摘要
为构建以J亚型禽白血病病毒(ALV-J)的LTR元件为启动子的真核表达载体,本研究将ALV-J LTR克隆至pBluescript II SK(+)载体中,并在5'LTR和3'LTR之间分别插入报告基因(eGFP)和新城疫病毒(NDV)的NP基因,得到重组质粒pSK-LTR-GFP和pSK-LTR-NP。将上述重组质粒分别转染293T细胞、BHK-21细胞和鸡胚成纤维细胞(CEF),应用RT-PCR、western blot和荧光显微镜检测目的基因的表达。结果表明,无论是在哺乳动物细胞(BHK-21和293T)还是在禽原代细胞中,LTR均能够启动外源基因(eGFP和NP)良好转录和表达。流式细胞技术分析显示,报告基因在293T细胞中的表达在48 h达到峰值。本研究为利用ALV-J LTR元件构建输送或表达外源基因的质粒载体奠定了基础。
To construct the eukaryotic expression vector used the long terminal repeat （LTR） of avian leukosis virus subgroup J （ALV-J） as a promoter, LTRs were cloned into pBluescript II SK＋ vector, subsequently, eGFP gene and NDV NP gene were inserted into 5＇LTR and 3＇LTR, respectively. The resultant recombinant plasmids were named pSK-LTR-GFP and pSK-LTR-NP. The two recombinant plasmids were transfeeted into 293T cell, BHK-21 cell and chicken embryonic fibroblast cell, and the exogenous genes were successfully expressed by detecting with indirect immunofluorescence assay and westem blot. The results showed that LTR could effeciently initiate the transcription and expression of foreign genes （eGFP and NP）. The flow cytometry results indicated that the expression level of reporter gene reached peak at 48 hours in 293T cell. This study would facilitate for developing a plasmid vector used ALV-J LTR for delivering and/or expressing exogenous gene.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2013年第10期795-798,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(31101848)
公益性农业科研专项(201003012
0080319)
国家863计划(2011AA10A200)
安徽省科技攻关战略性新兴产业项目(11010302119)
安徽省家禽产业体系资助项目
关键词
禽白血病病毒
启动子
LTR
avian leukosis virus subgroup J
promoter, LTR
