.(06):140-141.
[23]Jansen,R.,Bdaire,J.,Kamp,E.M.,et al.The CAMP effect of Actinobacillus pleuropneumoniae is caused by Apx toxins.FEMS Microbiol Lett,1995.126(2):139-43.
[24]Nielsen,R.,Andresen,L.O.,Plambeck,T.,et al.Serological characterization of Actinobacillus pleuropneumoniae biotype 2 strains isolated from pigs in two Danish herds.Vet Microbiol,1997.54(1):35-46.
[25]Jacobsen,M.J.,Nielsen,J.P.,Nielsen,R.Comparison of virulence of different Actinobacillus pleuropneumoniae serotypes and biotypes using an aerosol infection model.Vet Microbiol,1996.49(3-4):159-68.
[26]Dubreuil,J.D.,Jacques,M.,Mittal,K.R.,et al.Actinobacillus pleuropneumoniae surface polysaccharides:their role in diagnosis and immunogenicity.Anim Health Res Rev,2000.1(2):73-93.
[27]Nielsen,R.Haemophilus pleuropneumoniae(Actinobacillus pleuropneumoniae).Serotypes 8,3 and 6.Serological response and cross immunity in pigs.Nord Vet Med,1985.37(4):217-27.
[28]Baltes,N.,Tonpitak,W.,Gerlach,G.F.,et al.Actinobacillus pleuropneumoniae iron transport and urease activity:effects on bacterial virulence and host immune response. Infect Immun,2001.69(1):472-8.
[29]Choi,C.,Kwon,D.,Min,K.,et al.Detection and localization of ApxI,-Ⅱ and -Ⅲ genes of Actinobacillus pleuropneumoniae in natural porcine pleuropneumonia in natural porcine pleuropneumonia by in situ hybridization.Vet Pathol,2001.38(4):390-5.
[30]Frey,J.,Bosse,J.T.,Chang,Y.F.,et al.Actinobacillus pleuropneumoniae RTX-toxins:uniform designation of haemolysins,cytolysins,pleurotoxin and their genes.J Gen Microbiol,1993.139(8):1723-8.
[31]Inzana,T.J.,Todd,J.,Veit,H.P.Safety,stability,and efficacy of noncapsulated mutants of Actinobacillus pleuropneumoniae for use in live vaccines.Infect Immun,1993.61(5):1682-6.
[32]Ward,C.K.,Lawrence,M.L.,Veit,H.P.,et al.Cloning and mutagenesis of a serotype-specific DNA region involved in encapsulation and virulence of Actinobacillus pleuropneumoniae serotype 5a:concomitant expression of serotype 5a and 1 capsular polysacchaddes in recombinant A.pleuropneumoniae serotype 1.Infect Immun,1998.66(7):3326-36.
[33]王春来,刘思国,王牟平,等.猪传染性胸膜肺炎放线杆菌血清型7型25-4株ApxⅡCA基因的克隆和基因缺失;中国预防兽医学报,2004。(02):94-97.
[34]张立昌。猪传染性胸膜肺炎研究进展。养猪,2001.(01):40-42。
[35]Frey,J.Virulence in Actinobacillus pleuropneumoniae and RTX toxins.Trends Microbiol,1995.3(7):257-61.
[36]Gygi,D.,Nicolet,J.,Hughes,C.,et al.Functional analysis of the Ca(2+)-regulated hemolysin Ⅰ operon of Actinobacillus pleuropneumoniae serotype 1.Infect Immun,1992.60(8):3059-64.
[37]Lo,R.Y.Molecular characterization of cytotoxins produced by Haemophilus,Actinobacillus,Pasteurella.Can J Vet Res,1990.54 SupplS33-5.
[38]Schaller,A.,Kuhnert,P.,de la Puente-Redondo,V.A.,et al.Apx toxins in Pasteurellaceae species from animals.Vet Microbiol,2000.74(4):365-76.
[39]Jansen,R.,Briaire,J.,Kamp,E.M.,et al.Structural analysis of the Actinobacillus pleuropneumoniae-RTX-toxin Ⅰ(ApxⅠ)operon.Infect Immun,1993.61(9):3688-95.
[40]Seah,J.N.,Frey,J.,Kwang,J.The N-terminal domain of RTX toxin ApxI of Actinobacillus pleuropneumoniae elicits protective immunity in mice.Infect Immun,2002.70(11):6464-7.
[41]李敏,姜国彦,彭永刚,等.猪传染性猪胸膜肺炎APXⅣA毒素特征的研究进展.畜牧兽医科技信息,2004.(09):11-12
[42]Frey,J.,Kuhnert,P.,Villiger,L.,et al.Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins.Res Microbiol,1996.147(5):351-61.
[43]van den Bosch, H., Frey, J. Interference of outer membrane protein PalA with protective immunity against Actinobacillus pleuropneumoniae infections in vaccinated pigs. Vaccine, 2003. 21 (25-26):3601-7.
[44]Gerlach, G F., Anderson, C, Klashinsky, S., et al. Molecular characterization of a protective outer membrane lipoprotein (OmlA) from Actinobacillus pleuropneumoniae serotype 1. Infect Immun, 1993. 61 (2):565-72.
[45]Van Overbeke, I., Chiers, K., Ducatelle, R., et al. Effect of endobronchial challenge with Actinobacillus pleuropneumoniae serotype 9 of pigs vaccinated with a vaccine containing Apx toxins and transferrin-binding proteins. J Vet Med B Infect Dis Vet Public Health, 2001.48 (1):15-20.
[46]Bosse, J. T., Machines, J. I. Genetic and biochemical analyses of Actinobacillus pleuropneumoniae urease. Infect Immun, 1997. 65 (11):4389-94.
[47]Gottschalk, M., De Lasalle, F., Radacovici, S., et al. Evaluation of long chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 5 for the serodiagnosis of swine pleuropneumonia. Vet Microbiol, 1994. 38 (4):315-27.
[48]Radacovici, S., Gottschalk, M., Dubreuil, J. D. Lipopolysaccharides of Actinobacillus pleuropneumoniae (serotype 1): a readily obtainable antigen for ELISA serodiagnosis of pig pleuropneumonia. Vet Microbiol, 1994.39 (3-4):219-30.
[49]Andresen, L. O., Klausen, J., Barfod, K., et al. Detection of antibodies to Actinobacillus pleuropneumoniae serotype 12 in pig serum using a blocking enzyme-linked immunosorbent assay. Vet Microbiol, 2002. 89 (1):61-7.
[50]Klausen, J., Andresen, L. O., Barfod, K., et al. Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum. Vet Microbiol, 2001. 79 (1):11-8.
[51]Nielsen, R., Plambeck, T., Foged, N. T. Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 8. Vet Microbiol, 1993.34(2): 131-8.
[52]Bosse, J. T., Friendship, R., Rosendal, S., et al. Development and evaluation of a mixed-antigen ELISA for serodiagnosis of Actinobacillus pleuropneumoniae serotypes 1, 5, and 7 infections in commercial swine herds. J Vet Diagn Invest, 1993. 5 (3):359-62.
[53]Grondahl-Hansen, J., Barfod, K., Klausen, J., et al. Development and evaluation of a mixed long-chain lipopolysaccharide based ELISA for serological surveillance of infection with Actinobacillus pleuropneumoniae serotypes 2, 6 and 12 in pig herds. Vet Microbiol, 2003. 96 (1):41-51.
[54]Leiner, G, Franz, B., Strutzberg, K., et al. A novel enzyme-linked immunosorbent assay using the recombinant Actinobacillus pleuropneumoniae ApxII antigen for diagnosis of pleuropneumonia in pig herds. Clin Diagn Lab Immunol, 1999. 6 (4):630-2.
[55]Nielsen, R., van den Bosch, J. F., Plambeck, T., et al. Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae.Vet Microbiol,2000.71(1-2):81-7.
[56]王淑敏,冼琼珍,温广杰。PPA-ELISA检测猪传染性胸膜肺炎抗体方法的建立。黑龙江畜牧兽医,2003。(01):37-38.
[57]逯忠新,鲁炳义,赵萍。用间接血凝试验检测猪传染性胸膜肺炎.中国兽医科技,1999。(10):25-27。
[58]Inzana,T.J.Simplified procedure for preparation of sensitized latex particles to detect capsular polysaecharides:application to typing and diagnosis of Actinobaeillus pleuropneumoniae.J Clin Microbiol,1995.33(9):2297-303.
[59]Mittal,K.R.,Higgins,R.,Lariviere,S.Evaluation of slide agglutination and ring precipitation tests for capsular serotyping of Haemophilus pleuropneumoniae.J Clin Microbiol,1982.15(6):1019-23.
[60]Mittal,K.R.,Higgins,R.,Lariviere,S.An evaluation of agglutination and coagglutination techniques for serotyping of Haemophilus pleuropneumoniae isolates.Am J Vet Res,1987.48(2):219-26.
[61]Chiers,K.,Van Overbeke,I.,Donne,E.,et al.Detection of Actinobacillus pleuropneumoniae in cultures from nasal and tonsillar swabs of pigs by a PCR assay based on the nucleotide sequence of a dsbE-like gene.Vet Mierobiol,2001.83(2):147-59.
[62]Frey,J.,Beck,M.,van den Bosch,J.F,et al.Development of an efficient PCR method for toxin typing of Actinobacillus pleuropnettmoniae swains.Mol Cell Probes,1995.9(4):277-82.
[63]Gram,T.,Ahrens,P Improved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nueleotide sequence of an outer membrane lipoprotein.J Clin Microbiol,1998.36(2):443-8.
[64]Jessing,S.G,Angen,O.,Inzana,T.J.Evaluation of a multiplex PCR test for simultaneous identification and serotyping of Actinobacillus pleuropneumoniae serotypes 2,5,and 6.J Clin Microbiol,2003.41(9):4095-100.
[65]Lo,T.M.,Ward,C.K.,Inzana,T.J.Detection and identification of Actinobacillus pleuropneumoniae serotype 5 by multiplex PCR.J.Clin Microbiol,1998.36(6):1704-10.
[66]Rayamajhi,N.,Shin,S.J.,Kang,S.G.,et al.Development and use of a multiplex polymerase chain reaction assay based on Apx toxin genes for genotyping of Actinobacillus pleuropneumoniae isolates.J Vet Diagn Invest,2005.17(4):359-62.
[67]Savoye,C.,Jobert,J.L.,Berthelot-Herault,F.,et al.A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental conditions.Vet Microbiol,2000.73(4):337-47.
[68]Sthitmatee,N.,Sirinarumitr,T.,Makonkewkeyoon,L.,et al.Identification of the Actinobacillus pleuropneumoniae serotype using PCR based-apx genes.Mol Cell Probes,2003.17(6):301-5.
[69]Sirois,M.,Lemire,E.G.,Levesque,R.C.Construction of a DNA probe and detection of Actinobacillus pleuropneumoniae by using polymerase chain reaction.J Clin Microbiol,1991.29(6):1183-7.
[70]Hennessy,K.J.,Iandolo,J.J.,Fenwick,B.W.Serotype identification of Actinobacillus pleuropneumoniae by arbitrarily primed polymerase chain reaction.J Clin Microbiol,1993.31(5):1155-9.
[71]庞耀珊,谢芝勋,刘加波,等.二温式PCR检测猪传染性胸膜肺炎放线杆菌方法的建立与应用.广西农业科学,2006。(02):203-205.
[72]李树清,易建平,陈志飞,等.复合PCR鉴定胸膜肺炎放线杆菌方法的建立及初步应用.微生物学报,2005.(06):966-969.
[73]李树清,易建平,胡永强,等.猪传染性胸膜肺炎放线杆菌的套式PCR检测.中国预防兽医学报,2005.(03):217-222。
[74]Jaglic,Z.,Svastova,P.,Rychlik,I.,et al.Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxⅣA gene and by ribotyping.Vet Microbiol,2004.103(1-2):63-9.
[75]Tumi,C.,Blackall,P.J.An evaluation of the apxⅣA based PCR-REA method for differentiation of Actinobacillus pleuropneumoniae.Vet Microbiol,2007.121(1-2):163-9.
[76]Angen,O.,Jensen,J.,Lavdtsen,D.T.Evaluation of 5' nuclease assay for detection of Actinobacillus pleuropneumoniae.J Clin Microbiol,2001.39(1):260-5.
[77]李树清,易建平,李健,等.胸膜肺炎放线杆菌的实时荧光PCR检测.畜牧兽医学报,2005.(09):969-973.
[78]Cho,W.S.,Choi,C.,Chae,C.In situ hybridization for the detection of the apxⅣ gene in the lungs of pigs experimentally infected with twelve Actinobacillus pleuropneumoniae serotypes.Vet Res,2002.33(6):653-60.
[79]刘杰。猪传染性胸膜肺炎油剂灭活疫苗.畜牧兽医科技信息,2003。(02):49
[80]Hensel,A.,Huter,V.,Katinger,A.,et al.Intramuscular immunization with genetically inactivated(ghosts)Actinobacillus pleuropneumoniae serotype 9 protects pigs against homologous aerosol challenge and prevents carder state.Vaccine,2000.18(26):2945-55.
[81]Liao,C.W.,Cheng,I.C.,Yeh,K.S.,et al.Release characteristics of microspheres prepared by co-spray drying Actinobacillus pleuropneumoniae antigens and aqueous ethyl-cellulose dispersion.J Microencapsul,2001.18(3):285-97.
[82]Liao,C.W.,Chiou,H.Y.,Yeh,K.S.,et al.Oral immunization using formalin-inactivated Actinobacillus pleuropneumoniae antigens entrapped in microspheres with aqueous dispersion polymers prepared using a co-spray drying process.Prev Vet Med,2003.61(1):1-15.
[83]Prideaux,C.T.,Lenghaus,C.,Krywult,J.,et al.Vaccination and protection of pigs against pleuropneumonia with a vaccine strain of Actinobacillus pleuropneumoniae produced by site-specific mutagenesis of the ApxⅡ operon.Infect Immun,1999.67(4):1962-6.
[84]Tonpitak,W.,Baltes,N.,Hennig-Pauka,I.,et al.Construction of an Actinobacillus pleuropneumoniae serotype 2 prototype live negative-marker vaccine.Infect Immun,2002.70(12):7120-5.
[85]Maas,A.,Meens,J.,Baltes,N.,et al.Development of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae infection.Vaccine,2006.24(49-50):7226-37.
[86]贝为成,陈焕春,何启盖,等。猪传染性胸膜肺炎毒素基因工程弱毒活疫苗的研究,全国规模化猪场疫病控制与净化资料专辑,2002:29-32
[87]贝为成,何启盖,方六荣,等。猪传染性胸膜肺炎放线杆菌毒素apxⅡ基因无药物抗性标记突变株HBC--/GFP-+的构建及其生物学特性研究.生物工程学报。2004。(05):719-724
[88]刘霞,刁有祥,禚宝山.猪传染性胸膜肺炎的研究进展。山东农业大学学报(自然科学版),2006.(01):152-156.
[89]Furesz,S.E.,Mallard,B.A.,Bosse,J.T.,et al.Antibody- and cell-mediated immune responses of Actinobacillus pleuropneumoniae-infected and bacterin-vaecinated pigs.Infect Immun,1997.65(2):358-65.
[90]郭洋,刘思国,王春来等。胸膜肺炎放线杆菌菌研究进展。现代畜牧兽医,2006.06:61-64。
[91]王春来,刘思国,邵美丽等.重组ApxⅡA对胸膜肺炎放线杆菌灭活疫苗免疫效果的影响。中国兽医科学,2006.36(06):434-438.
[92]Goethe,R.,Gonzales,O.F,Lindner,T.,et al.A novel strategy for protective Actinobacillus pleuropneumoniae subunit vaccines:detergent extraction of cultures induced by iron restriction.Vaccine,2000.19(7-8):966-75.
[93]Tumamao,J.Q.,Bowles,R.E.,van den Bosch,H.,et al.An evaluation of the role of antibodies to Actinobacillus pleuropneumoniae serovar 1 and 15 in the protection provided by sub-unit and live streptomycin-dependent pleuropneumonia vaccines.Aust Vet J,2004.82(12):773-80.
[94]Tumamao,J.Q.,Bowles,R.E.,van den Bosch,H.,et al.Comparison of the efficacy of a subunit and a live streptomycin-dependent porcine pleuropneumonia vaccine.Aust Vet J,2004.82(6):370-4.
[95]梁雨,田淑芳,刘文军等。核酸免疫可特异地诱发小鼠对乙型肝炎病毒表面抗原S+PreS1免疫反应.病毒学报,1996.(3):193-199
[96]李建伟,赵晓岩,杨建德等.鸡马立克氏病DNA疫苗免疫的初步研究.中国畜禽传染病,1996。(5):9-12.
[97]陈化兰,于康震.DNA免疫诱导鸡对禽流感病毒的免疫保护反应。中国农业科学,1998。31(5):63-68.
[98]贝为成,严琳,何启盖等.猪传染性胸膜肺炎放线杆菌毒素apx ⅡA基因的真核表达及其核酸疫苗.农业生物技术学报,2005。13(5):616-619。
[99]Frey,J.,Nicolet,J.Immunological properties of Actinobacillus pleuropneumoniae hemolysin I.Vet Microbiol,1991.28(1):61-73.
[100]Byrd,W.,Hooke,A.M.Immunization with temperature-sensitive mutants of Actinobacillus pleuropneumoniae induces protective hemolysin-neutralizing antibodies in mice.Curr Microbiol,1997.34(3):149-54.
[101]Haesebrouck,F.,Chiers,K.,Van Overbeke,I.,et al.Actinobacillus pleuropneumoniae infections in pigs:the role of virulence factors in pathogenesis and protection.Vet Microbiol,1997.58(2-4):239-49.
[102]Fischer,B.,Sumner,I.,Goodenough,P.Isolation and renaturation of bio-active proteins expressed in Escherichia coli as inclusion bodies.Arzneimittelforschung,1992.42(12):1512-5.
[103]Burdyehova,R.,Ryehtera,M.,Horvath,R.,et al.Expression of Aetinobaeillus pleuropneumonia gene coding for Apx Ⅰ protein in Eseherichia coli.FEMS Mierobiol Lett,2004.230(1):9-12.
[104]王冬梅,刘磊,逯忠新等。猪胸膜肺炎放线杆菌apxⅠA基因的克隆及其原核表达.中国兽医科技,2005.35(5):373-376.
[105]王芳,何孔旺,邱索平,等.猪传染性胸膜肺炎放线杆菌毒素Ⅰ基因的克隆、序列分析及表达。中国预防兽医学报,2006.(02):121-123.
[106]Frey,J.,van den Bosch,H.,Segers,R.,et al.Identification of a second hemolysin (HlyⅡ)in Actinobacillus pleuropneumoniae serotype 1 and expression of the gene in Eseherichia coli.Infect Immun,1992.60(4):1671-6.
[107]Shin,S.J.,Bae,J.L.,Cho,Y.W.,et al.Expression of apxlA of Actinobacillus pleuropneumoniae in Saccharomyces cerevisiae.J Vet Sci,2003.4(3):225-8.
[108]Cregg,J.,Cereghino,J.,Shi,J.,et al.Recombinant protein expression in Pichia pastoris.Molecular Biotechnology,2000.16(1):23-52.
[109]赵翔,霍克克。毕赤酵母的密码子用法分析。生物工程学报,2000。16(3):308-311.
[110]Gustafsson,C.,Govindarajan,S.,Minshull,J.Codon bias and heterologous protein expression.Trends Biotechnol,2004.22(7):346-53.
[Ill]Sharp,P.M.,Tuohy,T.M.,Mosurski,K.R.Codon usage in yeast:cluster analysis clearly differentiates highly and lowly expressed genes.Nucleic Acids Res,1986.14(13):5125-43.
[112]钱平,李祥敏,仇华吉等.外源基因在甲醇酵母Pichia pastoris中的表达策略.动物 医学进展,2002.23(6):24-27。
[ll3]Fidler,A.E.,Lin,J.S.,Lun,S.,et al.Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris.J Mol Endocrinol,2003.30(2):213-25.
[114]贝为成,严琳,何启盖,等。猪传染性胸膜肺炎放线杆菌毒素apxⅡA基因的真核表达及其核酸疫苗.农业生物技术学报,2005。(05):616-619
[115]逯忠新,赵卫平,赵萍,等.猪胸膜肺炎放线杆菌apxⅡA基因原核表达载体的构建及其表达.中国兽医科技,2004.(08):36-38。
[116]袁秀芳,王一成,徐丽华,等.猪传染性胸膜肺炎放线杆菌apx Ⅱ基因特异片段的克隆和表达.浙江农业学报,2007.(01):6-10.
[117]黄红亮,周锐,陈美玲,等.胸膜肺炎放线杆菌毒素apxⅣA基因的克隆与表达及间接ELISA方法的建立。生物工程学报,2005.21(2):294-299.
[ll8]Dreyfus,A.,Schaller,A.,Nivollet,S.,et al.Use of recombinant ApxⅣ in serodiagnosis of Actinobacillus pleuropneumoniae infections,development and prevalidation of the ApxⅣ ELISA.Vet Mierobiol,2004.99(3-4):227-38.
[119]徐义刚,崔丽春,葛俊伟,等.猪瘟病毒T细胞表位E290多肽与猪细小病毒Vp2蛋白在干酪乳杆菌中的共表达及免疫小鼠特异性抗体的测定.微生物学报,2007。(04):667-672.
[120]范京惠,左玉柱,王玉玲,等.猪瘟病毒T细胞表位与猪细小病毒Vp2融合基因在重组杆状病毒中的表达。中国兽医科学,2007.(08):661-665.
[121]曾黎平,王萍,乌慧玲,等。新城疫病毒F和hn核心片段在杆状病毒中的表达.江西农业大学学报,2005。(01):114-118.
[122]郭设平,刘思国,王春来,等.牛分枝杆菌mpb64和Ag85B融合基因在大肠埃希氏菌中的原核表达.中国兽医科学,2005.(12):946-949
[123]郭设平,刘思国,张秀华,等.牛分枝杆菌抗原mpb70、Mpb83和esat-6的融合表达及重组蛋白的初步应用.畜牧兽医学报,2006.(07)。676-680
L124]吴波,张书环,邓铨涛,等.四种牛分枝杆菌特异性蛋白融合表达及在牛结核病诊断中的临床应用.中国奶牛,2007.(10):16-19.
[125]Cockle,P.J.,Gordon,S.V.,Hewinson,R.G,et al.Field evaluation of a novel differential diagnostic reagent for detection of Mycobacterium bovis in cattle.Clin Vaccine Immunol,2006.13(10):1119-24.
[126]刁有祥,丁家波,姜世金,等。猪传染性胸膜肺炎病料中pcv-2和pnsv的Pcr检测.微生物学报,2005。(03):397-400。
[127]赵朝霞.一起猪圆环病毒、繁殖与呼吸综合征、传染性胸膜肺炎混合感染的病例报 道.中国动物检疫,2007.(01):36-37.
[128]施开创,郭鑫,杨汉春,等.猪繁殖与呼吸综合征病毒和猪圆环病毒2型基因重组质粒实时荧光定量Pcr标准曲线的构建.中国兽医杂志,2007。(04):10-12。
[129]精编分子生物学实验指南.北京,科学出版社,1998:39
[130]花群义,金宁一,周晓黎,等。应用实时荧光定量TaqManRT-PCR检测口蹄疫病毒。中国兽医学报,2005.(02):116-119
[131]罗玉均,张桂红,陈建红,等.Taqman探针荧光定量PCR快速检测猪圆环病毒2型.中国兽医杂志,2007.(01):77-79
[132]石立立,顾潮江,张倩,等.应用改良的实时TaqMan荧光定量RT-PCR技术检测口蹄疫病毒及其3D基因转录水平.中国病毒学,2006。(05):449-453
[133]宋志军,宋长绪,杨增岐,等。猪生殖与呼吸综合征病毒TaqMan荧光定量RT-PCR 检测方法的建立.中国兽医科学,2006.(02):98-102
[134]孙晓智,石鑫,吴绍强.荧光定量PCR技术及其在动物检疫中的应用.中国动物检疫,2006.(12):46-48
[135]索化夷,汤承,岳华,等。实时荧光定量TaqMan-PCR检测鸭瘟病毒方法的建立。中国预防兽医学报,2006.(03):332-335
[136]王忠发,邵俊斌,沃健儿,等.Taqman实时荧光定量PCR快速检测白斑综合症病毒的方法研究。中国卫生检验杂志,2005.(06):663-665
[137]张贺,李波,周虚,等。实时荧光定量PCR技术研究进展及应用。动物医学进展,2006.(S1):665-668
[138]张险朋,兰婷妹,赖笑娴。实时荧光PCR技术在动物疫病诊断中的应用.动物医学进展,2006.(03):102-105
[139]张福良,宋长绪,杨鸣琦,等.猪圆环病毒2型荧光定量PCR检测方法的建立.中国兽医学报,2006。(03):248-250.
[140]张鹤晓,赖平安,高志强,等.荧光RT-PCR检测活禽和禽产品中新城疫病毒中强毒株的研究。中国兽医杂志,2006.(03):1-5
[141]秦智锋,吕建强,肖性龙,等.禽流感H5、H7、H9亚型多重实时荧光RT-PCR检测方法的建立。病毒学报,2006。(02):131-136
[142]陈博文,罗宝正,陈竞帆,等。多重荧光PCR检测Ⅱ型猪链球菌方法的建立及应用。中国兽医学报,2007.(03):355-358。
[143]张蔚,潘劲草,孟冬梅,等.多重实时PCR检测产毒素性霍乱弧菌和副溶血弧菌.微生物学通报,2007。(05):950-954.