猪繁殖与呼吸综合征病毒变异株ORF6基因的克隆与原核表达研究  

Cloning and Expression in E.coli of ORF6 Gene of Porcine Reproductive and Respiratory Syndrome Virus Variant

作　　者：刘永宏[1] 刘月焕[2] 王凤龙[1] 韩春华[2] 林健[2] 王金玲[1] 杨龙峰 赵丽[1] 张秋雨 张永富[5] 

机构地区：[1]内蒙古农业大学动物科学与医学学院,内蒙古呼和浩特010018 [2]北京市农林科学院畜牧兽医研究所,北京100089 [3]北京市延庆县动物卫生监督管理局,北京102100 [4]北京庄笛浩禾生物医学科技有限公司,北京1000431 [5]内蒙古民族大学动物科技学院,内蒙古通辽028000

出　　处：《中国动物检疫》2009年第7期29-32,共4页China Animal Health Inspection

基　　金：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

摘　　要：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。A pair of primers were designed and synthesized according to the ORF6 gene sequences of American genotype porcine reproductive and respiratory syndrome virus （PRRSV） in GenBank.The ORF6 gene （M gene） of porcine reproductive and respiratory syndrome virus （PRRSV） was amplified by RT-PCR. The amplified gene fIagment was then cloned into the prokaryotic expression vector pET-32a （＋）,the recombinant plasmid was transformed into host cell DH5a ,the selected positive clones were subjected to sequence analysis of the inserted ORF6 gene after double enzyme digestion and PCR identification.The result showed that the construction of the prokaryotic expression vector for ORF6 gene was successful. The deduced amino acids of the cloned ORF6 gene showed 96.0 %-100 % to LV. The phylogenetic trees revealed the strain was clustered within North American genotype. homology to North American strain and 79.9%. The results of SDS-PAGE and Western-blot indicated that the M protein gene cloned in the HIS-Taq was expressed in a high level and the recombinant fusion protein, which was about 39kDa had immunologically reactive activity.

关 键 词：猪繁殖与呼吸综合征病毒 变异株 ORF6基因 克隆 原核表达 

分 类 号：S852.659.6[农业科学—基础兽医学]