斑点叉尾鮰病毒“自杀性”DNA疫苗的构建及其体外表达检测  

Construction and Expression Characteristic in vitro of Channel Catfish Virus Suicide DNA Vaccine

作　　者：熊海林[1] 尹殿卯[1] 王卫民[1] 王敏[1] 李莉娟[1] 刘玉林[1] 刘学芹[1] 吴兵[1] 黄锋涛[1] 

机构地区：[1]华中农业大学水产学院农业动物遗传育种与繁殖教育部重点实验室,湖北武汉430070

出　　处：《水生态学杂志》2011年第1期84-88,共5页Journal of Hydroecology

基　　金：国家自然科学基金(30901118);教育部新教师基金(20070504047);湖北省自然科学基金(2008CBB099);华中农业大学教师启动基金(2006XRC047)

摘　　要：根据CCVORF6基因序列,设计合适的引物,扩增ORF6基因,分别将其克隆到"自杀性"DNA疫苗载体pS-FV与常规DNA疫苗载体pcDNA3.1(+)中,转化感受态细胞DH5α后提取质粒,构建斑点叉尾鮰(Ictalurus punc-tatus)"自杀性"DNA疫苗ps-ORF6与常规DNA疫苗pcd-ORF6,利用转化后的大肠杆菌菌液为模板进行PCR扩增、提取质粒酶切鉴定以及序列测定等方法证实重组质粒构建正确。将重组质粒转染人胚肾细胞(293T),间接免疫荧光试验表明ORF6均获得表达,但"自杀性"DNA疫苗的表达效果不如常规DNA疫苗,该研究为这2种疫苗进一步的鱼体试验奠定了基础。In this study,suitable primers were designed according to channel catfish virus（CCV）ORF6 gene sequence.The gene was cloned into suicide DNA vaccine vector pSFV and conventional DNA vaccine vector pcDNA3.1（＋）respectively.After transformation into E.coli DH5α and extraction plasmids,the suicide DNA vaccines ps-ORF6 and conventional DNA vaccine pcd-ORF6 were constructed.The CCV ORF6 gene was correctly inserted into vector respectively,confirmed by PCR,double enzymes digestion and DNA sequencing.The recombinant plasmids were transfected into 293T cells.Indirect immunofluorescence assay showed that ORF6 were expressed in both vaccines,but the express level of the suicide DNA vaccine was not as good as the conventional DNA vaccine.The study provides the basis for the further fish test of two vaccines.

关 键 词：斑点叉尾鮰病毒 “自杀性”DNA疫苗 构建 表达 

分 类 号：Q343.1[生物学—遗传学]