PRRSV JL/07/SW毒株GP5基因克隆与真核表达  

Cloning and expression of GP5 gene JL/07/SW strain of porcine reproductive and respiratory syndrome virus

作　　者：白晶[1,2] 剡根强[1] 丁壮[2] 陈创夫[1] 李志杰[2] 丛彦龙[2] 母连志[2] 

机构地区：[1]新疆石河子大学动物科技学院,新疆石河子832000 [2]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医学报》2010年第5期569-572,共4页Chinese Journal of Veterinary Science

基　　金：广东省农业攻关项目(2008A020100020)

摘　　要：根据PRRSV VR-2332毒株核苷酸序列,设计针对GP5基因的特异引物,以PRRSVJL/07/SW毒株细胞培养物为模板,利用RT-PCR方法,成功的扩增出GP5基因,将该基因与真核表达载体pCI-neo连接,转染Marc-145细胞中,用间接免疫荧光试验、SDS-PAGE和western-blot鉴定目的蛋白的表达。结果:RT-PCR分别扩增出GP5基因大小与预期一致,与国际PRRSV美洲型VR-2332毒株的GP5基因序列同源率为89.2%;间接免疫荧光、SDS-PAGE及Western blot试验证明GP5蛋白在Marc-145细胞中成功表达;同时利用pCI-neo真核表达载体在Marc-145细胞中成功表达了PRRSVJL/07/SW毒株GP5基因,为下一步研究蛋白的功能奠定了基础。According to the PRRSV VR-2332 strain nucleotide sequence reported in the GenBank.One pair of primer was designed in order to amplify PRRSV JL/07/SW strain GP5 gene.The GP5 gene of PRRSV JL/07/SW strain was successfully amplified by RT-PCR from the cell cultures of PRRSV Jl/07/SW.The GP5 gene was ligated with pCI-neo and transfected into the cell of Marc-145,and the expressed product was identified by indirect IFA、SDS-PAGE and western-blot.Results The GP5 gene were consistent with this expected,with 89.2% of homology to the PRRSV Europe-type VR-2332 strain;restiction analysis and sequencing result proved that both cloning and expression vectors were constructed correctly.Indirect IFA 、SDS-PAGE and western blot are showed that the protein of GP5 was successfully expressed in Marc-145 cells;and the GP5 genes were successfully expressed in Marc-145 cells by using eukaryotic expression vector pCI-neo,which provides a foundation of further study on function of structural protein of PRRSV JL/07/SW.

关 键 词：PRRSV GP5基因 真核表达 

分 类 号：S852.65[农业科学—基础兽医学]