重组猪瘟病毒E2基因逆转录病毒载体的构建及其表达活性  

Retroviral vector mediated in vitro expression of classical swine fever virus E2 gene and stability analysis

作　　者：田宏[1] 刘湘涛[1] 林彤[1] 吴锦艳[1] 龚真莉[1] 郑海学[1] 代兴国[1] 孙世琪[1] 尚佑军[1] 谢庆阁[1] 张彦明[2] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室,甘肃兰州730046 [2]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《中国兽医学报》2007年第4期460-463,共4页Chinese Journal of Veterinary Science

基　　金：国家863高技术研究发展计划(2003AA241110)

摘　　要：从实验感染兔脾组织中提取总RNA,应用RT-PCR得到了CSFV C-株结构蛋白E2基因,定向克隆于逆转录病毒载体pBABEpuro,经PCR、酶切和序列分析鉴定,获取阳性重组质粒。将该重组质粒与水疱性口炎病毒载体pVSV-G共转染GP2-293细胞,收获假型病毒,并在Polybrene的介导下感染PK15细胞,嘌呤霉素筛选表达猪瘟E2基因的细胞株。通过细胞传代并结合PCR、免疫荧光及ELISA检测表明,所筛的细胞细胞系能稳定表达猪瘟E2基因,而且表达产物具有良好的生物学活性。CSFV E2 were amplified by RT-PCR using specific primers. The amplified fragment was cloned into pBABE puro vector for sequence analysis,and yield the recombinant plasmid pBABE puro-E2. Then cotransfected pBABE puro-E2 and pVSV-G into GP2-293 packaging cells by liposomes,and harvest the recombinant retrovirus, meanwhile infected PK15 cell by recombinant retroviruses under polybrene. Immunofluorescence,ELISA and PCR analysis showed that the foreign gene were integrated into the chromosome of transfected PK15 cells,and would be stably and highly expressed.

关 键 词：猪瘟C株E2基因 重组逆转录病毒载体 PK15细胞 基因表达 

分 类 号：S852.651[农业科学—基础兽医学]