刘永宏,刘月焕,王凤龙,韩春华,林健,王金玲,杨龙峰,赵丽,张秋雨,张永富

• 1:内蒙古农业大学动物科学与医学学院
• 2:北京市农林科学院畜牧兽医研究所
• 3:北京市延庆县动物卫生监督管理局
• 4:北京庄笛浩禾生物医学科技有限公司

摘要(Abstract)：

根据GenBank中美洲型猪繁殖与呼吸综合征病毒(PRRSV)的ORF6基因序列,设计合成一对特异性引物,应用RT-PCR方法扩增出PRRSV的ORF6基因(M基因)。将所扩增片段克隆入原核表达载体pET-32a(+),pET-ORF6重组质粒转化DH5a宿主菌后,经双酶切、PCR鉴定后挑选阳性克隆测序鉴定并对插入的ORF6基因序列进行分析。结果表明,ORF6基因的原核表达载体构建成功。ORF6基因推导的氨基酸与美洲型相应基因的同源性为96.0%～100%,与LV株的同源性为79.9%,系统进化树表明该PRRSV属于美洲型。将构建成功的重组质粒pET-ORF6转化BL21,诱导后经SDS-PAGE和Western-blot分析表明:克隆在HIS标签的膜基质蛋白基因与HIS获得了高效表达,表达的融合蛋白HIS-M分子量约为39kDa,并且有免疫学反应活性。

关键词(KeyWords)： 猪繁殖与呼吸综合征病毒;变异株;ORF6基因;克隆;原核表达

Abstract:

Keywords:

基金项目(Foundation): 北京市农林科学院青年基金项目(2007020122);; 北京市科委猪高致病性蓝耳病防控技术子课题

作者(Author): 刘永宏,刘月焕,王凤龙,韩春华,林健,王金玲,杨龙峰,赵丽,张秋雨,张永富

参考文献(References)：

• [1]Wensvoort G.Lelystad virus and the porcine epidemic abortion and res-piratory syndrome[J].Vet Res,1993,24:117-124.
• [2]Cho J G,Dee S A.Porcine epidemic abortion and respiratory syndrome virus[J].Theriogenology,2006,66:655-662.
• [3]Keffaber K K.Reproductive fail-ure of unknown etiology[J].Am Assoc Swine Pract Newsl,1989,(1):1-9.
• [4]Wensvoort G,Terpstra C,Pol J M A,et a1.Mystery swine disease in the Netherlands:the isolation of Lelystad virus[J].Vet Q,1991,13:121-130.
• [5]Benfield D A,Nelson E,Collins J E.Characterization of swine infertility and respiratory syndrome(SIRS)virus(isolate ATCC VR-2332)[J].Vet Diagn Investig,1992,4:127-133.
• [6]Collins J E,Benfield D A,Chris-tianson W T,et a1.Isolation of swine in-fertility and respiratory syndrome virus(isolate ATCC VR-2332)in North America and experimental reproduc-tion of the disease in gnotobiotic pigs[J].Vet Diagn Investig,1992,4:117-126.
• [7]郭宝清,陈章水,刘文兴,等.Isolation of porcine reproductive and respiratory syndrome virus from abor-tion fetus suspected by PRRS[J].中国畜禽传染病,1996,18(2):l-4.
• [8]Conzelmann K K,Visser N,Van Woensel P,et al.Molecular characteri-zation of porcine reproductive and res-piratory disease virus,a member of the arterivirus group[J].Virol,1993,193:329-339.
• [9]Cavanagh D.Nidovirales:a new or-der comprising Coronaviridae[J].Arch Virol,1997,142:629-633.
• [10]Nelson C J,Murtaugh M P,Faaberg K S.Porcine reproductive and respiratory syndrome virus comparison:Divergent evolution on two continents[J].Virol,1999,73:270-280.
• [11]Wissink E H,van Wijk H A,Kroese M V,et al.The major envelope protein,GP5,of a European porcine re-productive and respiratory syndrome virus contains a neutralization epitope in its N-terminal ectodomain[J].J Gen Virol,2003,84(6):1535-1543.
• [12]Sur J H,et al.In vivo detection of porcine reproductive and respiratory syndrome virus RNA by in situ hy-bridization at different times postinfec-tion[J].J Clin Microbiol,1996,34(9):2280-2286.
• [13]Key K F,Guenette D K,Yoon K J,et al.Development of a heteroduplex mobility assay to identify field isolates of porcine reproductive and respiratory syndrome virus with nucleotide se-quences closely related to those of modified live-attenuated vaccines[J].J Clin Microbiol,2003,41(6):2433-2439.
• [14]Saalmüller A,Hirt W,Maurers,et al.Discrimination between two subsets of porcine CD8+cytolytic T lympho-cytes by the expression of CD5antigen[J].Immunol,1994,81(4):578-583.
• [15]Sur J H,Doster A R.Evidence for the localization of porcine reproductive andrespiratorysyndromevirus(PRRSV)antigen and RNA in ovarian follicles in gilts[J].Vet Patho,1998,35(6):506-514.
• [16]J.萨姆布鲁克,D.W.拉赛尔.分子克隆实验指南[M].黄培堂,等译.第3版.北京:科学出版社,2003.
• [17]Tian K G,et al.Emergence of Fatal PRRSV Variants:Unparalleled Outbreaks of Atypical PRRS in China and Molecular Dissection of the U-nique Hallmark[J].PLoS ONE,2007,2(6):e526.