摘要
根据GenBank数据库中弓形虫RH株GRA6基因序列设计1对引物,采用PCR技术从弓形虫GJS株基因组DNA中扩增GRA6基因,导入真核表达载体pcDNA3.1/CT-GFP-TOPO,转化大肠杆菌DH5α,经酶切、PCR及测序鉴定,将重组质粒转染BHK-21细胞。GFP标签证明质粒DNA成功转染到细胞中并得以表达,通过Western-blot分析,细胞裂解液中有一条约52 ku的条带,可被山羊抗弓形虫超免疫血清识别,大小与预测值相符。表明真核表达质粒pcDNA3.1/CT-GFP-TOPO-GRA6中的GRA6基因在BHK-21细胞中获得表达且表达产物具有抗原性。
One pair of specific primers was designed according to the published dense granule antigen 6（GRA6） gene sequence of Toxoplasma gondii RH strain in GenBank.With the primers,the gene encoding GRA6 protein was amplified by PCR from the genomic DNA of T.gondii GJS strain and then cloned into the eukaryotic expression vector pcDNA3.1/CT-GFP-TOPO.The recombinant plasmid pcDNA3.1/CT-GFP-TOPO-GRA6 was identified by PCR,restriction enzyme digestion and sequencing analysis.Then,the plasmid pcDNA3.1/CT-GFP-TOPO-GRA6 was transfected into BHK-21 cells mediated by Lipofectamine 2000.GFP showed that the recombinant plasmid was successfully expressed in BHK-21 cells.Western-blot analysis showed that a band approximately 52ku in size in BHK-21 cell lysate was recognized by the goat hyperimmune serum against T.gondii,indicating that the GRA6 protein expressed transiently in the cells had antigenicity.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2010年第4期363-367,共5页
Chinese Veterinary Science
基金
国家科技支撑计划项目(2007BAD40B05)
公益性行业(农业)科研专项经费项目(200803017)
甘肃省重大专项科技项目(2GS063-A43-013)
关键词
刚地弓形虫
致密颗粒6
基因克隆
真核表达
Toxoplasma gondii
dense granule antigen 6（GRA6）
gene cloning
eukaryotic expression
